人角膜及角膜缘上皮细胞分化标记的检测

目的检测分化标记在人角膜及角膜缘上皮细胞的表达,以了解角膜及角膜缘细胞分化状态,旨在发现新的角膜上皮干细胞的阴性标记。方法获取人角膜及角膜缘组织,对冰冻切片及整个角膜组织行免疫荧光染色检测分化标记钙粘连素E、角蛋白3(CK3)、角蛋白12(CK12)、缝隙连接蛋白43、巢蛋白(nestin)和包壳蛋白(involucrin)的表达,经荧光显微镜及激光扫描共焦电镜观察,并行半定量RT-PCR以检测其相关分化标记基因的表达。结果分化标记CK3、CK12、缝隙连接蛋白43、巢蛋白和包壳蛋白在角膜和角膜缘上皮的表层细胞表达,角膜缘基底细胞不表达。激光扫描共焦电镜观察及RT-PCR结果显示角膜缘基底上皮细胞不表达细胞CK3、连接蛋白43和巢蛋白,而角膜上皮细胞则明显表达。结论角膜及角膜缘表层上皮较为成熟分化,而角膜缘基底细胞具有未分化细胞的特征,很可能是干细胞的部位。

Identification of undifferentiation markers on human corneal and limbal epithelia

ObjectiveTo identify the expression of undifferentiation markers on human corneal and limbal epithelial cells,and the differentiation status of corneal and limbal epithelial cells in order to find negative markers of corneal epithelial stem cells.MethodsImmuno-fluorescence staining was performed in cornea-limbus sections and whole cornea mounting,and fluorescence microscopy and laser scan confocal microscopy(LSCM) were used to evaluate the expression of differentiation markers as(E-cadherin),cytokeratin 3(CK3),cytokeratin 12(CK12),Connexin(Cx)43,nestin,and involucrin.Semiquantitative RT-PCR was used to evaluate gene expression of differentiation markers in corneal and limbal epithelia cells.ResultsDifferentiation markers as CK3,CK12,Cx43,nestin,E-cadherin and involucrin stained only the superficial cells of cornea and limbal epithelia,while no stainings was found on limbal basal cells.Accordingly LSCM and RT-PCR results showed that mRNA of CK3,Cx43 and nestin were only detected in corneal epithelia cells.ConclusionThe superficial cells of limbus and cornea are of mature status,while limbal basal cells show some undifferentiated feature,which indicates the location of stem cells.