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
Summary
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.
Abstract
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。
Introduction
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。
Protocol
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。 <p cla…
Representative Results
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）?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| minimum essential medium | Thermo Fisher Scientific | 11095-080 | |
| Optisol-GS | Bausch & Lomb | 50006-OPT | |
| ABAM antibiotic-antimycotic mixture | Thermo Fisher Scientific | 15240062 | |
| calf skin collagen | Sigma-Aldrich | C9791 | |
| agar, tissue culture grade | Sigma-Aldrich | A1296 | |
| n-heptanol | Sigma-Aldrich | 72954-5ML-F | |
| O.C.T. compound | VWR International | 25608-930 | |
| Dispase II | Roche Applied Science | 4942078001 | |
| keratinocyte serum-free medium (KSFM) | Thermo Fisher Scientific | 17005042 | |
| EpiLife medium with calcium | Thermo Fisher Scientific | MEPI500CA | |
| N2 medium supplement, 100x | Thermo Fisher Scientific | 17502-048 | |
| B27 medium supplement, 50x | Thermo Fisher Scientific | 17504-044 | |
| human keratinocyte growth supplement, 100x | Thermo Fisher Scientific | S-001-5 | |
| trypsin 0.25% – EDTA 0.02% with phenol red | Thermo Fisher Scientific | 25200056 | |
| trypsin 0.25% with phenol red | Thermo Fisher Scientific | 15050065 | |
| soybean trypsin inhibitor | Sigma-Aldrich | T6414 | |
| fetal bovine serum | Thermo Fisher Scientific | 26140079 | |
| insulin-transferrin-selenite supplement (ITS) | Sigma-Aldrich | I3146-5ML | |
| antibody to keratin 14 | Santa Cruz Biotechnology | sc-53253 | |
| antibody to keratin 15 | Santa Cruz Biotechnology | sc-47697 | |
| antibody to keratin 17 | Santa Cruz Biotechnology | SC-58726 | |
| antibody to ΔNp63α | Santa Cruz Biotechnology | sc-8609 | |
| antibody to PAX6 | BioLegend | PRB-278P-100 | |
| antibody to nidogen-1 | R&D Systems | MAB2570 | |
| antibody to integrin α3β1 | EMD Millipore | MAB1992 | |
| human fibronectin | BD Biosciences | 354008 | |
| human laminin | Sigma-Aldrich | L4445 | |
| human type IV collagen | Sigma-Aldrich | C6745-1ML | |
| adenovirus expressing MMP-10 shRNA | Capital BioSciences | custom made | |
| adenovirus expressing cathepsin F shRNA | Capital BioSciences | custom made | |
| adenovirus expressing scrambled shRNA and GFP | Capital BioSciences | custom made | |
| adenovirus expressing c-met | OriGene (plasmid) | SC323278 | |
| adenovirus expressing GFP | KeraFAST | FVQ002 | |
| sildenafil citrate, 25 mg | Pfizer | from pharmacy | |
| epidermal growth factor | Thermo Fisher Scientific | PHG0311 | |
| poly-L-lysine | Sigma-Aldrich | P4707 | |
| polybrene | Sigma-Aldrich | 107689-10G | |
| ViraDuctin | Cell Biolabs | AD-200 | |
| ibiBoost | ibidi, Germany | 50301 | |
| phosphate buffered saline (PBS) | Thermo Fisher Scientific | 10010049 | |
| Corning round end spatula | Dow Corning | 3005 | |
| 60-mm petri dishes | Thermo Fisher Scientific | 174888 | |
| Nunc Lab-Tek II multiwell chamber slides | Sigma-Aldrich | C6807 | |
| 200 microliter pipet tips | Bioexpress | P-1233-200 | other suppliers available |
| inverted microscope | Nikon | Diaphot | other suppliers/models available |
| humidified CO2 incubator | Thermo Fisher Scientific | 370 (Steri-Cycle) | other suppliers/models available |
| fluorescent microscope | Olympus, Japan | BX-40 | other suppliers/models available |
| dissecting stereo microscope | Leica, Germany | S4 E | other suppliers/models available |
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Cite This Article
Kramerov, A. A., Saghizadeh, M., Ljubimov, A. V. Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells. J. Vis. Exp. (110), e54058, doi:10.3791/54058 (2016).