体外培养人角膜缘上皮细胞抑制激活态角膜基质细胞的生长

背景：角膜受到损伤后,角膜基质细胞激活转变为成纤维细胞,引起角膜基质瘢痕化,导致视力下降甚至丧失。 目的：观察角膜不同部位上皮细胞与角膜基质细胞的相互作用,探索角膜缘上皮细胞群能否抑制激活态角膜基质细胞的生长。 方法：采用酶消化及机械外力相结合的方法获取人角膜中央、角膜旁中央及角膜缘处角膜上皮细胞与浅层角膜基质细胞,进行体外培养。相差显微镜下观察细胞形态及生长变化。待培养角膜上皮细胞与基质细胞发生接触抑制时,记作“0 周”,采用免疫荧光染色技术检测培养细胞中PCNA及p63蛋白的表达。 结果与结论：培养的角膜上皮细胞与成纤维细胞发生接触抑制时,两种细胞间有明显分界线。角膜缘组上皮细胞中PCNA及p63蛋白均有较高的表达;角膜旁中央组PCNA有较高的表达,p63蛋白阴性表达;角膜中央组PCNA表达较低,p63蛋白阴性表达;从鉴定结果中可以得出只有角膜缘组中存在一定比例的角膜缘上皮干细胞。角膜缘组上皮细胞逐渐包围并化解成纤维细胞,在相互作用4周后,成纤维细胞聚集成死细胞团,缺乏角膜缘干细胞的中央组及旁中央组中成纤维细胞生长面积增加,上皮细胞生长受到抑制甚至死亡。说明体外培养的角膜缘上皮细胞群可以抑制激活态角膜基质细胞的生长。

Growth of actived keratocytes inhibited by limbal epithelial cells cultured in vitro

BACKGROUND:When cornea injured, it can induce the phenotypic transition of keratocyte into the fibroblast, which causes the formation of the scar in the stroma and the loss of the vision. OBJECTIVE:To observe the interaction between the corneal epithelial cell in different regions and the actived keratocyte and to explore that whether the limbal epithelial cell can inhibit the growth of the actived keratocyte. METHODS:Enzyme digestion and mechanical dissociation were adopted to collect corneal epithelial cells and the keratocyte from central cornea, paramedian cornea, limbus, and then cultured in vitro. The changes of cell morphology and growth were recorded by phase contrast microscopy. The phenotype and proliferation capacity of the cultured cells were identified by immunofluorescent staining with antibodies for p63 and proliferating cell nuclear antigen (PCNA). RESULTS AND CONCLUSION:When the contact inhibition occured between corneal epithelial cells and fibroblasts, there was a define boundaries to divide them into two sections. In the limbal group, the positively expressing rates in the cultured corneal epithelial cells were higher for PCNA and p63, in the paramedian group the expression of PCNA is also higher and the expression of p63 was negative. In the central group, the expression of PCNA is lower and p63 was negative expression. Only the limbal group has a certain proportion of limbal epithelial stem cells. In limbal group the growth of fibroblasts was inhibited by limbal epithelial cells, after interaction four weeks, fibroblasts were degenerated into dead cell clusters. Because of the limbal stem cells deficiency in central group and paramedian group, the area of fibroblasts was increasing obviously, but the growth of corneal epithelial cells was inhibited and some were displaced by fibroblasts. It is indicated that limbal epithelial cells can inhibit the growth of actived keratocytes and degenerate them in vitro culture.