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目的体外组装补体膜攻击复合物(membrane attack complex,MAC),建立肾小管上皮细胞HK-2亚溶破模型,为进一步探讨小管间质的免疫性损伤效应奠定基础。方法用酵母多糖激活急性期患者血清制备补体优球蛋白C56,以新鲜正常人血清(NHS)作为C7~C9来源,体外组装s MAC建立HK-2亚溶破模型,LDH检测评价细胞亚溶破剂量,激光共聚焦显微镜(LSCM)鉴定s MAC沉积,流式细胞仪检测s MAC对HK-2表面HLA-DR分子表达的影响。结果确定C561:480,NHS 1:20为HK-2最适亚溶破量;LSCM显示s MAC沉积于HK-2细胞表面;不同时相观察s MAC刺激后HK-2细胞HLA-DR表达量逐渐升高,HLA-DR比对照组显著增加(P<0.01)。结论成功建立了肾小管上皮细胞补体膜攻击复合物亚溶破模型,补体活化可能导致TEC本身参与了免疫炎症效应。
OBJECTIVE: To establish a membrane attack complex (MAC) in vitro and to establish HK-2 sub-lysis model of renal tubular epithelial cells, which will lay a foundation for further study on the immunologic injury of tubulointerstitium. Methods Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of complement eru cylin C56 in the serum of patients with acute phase infection. Fresh normal human serum (NHS) was used as a source of C7 ~ C9. The expression of HLA-DR on HK-2 surface was detected by flow cytometry after laser scanning confocal microscopy (LSCM) for s MAC deposition. The results showed that C561: 480 and NHS 1:20 were the best subsolid-lysates of HK-2. LSCM showed that s MAC was deposited on the surface of HK-2 cells. The expression of HLA-DR in HK-2 cells Gradually increased, HLA-DR than the control group increased significantly (P <0.01). Conclusion The sub-lysis model of human renal tubular epithelial cell complement membrane attack complex has been established successfully. Complement activation may cause TEC itself to participate in the immunoinflammatory effect.