我们感谢的萨沙赖希曼和多米尼克Santiard男爵为他们的帮助，在编辑RNA纯化协议。

1。日报：过程来恢复从外科块标本在实验室<ol><li>从快递公司的进口合同。 </li><li>填写10（或25）的运输形式实验室实验室的联络人（电话号码和电子邮件地址）的邮政地址。 </li><li>准备10（或25）样品编号从1到10（或25）的形式。这些形式包括专用空间通知）匿名鉴定的病人，B）的手术日期和c）任何额外的言论外科医生想补充。准备10（或25）软垫信封（150 x 210毫米）。 </li><li>用无RNA酶试剂25毫升（或65）6 M盐酸胍酸二乙酯（DEPC）处理过的H 2 O（GHCL）。 </li><li>填写10或25号5毫升无菌聚乙烯圆底管GHCL溶液2.4毫升。 </li><li>二手氩气，以填补这些管子的顶端部分，比按TI机已上的盖，以防止存储在过去几年，在手术机柜GHCL溶液氧化。 </li><li>介绍，每5毫升管50毫升无菌聚丙烯锥形筒底部用螺丝帽。使用一块干净的纸巾，保持管5毫升到50毫升管，管关闭。 </li><li>将50毫升管聚苯乙烯机架和机架到一个纸箱软垫信封，航运形式，样品形式。 </li><li>粘贴的纸板箱的盖子里面的说明（参见：1.12-1.19）​​，以方便他们阅读。 </li><li>通过邮件发送的纸箱在医院的联络人。 </li></ol>在医院<ol start="11"><li>把纸箱从手术室手术柜。注意注意：如果过程涉及免疫系统受损的患者，纸板不应该被带到手术室。 </li><li>在手术过程中，将视网膜SPECIM连接到编号为5毫升无菌聚丙烯填充GHCL的解决方案。关闭严密管。 </li><li>返回管简略。 </li><li>将试管的血管摇臂10分钟。 </li><li>填写随附的样品编号的形式。 </li><li>报告到相应编号的50毫升管的识别号码。 </li><li>介绍与手术标本管5毫升到50毫升管，取代纸巾，并拧紧管。 </li><li>引入管和填充样品形式把它在一个随附软垫信封。关闭它。 </li><li>拿起致电快递公司。 </li></ol> 2。 RNA纯化在实验室<ol><li>均质GHCL溶液用均化1分钟的聚丙烯与5毫升管中的试样移动的同时向上和向下的管子。 </li><li>加入270微升的2M乙酸钾pH值为5.0。管放置在振动筛上，在其大力摇晃10分钟水平位置（420分钟-1）。 </li><li>在5,000 rpm（6,500 XG）在20℃下离心10分钟</li><li>顺利地吸出可溶部分，而不会干扰沉淀。 </li><li>转移到一个14毫升的无菌试管。 </li><li>添加5.3毫升100毫米的Tris pH值8.0，N-月桂酰肌氨酸1％。 </li><li>添加3.2克氯化铯（CSCL），涡旋混合管。 </li><li>在一个无菌的11毫升的聚异分的离心管中，添加1.8毫升的氯化铯/乙二胺四乙酸（EDTA）。 </li><li>用10毫升无菌巴斯德移液管，管的边缘上缓慢滑动，以避免干扰的密度胶垫RNA溶液转移到1.8毫升氯化铯/ EDTA。 </li><li>入转子，将反应管（第二管中，如有必要，视网膜无缓冲区）。 </li><li> （225,000 XG）在32000转离心24小时，在20°C </li><li>用无菌巴斯德吸管取出优越的解决方案的一部分，并丢弃。 </li><li>逐步移除，同时检查时刻与第二无菌巴斯德吸管DNA（粘性）时吸气，并将其丢弃。 </li><li>小心不要释放RNA沉淀与第三无菌巴斯德吸管中取出剩余的溶液。 </li><li>第试管底部，用手术刀火焰消毒，然后把管倒挂一个无菌的目光上的剩余部分。 </li><li>扭转管和160微升GHCL微妙冲洗。 </li><li>让制粒干燥10分钟。 </li><li>将沉淀重悬于150微升（10mM的Tris pH值7.5 - 1mM EDTA的 - 0.1％SDS）。 </li><li>将溶液转移到2毫升无菌离心管中，然后收获剩余的30微升（10毫米的Tris pH值7.5 - 1毫米EDTA - 0.1％SDS）沉淀。 </li><li>加入150μl中（10mM Tris pH值7.5 - 1mM EDTA）中。 </li><li>加入30μl的3M醋酸钠pH值为5.0，涡管。 </li><li>加入900μl的100％乙醇（-20℃），涡管。 </li><li>将融化的冰管30分钟。 </li><li>岑trifuge管30分钟，以15,000 rpm于4℃。 </li><li>吸微妙可溶部分，并丢弃。 </li><li>加入500μl70％乙醇（RT），涡旋管。 </li><li>离心管20分钟，以15,000 rpm（18,000 XG）在4℃下</li><li>重复漂洗步骤（70％乙醇）。 </li><li>短暂离心，消除剩余的乙醇与P200的移液器。 </li><li>让沉淀空气干燥10分钟。 </li><li>悬浮颗粒在50μLDEPC处理的H 2 O </li><li>大力涡旋混合。 </li><li>孵育15分钟，在45℃的水浴中。 </li></ol> 3。 RNA通过凝胶电泳分析<ol><li>倒入琼脂糖凝胶化学罩内。 </li><li>在无菌1.5毫升离心管中，加入2μl的RNA进行分析和样品制备缓冲液6.4微升。 </li><li>在第二个1.5毫升试管中，加入3μl的RNA标准品和样品制备缓冲液9.6微升。 </li><li>加热管15分钟，在65℃下，把它们放在冰上。 </li><li>无染料的RNA样品管和1微升的EB样缓冲液与染料中的RNA标准品管中加入1μl溴化乙锭（EB）的上样缓冲液。 </li><li>运行的凝胶在80 - 100伏的专家在运行缓冲液的化学罩，直到其中的染料溴酚蓝到达凝胶底部的2/3的。 </li><li>冲洗凝胶，用250毫升去DEPC处理2X SSC两次15分钟。 </li><li>在紫外光照射下，以数字化图像。 </li><li>计算上带（23S rRNA的）和低频（16S rRNA基因）之间的比率。 </li></ol> 4。最终纯化的RNA <ol><li>的RNA样品的体积调整至100μlDEPC处理过的H 2 O（如有必要）。 </li><li>添加100微升酸酚（1毫升苯酚DEPC处理的H 2 O + 130微升50毫米醋酸钠，pH值5.2），涡大力饱和。“ </li><li> （15,000 XG），在室温下以13000rpm离心10分钟。 </li><li> Recoveŗ仔细和转移为一种新型无菌1.5毫升离心管中水相（上期）。 </li><li>加入10μl的3M醋酸钠，pH值5.2和250μl的100％乙醇（-20℃）。 </li><li>孵育管30分钟到融化的冰。 </li><li>离心管30分钟，每分钟14000转（18,000 XG）在4℃下</li><li>仔细吸可溶部分，并丢弃。 </li><li>添加500μl的70％乙醇（室温）下，涡管。 </li><li>离心管20分钟，每分钟14000转（18,000 XG）在4℃下</li><li>重复漂洗步骤（70％乙醇）。 </li><li>短暂离心，消除剩余的乙醇与P200的移液器。 </li><li>让空气干燥沉淀10分钟。 </li><li>到50微升DEPC处理过的H 2 O 中重悬沉淀</li><li>管15分钟，在45℃下孵育</li><li> 2微升RNA样品上，测量在260 nm和280nm处的吸光率（Abs）。的比例计算Abs260/Abs280。 </li> &lt;LI&gt;商店的RNA样品在-80°C（稳定数年）。 </li></ol> 5。解决方案和清洁程序<ol><li> 2 M醋酸钾，pH值5.0：溶解19.6醋酸钾为70毫升DEPC处理的H 2 O到用1M NaOH中浸泡15分钟，然后用DEPC处理过的H 2 O冲洗5次清洁的pH计探针用醋酸调节pH值至5.0，然后调整音量至100 mL DEPC处理的H 2 O </li><li> ，pH值8.0的Tris-HCl 100mM的N-月桂酰肌氨酸1％：将2克N-月桂酰肌氨酸（下罩）的0.5M Tris-碱pH值8.0〜40毫升。 DEPC处理的H 2 O调节音量至200毫升）中海集运/ EDTA：中海集运96克50毫升10毫米EDTA pH值7.5编写DEPC处理的H 2 O高压灭菌，并调整体积至100毫升，用DEPC处理过的H 2 O的检查pH值&lt;7.5。 </li><li>琼脂糖凝胶1％：将1克琼脂糖在62毫升DEPC处理的H 2 O在微波炉中煮。添加18毫升为12.3 M甲醛，20毫升5×MOPS。倒入化学罩内。 </li><li>样品制备缓冲器：要准备即席。混合化学罩8微升5×MOPS，16微升12.3中号甲醛，甲酰胺和40μl内。 </li><li> EB样缓冲液（不含染料）：要准备的即席。入20μl的80％的甘油，用DEPC处理过的H 2 O制备，加入4微升10毫克/毫升的溴化3,8 -二氨基-5 -乙基-6 -苯基菲啶溴化</li><li> EB样缓冲液（染料）：要准备的即席。入10μl的80％甘油中含有0.25％的红外谱图严重混叠氰DEPC-处理过的H 2 O，0.25％溴酚蓝，加入2μl10毫克/毫升的溴化3,8 -二氨基-5 -乙基-6 -苯基菲啶溴化</li><li>电泳缓冲液：60毫升5倍MOPS，增加12.3中号甲醛54毫升和186毫升DEPC处理的H 2 O </li><li>用DEPC处理过的H 2 O：0.1％体积/体积焦碳酸二乙酯2小时孵育蒸馏水中，在37℃，同时搅拌。高压灭菌消毒。 </li><li>清洁清洗液中加入25毫升（RBS）均质：1升的DEPC处理的H 2 O，并浸入轴搅拌1分钟。孵育2小时，在60℃下彻底冲洗成铝DEPC处理的H 2 O将仪器放入仪器箱。包装盒。高压灭菌消毒。 </li><li>清洁电泳设备：清洗装置，托盘和15井梳。孵育15分钟，在3％的过氧化氢。用100％乙醇冲洗一次。空气干燥。 </li><li>清洁玻璃菜：清洁玻璃板菜RBS 2％所有的夜晚，然后在200℃灭菌前用蒸馏水冲洗</li></ol>

<ol>
	<li>Mitry, D., Charteris, D. G., 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+epidemiology+of+rhegmatogenous+retinal+detachment:+geographical+variation+and+clinical+associations.">The epidemiology of rhegmatogenous retinal detachment: geographical variation and clinical associations.</a> The British Journal of Ophthalmology. 94 (6), 678 (2010).</li><li>Green, R. E., Krause, J., 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=A+draft+sequence+of+the+Neandertal+genome.">A draft sequence of the Neandertal genome.</a> Science. 328 (5979), 710 (2010).</li><li>Glisin, V., Crkvenjakov, R., 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=Ribonucleic+acid+isolated+by+cesium+chloride+centrifugation.">Ribonucleic acid isolated by cesium chloride centrifugation.</a> Biochemistry. 13 (12), 2633 (1974).</li><li>Van Gelder, R. N., von Zastrow, M. E., 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=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 87 (5), 1663 (1990).</li><li>Cavalieri, L. F., Yamaura, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+tRNAs+as+inhibitors+of+viral+reverse+transcription+in+vitro.">coli tRNAs as inhibitors of viral reverse transcription in vitro.</a> Nucleic Acids Research. 2 (12), 2315 (1975).</li><li>Sawadogo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+inhibition+of+yeast+RNA+polymerases+A+and+B+by+tRNA+and+alpha-amanitin.">On the inhibition of yeast RNA polymerases A and B by tRNA and alpha-amanitin.</a> Biochemical and biophysical research communications. 98 (1), 261 (1981).</li><li>Delyfer, M. N., Raffelsberger, W., 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=Transcriptomic+analysis+of+human+retinal+detachment+reveals+both+inflammatory+response+and+photoreceptor+death.">Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death.</a> PloS ONE. 6 (12), e28791 (2011).</li><li>Kalathur, R. K., Gagniere, N., 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=RETINOBASE:+a+web+database,+data+mining+and+analysis+platform+for+gene+expression+data+on+retina.">RETINOBASE: a web database, data mining and analysis platform for gene expression data on retina.</a> BMC Genomics. 9, 208 (2008).</li><li>Nakazawa, T., Hisatomi, T., 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=Monocyte+chemoattractant+protein+1+mediates+retinal+detachment-induced+photoreceptor+apoptosis.">Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (7), 2425 (2007).</li><li>Leveillard, T., Sahel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod-derived+cone+viability+factor+for+treating+blinding+diseases:+from+clinic+to+redox+signaling.">Rod-derived cone viability factor for treating blinding diseases: from clinic to redox signaling.</a> Science Translational Medicine. 2 (26), 26ps16 (2010).</li><li>Chalmel, F., Leveillard, T., 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=Rod-derived+Cone+Viability+Factor-2+is+a+novel+bifunctional-thioredoxin-like+protein+with+therapeutic+potential.">Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential.</a> BMC Molecular Biology. 8, 74 (2007).</li></ol>

什么都没有透露。

从手术块组织恢复发展的过程一直视网膜脱离的转录组分析的关键。人们应该注意到，这种类型的手术眼科医生经营，很少有时间参加一个生物研究计划，当他们在紧急情况和实践。此retinectomy也随机在每个服务，让简单的​​方法来达到统计上的数字是与网络。在这样的网络中，标准化组织集合是必不可少的成功的生物分析。通过提供的材料，非常容易使用和明确的指示，可在室温下储存在手?...

视网膜脱离（RD）治疗的主要目标是找到一种方法，以限制视细胞损伤和视网膜炎症产生的光感受器的视网膜色素上皮细胞的分离。道期间，视网膜色素上皮细胞被激活，迁移，脱分化，增殖的表面脱离的视网膜，施加收缩力，导致并发症。转录组分析的RD是一种识别靶基因表达修改后RD，因此未来的治疗性分子，可以提高最终视觉效果与手术的组合。脱氧核糖核酸（DNA），核糖核酸（RNA）是不稳定的，这是众所周知的，后者被广泛用于遗传研究的，其稳定性允许的来自古生物学样品中的超过30,000岁2尼安德特人基因组的测序。转录成信使RNA的基因组DNA中的基因是主要在基因表达的过程中，该mRNA是不稳定的，以构成信号。 RNA是非常迅速降解RNA酶酶终止信号。从生物体组织分离时，RNA是非常经常退化的表达之前在研究实验室研究。降解的RNA是不适合用于分析基因的表达。由于实验室工作人员不能参加手术，我们开发了一个过程，是很容易的，只需要外科医生恢复组织到合适的无RNA酶的解决方案。组织中的RNA中是稳定的，可以分析在该溶液中在室温下72小时后，没有任何降解的迹象。从标本中的RNA中纯化由一个标准化的方法涉及在氯化铯密度梯度超速离心分离后，被转移到实验室3。然后，RNA的质量，通过琼脂糖凝胶电泳，并通过RT-PCR评估的。 RNA纯化协议根据它们的密度是不同的DNA和RNA，并保证不被污染的RNA的DNA分子在基因表达研究中，将产生人造的信号的分离的分子的优点。此外，转移RNA（tRNA的），这是定量细胞内含量最丰富的RNA，分离到相同的物理属性从核糖体RNA（核糖体rRNA）和信使RNA（mRNA的），这两个最后一个是纯化过程中的最终产品。从制剂中的tRNA的去除是非常有用的，因为大多数的微阵列分析方案涉及使用反转录酶和RNA聚合酶而抑制tRNA的4-6。纯化的RNA的手术标本，使用标准协议的标记，杂交的微阵列芯片的结果进行了分析使用两个互补的方法，假发现率的方法，并使用了一种基于互信息与可视捷思上基于Web服务器Retinobase的7,8。

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Aventi J-E</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotor</td>
 <td>Beckman Coulter</td>
 <td>JS-5.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Ultracentrifuge</td>
 <td>Beckman Coulter</td>
 <td>LE 80K</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotor</td>
 <td>Beckman Coulter</td>
 <td>SW41</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>Biorad</td>
 <td>Pac 3000</td>
 <td></td>
 </tr>
 <tr>
 <td>PolytronTM</td>
 <td>Kinematica</td>
 <td>PT 2100</td>
 <td>Supplied with PT-DA 2105:2</td>
 </tr>
 <tr>
 <td>Agarose gel electrophoresis device</td>
 <td>Biorad</td>
 <td>MiniGel Cell GT</td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging System </td>
 <td>Biorad</td>
 <td>GelDoc-It Imager</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml sterile polyethylene tube</td>
 <td>Greiner</td>
 <td>115261</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile polyallomer centrifuge tube </td>
 <td>Beckman Coulter</td>
 <td>331372</td>
 <td></td>
 </tr>
 <tr>
 <td>Chemical reagents</td>
 <td>Sigma</td>
 <td></td>
 <td>Molecular biology grade (RNase and DNase free products)</td>
 </tr>
 <tr>
 <td>Cleaning Solution </td>
 <td>VWR </td>
 <td>RBS</td>
 <td></td>
 </tr>
 <tr>
 <td>Microarray chip</td>
 <td>Affymetrix</td>
 <td>Human U133 plus 2 array</td>
 <td></td>
 </tr>
 </tbody>
</table>

transcriptomic analysis of human retinal surgical specimens using journal

视网膜脱离（RD）介绍视网膜色素上皮（RPE）的视网膜神经上皮层的分离。的视网膜色素上皮的光敏感的神经元，光感受器的正常功能是至关重要的。从视网膜色素上皮脱离，视网膜创建一个物理间隙充满了细胞外液。 RD启动细胞和分子的不良事件，严重影响视网膜神经上皮层和视网膜色素上皮，因为生理的离子和代谢产物的交换严重扰动。果为视野脱离自一个的快速reapposition的两种组织的查询结果恢复视力1的持续时间有关。 RD的治疗完全是手术。去除玻璃体凝胶（玻璃体切除术）之后是除去非必需的部分分离的区域，有利于视网膜脱离的视网膜周围。删除视网膜标本是无主财产 （无），因此通常被丢弃。要恢复RNA从这些手术标本，我们开发的程序允许RNA保护杂志在从手术块转移到实验室。而且，我们标准化的协议通过氯化铯超速离心纯化RNA，以确保适用于全局基因表达分析，纯化的RNA。通过RT-PCR和微阵列分析的RNA的质量进行了验证。对数据的分析示出了同时参与炎症和光感受器变性过程中RD。

作者：（ 图1）的程序允许我们从外科手术的块恢复视网膜标本纯化的RNA，并分析视网膜脱离的转录。视网膜脱离的查询结果中的单核细胞趋化CCL2，MCP1基因的上调和杆感光体转导基因GNAT1短波锥视蛋白OPN1SW和homeogene CRX（ 图2）的表达减少。 的CCL2的感应产生的炎症9，而同时减少，GNAT1，OPN1SW CRX是光感受器变性的结果，两个?...

Watch this Scientific Journal Video about Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl at JoVE.com

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

我们使用了视网膜从retinectomy样本为转录组分析视网膜脱离。我们开发了一种程序，允许RNA保护手术块和实验室之间。我们的标准化的协议通过氯化铯超速离心法，以保证纯化的RNA是适合于微阵列分析中纯化RNA。

基因表达谱分析人视网膜手术标本杂志

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

transcriptomic-analysis-human-retinal-surgical-specimens-using

Research

JoVE Journal

Medicine

7.7K 次观看.  Institut National de la Santé et de la Recherche Médicale. 我们使用了视网膜从retinectomy样本为转录组分析视网膜脱离。我们开发了一种程序，允许RNA保护手术块和实验室之间。我们的标准化的协议通过氯化铯超速离心法，以保证纯化的RNA是适合于微阵列分析中纯化RNA。

视频: Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

一种用于兔视网膜下手术分步协议

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

使用体外活体细胞成像技术研究脂纳米粒子化疗

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

应用甲醛固定人尸体模型植入皮层微电极阵列的外科训练

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in pastoral communities. It is difficult to differentially diagnose and usually leads to irreversible neurologic deficits and spinal deformities. The initial diagnosis of BS is based on clinical findings and radiographic assessments, and the confirmed diagnosis should be established by the isolation of Brucella species from the blood and/or the standard tube agglutination test. Differential diagnosis of multifocal BS from either degenerative disc diseases or tuberculosis is especially highlighted. The surgical approach, either endoscopic or open, is demonstrated in detail, accompanied by radiographic evidence of structural compression or severe instability. Further, the crucial surgical steps, including single-stage transforaminal decompression, debridement, interbody fusion, and internal fixation, are explained. Moreover, perioperative care and postoperative rehabilitation are also addressed. Taken together, this clinical algorithm presents a practical guide that has yielded substantially satisfactory outcomes in the past decades, which can also be introduced for large-scale application to manage human BS, especially in endemic regions.

We describe a clinical algorithm, based on decades of front-line experience of diagnosis and surgical treatment of human Brucellar spondylodiscitis in the largest medical center of the China's Xinjiang Pastoral Area.

人类布鲁塞拉性皮诺皮诺皮炎的诊断和手术治疗

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

睡眠美人转座转染人视网膜色素上皮细胞氧化应激的诱导与分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...