笔者想感谢艾萨克·桑布拉诺博士及其工作人员在曼彻斯特眼科医院眼库的人供体眼组织的供应，以及生命科学组织学机构为H＆E染色图像学院的皮特·沃克先生。特别感谢罗杰·梅多斯先生对他的帮助与显微镜。 SJC是一个医学研究理事会的接受者（MRC）职业发展奖学金（MR / K024418 / 1），作者也承认，从MRC（G0900538和K004441）等近期的研究经费，争取视力（1866年）和黄斑社会。

从曼彻斯特皇家眼科医院眼库下去除角膜移植获得人体眼组织同意用于研究目的。利用研究人体组织是按照人体组织法案（2004）的指导方针，并与大学研究伦理委员会的批准（参考11305）。 1.丰富全布鲁赫的膜<ol><li>围绕清洁工作空间和解剖显微镜用1％消毒剂下方（体积/体积）中，然后用70％（体积/体积）乙醇擦拭。 </li><li>确保以下的手:一种新的一次性手术刀，两人育细胞刮刀（组织培养中常用）三对镊子（此处列出的A，B和C，见材料清单）。 </li><li>将眼地球到一次性培养皿，保持盖以备后用。使用新的一次性手术刀，除去残留的视神经，这可能会干扰建立一个扁平月UNT的组织。其次，提出四点切口等间隔创建四个象限，如果在进行第3部分（黄斑布鲁赫的膜浓缩）不通过黄斑区切。 <ol><li>确保该切口穿过的眼组织的整个深度延伸 - 从最里面的视网膜神经上（NSR），通过脉络膜和巩膜（眼睛的白色）。继续切口周围的眼罩视神经的保证金眼球的方式。这将允许眼罩被变平（参见图1B）。 </li></ol></li><li>保持使用镊子甲扁平眼罩的一个象限（防止脉络膜分离），可使用较大的镊子乙夹住玻璃体和底层NSR和拉出此组织离视网膜色素上皮细胞（RPE）开始在外围，移向中心打开的眼罩，然后到相对侧。一般，但并不总是既玻璃体和NSR分离为一体。使用scalpel至切除锚定在视盘任何残留NSR。 </li><li>同样，在出任组织的一个象限的地方使用镊子A，用无菌细胞刮刀从眼罩的整个内表面轻轻打跑RPE单层。这些细胞容易分离，并且可以被看作是深褐色颜料团块。用PBS冲洗从眼罩轻轻洗去脱落的视网膜色素上皮细胞。 </li><li>接下来，使用镊子A到所有4个象限的眼睛轻轻剥离覆暗红色的组织（包括两个Bruch膜和脉络膜）。将切除的组织到一个干净的菜。 </li><li>用一个细胞刮刀的扁平边缘以保持切除的组​​织到位，使用第二个刮刀轻轻刮去组织。打开组织上，重复，去掉尽可能多的多余的材料越好。最终，半透明膜仍将:这是粗略地丰富了布鲁赫的膜。至少20X放大率的使用解剖显微镜的是至关重要的帮助去除脉络布鲁赫的膜和充实。 </li><li>使用巴斯德吸管，简要地冲洗Bruch膜用超纯水将所述膜至1.5 ml微量离心管中之前，以除去残余脉络膜和血液。 </li><li>暂停富集Bruch膜在约1 ml的超纯水和涡流简要（约15秒），以帮助溶解任何污染的细胞物质（ 参见图2）。离心样品在500×g离心30秒以沉淀膜​​，吸上清。 注:其余富集Bruch膜现在准备溶解以后或存储（如在第2下面描述）在-80℃下以供使用。 </li></ol> 2.全溶富Bruch膜和Western Blot分析<ol><li>浸泡在500μl8M的乌尔组织提取丰富布鲁赫的膜蛋白EA 6小时在RT在1.5ml微离心管（图3A）。 </li><li>以10,000×g离心10分钟，离心溶解样品以沉淀任何残留的细胞外物质。除去上清液，并将其转移到3500沓MWCO透析膜，然后将样品在1升的PBS中在4℃至透析16小时。 </li><li>对于每个样品，加入40微升蛋白质分离珠和旋转样品/珠混合物（20rpm下在旋转混合器）在RT 30分钟。 </li><li>离心样品以10,000×g离心5分钟以沉淀珠。除去上清液和溶解蛋白质结合珠粒于30μl2×上样缓冲液（65.8毫Tris-盐酸，pH为6.8，2.1％SDS中，26.3％（重量/体积）甘油，0.01％溴酚蓝，3％β巯基乙醇）。 </li><li>孵育样品在100℃下进行10分钟，然后离心以10,000×g离心5分钟以沉淀剩余的珠。 </li><li>加载包含所释放的蛋白质上，以一个预制上清液的SDS-页面梯度凝胶（见材料清单）的蛋白质分离。 </li><li>可视化使用共同的染色方案，例如，考马斯蓝分离的蛋白条带。 <ol><li>简言之，加入20毫升亮蓝染色（0.1％亮蓝（重量/体积），50％甲醇，10％冰乙酸，40％水）的凝胶，并孵育30分钟，在室温温和搅拌。 </li><li>丢弃染色液并用去离子水洗涤染色的凝胶直至背景染色被充分减小，以使染色蛋白带可视化。 </li><li>或者，转印分离蛋白带至硝酸纤维素为西方分析，如下所述。 </li></ol></li><li>转移分离的蛋白质条带至硝化纤维膜，在80毫安使用在转移缓冲液半干转印装置（25毫摩尔Tris，192 mM的甘氨酸，10％[体积/体积]甲醇）2.5小时。 </li><li>阻挡膜在10毫升PBS中，10％（重量/体积）奶粉和0.02％（重量/体积）BSA的16小时，在4℃日之前Ë另外的蛋白质检测所需的主要抗体。 注意:如这里的示例， 图3C示出了溶解Bruch膜的代表性免疫印迹从三个独立供体探查补体级联，因子H样蛋白1（FHL-1）的一个调节器。 </li></ol>黄斑布鲁赫的膜3富集<ol><li>按照步骤1.1 - 1.3如前所述。 </li><li>虽然NSR仍然就位，定位中心凹黄色着色，代表该中央黄斑区（ 见图 4）。使用无菌6毫米活检穿孔器，并将其放置在黄斑区上方，坚决削减黄斑。 <ol><li>保持活检打孔的地方，做一个单独的切口与眼罩的边缘手术刀活检凸模刃口边缘，然后使用镊子B，剥离周围的眼球材料，实现了清洁活检。 </li></ol></li><li>使用镊子C，删除组织来源于清洁培养皿盖的冲头和地点6毫米光盘。放置在解剖显微镜下的培养皿的盖子和黄斑一拳。 </li><li>拆下细NSR盘（检查是否有黄色中心凹染色来推断活检的准确度）。使用无菌细胞刮刀轻轻取出视网膜色素上皮细胞。 </li><li> 通过使用镊子A的尖端巩膜固定黄斑光盘，慢慢剥去脉络膜和使用镊子C.同样Bruch膜复合物，使用细胞刮棒从Bruch膜除去不需要的脉络膜和血液物质。 </li><li>放置黄斑Bruch膜至1.5 ml微量离心管中，并暂停在1ml超纯水中。短暂涡旋（约15秒），以帮助溶解任何污染的细胞物质。离心样品在500×g离心30秒以沉淀膜​​，吸上清。 注意:富集Bruch膜从黄斑分离现在可用于蛋白质组的使用alysis用户首选消化协议。 </li></ol>

<ol>
	<li>Coleman, H. R., Chan, C. -. C., Ferris, F. L., Chew, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> Lancet. 372, 1835-1845 (2008).</li><li>Wong, W. 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=Global+prevalence+of+age-related+macular+degeneration+and+disease+burden+projection+for+2020+and+2040:+A+systematic+review+and+meta-analysis.">Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: A systematic review and meta-analysis.</a> Lancet Glob Health. 2 (2), e106-e116 (2014).</li><li>Coleman, A. 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=Impact+of+age-related+macular+degeneration+on+vision-specific+quality+of+life:+Follow-up+from+the+10-year+and+15-year+visits+of+the+study+of+osteoporotic+fractures.">Impact of age-related macular degeneration on vision-specific quality of life: Follow-up from the 10-year and 15-year visits of the study of osteoporotic fractures.</a> Am J Ophthalmol. 150 (5), 683-691 (2010).</li><li>Hageman, G. 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=A+common+haplotype+in+the+complement+regulatory+gene+factor+H+(HF1/CFH)+predisposes+individuals+to+age-related+macular+degeneration.">A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.</a> Proc Natl Acad Sci U S A. 102 (20), 7227-7232 (2005).</li><li>Klein, R. 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=Complement+factor+H+polymorphism+in+age-related+macular+degeneration.">Complement factor H polymorphism in age-related macular degeneration.</a> Science. 308 (5720), 385-389 (2005).</li><li>Haines, J. 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=Complement+factor+H+variant+increases+the+risk+of+age-related+macular+degeneration.">Complement factor H variant increases the risk of age-related macular degeneration.</a> Science. 308 (5720), 419-421 (2005).</li><li>Edwards, A. O., Ritter, R., Abel, K. J., Manning, A., Panhuysen, C., Farrer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+factor+H+polymorphism+and+age-related+macular+degeneration.">Complement factor H polymorphism and age-related macular degeneration.</a> Science. 308 (5720), 421-424 (2005).</li><li>Fritsche, L. 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=Seven+new+loci+associated+with+age-related+macular+degeneration.">Seven new loci associated with age-related macular degeneration.</a> Nat Genet. 45 (4), 433-439 (2013).</li><li>Clark, S., Bishop, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Factor+H+and+Related+Proteins+in+Regulating+Complement+Activation+in+the+Macula,+and+Relevance+to+Age-Related+Macular+Degeneration.">Role of Factor H and Related Proteins in Regulating Complement Activation in the Macula, and Relevance to Age-Related Macular Degeneration.</a> J Clin Med. 4 (1), 18-31 (2014).</li><li>Booij, J. C., Baas, D. C., Beisekeeva, J., Gorgels, T. G. M. F., Bergen, A. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+nature+of+Bruch&#8217;s+membrane.">The dynamic nature of Bruch&#8217;s membrane.</a> Prog Retin Eye Res. 29 (1), 1-18 (2010).</li><li>Clark, S. 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=Identification+of+factor+H-like+protein+1+as+the+predominant+complement+regulator+in+Bruch&#8217;s+membrane:+implications+for+age-related+macular+degeneration.">Identification of factor H-like protein 1 as the predominant complement regulator in Bruch&#8217;s membrane: implications for age-related macular degeneration.</a> J Immunol. 193 (10), 4962-4970 (2014).</li><li>Sim, E., Palmer, M. S., Puklavec, M., Sim, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+the+complement+control+protein+factor+H+(beta+1+H).">Monoclonal antibodies against the complement control protein factor H (beta 1 H).</a> Biosci Rep. 3 (12), 1119-1131 (1983).</li><li>Ripoche, J., Day, A. J., Harris, T. J., Sim, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+amino+acid+sequence+of+human+complement+factor+H.">The complete amino acid sequence of human complement factor H.</a> Biochem J. 249 (2), 593-602 (1988).</li><li>Moore, D. J., Clover, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+age+on+the+macromolecular+permeability+of+human+Bruch&#8217;s+membrane.">The effect of age on the macromolecular permeability of human Bruch&#8217;s membrane.</a> Invest Ophthalmol Vis Sci. I. 42 (12), 2970-2975 (2001).</li><li>McHarg, S., Clark, S. J., Day, A. J., Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration+and+the+role+of+the+complement+system.">Age-related macular degeneration and the role of the complement system.</a> Mol Immunol. 67 (1), 43-50 (2015).</li></ol>

The authors declare that they have no competing financial interests.

这里，我们描述Bruch膜的富集进行进一步的分析，允许随后的分析，包括Western印迹11，质谱和Bruch膜的利用改性尤斯灌流室11,14的扩散性质。关键步骤包括:a）眼的内表面的透彻刮削和洗涤以除去所有的RPE细胞b）该底层脉络膜的重复和仔细刮削而不会引起布鲁赫瓦解和的切除布鲁赫c）如超纯水洗涤以确保残余细胞的裂解。此外，如果富集Bruch膜是将要用于蛋白组学分析，蛋白质提取由尿素处理是最有效的在打破该弹性组织相比，其它方法如机械均化。丰富Bruch膜的组织学显示脉络膜的ECM不会被删除。这是恩存在意外作为pentilaminar Bruch膜的外层是由脉络膜毛细血管内皮细胞基底膜形成。事实上，它可能是有利的脉络膜毛细血管的ECM保持为发生变化这在AMD的包括末端补复杂/膜攻击复合物（MAC）15的沉积。应当指出的是，对于Bruch膜的详细的组织学分析，其结构将能更好地保留，如果切片在巩膜，脉络膜，和Bruch膜的存在。事实上， 在图2中呈现的组织学完全是意在展示富集从本手稿中描述的技术产生的程度。 黄斑布鲁赫富集需要小心剥离眼罩，确保NSR和尤其中央凹不受损坏;否则鉴定并因此隔离黄斑是不可能的。该Tissu酒店的完整性e是同样重要的是这个过程，它因此，建议使用下48小时验尸时间样本。需要富集的程度捐助者之间的变化，并且可受供体年龄，验时间，以及任何眼睛病理的存在。利用丰富的术语是故意在确认该技术的限制，这是那个&#39;净化&#39;或Bruch膜的使用这种方法&#39;隔离&#39;根本是不可能的。事实上，由于脉络膜的ECM并入Bruch膜有完全分离是不可行的。 理解的各眼组织的生化改变是在了解眼睛疾病进展至关重要。 Bruch膜的这种独特的丰富有利于这种认识;正在进行的研究是用质谱在AMD的不同阶段，研究丰富了黄斑布鲁赫的膜蛋白质组并在样本从受试者不同基因型始发改善的AMD的分子病理学的理解。已经在这里描述的技术已成功地用于鉴定FHL-1作为在上富集Bruch膜Bruch膜11，并进一步研究的主要补体调节器可能导致更多的新的见解的AMD的分子病理学。

在眼科研究中使用的验尸人眼组织对眼睛疾病的发病机制的认识的宝贵资源。人类供体眼分析作出了挑剔的支撑年龄相关性黄斑变性（AMD），这是失明的在西方世界1主要原因机制作出了重要贡献。从全球来看，AMD占所有失明的约8.7％，并与人口日益老龄化，预计影响到1.96亿人，到2020年2，AMD会导致中央视力的丧失，对病人的独立性和生活质量产生深远的影响3。早期AMD的特征在于玻璃疣的形成和它可以发展为地图样萎缩（有时被称为"干"AMD）或脉络膜新生血管形成（也称为新血管或"湿"的AMD）。虽然抗VEGF注射可稳定或改善视力的新生血管性AMD是我不是治愈性，并且在目前，还没有治疗存在地图样萎缩。 虽然某些遗传变型已被示出修改的AMD风险4 - 8，很少有关于这些变体如何影响黄斑在生化水平。在AMD发病的重要站点是Bruch膜。这种结构和各种研究中的玻璃膜疣的形式已经和周围Bruch膜在AMD 9提供补体激活的证据。布鲁赫膜是细胞外基质，从脉络膜分离的视网膜色素上皮（RPE）细胞的片材，并且大约4微米厚。布鲁赫膜并入脉络膜，一层包含有孔的毛细管和外部此脉络膜是含有粗的血管10（图1A）层。 布鲁赫的膜是额外的一个pentilaminar片蜂窝基质（ECM），以及这种方法详述如何隔离Bruch膜，随着底层脉络膜毛细血管的ECM（以下称富含Bruch膜），这在很大程度上是脱细胞，无红血细胞。富布鲁赫的膜，然后用于免疫印迹和组织学实验。此外，方法用于从眼睛的黄斑区域富集Bruch膜仅仅被描述，这将是特别感兴趣的是那些研究的AMD的生物化学方面的。

<table><tbody><tr><td>Auxillary objective 0.5X</td><td>GT-Vision Ltd</td><td>3638</td><td>Useful (but not essential) for lower magnification imaging of whole eye flat-mount</td></tr><tr><td>Biopsy Punch 6 mm</td><td>Oncall Medical Supplies</td><td>SCH-33-36</td><td>6mm Biopsy Punches</td></tr><tr><td>Bovine serum albumin</td><td>Sigma - Aldrich</td><td>A3059</td><td></td></tr><tr><td>Bromophenol Blue</td><td>Sigma - Aldrich</td><td>B0126</td><td></td></tr><tr><td>Cell scrapers</td><td>Sarstedt</td><td>83.183</td><td>For membrane enrichment</td></tr><tr><td>CyroPure Cyrovials</td><td>Sarstedt</td><td>72.38</td><td></td></tr><tr><td>Disposable Scalpels</td><td>Fisher Scientific</td><td>12387999</td><td>Sterile, individually wrapped Swann-Morton scalpels.</td></tr><tr><td>Dual Gooseneck LED spot lights on 30 cm arms</td><td>GT-Vision Ltd</td><td>0153</td><td>Flexible light source essential for dissection and imaging</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma - Aldrich</td><td>D8537</td><td>Used to prevent macula drying out and during membrane enrichment.</td></tr><tr><td>(A) Forceps</td><td>Scientific Laboratory Supplies</td><td>INS4280</td><td>(A) Fine (100 mm) and straight</td></tr><tr><td>(B) Forceps</td><td>Scientific Laboratory Supplies</td><td>INS4272</td><td>(B) Broad and blunt (150 mm). Useful for lifting large portions of sample</td></tr><tr><td>(C) Forceps</td><td>Scientific Laboratory Supplies</td><td>INS4323</td><td>(C) Straight fine points</td></tr><tr><td>Glycerol</td><td>Sigma - Aldrich</td><td>G1345</td><td></td></tr><tr><td>Glycine</td><td>Sigma - Aldrich</td><td>B0126</td><td></td></tr><tr><td>GXCAM 5 digital colour camera &amp;amp; GT-Vision software</td><td>GT-Vision Ltd</td><td>1122</td><td></td></tr><tr><td>Methanol</td><td>Sigma - Aldrich</td><td>M/4000/17</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Life Technologies</td><td>NP0007</td><td></td></tr><tr><td>NuPAGE Novex 4 - 12% Bis-Tris Protein Gels</td><td>Life Technologies</td><td>NP0322BOX</td><td></td></tr><tr><td>Petri dish</td><td>Sterilin</td><td>101VR20</td><td>90 mm, used as a dish for samples</td></tr><tr><td>Protein isolation beads (Strataclean resin)</td><td>Agilent</td><td>400714-61</td><td></td></tr><tr><td>SDS</td><td>Bio-Rad</td><td>161-0301</td><td></td></tr><tr><td>Stereozoom microscope GXMXTL3T (magnification range 7X - 45X)</td><td>GT-Vision Ltd</td><td>5595</td><td>Stereozoom microscope attached to boom stand for ease of use during dissection</td></tr><tr><td>Tris-HCl</td><td>Sigma - Aldrich</td><td>T3253</td><td></td></tr></tbody></table>

enrichment of bruch's membrane from human donor eyes

年龄相关性黄斑变性（AMD）是在发达国家视力障碍的主要原因。该疾病本身表现由中心视网膜的，称为黄斑的破坏，从而导致中心视力的丧失。早期AMD的特征在于小，淡黄病变称为软脉络膜小疣，可以进步的存在到晚期AMD如地理萎缩（干性AMD）或新血管形成（湿性AMD）。虽然临床变化很好的描述，并赋予AMD风险遗传影响的认识越来越以往任何时候都更加细致，一个领域缺乏重大进展是遗传风险的生物化学后果的认识。这部分是由于难以理解Bruch膜，非常薄的细胞外基质，它作为材料的从血液供给一个生物过滤器和视网膜色素上皮（RPE）细胞单层驻留的一个支架的生物化学。玻璃膜疣˚F内Bruch膜和它们的存在ORM破坏营养流向RPE细胞。仅通过调查Bruch膜的蛋白质组成，而事实上其他蛋白质与它如何交互，可以研究人员希望解开支撑玻璃膜疣形成，发展的AMD和随后的视力丧失的生化机制。本文详细方法用于富集要么整个Bruch膜，或者仅仅从黄斑区，因此，它可用于下游生化分析，并提供了这是如何已经改变Bruch膜生物化学的理解实施例。

上面描述的协议以产生富集Bruch膜导致Bruch膜，脉络膜的ECM的分离和除去大部分细胞/细胞碎片包括所有的视网膜色素上皮（图2）的。富集Bruch膜在水中的洗涤是在裂解任何剩余脉络膜细胞的关键步骤。杀脉络膜期间施加的压力出现压缩一些的少数剩余细胞核成被定义为Bruch膜（图2B）的区域。 富集Bruch膜的增溶允许其蛋白质含量的下游分析。蛋白质条带从溶解富集Bruch膜使用4分离 - 12％梯度SDS-PAGE凝胶，而没有用在自己的权利，以确定蛋白质含量，的确证明这种技术，以减少污染的血液蛋白质，AR的能力Ë非特异性结合（ 图3B）。在这方面，从蛋白质凝胶运行一个显着的缺席是人血清白蛋白的一个相当大的频段（〜66 kDa的），其可以以其他方式进行后续免疫印迹更难以解释:富集Bruch膜的组织学分析显示于图2还支撑本。实际上，作为工作部分先前描述11，溶解布鲁赫从一系列单独供体的膜进行探测的一个通过Western印迹（图3C），使用特异性抗体的蛋白质。在这里所示的情况下，抗体（称为OX23）12针对先天免疫的155 kDa的调节器，被称为补体因子H（FH）中，使用和证实从三个独立的较低分子量带的Bruch膜样品中的存在供体（ 图3C）。随后这原来是因子H样蛋白1（FHL-1）中，49 kDa蛋白与FH和来自相同基因13的剪接变异产生的。 FHL-1似乎是存在于Bruch膜11主要补体调节剂。 <img alt="图1" src="/files/ftp_upload/53382/53382fig1.jpg" /> 图1.解剖Bruch膜的人眼和充实。（A）Bruch膜是一种细胞外基质屏障。脉络膜包含一层有孔毛细血管连续合并到布鲁赫膜（称为脉络膜），并且这些分离的脉络膜的其余部分（含大血管）从所述视网膜色素上皮细胞（RPE）细胞单层，其生理上支承感光细胞在视网膜神经上皮层（NSR）。（B）的四个切口进入人眼杯是由允许它被打开，以创建平面安装。该玻璃体可以与使用镊子除去。 NSR的可能脱落连同玻璃体，但也可以利用镊子除去。甲细胞刮刀用于从Bruch膜除去RPE细胞层，同时它仍然固定在脉络膜和巩膜。在Bruch膜/脉络膜复合物可以剥离远离巩膜，并用新的细胞刮刀，外脉络膜可以去掉。在超纯水最后清洗裂解所有剩余的细胞物质。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53382/53382fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53382/53382fig2.jpg" /> 图2.组织学Bruch膜和前（A） 的脉络。H＆E染色切片及售后服务 （二）从Bruch膜和洗涤超纯WA刮去外脉络膜结构之三，以产生丰富Bruch膜。比例尺代表20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53382/53382fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53382/53382fig3.jpg" /> 图3.增溶作用富集布鲁赫膜对蛋白质含量分析。（A）的蛋白质可以从富集Bruch膜中提取由培养在8M尿素的6小时，在室温，被透析的PBS之前。对于Western分析，从整体Bruch膜透析蛋白可以使用蛋白隔离珠用的Laemmli还原样缓冲液煮沸分离和洗脱。（B）的代表性的考马斯蓝染色的SDS-PAGE凝胶，显示出既纯净和稀释溶解Bruch膜（BM）的使用方法D，形旁切的（A）（C）溶解布鲁赫的膜从3个人捐助者的代表性免疫印迹（泳道1 - 3）。探查H因子样蛋白1（FHL-1） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53382/53382fig3large.jpg" target="_blank">请点击这里查看大图这个数字。</a> <img alt="图4" src="/files/ftp_upload/53382/53382fig4.jpg" /> 图4.分离富集布鲁赫膜从黄斑的。视网膜黄斑区域是一个5毫米面积可由覆中央凹的黄色着色易于识别，而且由于靠近相邻的视盘。使用凹作为导向，放置在平坦安装人眼一个6毫米活检穿孔切除一个6毫米黄斑组织块。除去覆NSR和从切黄斑刮去RPE细胞。剩下的Bruch膜/脉络膜复合物才能使折射率n去皮远离巩膜，彻底刮除，除去脉络，剩下的裂解通过浸没在超纯水中细胞物质。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53382/53382fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Enrichment of Bruch's Membrane from Human Donor Eyes at JoVE.com

Enrichment of Bruch's Membrane from Human Donor Eyes

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

从人类供体眼富集布鲁赫的膜

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

enrichment-of-bruch-s-membrane-from-human-donor-eyes

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.0K 次观看.  University of Manchester. Identifying proteins specifically associated with Bruch’s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

视频: Enrichment of Bruch's Membrane from Human Donor Eyes

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

科研

发育生物学

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

使用睡美人转座子系统对原代人色素上皮细胞进行基于电穿孔的遗传修饰

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

医学

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

用于 AMD 研究的基于 OCT 的人类供体眼多模态成像

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

作者聚焦:基于离体 OCT 的人类供体眼多模态成像，用于年龄相关性黄斑变性的研究  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...

神经科学

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

生物学

分离、培养和鉴定人角膜缘生态位细胞的方案

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

作者聚焦:标准化角膜缘生态位细胞 （LNC） 分离和表征以支持广泛的 LNC 研究 

Here, we present a protocol to isolate and identify the human limbal niche cells.

角膜缘生态位细胞的分离和鉴定

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

从人类供体眼富集布鲁赫的膜

摘要

探索更多视频

此视频中的章节

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

使用睡美人转座子系统对原代人色素上皮细胞进行基于电穿孔的遗传修饰

新型人类组织特征视网膜培养技术的特征

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

角膜缘生态位细胞的分离和鉴定

从人类供体眼富集布鲁赫的膜

摘要

探索更多视频

此视频中的章节

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

使用睡美人转座子系统对原代人色素上皮细胞进行基于电穿孔的遗传修饰

新型人类组织特征视网膜培养技术的特征

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

角膜缘生态位细胞的分离和鉴定

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性