Normalization of Wound Healing and Diabetic Markers in Organ Cultured Human Diabetic Corneas by Adenoviral Delivery of c-Met Gene
Normalization of Wound Healing and Diabetic Markers in Organ Cultured Human Diabetic Corneas by Adenoviral Delivery of c-Met Gene
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DOI：
10.1167/iovs.09-4569
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发表时间：
2010-04-01
影响因子：
4.4
通讯作者：
Ljubimov, Alexander V.
中科院分区：
文献类型：
--
作者：
Saghizadeh, Mehrnoosh;Kramerov, Andrei A.;Ljubimov, Alexander V.
目的.糖尿病角膜显示基底膜和整合素标志物改变，蛋白酶表达增加，肝细胞生长因子（HGF）受体、c-met原癌基因减少，伤口愈合受损。在人器官培养的角膜中测试重组腺病毒（rAV）驱动的c-met过表达用于校正糖尿病异常。对从23名患有长期糖尿病的供体（5名患有糖尿病性视网膜病）死后获得的46个人角膜进行器官培养，并在巨细胞病毒启动子下用表达rAV的c-met基因（rAV-cmet）以每个角膜约10（8）个噬斑形成单位转导48小时。每个对照对侧角膜接受对照rAV（仅表达β-半乳糖苷酶基因或载体的rAV）。再孵育4至5天后，用正庚醇形成5 mm上皮伤口，并监测愈合情况。随后通过免疫组织化学和蛋白质印迹分析对角膜进行分析。通过免疫染色、磷酸激酶抗体阵列、蛋白质印迹分析和抑制剂分析检测信号分子的表达和作用。rAV-cmet转导导致c-met（总的、细胞外的和磷酸化的）的上皮染色增加，并且与rAV-载体-转导的对照对侧角膜相比，选择的糖尿病标志物的模式正常化。与rAV载体转导的角膜相比，c-met转导的糖尿病角膜中的上皮伤口愈合时间减少了两倍，并且变得与正常相似。c-Met的作用显然涉及p38丝裂原活化蛋白激酶的活化增加。c-Met转导并没有改变紧密连接蛋白的模式，表明上皮屏障功能没有改变。rAV驱动的c-met转导进入糖尿病角膜似乎恢复HGF信号传导，使糖尿病标志物模式正常化，并加速伤口愈合。c-Met基因治疗可用于矫正人类糖尿病角膜异常。（Invest Ophthalmol维斯科学。2010; 51：1970-1980）DOI：10.1167/iovs.09-4569
PURPOSE. Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor (HGF) receptor, c-met protooncogene, 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. (Invest Ophthalmol Vis Sci. 2010; 51:1970-1980) DOI: 10.1167/iovs.09-4569