胰蛋白酶对视网膜色素上皮细胞膜MHCⅡ分子表达的影响

目的 探讨不同浓度胰蛋白酶联合EDTA和机械刮擦等措施，分离培养的视网膜色素上皮(RPE)细胞并保持细胞膜HLA—DR分子活性的最佳条件。方法 常规培养人第3—6代RPE细胞，终浓度为500U／ml的IFN—γ刺激诱导培养4d，采取0．125%胰蛋白酶联合0．01％EDTA或机械刮擦等方法分离细胞；台盼蓝拒染活细胞计数；活细胞HLA—DR间接免疫荧光染色，流式细胞术分析；荧光显微镜观察。结果 0．125%胰蛋白酶 0．01％EDTA 机械刮擦组分离活细胞率最高；FCM检测HLA—DR免疫荧光染色细胞阳性率，0．125％胰蛋白酶 0．01％EDTA 机械刮擦组、机械刮擦组与对照组存在显著性差异；相对单细胞平均荧光强度，机械刮擦组与0．125％胰蛋白酶 0．01％，EDTA 机械刮擦组最高；荧光显微镜下可见机械刮擦组、0．125％胰蛋白酶 0．01％EDTA 机械刮擦组RPE细胞形态保持较好。结论 较低浓度胰蛋白酶结合EDTA联合机械刮擦方法能较好地保持RPE细胞膜结构及其糖蛋白的功能状态。

Effects of trypsin on HLA-DR molecular expression in cultured RPE cell membrane

Objective To investigate the methods of dissociation of cultured retinal pigment epithelium ( RPE) cell and the protection of its membrane glycoprotein. Mehtods The subcultured passage 3 ~ 6 human RPE cells were stimulated with final concentration 500 U /ml IFN-gamma for 4 days and were digested with 0. 125% or 0. 25% trypsin combined with 0. 01% EDTA or scraping. The death of RPE cells was confirmed with 0. 4% trypan blue exclusion and indirect immunofluorescence staining,and flow cytometry( FCM) assay was used for the analysis of MHC II expression. Results The survive rate of RPE cell was higher in 0. 125% trypsin + 0.01% EDTA + scrap group and 0. 125% trypsin +0.01% EDTA group in comparison with other groups(P < 0. 01) ,but no statistical significance between two groups. FCM assay of the positive HLA-DR in RPE cell were significantly higher in 0. 125% trypsin +0. 01% EDTA + scraping group and scraping group in comparison with control group ( P <0. 05 ). The scraping group and 0. 125% trypsin +0.01% EDTA + scraping group showed higher fluorescent intensity than control group (P < 0. 05). Integrity of RPE cell was maintained when observed under immunofluorescence microscope in scraping group,0. 125% trypsin + 0.01% EDTA group and 0. 125% trypsin + 0.01% EDTA + scraping group. Conclusion Lower concentration of trypsin combined with EDTA and scraping can effectively protect cell membrane structure and function.