Name: adenoviral gene therapy for diabetic keratopathy: effects on wound healing and stem cell marker expression in human organ-cultured corneas and limbal epithelial cells
Uploaded: 2016-04-07T15:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epitheli

We gratefully acknowledge financial support by NIH/NEI R01 EY13431 (AVL), CTSI grant UL 1RR033176 (AVL), and grants from the Regenerative Medicine Institute, Cedars-Cedars Medical Center.

国家疾病研究交换（NDRI，费城，宾夕法尼亚州）提供的同意验尸的健康和糖尿病人的眼睛和角膜。 NDRI的人体组织收集协议是由管理委员会，并接受卫生监督全国学院批准。这项研究已批准的雪松 - 西奈医学中心机构审查委员会（IRB）豁免协议EX-1055下进行的。合作角膜外科医生，博士。 E. Maguen和Y拉比诺维茨，提供丢弃角巩膜缘干细胞富集角膜上皮的文化隔离。该研究已批准的IRB协议Pro00019393下进行的。 1.人角膜器官培养注:正常和糖尿病患者角膜或整个眼睛的角膜冷冻存储介质接收（ 如 GS的Optisol）从国家供应商NDRI死后48小时内。 <ol><li>用镊子取下存储容器的眼角膜，并把它们洗干净我的两倍n中的抗生素 - 抗真菌剂（ABAM）混合物。在整个眼睛的情况下，从弯曲剪刀剪地球仪角膜出来。至少留出5毫米结膜缘，也洗了眼角膜在ABAM。 </li><li>备被放置在角膜凹的混合物。它由无血清的最低必需培养基（MEM）含有ABAM，1毫克/毫升小牛皮肤胶原蛋白（从10毫克/毫升的储备溶液制成在0.1N乙酸），1％琼脂，和胰岛素转铁蛋白 - 亚硒酸盐的补充7。 </li><li>微波该混合物进行杀菌和琼脂溶解沸腾。这可能需要1分钟，一切就会溶解。离开管盖部分打开，因为混合溢出后很容易沸腾。由于这个原因，可能需要几个沸腾循环，每10-15秒。冷却混合物至37-39℃。 </li><li>将角膜上皮面朝下在无菌60毫米菜肴。填充约为0.5毫升1.2中描述的混合物的角膜凹。该混合物凝结在2分钟内的眼角膜。 </li><li>放置角膜琼脂侧在无菌60 mm培养皿向下，并添加含MEM与ABAM充分介质，和胰岛素转铁蛋白-亚硒酸盐补充7（37℃），以在角膜缘为空气-液体界面培养大约保持其水平。中等量为每盘7-8毫升。 </li><li>放置与角膜的菜与水锅加湿5％CO 2培养箱在35℃（正常角膜温度）（这样，湿度水平保持在或高于95％）。添加在上皮每天100微升介质（两滴）滋润角膜。 </li><li>监控角膜形态，并在透射光用一个标准显微镜用4X目标上皮细胞微观活力。每天这样做一次。 </li><li>改变介质每三天。角膜可为至少三周进行培养。对于基因治疗实验，培养角膜为3-5天，然后transducË与利息（4.1-4.4）的基因，保留取决于转基因和受伤口愈合协议（2.1-2.4），另一个3-5天。 </li></ol> 2.上皮伤口愈合注意:在糖尿病患者的角膜，因为易碎上皮基底膜的过程中，不以正常眼角膜发生分离的机械刮除上皮清创是不可行的。因此，为了保持正常和糖尿病角膜伤口愈合的相似的条件下，化学去除用正庚上皮的使用19。此过程将删除上皮但保留在正常和糖尿病角膜既完整基底膜后面。 <ol><li>要执行中央的上皮清创20，将浸泡在正庚角膜中央前表面上75秒一个5毫米的滤纸片。 </li><li>取下过滤器，并在充满介质洗眼角膜。填用0.5ml琼脂 - 胶原凹混合文化为1.4。清创后，上皮细胞通常死亡，脱落，留下完整的微观基底膜7。 </li><li>使用8.5毫米磁盘来创建与只缘基因治疗实验中较大的上皮伤口。这将确保角膜缘干细胞在愈合过程中的参与。 </li><li>微观监控角膜愈合。每天做的4X和10X的照片，直到上皮缺损完全愈合。愈合过程可以根据状态（正常或糖尿病）和伤口大小取为2至15天。 注意:在愈合过程中，黑蜘蛛状细胞在顶端焦平面观察。这些细胞间质细胞凋亡的角膜上皮去除4后死亡。愈合被确定时，这些死细胞是由愈合上皮杂草丛生和不再可见（ 图2，小图）是完整的。 </li><li>愈合后完成后，切开角膜的一半，它们嵌入华侨城化合物和工艺上冰冻切片间接免疫荧光法或Western印迹16,21。 </li></ol> 3.干细胞富集培养的角膜缘细胞与维护的分离注:从角巩膜缘准备主角膜缘上皮干细胞（LESC）富集的文化。从健康献血者到来，废弃后从医生合作，在标准角膜存储介质（ 如的Optisol）接受角膜移植的轮辋。否则，LESC富集的文化可以从正常和糖尿病整个角膜切除轮辋索取或从NDRI接受角膜存储介质金球奖。 <ol><li>如果使用在整个角膜或地球仪，第一隔离角膜缘区域。要做到这一点，放在无菌塑​​料菜上皮侧角膜起来，并用9毫米环钻（圆形角膜刀）取下中心区。丢弃此核心部分。然后用13毫米环钻切除角膜缘区和丢弃外侧结膜的一部分。为了分离细胞使用的面包圈状角膜缘。细胞分离之前，除去从角膜的内侧的内皮细胞和虹膜的粘附残余相反用无菌棉签表面上皮。 </li><li>使用中性蛋白酶隔离治疗角膜缘细胞片。孵育2.4 U / ml的分散酶II的每个角巩膜缘中，在37℃在补充有10％胎牛血清（FBS）的2小时221.5毫升角质细胞无血清培养基（KSFM）。 </li><li>轻轻地解剖立体显微镜下的角膜缘上皮细胞片关闭边缘与镊子和离解的细胞在1ml 0.25％胰蛋白酶的 - 在室温下30分钟0.02％EDTA的溶液中。 </li><li>清洗细胞在10ml培养基（ 例如 ，用EpiLife）的，并在台式离心机中以300 xg离心沉淀他们在室温下5分钟。 </li><li>悬浮日E细胞在培养基中:用N，B27和人类角质形成细胞生长补充剂，和10ng / ml的表皮生长因子（EGF）培养基。 </li><li>种子细胞在涂覆有人基底膜蛋白包括纤维连接蛋白，IV型胶原，层粘连蛋白和（FCL）的混合物在烧瓶3,000-5,000细胞/ ml或60毫米培养皿，在0.5-1微克/厘米2，在KSFM中。 </li><li>培养的细胞在加湿（95％或更高湿度） 的 CO 2培养箱中于37℃，直到它们形成完全汇合单层。这可能需要1-2周。 注:定期由确认阳性免疫细胞化学染色为假定LESC标志物PAX6，角蛋白（K）14，K15，K17，和ΔNp63α小区标识。染色的细节，包括初级抗体已经出版8,16-18,22。 </li><li> （5稀释的0.25％的溶液1）10分钟，在37℃至通道中的融合细胞，用1ml 0.05％胰蛋白酶溶液处理它们。加入5 ml大豆TRypsin抑制剂溶液（10毫克/毫升）稀释1:4的培养基中，离心所述细胞在3.5。 </li><li>在3ml稀释大豆胰蛋白酶抑制剂溶液的洗细胞沉淀。板菜细胞到FCL涂层（见3.7）或玻璃玻片以2×10 4个细胞/ ml。 </li></ol> 4.器官培养角膜腺病毒转导注意:重组腺病毒（AV）包括AV-载体（无基因插入），AV-CMET（用的c-met基因的开放阅读框），AV-shM10（带的shRNA对MMP-10），和AV-SHCF（用shRNA的组织蛋白酶F）。它们的E1 / E3缺失的5型的AV的主要立即早期巨细胞病毒启动子的控制下表达基因。在AV-CMET病毒使用的是音像载体pAd / CMV / V5-DEST 16产生的。在AV-shRNA的病毒短发夹RNA序列的亚克隆定制生成具有HH1子和GFP标签序列从iLenti-EGFP载体沿成不能复制（-E1 / -E3）呼玛ñAV型5基因组用腺-4的表达系统17。 <ol><li>转导每对与AV-载体和另一个角膜的一个器官培养角膜，具有基因表达，调节AV。使用AV-CMET每角膜0.8至1.25×10 8噬斑形成单位（pfu）的培养基中48小时，在37℃保持角膜培养基表面下的每个在24孔培养皿的孔中。使用AV-shRNA的病毒，每1-2角膜×10 8 PFU。 </li><li>使用转导的病毒在补充75微克/毫升枸橼酸西地那非充满介质。此试剂激活小窝运输促进病毒摄取上皮细胞21。由于在水溶液中西地那非半衰期短，相同量以后重新添加到培养基中4-5小时。标准治疗是48小时。 </li><li>角膜转移到新的菜肴在介质中的圆端无菌刮刀和文化没有AV保持在角膜缘中等水平。附加后itional 4-8天在液体 - 空气界面，过程为各种分析或测试上皮伤口愈合对AV-处理的角膜（见上文）。通过增加对角膜的前100微升介质（两滴）定期滋润培养过程中角膜。 </li><li>仅用于角膜缘细胞的转导，孵育用在含有角膜缘中等水平的空气 - 液体界面的角膜的病毒，以避免中央上皮转导。 </li></ol> 5.培养角膜缘细胞的腺病毒转导<ol><li>保持在介质用10ng / ml的表皮生长因子上涂覆有以0.5微克/厘米2的整箱混合物塑料餐具的LESC富集培养物（见3.5）。 </li><li>转导已达到与该AV 70-80％汇合窝藏绿色荧光蛋白基因（AV-GFP）或者AV加扰的范围的感染多重性的shRNA-GFP（MOI:1-300 PFU /细胞）培养的细胞在与2毫微克/毫升的EGF的培养基。执行转导n，用于在37℃下在随后20小时在0.5ml培养基最小体积（0.2ml）中4小时。不需要用于细胞培养转导的小窝运输活化剂西地那非。 </li><li>使用以下试剂促进该AV结合到细胞表面，以提高该AV转导效率的一个:或者聚阳离子（1微克/毫升多聚L-赖氨酸或5微克/毫升聚凝胺），或6微升/毫升转导试剂（ 例如 ，ViraDuctin）或20微升/毫升转导增强子（ 例如 ，ibiBoost）。 </li><li>更换培养基为一个没有该AV后，孵育培养细胞4天，在加湿的 CO 2培养箱中并使用倒置荧光显微镜评估GFP表达水平。 </li><li>使用划痕伤口愈合试验在AV转导的培养，以评估细胞迁移。生长中的细胞的多孔室载玻片。通过用200μl的圆周划伤细胞在一条直线的直线运动使在一个单层的创伤佩特提示。伤人后，一个新的之一删除分离的细胞改变介质。 </li><li>每天拍摄伤口与连接到在4X放大倒置显微镜的数码相机。记录时间愈合完成时（伤口边缘进入沿整个伤口接触）。 </li></ol>

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The authors declare no financial interests.

角膜似乎是用于基因治疗的理想组织，由于其表面的位置，其中的基因递送，以及疗效和副作用的评价，是容易的。然而，这种功能强大的方法的临床翻译仍然缓慢，由于对角膜疾病的遗传原因和基因治疗靶24稀少的信息。糖尿病并发症，包括角膜改变可以在本质上，换算成代谢存储器9大多后生。出于这个原因，在糖尿病组织和细胞保持其异常体外 ，并且可以在器官和组织培养进行研究。另外，糖尿病引起的基因表达的水平和模式可以由基因治疗来校正特定的变化。我们已经确定了几个标记物，糖尿病角膜上皮，包括干细胞区室-5,6,8-改变，和所用的人角膜器官培养7,8通过正常化基因治疗的标志物表达，并在糖尿病角膜损害上皮伤口愈合。 我们选择，因为它的强烈的转基因表达，其非整合性质，其为上皮细胞，这是优于腺相关病毒（AAV）25的质量选择性的AV作为基因递送载体。角膜器官培养的AV传导有几个关键步骤。角膜应在48小时尸检递送到实验室，以最小化该存储过程中发生上皮的损失。为了增加病毒的摄取，转导21中添加小窝运输助推器，如西地那非，它是重要的。当验证在蛋白表达的转导作用，利用几个标记建议（ 图3），作为具体的干细胞标记物是不明确鉴定。当使上皮的伤口，有必要使用彻MICAL清创由于手动刮期间导致其脱离糖尿病上皮基底膜的脆弱性。当上皮继续生长在他们（ 图2）愈合过程的仔细日常监测需要愈合的客观标准，如死了上皮下角膜基质从视野中消失。最后，由于一些AV和/或绿色荧光蛋白毒性到主角膜缘细胞，重要的是要降低病毒剂量，在采用无毒转导的助推器，如聚阳离子的同时，以确保有效的和安全的转导（ 图5 - 7）。 基因治疗系统可以被修改为使用动物器官培养的角膜。它们比人类便宜得多，可以在数量上的常规碱获得，并且可以是适合用于筛选， 例如 ，伤口愈合调节药物。然而，人的角膜提供更广泛的潜在可能性这项研究由于大量试剂，尤其是抗体。在非糖尿病动物的角膜的药物筛选的情况下，清创术可以机械完成。如果某个标记的抗体不会在某些人角膜很好地工作，这可能是由于个体差异和/或疾病的持续时间或严重性。在这种情况下，可推荐检查其他标记。正庚醇，以除去细胞的能力可能显著贮存期间随时间减少，这可能是由于氧化。如果出现这种情况，一个新的，以前未开封，小瓶可能需要使用。如果与昂贵人角膜的工作，进行第二轮清创的可尝试。西地那非具有在水溶液中半衰期短;因此，它必须被新鲜执行角膜传导每次制备。培养角膜细胞可能取决于捐赠者的年龄的成功;从年轻供细胞通常生长更好。一个单一的基因治疗剂可能不会引发signif着的效果;在这种情况下，这样的试剂的组合可能是最佳的17。如果主角膜细胞显示毒性的迹象，它降低病毒和聚阳离子（ 例如 ，聚-L-赖氨酸）是重要的剂量，同时保持有效的转导，以尽量减少毒性作用。 虽然糖尿病器官培养重现疾病和改变的基因表达的许多迹象，它们是神经支配和缺乏免疫细胞的供应，这可能会影响伤口愈合，干细胞。在这方面，动物模型可以提供一些优势，虽然动物没有重现的人类疾病体征全谱。其它基因治疗的目标存在，可有效正常化糖尿病角膜。这种目标的例子可以是微小RNA，特别是的mir-146a的或miR-424 23。具有特定微小RNA的抑制所使用的靶基因修饰的组合可以潜在地产生一种更有效的有益效果。 Anoth器的限制包括与所述局部施用的AV（ 例如 ，在眼滴剂的形式）的角膜相对长的温育，以确保最大的病毒摄取的必要性。这可能造成在诊所中的问题，虽然胶带眼睑可在延长的孵育帮助。在病毒溶液使用西地那非低浓度会加速该AV摄取。在AV传导效果是短暂的，这可能会限制在基于AV - 基因治疗的疗效的潜力。虽然它持续足够长的时间以校正糖尿病伤口愈合放慢，持续的上皮干细胞的正常化，可能需要更长的持久的影响，如由基于AAV的基因治疗24提供。应当提到的是免疫原性早期的AV载体的一个严重的缺点。然而，新一代的重组AV包括"无胆"的病毒在很大程度上是免费的这一副作用，增加他们被强大的translati前景Onal地区基因治疗车辆26。 这可以是用于伤口愈合16,17,21重要各种途径的小分子调节剂，27-30是在实验模型及临床使用。然而，它们不靶向特定的细胞类型，可有不同的细胞不同的效果，并且可以只对糖尿病角膜病变31,32的一些方面起作用。基因治疗提供了可能性以改变其在特定细胞所需的方向，这是我们在这一工作完成同时使用基因的过度表达（C-MET）和沉默（MMP-10和组织蛋白酶F），转导的上皮细胞中的基因表达。该方法还允许调制的特定疾病的标记基因的表达，一个个或组合5,6。我们的实验表明，即使在三个的AV窝藏不同的基因表达调节剂相结合的情况下，所有三个基因受到影响如预期的，并且治疗是当单独17使用每个AV比更有效。 在严重的糖尿病性角膜病持续性上皮缺损或在玻璃体切割或激光光凝不全上皮愈合，更换不正常的角膜缘干细胞可以是基因治疗的替代方案。在这些情况下，当在体外基因治疗培养和扩增角膜缘上皮细胞可在患病的角膜移植。我们的数据显示，糖尿病患者角膜缘培养的细胞显示干细胞标志物的表达作为体外角膜使他们的基因治疗好目标类似的差异。然而，这种治疗需要优化，由于这些细胞所用的病毒载量和转染试剂的高灵敏度。我们能够显示，阳离子转染试剂确保了在培养的糖尿病角膜上皮感兴趣的基因的高表达效率和安全分娩。 ŧHESE结果可能铺平道路，为扩大LESC了严重的糖尿病角膜病变的情况下，未来的移植目的在体外成功的基因治疗的方式。由于遗传性和获得LESC不足也有类似糖尿病33,34角膜神经的减少有关，健康LESC富集培养到病变角膜移植可能缓解糖尿病神经病变的角膜也。

糖尿病患者角膜病变主要导致上皮变性（角膜）和神经（神经病）的变化。它通常是由上皮伤口愈合和角膜神经减少1-4的异常表现。据估计，60％-70％的糖尿病患者有不同的角膜问题1,3。我们的研究已经确定了几个标志物蛋白在人类糖尿病性角膜包括的c-met原癌基因（肝细胞生长因子受体）的下调改变的表达和基质金属蛋白酶10（MMP-10）和组织蛋白酶F 5，6的上调。我们也显著记录在人类糖尿病性角膜下降几个推定上皮干细胞标记物的表达。 在以前的研究中，我们开发了一种基于腺病毒的基因治疗使用人类糖尿病性角膜器官培养系统，该系统示出了伤口愈合缓慢正常化糖尿病改变的标志物的水平，糖尿病标记的变化，和干类似于体外角膜7,8-细胞标记物表达的减少。变化的这个持续性似乎是由于后生代谢存储器9中的存在。该培养体系进一步用于基因治疗。此疗法的目标是从标记选择了与糖尿病角膜（ 的c-met原癌基因），或增加的表达（MMP-10和组织蛋白酶F）的任一减少的表达。 腺病毒（AV）疗法在整个器官培养的角膜或只角巩膜周缘隔室使用。这个车厢港口上皮干细胞的更新角膜上皮和积极参与伤口愈合4,10-15。这里，提供了用于正常和糖尿病的人角膜器官培养，上皮伤口愈合，分离和干细胞富集的角膜缘细胞培养物的表征，和腺病毒细胞和角膜转导协议。我们的结果表明，该疗法对糖尿病角膜用于未来可能的移植正常化标志物的表达和伤口愈合的可行性。他们还表明，联合治疗是恢复正常标记图案和上皮的愈合，糖尿病角膜16-18最有效的方式。

<table><tbody><tr><td>minimum essential medium</td><td>Thermo Fisher Scientific</td><td>11095-080</td><td></td></tr><tr><td>Optisol-GS&amp;nbsp;</td><td>Bausch &amp;amp; Lomb</td><td>50006-OPT</td><td></td></tr><tr><td>ABAM antibiotic-antimycotic mixture</td><td>Thermo Fisher Scientific</td><td>15240062</td><td></td></tr><tr><td>calf skin collagen&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C9791</td><td></td></tr><tr><td>agar, tissue culture grade</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A1296</td><td></td></tr><tr><td>n-heptanol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>72954-5ML-F</td><td></td></tr><tr><td>O.C.T. compound&amp;nbsp;</td><td>VWR International</td><td>25608-930</td><td></td></tr><tr><td>Dispase II&amp;nbsp;</td><td>Roche Applied Science</td><td>4942078001</td><td></td></tr><tr><td>keratinocyte serum-free medium (KSFM)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>17005042</td><td></td></tr><tr><td>EpiLife medium with calcium</td><td>Thermo Fisher Scientific</td><td>MEPI500CA</td><td></td></tr><tr><td>N2 medium supplement, 100x</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td></td></tr><tr><td>B27 medium supplement, 50x</td><td>Thermo Fisher Scientific</td><td>17504-044</td><td></td></tr><tr><td>human keratinocyte growth supplement, 100x</td><td>Thermo Fisher Scientific</td><td>S-001-5</td><td></td></tr><tr><td>trypsin 0.25% - EDTA 0.02% with phenol red</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>trypsin 0.25% with phenol red</td><td>Thermo Fisher Scientific</td><td>15050065</td><td></td></tr><tr><td>soybean trypsin inhibitor&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T6414</td><td></td></tr><tr><td>fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>26140079</td><td></td></tr><tr><td>insulin-transferrin-selenite supplement (ITS)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>I3146-5ML</td><td></td></tr><tr><td>antibody to keratin 14</td><td>Santa Cruz Biotechnology</td><td>sc-53253</td><td></td></tr><tr><td>antibody to keratin 15</td><td>Santa Cruz Biotechnology</td><td>sc-47697</td><td></td></tr><tr><td>antibody to keratin 17</td><td>Santa Cruz Biotechnology</td><td>SC-58726</td><td></td></tr><tr><td>antibody to &amp;Delta;Np63&amp;alpha;</td><td>Santa Cruz Biotechnology</td><td>sc-8609</td><td></td></tr><tr><td>antibody to PAX6</td><td>BioLegend</td><td>PRB-278P-100</td><td></td></tr><tr><td>antibody to nidogen-1</td><td>R&amp;amp;D Systems</td><td>MAB2570</td><td></td></tr><tr><td>antibody to integrin &amp;alpha;3&amp;beta;1</td><td>EMD Millipore</td><td>MAB1992</td><td></td></tr><tr><td>human fibronectin</td><td>BD Biosciences</td><td>354008</td><td></td></tr><tr><td>human laminin</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L4445</td><td></td></tr><tr><td>human type IV collagen</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C6745-1ML</td><td></td></tr><tr><td>adenovirus expressing MMP-10 shRNA</td><td>Capital BioSciences</td><td>custom made</td><td></td></tr><tr><td>adenovirus expressing cathepsin F shRNA</td><td>Capital BioSciences</td><td>custom made</td><td></td></tr><tr><td>adenovirus expressing scrambled shRNA and GFP</td><td>Capital BioSciences</td><td>custom made</td><td></td></tr><tr><td>adenovirus expressing c-met</td><td>OriGene (plasmid)</td><td>SC323278</td><td></td></tr><tr><td>adenovirus expressing GFP</td><td>KeraFAST</td><td>FVQ002</td><td></td></tr><tr><td>sildenafil citrate, 25 mg</td><td>Pfizer</td><td>from pharmacy</td><td></td></tr><tr><td>epidermal growth factor&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>PHG0311</td><td></td></tr><tr><td>poly-L-lysine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P4707</td><td></td></tr><tr><td>polybrene</td><td>Sigma-Aldrich&amp;nbsp;</td><td>107689-10G</td><td></td></tr><tr><td>ViraDuctin</td><td>Cell Biolabs</td><td>AD-200</td><td></td></tr><tr><td>ibiBoost</td><td>ibidi, Germany</td><td>50301</td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>Thermo Fisher Scientific</td><td>10010049</td><td></td></tr><tr><td>Corning round end spatula&amp;nbsp;</td><td>Dow Corning</td><td>3005</td><td></td></tr><tr><td>60 mm Petri dishes</td><td>Thermo Fisher Scientific</td><td>174888</td><td></td></tr><tr><td>Nunc Lab-Tek II multiwell chamber slides&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C6807</td><td></td></tr><tr><td>200 &amp;mu;l pipet tips</td><td>Bioexpress</td><td>P-1233-200</td><td>other suppliers available</td></tr><tr><td>inverted microscope&amp;nbsp;</td><td>Nikon</td><td>Diaphot</td><td>other suppliers/models available</td></tr><tr><td>humidified CO&lt;sub&gt;2&lt;/sub&gt; incubator&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>370 (Steri-Cycle)</td><td>other suppliers/models available</td></tr><tr><td>fluorescent microscope</td><td>Olympus, Japan</td><td>BX-40</td><td>other suppliers/models available</td></tr><tr><td>dissecting stereo microscope</td><td>Leica, Germany</td><td>S4 E</td><td>other suppliers/models available</td></tr></tbody></table>

adenoviral gene therapy for diabetic keratopathy: effects on wound healing and stem cell marker expression in human organ-cultured corneas and limbal epithelial cells

该协议的目的是描述分子改变在人类糖尿病性角膜并展示如何能够通过在器官培养的角膜的腺病毒基因治疗得到缓解。糖尿病角膜病是角膜神经和上皮伤口愈合的频繁异常糖尿病的并发症。我们也记录在人类糖尿病几个角膜上皮推测干细胞标记显著改变的表达。为了减轻这些变化，腺病毒的基因治疗用的c-met原癌基因表达的上调和/或蛋白酶基质金属蛋白酶10（MMP-10）和组织蛋白酶F.本疗法的下调成功实施糖尿病角膜伤口加速愈合即使只有角膜缘干细胞室转导。最好的结果是用联合治疗获得。对于标准化的干细胞可能病人移植的例子也提出了优化*的在使用聚阳离子促进干细胞富集的文化基因转导。这种方法不仅可用于选定的基因而且对角膜上皮创伤愈合的其他介质是有用的和干细胞功能。

我们以前表明，在角膜器官培养，糖尿病标记物（ 例如 ，基底膜蛋白和整联α3β1）的正常和糖尿病角膜之间和伤口愈合中的表达的差异将被保留。此培养系统进行基因治疗，旨在正常化糖尿病改变的标记物，c-Met的MMP-10的水平，以及组织蛋白酶F. 当整个角膜上皮用对AV-CMET或AV-shRNA的对MMP-10或组织蛋白酶F（单独或组合）转导，上皮伤口愈合显著更快完成（P &lt;0.03通过成对学生t检验比较AV-载体）高于从同一供体16,17对AV-载体转导的同胞角膜。在AV-CMET减少了一半，而没有显著从正常角膜愈合不同愈合时间（Figu重新1）。在AV-shM10和AV-SHCF有较小的影响。然而，在AV-CMET用AV-shM10和AV-SHCF（组合）的组合所产生的最强的效果完全正常化上皮愈合 ​​时间（ 图1）。这减少愈合时间伴随着糖尿病（基底膜和整合）的标准化模式和遏制糖尿病角膜8,16,17细胞标记物。 上皮干细胞是主要的伤口愈合参与者4。因此，我们研究角膜缘基因靶向治疗的干细胞室是否会成为糖尿病角膜有利。为此，仅糖尿病角膜的角膜缘份由AV-CMET或组合和大转导，创建8.5毫米伤口。在AV-CMET转角膜显著愈合快（P &lt;0.001通过配对t检验），比AV-载体转导的同胞眼角膜（ 图URE 2）。的组合处理导致同样的结果18。基因治疗增加糖尿病抑制标记物，如整联α3β1和基底膜成分巢蛋白-1（ 图3，左列，二合一）的表达。重要的是，在整个角膜传导8,17，与任一对AV-CMET或组合的角膜缘的基因治疗导致推定LESC标记包括K15和ΔNp63α的表达显着增加，相对于对AV-载体转导角膜（ 图3 ，右列，无论是c-met和COMBO）。这些数据支持干细胞正常化的对糖尿病角膜的基因治疗加速伤口愈合的作用，并表明我们的方法糖尿病角膜疾病治疗的可行性。 接下来我们开始研究培养的LESC富集角膜缘上皮细胞的基因治疗效果。文化正常和糖尿病角膜缘上皮细胞的建立和染色推定LESC标记。许多细胞染色阳性（ 图4）。在糖尿病培养物中的染色强度始终比在正常培养物（作为一个例子显示在图4中K15），这是非常相似的情况中，糖尿病与正常体外角膜8弱。这些实验证实所得到的细胞培养物作为适用于基因治疗。 具有端粒酶永生化的角膜上皮细胞系初步实验（见23）表明，细胞容易受可用的AV构建体转导（此处未示出数据）。但是，主缘培养被证明是更敏感的病毒载量和/或转导试剂，迫使转导协议的最优化。通过培养的角膜缘细胞转导在AV-GFP（ 图5）依赖于感染复数（MOI; 30-300范围PFU /细胞），导致在高MOI（在&gt; 80％的细胞表达GFP）相当高的水平传导。然而，较高的MOI也有毒的，从而导致细胞死亡或严重受损细胞迁移入刮伤后3-4天转导的（ 图6）。在较低MOI（10-30 PFU /细胞），产生了较少的AV传导或无细胞毒作用，但造成了一些表达GFP的细胞（5-15％）的降低。 我们接下来试图提高转导效率，而不通过使用转导增强试剂，其通常方便的AV结合到细胞表面损害细胞活力。他们通过在MOI 10-30 AV表现出角膜缘上皮细胞转导不同的效率改进。两个聚阳离子，聚-L-赖氨酸和聚凝胺，是无毒的和最有效的导致GFP阳性细胞的数目3-4倍的增加（ 图7，顶行）。 ibiBoost也是有效的，但有轻微毒性，和ViraDuctin是无效和相当的毒性（ 图7，底行）。因此，聚阳离子提供安全和有效的方式在该AV传导，以提高兴趣表达的基因，并应被用于基因表达和伤口愈合的实验。 <img alt="图1" src="/files/ftp_upload/54058/54058fig1.jpg" /> 图1:c-Met 的基因转导导致显著减少角膜上皮的创面愈合时间（平均±SEM）。此外，联合基因治疗（组合）与AV窝藏的c-met基因的shRNA对MMP-10和蛋白酶˚F基因完全正常化上皮伤口愈合时间。统计学意义（P值）由配对t检验与respectiv建立比较<html> Ë载体治疗。条形代表平均值的标准差。 <a href="https://www.jove.com/files/ftp_upload/54058/54058fig1large.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/54058/54058fig2.jpg" /> 图2:在一对糖尿病患者的角膜的8.5毫米上皮的伤口愈合。上排，矢量转导的角膜;愈合in 3天完成。下排，AV-CMET转家伙角膜;愈合5天完成。箭头显示的伤口（W）的边缘。 *，非愈合的部分（也示于高倍率插入图），与蛛形apoptosed基质角膜。它们是可见直至上皮层生长在它们的顶部。栏= 50微米。相衬。 <a href="https://www.jove.com/files/ftp_upload/54058/54058fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ove_content"FO:保together.within页="1"&gt; <img alt="图3" src="/files/ftp_upload/54058/54058fig3.jpg" /> 图3:角膜缘基因治疗后的不同标记物的免疫染色糖尿病角膜部分。两个AV-CMET和组合治疗导致糖尿病标记（整合α3β1和巢蛋白-1）和推定的干细胞标志物（K15和ΔNp63α）显着增加角膜染色。在巢蛋白-1面板箭头标记角膜上皮基底膜。酒吧= 30微米。 E，上皮细胞; S，间质。 <a href="https://www.jove.com/files/ftp_upload/54058/54058fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54058/54058fig4.jpg" /> 图4:推定LESC标记K15主角膜缘上皮培养物的免疫细胞化学染色。上排，正常的文化;大多数细胞笑W¯¯强阳性K15。下排，糖尿病文化;大多数细胞只显示弱阳性。右侧面板都带有DAPI核复染。正常和糖尿病的细胞具有相同的曝光时间进行拍摄。酒吧= 20微米。 <a href="https://www.jove.com/files/ftp_upload/54058/54058fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54058/54058fig5.jpg" /> 图5:GFP-表达在转导的角膜缘上皮细胞的水平取决于AV-GFP的感染复数:（ 一 ）30 PFU /细胞; （ 二 ）120 PFU /细胞; （ 三 ）300 PFU /细胞在转导后3天。活细胞的照片示。酒吧= 300微米。 <a href="https://www.jove.com/files/ftp_upload/54058/54058fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p类="jove_content"FO: - together.within页保留="1"&gt; <img alt="图6" src="/files/ftp_upload/54058/54058fig6.jpg" /> 图6:细胞迁移是由划痕测试所揭示增加的AV浓度的不利影响:（1）对照; （ 二 ）20 PFU /细胞; （ 三 ）80 PFU /细胞; （ 四 ）120 PFU /细胞。活细胞的照片示。酒吧= 500微米。 <a href="https://www.jove.com/files/ftp_upload/54058/54058fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54058/54058fig7.jpg" /> 图7:在30 PFU /细胞带有AV-GFP转导的角膜缘上皮细胞。 （ 一 ）控制; （ 二 ）聚-L-赖氨酸; （C，D）转导试剂; 5天转导。转导试剂Caused导致细胞的舍入和死亡（四，相衬）一个显著的细胞毒性作用。活细胞的照片示。酒吧= 400微米。 <a href="https://www.jove.com/files/ftp_upload/54058/54058fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epitheli

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

adenoviral-gene-therapy-for-diabetic-keratopathy-effects-on-wound

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  Cedars-Sinai Medical Center. An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures. 

视频: Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

科研

医学

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

新洲形成的助手依赖型腺病毒载体介导的基因转移对糖尿病小鼠肝

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

神经科学

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

一种由工程成纤维细胞生长因子1刺激的Descemet剥离的人类角膜器官培养模型，仅具有加速愈合

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

小动物模型中腺相关病毒载体的结膜下给药

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

用于轴突再生研究的角膜感觉纤维损伤方案

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

作者聚焦：通过创新模型推进角膜神经支配研究 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

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基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

新洲形成的助手依赖型腺病毒载体介导的基因转移对糖尿病小鼠肝

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

创面愈合研究的前活体角膜器官培养模型

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

一种由工程成纤维细胞生长因子1刺激的Descemet剥离的人类角膜器官培养模型，仅具有加速愈合

小动物模型中腺相关病毒载体的结膜下给药

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略