人类永生化角膜内皮细胞的生物学特性

Biological features of immortalized human corneal endothelial cells

目的探讨人类永生化角膜内皮细胞在免疫反应中的分子表达及其刺激T淋巴细胞活化的能力。方法人类永生化角膜内皮细胞分别培养在含干扰素-γ(IFN-γ)(1000U/mL)和不包含IFN-γ(1000U/mL)的F99和M199培养液中,按照1∶1等比例混合并含有5%小牛血清。健康志愿者采集外周血并分离外周血单核细胞(PBMCs),应用锥虫蓝染色鉴定细胞的完整性和细胞数量。免疫组织化学染色法测定人类永生化角膜内皮细胞分别在有无IFN-γ诱导条件下的HLA-DP、DQ、DR抗原及CD40的表达。同时,体外共同培养人类永生化角膜内皮细胞和PBMCs,荧光活化细胞分类(FACS)法测定活化的T淋巴细胞中CD69的表达。结果 IFN-γ能够诱导HLA-DP、DQ、DR抗原和CD40在人类永生化角膜内皮细胞上的阳性表达,表现为细胞膜和细胞质中的红色染色。CD3和CD28抗体预处理的外周血单核细胞中,20%的CD69阳性T淋巴细胞活化,无IFN-γ诱导时有10%阳性T淋巴细胞,而IFN-γ诱导组中,有12%的阳性T淋巴细胞,说明共同培养人类永生化角膜内皮细胞和PBMCs体系中,CD69阳性淋巴细胞数量增加。结论外周血中的T淋巴细胞可以被人类永生化角膜内皮细胞活化,由此人类永生化角膜内皮细胞是具有免疫学特性潜能的抗原递呈细胞。

Background Regarding to immunological point, the tissue of eye, including cornea and retina, are in immunoprivileged location. So far, seldom reports concerned the biological features and characteristics of immortalized human corneal endothelial cells were reported. Objective In this study, immortalized human corneal endothelial cells were investigated to evaluate the capabilities presenting the molecule in immunological reaction and stimulate the activation of T lymphocytes. Methods Immortalized human corneal endothelial cells were cultivated in medium-F99 ＋ M199 （ 1：1 ） containing 5% calf serum with and without γ-IFN （1 000 U/mL） respectively. Peripheral blood monocytes were collected from the peripheral blood of healthy subjects and the integrity and numbers of monocytes were detected by trypan blue staining. Expressions of the human leukocyte antigen （HLA） -DP,-DQ,-DR and CD40 in immortalized human corneal endothelial cells were detected by immunohistoehemistry. Meanwhile,purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells for the fluorescence activated cell sorting （FACS） analysis. The expression of CD69 was determined by FACS analysis to evaluate the activated T lymphocytes. Results In γ-interferon inducement group,HLA-DP,-DQ,-DR and CD40 antigen were positively expressed in immortalized human corneal endothelial cells, showing the red staining on the surface of cells and cytoplasm,and no positive expression of CD40 antigen was seen in the ceils without γ-intefferon inducement. About 20% CD69 positive T lymphotes were seen in pre-treated peripheral blood mononuclear ceils with CD3 and CD28 antibody, 10% positive T lymphotes in without γ-interferon group and 12% positive T lymphotes in with γ-interferon group,indicating the numbers of CD69 positive lymphocytes were increased in the cocuhure system of immortalized human corneal endothelial cells and peripheral blood mononuclear cells. Conclusion T-lymphocytes in the peripheral blood can be activated by immortalized human corneal endothelial cells. It was assumed that immortalized human corneal endothelial cells function as potential antigen presenting cells in the immunological process.