Name: a primary human trophoblast model to study the effect of inflammation associated with maternal obesity on regulation of autophagy in the placenta
Uploaded: 2017-09-27T15:00:00.000+00:00
Duration: 11 min 44 s
Description: 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

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>

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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?...

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&amp;rsquo;s Modified Dulbecco&amp;rsquo;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&amp;alpha;</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>K&lt;sub&gt;2&lt;/sub&gt;EDTA 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-&amp;beta;-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?...

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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

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

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

产妇血管重构的评估在怀孕期间在小鼠子宫

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

科研

发育生物学

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

小鼠 体内 胎盘靶向CRISPR操作

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

遗传学

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

免疫与感染

使用基于流式的方法改善离体胎盘外植体培养

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting&#160;maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new&#160;possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

作者聚焦:通过胎盘组织中的单细胞测序和空间组织学推进研究 

Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments.

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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

摘要

探索更多视频

此视频中的章节

产妇血管重构的评估在怀孕期间在小鼠子宫

人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断

小鼠体内胎盘靶向CRISPR操作

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

分离小鼠原代滋养细胞及滋养细胞侵袭实验

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

摘要

探索更多视频

此视频中的章节

产妇血管重构的评估在怀孕期间在小鼠子宫

人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断

小鼠 体内 胎盘靶向CRISPR操作

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

分离小鼠原代滋养细胞及滋养细胞侵袭实验

小鼠体内胎盘靶向CRISPR操作