目的： 研究高糖环境对人角膜上皮细胞损伤修复的影响，初步探讨Cyclin D1蛋白在高糖培养的角膜上皮细胞创伤愈合中表达的意义。

方法： 模拟糖尿病角膜病变的高糖微环境，人角膜上皮细胞经复苏、培养传代后，分别设置等体积蒸馏水的DMEM完全培养基的正常对照组和含25mmol/L葡萄糖的DMEM完全培养基高糖处理组的两组细胞，细胞长满后进行划伤刺激，倒置相差显微镜下观察比较各组细胞生长状况及其变化，于不同时间点(0、12、24、48和72h)利用Western blot检测分析高糖培养的角膜上皮细胞中Cyclin D1蛋白的表达，qRT-PCR检测Cyclin D1 mRNA水平相对表达情况。

结果： 体外高糖处理条件下，人角膜上皮细胞划伤后修复速度减慢，漂浮细胞增多，细胞重新贴壁少，细胞间距增加，随着高糖处理时间的延长，细胞状态变差，生长速度慢； 正常组细胞损伤修复较快， 细胞密度增大，且形态规则，细胞膜光滑。Western blot 检测划伤刺激上调Cyclin D1的表达，但随着时间延长上调作用呈逐渐减弱，两组Cyclin D1最高表达量均出现在12h。高糖处理组Cyclin D1表达量低于正常对照组。qRT-PCR结果显示：高糖处理后，Cyclin D1 mRNA表达呈上调趋势，但随着高糖处理时间延长，上调作用减弱，且在48h处mRNA水平恢复至与对照组同一水平。

结论： 在角膜上皮细胞创伤愈合过程中，高糖呈负性调节，抑制Cyclin D1蛋白的表达，且与角膜上皮细胞增殖能力下降及凋亡相关。

METHODS: The high-glucose micro-environment of diabetic corneal lesions was simulated. After human corneal epithelial cells were resuscitated, cultured and passaged, a normal control group of DMEM complete medium of equal volume of distilled water and a high-glucose treated group of DMEM complete medium containing 25mmol/L glucose were set respectively. After the cells were overgrown, the cells were stimulated with scratches. The growth conditions and changes of the cells in each group were observed and compared under an inverted phase contrast microscope. Western glucose was used to analyze high glucose at different time points(0, 12, 24, 48, and 72h)Cyclin D1 Protein expression in cultured corneal epithelial cells. The qRT-PCR was used to analyze high glucose at different time points and each group Cyclin D1 mRNA expression.

RESULTS: Under the conditions of high glucose treatment in vitro, the repair rate of human corneal epithelial cells was slowed down after injury, floating cells increased, cells reattached less, and cell spacing increased. With the increase of high glucose treatment time, the cell state became worse and the growth rate slow; normal group repaired cell damage faster, increased cell density, regular morphology, and smooth cell membrane. Cyclin D1 expression was up-regulated by Western blot, but the up-regulation effect gradually weakened with time. The highest expression of Cyclin D1 in both groups appeared at 12h. The expression of Cyclin D1 in the high glucose treatment group was lower than that in the normal control group. The qRT-PCR results showed that after high glucose treatment, the expression of Cyclin D1 mRNA was up-regulated, but with the increase of high glucose treatment time, the up-regulation effect weakened, and the mRNA level recovered to the same level as the control group at 48h.

CONCLUSION: In the process of corneal epithelial cell wound healing, high glucose negatively regulates and inhibits the expression of Cyclin D1 protein, and is related to the decline of corneal epithelial cell proliferation and apoptosis.