高糖抑制角膜缘干细胞间质性及其迁移能力的研究

High glucose suppresses the corneal limbal stem cell migration and changes the cell phenotype

目的探讨高糖环境对角膜缘干细胞增殖、迁移能力及表面分子标志的影响。方法通过细胞免疫荧光、细胞增殖检测(CCK-8)、划痕和Transwell实验观察角膜缘干细胞在高糖环境下的迁移及增殖能力的改变。选取16只SPF级大鼠用链脲霉素进行糖尿病造模,14只正常SPF级大鼠作为对照组,刮除两组大鼠角膜上皮观察上皮创伤愈合情况,采用HE染色和免疫组织化学染色探讨糖尿病对角膜缘干细胞表面分子标志及细胞状氉态的影响。结果高糖可导致体外培养的角膜缘干细胞增殖减慢,24 h、48 h及72 h增殖率为0.728、0.345及0.395,较对照组明显降低(P<0.05),迁移功能障碍(48 h时正常对照组迁移率为100%,高糖组为17.6%);高糖组细胞表面分子β-catenin和vimentin的mRNA和蛋白表达水平降低、细胞内定位异常。糖尿病大鼠角膜上皮创伤后愈合延迟,角膜组织HE染色发现糖尿病大鼠角膜上皮层变薄,基底细胞结构紊乱、形态异常。角膜免疫组织化学染色发现与对照组相比,角膜基底细胞的vimentin和β-catenin蛋白表达明显降低。结论高糖可导致角膜缘干细胞迁移障碍和增殖抑制,其损伤机制与高糖导致角膜缘干细胞表面分子标志的丢失有关。

Objective To explore the influence of extracellular high glucose on the proliferation,migration and biomarkers of corneal limbal stem cells.Methods Establishment of a model of high glucose in cultured human limbal stem cells to observe and investigate the effects of extracellular high glucose on the proliferation and migration of corneal limbal stem cells by immunoflurescence,CCK-8 and Transwell assay,respectively.Totally 16 SPF rats were collected and induced diabetic model by streptozotocin as the high-glucose group,and the normal rats of the same age served as the control group.Corneal epidermises of rats in both groups were scraped to observe the repair of corneal epithelium.And the corneas were treated with HE staining and immunohistochemical staining to detect the expression of biomarkers of corneal limbal stem cells and the modality changes of cells.Results The proliferation rate of human limbal epithelial cells was significantly decreased when exposed to high glucose,and the rate at 24 h,48 h and 72 h was 0.728,0.345 and 0.395,respectively,which was markedly lower than that in the control group,with a significant difference(P<0.05);meanwhile the cell migration rate of the high-glucose group was 17.6%at 48 h,which was significantly slower than that of the control group(100%).And the inhibition was accompanied by the decreased expression ofβ-catenin and vimentin.Furthermore,the expression levels ofβ-catenin and vimentin mRNA and protein were down-regulated,with abnormal location,in the high-glucose group.And diabetic rats had poor corneal epithelial healing.The epithelial layer became thinner and the structures were disorganized in diabetic rats through HE staining.The immunohistochemical assay revealed the expression ofβ-catenin and vimentin of cornea limbal stem cells was down-regulated in high-glocose group when compared with the control group.Conclusion High glucose can significantly inhibit the proliferation and migration of cornea limbal stem cells,and its main damage mechanism is correlated with the abnormalities ofβ-catenin and vimentin.