作者感谢那些为这项研究捐出胎盘的妇女。我们还感谢站和产妇和胎儿研究小组协调收集胎盘的劳动和分娩部。我们感谢埃里克. 王博士和凯利, 他对实验方法和优化的支持和帮助。
这项工作由 NIH HD076259A (am) 和阿哈 GRNT29960007 (am) 资助。

胎盘是根据俄勒冈州波特兰健康和科学大学机构审查委员会批准的一项议定书从大学医院的劳动和分娩单位收集的, 并征得患者. 
 1. 胎盘组织的收集 <ol> <li> 准备 注: 所有遇到组织的设备都必须是无菌的。
	<ol> <li> 用灭菌在121和 #176 上对解剖设备进行消毒60分钟; C. </li>
		<li> 使用个人防护设备 (PPE): 实验室大衣/长袍/磨砂、手套和面罩, 戴口罩或护目镜. </li>
		<li> 打开水浴并设置为37和 #176; C. </li>
		<li> 暖两个50毫升圆锥管, 每个包含25毫升的完整介质 (Iscove 和 #39; s 修饰的 Dulbecco 和 #39; s 培养基补充 10% FBS 和1% 青霉素/链霉素, 表 3 ) 在37和 #176; C 水浴. </li>
		<li> 经患者知情同意后, 在剖宫产分娩后立即获得胎盘. </li>
		<li> 熟悉胎盘。脐带插入胎儿侧 (绒毛膜板), 血管可以从脐带插入部位放射出来。另一侧是母方, 或基板。它包括含有血管树的蜕膜和结构, 称为子叶. </li>
	</ol> </li> <li> 绒毛状组织的取样 注: 绒毛组织应在分娩后尽快从胎盘中取样, 最好在30分钟或更少的情况下。
	<ol> <li> 与绒毛膜板朝上, 消费2-3 全部分 (约2.5 厘米 x 2.5 厘米大小) 2-3 厘米, 从周围的胎盘随机, 使用镊子和剪刀 ( 图 1A ). 
		注意: 避免部分胎盘出现异常 (即白色钙化). </li>
		<li> 修剪掉绒毛膜和基底板以及任何大型血管. </li>
		<li> 将产生的长柔毛组织 ( 图 1B , 大约 80-120 g 总计) 放入温暖完整的介质中, 并在取样30分钟内开始滋养层隔离. 
		注意: 某些下游的化验和应用受到分娩、取样和滋养层隔离之间的潜伏期长短的影响。最好将这些期间尽可能短, 并在隔离之间保持一致. </li>
		<li> 使用步骤 1.2.1-1.2.2 中描述的技术, 从胎盘 (包括中心) 的5随机 1 cm x 1 厘米的绒毛状组织切片中取样. </li>
		<li> 将每个 1 cm x 1 厘米长柔毛组织样本切成4-5 小块 (大约30毫克), 在2毫升离心管中放置, 并在液体 N 中闪烁冻结 2 。存储在-80 和 #176; C 供以后分析使用. </li>
	</ol> </li> </ol> 2。从绒毛组织中分离滋养的研究 <ol> <li> 准备 注: 使用无菌设备并执行涉及层流罩中细胞的程序。
	<ol> <li> 解冻四冻结密度渐变 ( 表 1 , 请参阅4和 #176 的基本耗材、试剂和设备、辅助材料的表); C 在胎盘到达前的前一天晚上. 
		注意: 或者, 可以在隔离的当天进行密度渐变. </li>
		<li> 打开离心机并设置为20和 #176; C. 
		注: 除另有规定外, 所有离心步骤均达到最大加速度和减速. </li>
		<li> 准备 1x HEPES 缓冲盐溶液 (HBSS), 辅以 Ca +2 和 Mg +2 ( 表 3 ). </li>
		<li> 温热胰蛋白酶到37和 #176; C. </li>
		<li> 稀释 dnasei 到大约 2720 kilounits/毫升在无菌补充 HBSS. </li>
		<li> 暖50毫升新生小牛血清 (非华语) 到37和 #176; C. </li>
		<li> 将完整介质预热到37和 #176; C. </li>
		<li> 热冷冻介质 (90% FBS, 10% 亚砜, 表 3 ) 到37和 #176; C. 
		 警告: 亚砜是有毒的, 必须用手套处理. </li>
		<li> 准备消化缓冲 ( 表2和 3 ), 混合308毫升的补充 HBSS, 50 毫升的胰蛋白酶 (3751.7 BAEE 单位/毫升), 和0.5 毫升的 dnasei (379.4 kilounits/毫升) 在一个无菌瓶. 
		注意: 从绒毛组织中分离细胞所需的大约时间是 7 h. </li>
	</ol> </li> <li> 处理长柔毛组织 <ol> 在50毫升锥形管中冲洗每一片绒毛状组织, 填充室温磷酸盐缓冲盐水 (PBS)。重复和更换 pbs 的必要, 直到多余的血液被删除 (pbs 冲洗将是浅红色或粉红色时, 组织是彻底冲洗). </li>
		<li> 将长柔毛组织放置在无菌培养皿中, 并通过显微镜滑动将柔软的长柔毛组织从血管中取出, 以尽可能多地去除血管. </li>
		<li> 用剪刀将产生的长绒毛组织精细剁碎. </li>
	</ol> </li> <li> 长柔毛组织消化和粗隔离滋养 <ol> <li> 将切碎的绒毛状组织转移到无菌瓶中, 并根据胰蛋白酶的具体活动量计算出消化液。和 dnasei (165 毫升, 表 2 ). </li>
		<li> 在37和 #176 中孵育35分钟后; C 水浴与晃动在70转每分钟 (rpm), 倾斜消化瓶在它的边和允许未消化的片断组织在瓶的底部安定。小心地用血清吸管把上清液抽出, 避免被固定的组织. </li>
		<li> 将上清通过100和 #181; m 单元格过滤器均匀地放在50毫升锥形管之间. 
		注意: 为了节省时间, 建议在第二次消化时加入消化液 (110 毫升, 表 2) 到剩余的固定组织和恢复孵化, 如步骤2.3.2 所述. </li>
		<li> 轻轻地从试管底部的血清吸管中缓慢地分配 3-5 毫升的无张力上清液。在被劳损的上清 (含滋养) 和非控制性之间的半月板应该是可见的 ( 图 2A ). </li>
		<li> 在 1250 x g 上离心清, 15 分钟在20和 #176; C。由此产生的颗粒将包括在最下层层的红细胞, 其次是含有滋养层细胞的白细胞 ( 图 2B ). </li>
		<li> 对第二和第三个消解重复步骤 2.3.2 2.3.5 (分别添加110毫升和83.5 米, 用于组织瓶的消化液, 表 2 ). </li>
		<li> 一旦所有清都被离心, 重每个小球在5毫升的温暖完整的媒体, 然后将悬浮在一起. </li>
		<li> 将两个50毫升圆锥管和离心机在 1250 x g 之间平均拆分15分钟, 在20和 #176 处分离细胞悬浮液. </li>
		<li> 在6毫升温热详情中, 轻轻移除上清和重每一个细胞小球等媒体. </li>
	</ol> </li> <li> 密度离心式 <ol> <li> 将单元悬浮均匀地划分为四密度渐变 (每3毫升), 通过缓慢而仔细地分层在密度渐变顶部的单元悬浮, 并传递吸管. </li>
		<li> 离心密度梯度在 1250 x g 为 20 min 在20和 #176; C 以极小的加速度和减速。这将产生可区分的沉淀单元格带 ( 表 4 ). </li>
		<li> 在包含滋养层细胞的不透明带 (35 至 #160;-50% 副总经理) 到达 ( 表 4 ) 之前, 慢慢小心地移除密度梯度介质的顶端图层. </li>
		<li> 将滋养层带入两个50毫升锥形管中, 并用完整的热介质填充. </li>
		<li> 轻轻翻转管 3-6 倍, 混合和离心在 1250 x g 为15分钟, 在20和 #176; C. </li>
		<li> 在5毫升的温热完整介质中清除上清和重每个细胞颗粒。结合细胞悬浮和计数可行的细胞使用例和台盼蓝 (或优选的细胞计数法). </li>
	</ol> </li> <li> 计算具有例 <ol> <li> 的单元格, 通过移的血清吸管或通过轻轻地反转管数次, 将细胞悬浮液混合在一起. </li>
		<li> 将单元悬浮和台盼蓝等相等部分 (即20和 #181; L) 分别放入一个单独的管中, 然后轻轻混合. </li>
		<li> 室温孵育 1-2 分钟. </li>
		<li> 轻轻地免除 15-20 和 #181; 在片和例之间的细胞-台盼蓝混合物的 L, 并允许细胞通过毛细管作用在网格中扩散. </li>
		<li> 计数活细胞 (死细胞将被染色深蓝色) 在每一个四 4 x 4 象限的计数器。采用计数系统, 以确保单元格不会多次计数 (即不计算触及底部或左侧边界的单元格). 
		注: 每 4 x 4 象限应包含 50-150 单元格。太少或太多的细胞会导致细胞数量的过度或低估. </li>
		<li> 平均每个 4 x 4 象限中的单元格计数, 乘以 10 4 , 然后乘以稀释因子 (单元悬浮到台盼蓝) 来计算每毫升的细胞数. </li>
		<li> 将每个 mL 的单元格数乘以单元悬浮的总体积, 以计算总的细胞产量. 
		注: 约1亿细胞预计从隔离开始与80-120 克的绒毛组织. </li>
	</ol> </li> <li> 电镀单元格 <ol> <li> 板300万单元格/井 (3.3 x 10 5 单元每 cm 2 ) 在6井板 (2 毫升每井) 和轻轻地来回晃动和两侧均匀分布的细胞. 
		注意: 滋养需要组织培养处理过的钢板才能正确粘附。需要一个单层细胞来促进 syncytialization. </li>
		<li> 将层流罩中的镀膜细胞保持约30分钟, 使细胞均匀分布、沉淀, 并开始附着在井底, 然后放入孵化器. </li>
		在37和 #176 中为多达 72 h (每日媒体更改) 的区域性单元格. C 孵化器与 5% CO 2 和95% 湿度. 
		注意: 在显微镜下检查滋养每24小时 10 20x 的培养基。滋养不增殖, 不能传代。在 72 h 文化的过程中, 圆的个体滋养保险丝形成一个合 ( 图 3A 和 B ). </li>
	</ol> </li> <li> 冻结单元格 <ol> <li> 将未使用的单元格离心为 1250 x g, 10 分钟在20和 #176; C. </li>
		<li> 从颗粒中吸出尽可能多的介质. </li>
		<li> 在冻结介质中重颗粒 ( 表 3 ). </li>
		<li> 将等分冻结在-80 和 #176; C 在一个装满100% 异丙醇的冷冻容器中。将冻结的等分到第二天的液体 N 2 进行长期存储. 
		注意: 冷冻后细胞可以培养. </li>
	</ol> </li> <li> 解冻单元格 <ol> <li> 删除分的冻结单元格, 并在37和 #176 中进行解冻; 漩涡中的水浴。将分从水浴中取出, 然后再将其完全解冻到液体中. </li>
		<li> 立即将解冻的细胞转移到15毫升的锥形管上。开始放慢, 增加10毫升的完整媒体间歇混合. </li>
		<li> 反转管多次混合. </li>
		<li> 200 x g 离心10分钟在20和 #176; C. </li>
		<li> 在 2-5 毫升的温完整介质中吸出上清和重颗粒. </li>
		<li> 按前面所述计算单元格和板数. </li>
	</ol> </li> </ol> 3。肿瘤坏死因子和 #945 治疗原发性滋养, 细胞裂解的收集, 和西部印迹 <ol> <li> 准备 <ol> <li> 热完整介质到37和 #176; C. </li>
		<li> 制作10和 #181; 在完整的媒体和存储在-20 和 #176; 直到准备好使用. </li>
		<li> 制作1和 #181; 当准备好治疗细胞时, 在完整的培养基中, TNF 和 #945 的工作存量;连续稀释 TNF 和 #945; 工作库存到 10 4 pg/毫升, 10 3 pg/毫升, 500 pg/毫升, 250 pg/毫升, 和 125 pg/毫升在完整的媒体. 
		注: TNF 和 #945; 浓度 10 4 pg/毫升是适度的细胞毒。TNF 和 #945 的调整; 浓度测试将取决于所需的下游应用程序的具体情况. </li>
	</ol> </li> <li> 处理具有 tnf 和 #945 的单元格; <ol> 在培养 24 h 后, 从细胞中吸取培养基, 用2毫升的 tnf 和 #945 代替; 每口6孔板上的补充介质 (每种处理和车辆至少两个井)控件). </li>
		<li> 经过 24 h 的 tnf 和 #945;-暴露 (48 h 文化), 取代 tnf 和 #945; 补充媒体与完整的媒介. </li>
	</ol> </li> <li> 采集细胞和总蛋白 <ol> 在培养72小时后 (24 小时后清除 TNF 和 #945;), 吸液培养基, 用室温 PBS 轻轻冲洗细胞, 并添加80和 #181; 冰冷 Radioimmunoprecipitation 测定缓冲 (帕), 含有新鲜添加的蛋白酶和磷酸酶抑制剂 ( 表 3 ), 直接对每个井. </li>
		<li> 用刮板机将电池从盘中取出。将细胞转移到1.5 毫升离心管, 在治疗组内汇集水井. </li>
		<li> 溶解通过涡流管在高处至少三间隔十五年代. </li>
		<li> 在4和 #176 孵育细胞; C 与摇摆为 15 min. 
		注: 或者, 在步骤3.3.3 后, 细胞可以在冰上孵育15分钟, 通过弹管, 涡流, 或移的间歇搅拌</li>
		<li> 重复步骤 3.3.3 </li>
		<li> 离心管 1万 x g 为5分钟, 在4和 #176; C 到颗粒细胞碎片. </li>
		<li> 将上清液 (含细胞蛋白) 转移到新的1.5 毫升离心管, 并存储在-80 和 #176; C. 
		注意: 协议可以在这里停止并在以后恢复。建议多等分细胞蛋白样品, 避免多次冻融循环. </li>
		<li> 确定总蛋白浓度的首选方法, 如二辛可宁酸测定 (基本分析, 见表的必要用品, 试剂, 和设备, 补充材料). </li>
	</ol> </li> <li> SDS-页和西方印迹为卢比或感兴趣的蛋白质 注意: 根据制造商和 #39 的指示使用优选的实验室系统, 遵循西方印迹协议。
	<ol> <li> 在20-40 和 #181 之间加载; 在12% 丙烯酰胺凝胶上, 每井的样品缓冲 ( 表 3 ) 中的总蛋白 g。通过 SDS 页分离蛋白质 ( 表 3 ). </li>
		根据制造商和 #39 在传输缓冲区中的说明 ( 表 3 ), 将凝胶中的蛋白质转到聚偏氟 (PVDF) 膜上. </li>
		<li> 用0.1% 吐温 20 (TBST, 表 3 ) 将5% 奶粉中的膜孵育在室温下, 至少1小时的温度与摇摆. </li>
		<li> 在 TBST 的1% 奶粉中, 在一夜之间, 在4和 #176 中, 用一个卢比的主抗体 (见基本用品、试剂和设备, 补充材料的表) 孵育 1:500; C。
		<li> 在室温下轻轻地冲洗 3 x 5 分钟的 TBST 和 1 x 5 分钟的温度与摇摆. </li>
		<li> 孵育膜在 1:2000-1:5000 次抗体 (HRP 链接或首选可见共轭, 见表的基本用品, 试剂, 设备, 补充材料) 在5% 奶粉在 TBST 至少1小时室温与摇摆. </li>
		<li> 重复步骤3.4.5 中概述的洗涤过程, 并在成像系统上将印迹与适当的可视化 (即化学发光) 的基板形象化. </li>
		<li> 重复步骤3.4.5 中概述的洗涤过程, 并对薄膜进行 #946;-肌动蛋白或根据制造商和 #39 的指示进行的首选加载控制. </li>
		<li> 在首选图像分析软件上, 通过手工量化吸光度 (波段强度)、背景吸收减量和归一化到相应的加载控制波段强度来分析卢比的表达。根据需要进行统计分析, 以测试蛋白质水平的统计学意义变化. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gregor, M. F., Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+obesity.">Inflammatory mechanisms in obesity.</a> Annu. Rev. Immunol. 29, 415-445 (2011).</li><li>van Horssen, R., Ten Hagen, T. L. M., Eggermont, A. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+in+cancer+treatment:+molecular+insights,+antitumor+effects,+and+clinical+utility.">TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility.</a> Oncologist. 11 (4), 397-408 (2006).</li><li>Yogev, Y., Catalano, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy+and+obesity.">Pregnancy and obesity.</a> Obstet. Gynecol. Clin. North Am. 36 (2), 285-300 (2009).</li><li>Basu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregravid+obesity+associates+with+increased+maternal+endotoxemia+and+metabolic+inflammation.">Pregravid obesity associates with increased maternal endotoxemia and metabolic inflammation.</a> Obesity (Silver Spring). 19 (3), 476-482 (2011).</li><li>Muralimanoharan, S., Guo, C., Myatt, L., Maloyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+miR-210+expression+and+mitochondrial+dysfunction+in+the+placenta+with+maternal+obesity.">Sexual dimorphism in miR-210 expression and mitochondrial dysfunction in the placenta with maternal obesity.</a> Int J Obes (Lond). 39 (8), 1274-1281 (2015).</li><li>Myatt, L., Maloyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+and+placental+function.">Obesity and placental function.</a> Semin. Reprod. Med. 34 (1), 42-49 (2016).</li><li>Aune, D., Saugstad, O. D., Henriksen, T., Tonstad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+body+mass+index+and+the+risk+of+fetal+death,+stillbirth,+and+infant+death:+a+systematic+review+and+meta-analysis.">Maternal body mass index and the risk of fetal death, stillbirth, and infant death: a systematic review and meta-analysis.</a> JAMA. 311 (15), 1536-1546 (2014).</li><li>Eriksson, J. G., Kajantie, E., Osmond, C., Thornburg, K., Barker, D. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boys+live+dangerously+in+the+womb.">Boys live dangerously in the womb.</a> Am. J. Hum. Biol. 22 (3), 330-335 (2010).</li><li>Kliman, H. J., Knobil, E., Neill, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophoblast+to+human+placenta.">Trophoblast to human placenta.</a> Enc. Reprod. 4, 834-846 (1999).</li><li>Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+in+vitro+differentiation+of+cytotrophoblasts+from+human+term+placentae.">Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.</a> Endocrinology. 118 (4), 1567-1582 (1986).</li><li>Bax, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+changes+and+immunocytochemical+analysis+of+human+placental+trophoblast+during+short-term+culture.">Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short-term culture.</a> Placenta. 10 (2), 179-194 (1989).</li><li>Le Bellego, F., Vaillancourt, C., Lafond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+cytotrophoblast+cells+and+in+vitro+methods+for+studying+human+cytotrophoblast+cells'+calcium+uptake.">Isolation and culture of term human cytotrophoblast cells and in vitro methods for studying human cytotrophoblast cells' calcium uptake.</a> Methods Mol. Biol. 550, 73-87 (2009).</li><li>Muralimanoharan, S., Gao, X., Weintraub, S., Myatt, L., Maloyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+activation+of+placental+autophagy+in+obese+women+with+evidence+for+fetal+programming+from+a+placenta-specific+mouse+model.">Sexual dimorphism in activation of placental autophagy in obese women with evidence for fetal programming from a placenta-specific mouse model.</a> Autophagy. 12 (5), 752-769 (2016).</li><li>Matsunaga, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+Beclin+1-binding+proteins,+Atg14L+and+Rubicon,+reciprocally+regulate+autophagy+at+different+stages.">Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages.</a> Nat. Cell. Biol. 11 (4), 385-396 (2009).</li><li>Zhong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+regulation+of+autophagic+activity+by+Atg14L+and+Rubicon+associated+with+Beclin+1-phosphatidylinositol-3-kinase+complex.">Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex.</a> Nat. Cell. Biol. 11 (4), 468-476 (2009).</li><li>Yang, C. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+Protein+Rubicon+Mediates+Phagocytic+NADPH+Oxidase+Activation+in+Response+to+Microbial+Infection+or+TLR+Stimulation.">Autophagy Protein Rubicon Mediates Phagocytic NADPH Oxidase Activation in Response to Microbial Infection or TLR Stimulation.</a> Cell Host &amp; Microbe. 11 (3), 264-276 (2012).</li><li>Yang, C. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autophagy+Regulator+Rubicon+Is+a+Feedback+Inhibitor+of+CARD9-Mediated+Host+Innate+Immunity.">The Autophagy Regulator Rubicon Is a Feedback Inhibitor of CARD9-Mediated Host Innate Immunity.</a> Cell Host &amp; Microbe. 11 (3), 277-289 (2012).</li><li>Zi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rubicon+deficiency+enhances+cardiac+autophagy+and+protects+mice+from+lipopolysaccharide-induced+lethality+and+reduction+in+stroke+volume.">Rubicon deficiency enhances cardiac autophagy and protects mice from lipopolysaccharide-induced lethality and reduction in stroke volume.</a> J. Cardiovasc. Pharmacol. 65 (3), 252-261 (2015).</li><li>Mele, J., Muralimanoharan, S., Maloyan, A., Myatt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+mitochondrial+function+in+human+placenta+with+increased+maternal+adiposity.">Impaired mitochondrial function in human placenta with increased maternal adiposity.</a> Am. J. Physiol. Endocrinol. Metab. 307 (5), E419-E425 (2014).</li><li>Mor, G., Cardenas, I., Abrahams, V., Guller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+pregnancy:+the+role+of+the+immune+system+at+the+implantation+site.">Inflammation and pregnancy: the role of the immune system at the implantation site.</a> Ann N Y Acad Sci. 1221 (1), 80-87 (2011).</li><li>Resi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+inflammation+and+adipose+tissue+matrix+remodeling+precede+physiological+adaptations+to+pregnancy.">Molecular inflammation and adipose tissue matrix remodeling precede physiological adaptations to pregnancy.</a> Am. J. Physiol. Endocrinol. Metab. 303 (7), E832-E840 (2012).</li><li>Levine, B., Mizushima, N., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+immunity+and+inflammation.">Autophagy in immunity and inflammation.</a> Nature. 469 (7330), 323-335 (2011).</li><li>Virgin, H. W., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+genes+in+immunity.">Autophagy genes in immunity.</a> Nat. Immunol. 10 (5), 461-470 (2009).</li><li>Sagrillo-Fagundes, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Primary+Trophoblast+Cell+Culture+Model+to+Study+the+Protective+Effects+of+Melatonin+Against+Hypoxia/reoxygenation-induced+Disruption.">Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.</a> JoVE. (113), (2016).</li><li>Segovia, S. A., Vickers, M. H., Gray, C., Reynolds, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+obesity,+inflammation,+and+developmental+programming.">Maternal obesity, inflammation, and developmental programming.</a> Biomed. Res. Int. , 418975 (2014).</li><li>Komatsu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha+in+serum+of+patients+with+inflammatory+bowel+disease+as+measured+by+a+highly+sensitive+immuno-PCR.">Tumor necrosis factor-alpha in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR.</a> Clin. Chem. 47 (7), 1297-1301 (2001).</li><li>Matsunaga, K., Noda, T., Yoshimori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+Rubicon+to+cross+the+Rubicon.">Binding Rubicon to cross the Rubicon.</a> Autophagy. 5 (6), 876-877 (2009).</li></ol>

作者没有什么可透露的。

胎盘, 负责调节胎儿的生长, 在肥胖环境中表现出损害性功能6。尽管滋养的代谢要求很高, 但母体肥胖的胎盘表现出功能失调的线粒体呼吸6,19。胎盘代谢的变化可能有助于增加并发症的发病率和不良胎儿结局观察到肥胖怀孕3,6,7。在母体肥胖的胎盘中, 自噬也会受到损害。胎盘从肥胖怀孕与男性胎儿显示功能失调的噬通量证明通过积累的体13。维持最佳的噬通量是关键的细胞稳态和有缺陷的自噬在胎盘与母体肥胖可能发挥重要作用, 调解不良结局与肥胖怀孕。
在母体肥胖症中表现出的胎盘功能受损的确切原因尚不清楚, 可能源于炎症信号。肥胖和妊娠都是炎症状态, 其特征是循环 TNFα1、4、20、21。TNFα是一种自噬的激活剂22,23, 并可能调节胎盘中的噬通量的变化, 从肥胖怀孕。TNFα通常用于研究炎症对细胞的影响, 特别是在癌症的情况下2。在这里提出的协议适应这一方法研究的影响, 肥胖相关的炎症对胎盘的功能能力, 通过治疗原滋养与外源性 TNFα的文化。
该协议包括四主要组成部分: 从胎盘绒毛组织的取样, 从绒毛组织分离的原滋养, 原滋养的培养, 其次是 TNFα治疗, 并收集总细胞裂解其次免疫印迹分析法测定蛋白质表达的兴趣。为了隔离纯滋养, 至关重要的是, 绒毛膜和基底板在胎盘取样 (图 1B) 中从绒毛状组织中彻底剥离。还必须及时地处理绒毛组织 (即在分娩后30分钟内)。本议定书详述了三消化步骤的使用, 每一个持续35分钟, 以释放滋养从额外的细胞基质的长柔毛组织。长消解通过 trypsinization 细胞表面蛋白破坏细胞的风险。增加摘要的数量并没有明显增加可行的滋养的最终收益率。然而, 减少消解的时间和数量将导致细胞产量下降 (西蒙, 肉, 和 Maloyan, 未发布的数据)。这个协议可靠地生产大约1亿个细胞从 80-120 克长柔毛组织。
在隔离之间的密度梯度上观察到的不同滋养层带的数量有变异性。为了避免其他细胞类型的污染, 在密度梯度 (表 4) 中严格收集含有滋养的带是很重要的。此处详述的协议是从以前建立的方法中进行优化的, 它们从其他单元格类型10、1112中产生相对纯净的滋养。进一步纯化可以通过正负选择24实现。然而, 这种方法有降低可行滋养产量的风险。Cytokeratin-7 在原滋养中的免疫细胞化学分析在24和 72 h 的培养时间后, 这里所提出的程序证实了 syncytialization 核细胞质量的存在在72小时, 在滋养层的寿命中的一个标志性事件 (图 3A 和 B)。此外, 72 小时培养的原滋养中波形的免疫细胞化学分析发现极少污染成成纤维 (图 3C)。为了常规的目的, 滋养的纯度可以通过简单的形态学评估在文化中进行监测。在 24 h 文化时间, 圆的个体细胞控制文化。细胞经过72小时的 syncytialization 培养后, 形成融合细胞团簇。在这种培养中发现的最常见的污染细胞类型是成纤维细胞或内皮细胞膜, 分别拉长和多边形。这些特性可以用明亮的显微镜进行粗略的监测。
治疗原发性滋养与 TNFα提供了一个控制系统, 允许一个测量的影响, TNFα信号的调节关键通路在滋养。自然, 在肥胖产妇环境中, 除了 TNFα外, 还有其他因素在体内, 可能负责调节滋养层功能。这些包括但不限于荷尔蒙信号, 高脂血症, 和许多其他促炎因素25。不同的治疗组合将进一步重述肥胖的宫内环境前体以更加生理学的准确的方式。本协议中用于治疗滋养的外源 TNFα的浓度远远超过通常在体内循环的。血清浓度已报告达到 20 ng/毫升或更高的某些炎症条件26。为了获得现有的实验室方法 (即西方印迹) 的可测量的反应, 有必要扩大 TNFα浓度, 以揭示基因表达的变化和兴趣的途径。这些响应可能在较小的范围内存在在体内。然而, 它们仍可能对滋养的功能产生重大影响。暴露滋养高浓度的 TNFα运行的风险, 在基因表达和通路分析, 可能植根于细胞毒性效应的人工制品。在滋养中观察到 104 pg/mL TNFα补充培养基的细胞毒性, 如 LDH 细胞毒性测定 (未显示数据)。然而, 浓度在或低于 103 pg/毫升 TNFα没有产生任何可观的细胞死亡。
测试 TNFα浓度在更小的间隔和/或低于这里描述可能是最好的跟进数量聚合酶链反应 (qPCR) 用于基因表达分析, 这一技术很适合于检测基因表达的微小变化。虽然 qPCR 专门测试基因表达水平的转录水平, 西方印迹检测的最终水平的蛋白质产品, 可能可以执行他们的细胞任务 (s)。同时使用这两种分析技术是确定 TNFα治疗对基因表达水平调节的影响的有力方法, 同时也是为了确定表达水平的变化是否是转录的结果,后, 或修饰规则。炎症应激可影响滋养层的行为、形态学和 syncytialization。包括形态学评估 (即免疫细胞化学), 除了分子分析方法, 将提供更全面的分析 TNFα暴露对滋养层健康的影响。根据试验要求对 TNFα浓度进行了调整, 并进行了下游测定。
通过执行这里描述的协议, TNFα介导的炎症被发现上调的卢比表达的人滋养从精益怀孕与女性胎儿。在滋养层裂解的西部印迹显示两个不同的波段附近和高于预期的分子量 140 kDa (图 4A)。有证据表明, 较低的波段是非特异性的 (PD047 反卢比 pAb, MBL 数据表, http://ruo.mbl.co.jp/bio/g/dtl/A/？pcd=PD027#u-pub)。女性的滋养表现出上调表达的能力, 以回应 TNFα治疗浓度高达 250 pg/毫升。在高于此的浓度下, 卢比表达式水平降低到基线 (未经处理的控制), 甚至低于 (图 4B, n = 3)。鉴于治疗滋养从精益妊娠与 TNFα模拟炎症在肥胖的宫内环境, 这一结果是互补的发现, 卢比是显着上调在闪光冷冻绒毛组织活检从胎盘从肥胖怀孕与女性胎儿相比, 精益控制 (图 4C 和 D, 方差分析, n = 6 每 BMI 类, P & #60; 0.05)。
虽然噬通量似乎没有区别滋养从肥胖怀孕与女性胎儿相比, 精益控制, 滋养从肥胖怀孕与男性胎儿表现出活化的形成和积累的体13. 卢比是自噬的负调节器, 它的作用是停止 autophagosome-溶融合27。女性滋养可能上调表达的卢比作为补偿或保护机制, 以对抗 TNFα介导的炎症, 这可以激活自噬的22, 使他们保持精益的噬通量的设置孕妇肥胖症胎盘中的这种反应可能在肥胖的宫内环境中对女性胎儿的比男性更有影响。建立与母体肥胖相关的炎症环境的模型前体通过治疗原发性人滋养的 TNFα提供了一个平台, 研究肥胖宫内环境对关键通路调节的影响滋养.通过新的和组合疗法来综述肥胖孕产妇环境来修正这一议定书, 将为研究孕产妇肥胖对胎盘功能的影响提供一个令人振奋的途径。

肥胖是一种炎症状态, 其特点是慢性低度炎症, 来源于过量的脂肪组织和养分的供应。在肥胖症中, 促炎细胞因子在代谢组织中升高, 并在循环系统中具有系统性。一个强有力的证据显示, TNFα在肥胖的设置中显著升高, 而胰岛素抵抗和代谢功能障碍的影响是1。活化 TNFα也有助于疾病的发病机制, 如癌症和自身免疫, 使其成为一个有吸引力的治疗目标2。
肥胖的炎症是由妊娠加重的, 也是一个炎症状态3,4。以前的结果表明, 胎盘 TNFα的含量随着孕产妇的肥胖而增加。此外, TNFα治疗抑制雌性而非雄性滋养层细胞的线粒体呼吸, 表明 TNFα参与了以性二方式调节胎盘代谢的作用5。孕产妇肥胖与妊娠期间各种并发症的发病率增加有关, 包括死胎, 男性胎儿最易受感染3,6,7,8.由于其在母体-胎儿界面的关键作用, 在肥胖宫内环境中胎盘功能的变化对炎症信号的反应可能在调节肥胖妊娠结局中起重要作用。
细胞和合体在胎盘的绒毛状组织是关键的内分泌信号和营养和氧气交换之间的母亲和发育的胎儿9。绒毛细胞 (以下简称滋养) 功能能力的紊乱可能危及胎儿的健康和发育。本协议描述了一种从人类足月胎盘中提取绒毛组织的方法, 通过剥离绒毛膜和基底板, 以及一个优化的分离滋养原细胞培养的程序。该协议是从建立的方法, 涉及酶消化的绒毛组织, 释放细胞从胞外基质后, 差密度离心分离滋养10, 11,12。本议定书详细介绍了一种方法, 其中主要滋养来自胎盘从精益怀孕的治疗与培养基辅以 TNFα, 以模拟一个组成部分的炎症环境与孕产妇肥胖。最后, 本文介绍了一种从 TNFα处理后的滋养中提取总细胞裂解的方法, 并对其进行了探讨, 以检测基因表达的变化。
虽然此模型不重述致在子宫内环境中的整体, 但它提供了一个受控系统, 允许您解析 TNFα介导的炎症在滋养的反应中的个人贡献孕妇肥胖症这个模型既提供了发现或确认分子靶点的机会直接调控的 TNFα信号在滋养, 并且允许一个测试, 如果基因表达模式的变化观察到的在体内在胎盘与母体肥胖可能是 TNFα介导的炎症的结果。
本文所介绍的方法是为了测试 TNFα介导的炎症对人类滋养自噬的调节作用。滋养从肥胖怀孕与男性胎儿陈列被打乱的噬转交, 或者 autophagosome 成熟13。一种称为卢比的蛋白质 (运行域蛋白 Beclin1-interacting 和富含半胱氨酸的含), 这是本地化的溶酶体和晚期体, 最近被描述为 "刹车" 在噬的周转过程中, 因为它的功能作为一个负autophagosome 成熟的调节器14,15。事实上, 卢比是抑制自噬的一个罕见的例子, 它使它成为一个有价值的治疗目标。很少有关于卢比的病理生理学意义的信息, 除了它在先天免疫应答微生物16、17和心肌细胞保护18中的作用。使用这里描述的协议, 它发现, 卢比是上调在女性原发性滋养的反应, 以增加浓度的 TNFα高达 250 pg/毫升的治疗。在孕产妇肥胖时, 对卢比的调节可能会影响女性胎儿的体重。综述炎症与孕产妇肥胖相关前体通过暴露人滋养外源性 TNFα提供了一个平台, 研究肥胖宫内环境对关键通路调节的影响滋养和延伸, 胎盘功能。

<table>
	<tbody>
		<tr>
			<td>10X HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 (anhyd.)</td>
			<td>Sigma-Aldrich</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 (anhyd.)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease/Phosphatase inhibitors</td>
			<td>Thermofisher Scientific</td>
			<td>88668</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>Invitrogen</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15576-028</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Fisher Scientific</td>
			<td>BP166-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate.</td>
			<td>Fisher Scientific</td>
			<td>AAJ6228822</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#8217;s Modified Dulbecco&#8217;s Medium (IMDM)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Neonatal Calf Serum (NCS)</td>
			<td>Gibco</td>
			<td>26010-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (Pen/Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Formalin</td>
			<td>Fisher Scientific</td>
			<td>23-427-098</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3177-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>70013-032</td>
			<td></td>
		</tr>
		<tr>
			<td>K2EDTA vacutainer blood collection tubes</td>
			<td>BD</td>
			<td>366450</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (Density Gradient Media, DGM)</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 2.0 mL, natural</td>
			<td>Fisher Scientific</td>
			<td>22363352</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Corning</td>
			<td>25-900-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Mini-PROTEAN Tetra System</td>
			<td>Bio-Rad</td>
			<td>1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Mini Trans-Blot Cell</td>
			<td>Bio-Rad</td>
			<td>1658033</td>
			<td></td>
		</tr>
		<tr>
			<td>TGX FastCast Acrylamide Kit, 12%</td>
			<td>Bio-Rad</td>
			<td>1610175</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Protean 3 Multi-Casting Chamber</td>
			<td>Bio-Rad</td>
			<td>1654112</td>
			<td></td>
		</tr>
		<tr>
			<td>4X Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Bio-Rad</td>
			<td>1610718</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Nonfat Dry Milk</td>
			<td>Carnation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rubicon (D9F7) Rabbit mAb</td>
			<td>Cell Signalling Technology</td>
			<td>8465S</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-&#946;-Actin antibody produced in mouse</td>
			<td>Sigma-Aldrich</td>
			<td>A2228-100UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody</td>
			<td>Cell Signalling Technology</td>
			<td>7074S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signalling Technology</td>
			<td>7076S</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34578</td>
			<td></td>
		</tr>
	</tbody>
</table>

a primary human trophoblast model to study the effect of inflammation associated with maternal obesity on regulation of autophagy in the placenta

孕产妇肥胖与不良围产期结局的风险增加有关, 可能是由胎盘功能受损引起的, 部分原因是自噬的失调。肥胖妊娠胎盘中自噬调节因子表达的异常变化可能由与肥胖和妊娠有关的炎症过程调节。这里描述的是一个协议的绒毛组织和绒毛细胞的分离, 从一项人类胎盘的原代细胞培养。其次是一种方法模拟的炎症环境, 在肥胖的宫内环境中, 治疗原发性滋养从精妊娠与肿瘤坏死因子α (TNFα), 一个促炎细胞因子, 是升高的肥胖和怀孕.通过这里所描述的协议的实施, 发现暴露于外源性 TNFα调节了滋养的表达, 这是一种自噬的负调节, 来自于女性胎儿的精益妊娠。虽然在肥胖的宫内环境中的各种生物因素保持了在滋养中调节关键通路的潜能, 这个ex 体内系统特别适用于确定表达模式是否观察到了体内在人类胎盘中, 孕产妇肥胖是 TNFα信号的直接结果。最终, 这种方法提供了一个机会来分析与母体肥胖有关的炎症的调控和分子影响, 对滋养的自噬和其他重要的细胞通路有可能影响胎盘功能。

足月人胎盘从精益 (前身体质量指数 (BMI) 和 #60; 25) 在分娩后15分钟内采集和取样产妇, 并进行剖宫产 (无分娩)。胎盘检查没有钙化和典型的发展: 体重之间的300-600 克与脐带和膜移除, 圆形, 在 15-25 厘米直径, 和脐带插入中间胎盘.从胎盘 (图 1) 的2-3 样本中分离出基部和绒毛膜板的绒毛组织, 产生约100克的绒毛组织作为初级滋养层隔离的起始材料。在取样长柔毛组织的20分钟内, 分离原滋养的过程就开始了, 这里描述的是 0.8-1 x 108存活的细胞。细胞在6井文化板材被播种了在密度 3 x 106 (3.3 x 105细胞每 cm2)。经过24小时的培养, 细胞被检查在显微镜下的依恋和适当的滋养层形态学确认 (单个圆形细胞)。培养基被完全介质所取代, 其中含有一系列浓度的 TNFα在 125-104 pg/毫升之间, 因此至少有两口井包括每浓度和车辆控制 (仅限于完整介质)。
二十四小时后 TNFα处理 (48 h 文化), 所有 TNFα媒体被替换为完全媒介。没有观察到明显的细胞死亡, 由于治疗 TNFα在浓度或低于 103 pg/毫升。治疗与 104 pg/毫升 TNFα是适度的细胞毒和这种 TNFα浓度的细胞毒性作用没有坚持后, 媒体改变, 如证明乳酸脱氢酶 (LDH) 化验 (数据没有显示)。在72小时的培养基中, 在显微镜下检查细胞 syncytialization。syncytialization 和成纤维细胞污染的免疫组织化学显示滋养的相对纯净的隔离 (图 3)。细胞裂解被收获根据这里描述的协议, 屈服在3-8 µg/µL 的总蛋白的每准备由一个定量测定 (未显示的数据)。TNFα治疗女性滋养细胞裂解的免疫印迹分析显示, 在 TNFα浓度的反应中, 下调的浓度为 250 pg/毫升, 随后 TNFα的卢比表达量为上调。超过 250 pg/毫升 (图 4A 和 B, 104 pg/ml 排除在基于细胞毒性作用的分析之外)。同样, 在从胎盘的肥胖妊娠与精益控制相比, 上调在闪光冷冻绒毛组织活检中的卢比是显著的, 如西方印迹分析 (图 4C 和 D, n = 6每个 BMI 类, 方差分析, P 和 #60 的胎盘; 0.05)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">浓度 (%)</td>
			<td>90% 副总经理 (毫升)</td>
			<td>1x HBSS (毫升)</td>
			<td>层厚 (毫升)</td>
		</tr>
		<tr>
			<td>4x 渐变</td>
			<td>4x 渐变</td>
			<td>共34.5 毫升</td>
		</tr>
		<tr>
			<td>70</td>
			<td>14</td>
			<td>4</td>
			<td>4。5</td>
		</tr>
		<tr>
			<td>60</td>
			<td>8</td>
			<td>4</td>
			<td>3</td>
		</tr>
		<tr>
			<td>55</td>
			<td>7.33</td>
			<td>4.67</td>
			<td>3</td>
		</tr>
		<tr>
			<td>50</td>
			<td>3.34</td>
			<td>2.67</td>
			<td>3</td>
		</tr>
		<tr>
			<td>45</td>
			<td>6</td>
			<td>6</td>
			<td>3 (13.5 毫升标记)</td>
		</tr>
		<tr>
			<td>40</td>
			<td>5.33</td>
			<td>6.67</td>
			<td>3</td>
		</tr>
		<tr>
			<td>35</td>
			<td>4.67</td>
			<td>7.33</td>
			<td>3 (19.5 毫升标记)</td>
		</tr>
		<tr>
			<td>30</td>
			<td>8</td>
			<td>16</td>
			<td>6</td>
		</tr>
		<tr>
			<td>20</td>
			<td>2.67</td>
			<td>9.33</td>
			<td>3</td>
		</tr>
		<tr>
			<td>10</td>
			<td>1.33</td>
			<td>10.67</td>
			<td>3</td>
		</tr>
	</tbody>
</table>
表1。主要滋养密度离心密度梯度的制作规范。 从左到右, 第一列指定密度梯度介质 (副总经理, 见表的基本用品, 试剂, 和设备, 补充材料) 集中表示为副总经理在 HBSS 的百分比。第二列指定副总经理的数量, 而列三指定了使副总经理解决方案的适当百分比所需的 HBSS 量。四列指定要添加到 50 mL 锥形管中以生成渐变的卷, 从最稠密的层开始。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>胰蛋白酶</td>
			<td>HBSS</td>
			<td>dnasei</td>
			<td></td>
		</tr>
		<tr>
			<td>消化</td>
			<td>(活动总数;BAEE 单位)</td>
			<td>容积 (毫升)</td>
			<td>(活动总数;Kunits)</td>
			<td>总容积 /消化</td>
		</tr>
		<tr>
			<td>1</td>
			<td>619037 (23.01 毫升)</td>
			<td>141.76 毫升</td>
			<td>62594 (0.230 毫升)</td>
			<td>165毫升</td>
		</tr>
		<tr>
			<td>2</td>
			<td>412691 (15.34 毫升)</td>
			<td>94.51 毫升</td>
			<td>41729 (0.154 毫升)</td>
			<td>110毫升</td>
		</tr>
		<tr>
			<td>3</td>
			<td>313270 (11.65 毫升)</td>
			<td>71.74 毫升</td>
			<td>31676 (0.116 毫升)</td>
			<td>83.5 毫升</td>
		</tr>
		<tr>
			<td>总</td>
			<td>1345000 (50 毫升)</td>
			<td>308毫升</td>
			<td>136000 (0.5 毫升)</td>
			<td>358.5 毫升</td>
		</tr>
	</tbody>
</table>
表2。根据 dnasei 和胰蛋白酶的特异活性, 制备原发滋养层分离的消化液的规范。 从左到右, 第一列指定消解的数量, 第二列指定每个消化所需的胰蛋白酶活性, 第三列指定为适当的消化添加的补充 HBSS 的总体积,第四列指定每个消化所需的 dnasei 活动, 最后一列指定消化溶液的体积添加到胎盘组织中进行适当的消化。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HBSS (补充与 Ca+2和 Mg+2)</td>
			<td>示例缓冲区</td>
		</tr>
		<tr>
			<td>10% 10x HBSS</td>
			<td>90% 4X Laemmli 染料</td>
		</tr>
		<tr>
			<td>1.26 mM CaCl2 (anhyd)</td>
			<td>10% 2-基</td>
		</tr>
		<tr>
			<td>0.80 mM MgSO4 (anhyd)</td>
			<td></td>
		</tr>
		<tr>
			<td>20.77 毫米 HEPES</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 值为 7.4, 10N 氢氧化钠 使体积高达1升与无菌 ddH2O 无菌过滤器成无菌瓶</td>
			<td></td>
		</tr>
		<tr>
			<td>完整的媒体</td>
			<td>运行缓冲区</td>
		</tr>
		<tr>
			<td>删除11% 伏/v IMDM</td>
			<td>25毫米三垒</td>
		</tr>
		<tr>
			<td>添加 10% v/v FBS</td>
			<td>190毫米甘氨酸</td>
		</tr>
		<tr>
			<td>添加 1% 1万 u/毫升青霉素/链霉素 (100 u/毫升最终)</td>
			<td>0.1% SDS</td>
		</tr>
		<tr>
			<td></td>
			<td>pH 值为8。3</td>
		</tr>
		<tr>
			<td>冷冻介质</td>
			<td></td>
		</tr>
		<tr>
			<td>90% 伏/伏 FBS</td>
			<td>传输缓冲区</td>
		</tr>
		<tr>
			<td>10% 伏/五甲基亚砜</td>
			<td>25毫米三</td>
		</tr>
		<tr>
			<td></td>
			<td>190毫米甘氨酸</td>
		</tr>
		<tr>
			<td>消化缓冲</td>
			<td>20% 甲醇</td>
		</tr>
		<tr>
			<td>50毫升胰蛋白酶 (26900 BAEE 单位/毫升)</td>
			<td>pH 值为8。3</td>
		</tr>
		<tr>
			<td>0.5 毫升 dnasei (27.2万 K 单位/毫升)</td>
			<td></td>
		</tr>
		<tr>
			<td>带到358.5 毫升补充 HBSS</td>
			<td>tbs</td>
		</tr>
		<tr>
			<td></td>
			<td>20毫米三</td>
		</tr>
		<tr>
			<td>帕缓冲区</td>
			<td>150毫米氯化钠</td>
		</tr>
		<tr>
			<td>25毫米三盐酸</td>
			<td>pH 值为7。6</td>
		</tr>
		<tr>
			<td>5毫米 EDTA</td>
			<td></td>
		</tr>
		<tr>
			<td>150毫米氯化钠</td>
			<td>TBST</td>
		</tr>
		<tr>
			<td>0.1% SDS</td>
			<td>TBS 与0.1% 吐温20</td>
		</tr>
		<tr>
			<td>0.5% 胆酸钠</td>
			<td></td>
		</tr>
		<tr>
			<td>1% 海卫 X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>每10毫升帕缓冲剂1片蛋白酶/磷酸酶抑制剂</td>
			<td></td>
		</tr>
	</tbody>
</table>
表3。主要滋养的分离和培养所需的溶液, 其次是印迹。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>副总经理</td>
			<td>ml 标记</td>
			<td>单元格类型</td>
		</tr>
		<tr>
			<td>10</td>
			<td>31.5-34。5</td>
			<td>碎片</td>
		</tr>
		<tr>
			<td>20</td>
			<td>28.5-31。5</td>
			<td></td>
		</tr>
		<tr>
			<td>30</td>
			<td>22.5-28。5</td>
			<td></td>
		</tr>
		<tr>
			<td>35</td>
			<td>19.5-22。5</td>
			<td>滋养</td>
		</tr>
		<tr>
			<td>40</td>
			<td>16.5-19。5</td>
			<td></td>
		</tr>
		<tr>
			<td>45</td>
			<td>13.5-16。5</td>
			<td></td>
		</tr>
		<tr>
			<td>50</td>
			<td>10.5-13。5</td>
			<td>淋巴细胞</td>
		</tr>
		<tr>
			<td>55</td>
			<td>7.5-10。5</td>
			<td></td>
		</tr>
		<tr>
			<td>60</td>
			<td>4.5-7。5</td>
			<td>血红细胞</td>
		</tr>
		<tr>
			<td>70</td>
			<td>4.5 以下</td>
			<td></td>
		</tr>
	</tbody>
</table>
表4。密度离心法沉淀滋养。 从左到右, 第一列指定副总经理的百分比 (表 1), 第二列指定了在50毫升圆锥管上找到副总的相应百分比的 ml 标记, 第三列指定了什么单元格类型的沉积物在50毫升锥形管上的副总经理和 ml 标记的相应百分比。滋养沉积在 50-35% 副副总之间, 形成明显的不透明带。在这一范围内或下面收集副副产物会导致细胞碎片和其他细胞类型如淋巴细胞的污染。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56484/56484fig1.jpg"> 
图1。绒毛组织被分离从期限人的胎盘通过去除绒毛膜和基板。 
a)与绒毛膜板 (胎侧) 正面朝上, 从胎盘中切除一个完整的厚度样本。B)通过除去绒毛膜和基底板来获得绒毛组织的样本。<a href="//ecsource.jove.com/files/ftp_upload/56484/56484fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56484/56484fig2.jpg"> 
图2。消化液中的细胞在新生小牛血清中的离心会产生多层细胞颗粒。 
a)新生小牛血清 () 在50毫升锥形管的消化液中, 在细胞悬浮液的下面分层。B)离心 (a) 产生多层细胞颗粒。最底层的层深红色, 由红血球组成。上面的图层包括滋养, 颜色为白色或米色。在滋养层之上, 是在管的顶端, 然后是消化液 (上清)。<a href="//ecsource.jove.com/files/ftp_upload/56484/56484fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56484/56484fig3.jpg"> 
图3。原发性人滋养层培养物中 Syncytialization 和成纤维细胞含量的免疫分析。 
A) Cytokeratin-7 (红色) 在细胞24小时后的代表性图像。B) Cytokeratin-7 (红色) 在合体 72 h 后的代表性图像显示核大量的细胞融合。C)波形 (红色) 在合体72小时后的代表性图像。图像是在荧光显微镜下获得的, DAPI (蓝色) 核 counterstain。10X 放大倍率可视化。<a href="//ecsource.jove.com/files/ftp_upload/56484/56484fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigi alt="Figure 4" class=""></img alt="Figure 4" class="xfigi>mg "src ="/文件/ftp_upload/56484/56484fig4 
图4。TNFα对女性滋养的治疗反应中的卢比表达调控及从瘦体与肥胖妊娠到女性胎儿的内源性卢比表达。 主要滋养从足月胎盘与健康怀孕的孕妇携带的女性胎儿被隔离和治疗 125, 250, 500, 103, 和 104 pg/毫升 TNFα (或车辆控制)。A)用 TNFα治疗女性滋养层裂解的典型西方印迹。β-肌动蛋白被用作加载控制。B)对雌性滋养 TNFα治疗的卢比表达式进行了量化, 从西方的印迹和规范化的β-肌动蛋白。价值是平均卢比表达每 TNFα浓度±东南亚在 n=3 胎盘。C)从精益与肥胖妊娠 (F1-F12) 的裂解绒毛组织的闪光冷冻活检中, 对整个组织中的卢比进行西印迹。乳酸脱氢酶 a (LDHA) 被用作负载控制。D)从 (C) 的西方印迹中的卢比表达式被量化并归为 LDHA。值是平均每 bmi 分类±东南亚在 n=6 胎盘每 bmi 类 (方差分析, * P 和 #60; 0.05)。<a href="//ecsource.jove.com/files/ftp_upload/56484/56484fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta at JoVE.com

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

本文介绍了一种用于人胎盘绒毛组织的取样方法, 其次是分离细胞原代细胞培养。滋养治疗 TNFα重述炎症在肥胖的宫内环境和促进发现的分子靶调控的炎症在胎盘孕产妇肥胖。

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

a-primary-human-trophoblast-model-to-study-effect-inflammation

Universidad San Sebastian

Research

JoVE Journal

Medicine

11.3K 次观看.  Oregon Health and Science University. 本文介绍了一种用于人胎盘绒毛组织的取样方法, 其次是分离细胞原代细胞培养。滋养治疗 TNFα重述炎症在肥胖的宫内环境和促进发现的分子靶调控的炎症在胎盘孕产妇肥胖。

视频: A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

科研

医学

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

研究辅助供氧对囊性纤维化气道微生物组影响的模型的设计与开发

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

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