Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, California, USA.
Invest Ophthalmol Vis Sci. 2010 Apr;51(4):1970-80. doi: 10.1167/iovs.09-4569. Epub 2009 Nov 20.
Purpose. Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor (HGF) receptor, c-met proto-oncogene, and impaired wound healing. Recombinant adenovirus (rAV)-driven c-met overexpression in human organ-cultured corneas was tested for correction of diabetic abnormalities. Methods. Forty-six human corneas obtained postmortem from 23 donors with long-term diabetes (5 with diabetic retinopathy) were organ cultured and transduced with rAV-expressing c-met gene (rAV-cmet) under the cytomegalovirus promoter at approximately 10(8) plaque-forming units per cornea for 48 hours. Each control fellow cornea received control rAV (rAV expressing the beta-galactosidase gene or vector alone). After an additional 4 to 5 days of incubation, 5-mm epithelial wounds were created with n-heptanol, and healing was monitored. The corneas were analyzed afterward by immunohistochemistry and Western blot analysis. Signaling molecule expression and role was examined by immunostaining, phosphokinase antibody arrays, Western blot analysis, and inhibitor analysis. Results. rAV-cmet transduction led to increased epithelial staining for c-met (total, extracellular, and phosphorylated) and normalization of the patterns of select diabetic markers compared with rAV-vector-transduced control fellow corneas. Epithelial wound healing time in c-met-transduced diabetic corneas decreased twofold compared with rAV-vector-transduced corneas and became similar to normal. c-Met action apparently involved increased activation of p38 mitogen-activated protein kinase. c-Met transduction did not change tight junction protein patterns, suggesting unaltered epithelial barrier function. Conclusions. rAV-driven c-met transduction into diabetic corneas appears to restore HGF signaling, normalize diabetic marker patterns, and accelerate wound healing. c-Met gene therapy could be useful for correcting human diabetic corneal abnormalities.
糖尿病角膜显示出基底膜和整合素标志物的改变、蛋白水解酶表达增加、肝细胞生长因子（HGF）受体、c-met 原癌基因减少以及伤口愈合受损。已经测试了重组腺病毒（rAV）驱动的 c-met 过表达在人器官培养角膜中的作用，以纠正糖尿病异常。
从 23 名长期糖尿病（5 名患有糖尿病性视网膜病变）的已故供体中获得 46 个人眼角膜，进行器官培养，并在巨细胞病毒启动子下用 rAV 表达 c-met 基因（rAV-cmet）转导，每个角膜约 10（8）个噬菌斑形成单位，持续 48 小时。每个对照同眼接受对照 rAV（表达β-半乳糖苷酶基因或载体）。孵育 4 至 5 天后，用正庚烷创建 5mm 的上皮伤口，并监测愈合情况。随后通过免疫组织化学和 Western blot 分析分析角膜。通过免疫染色、磷酸激酶抗体阵列、Western blot 分析和抑制剂分析检查信号分子表达和作用。
rAV-cmet 转导导致 c-met（总、细胞外和磷酸化）上皮染色增加，并与 rAV-载体转导的对照同眼相比，正常化了选择性糖尿病标志物的模式。与 rAV-载体转导的角膜相比，c-met 转导的糖尿病角膜上皮伤口愈合时间缩短了两倍，并且变得与正常相似。c-Met 作用显然涉及 p38 丝裂原活化蛋白激酶的激活增加。c-Met 转导没有改变紧密连接蛋白模式，表明上皮屏障功能没有改变。
rAV 驱动的 c-met 转导到糖尿病角膜中似乎恢复了 HGF 信号，使糖尿病标志物模式正常化，并加速了伤口愈合。c-met 基因治疗可能对纠正人糖尿病角膜异常有用。
