体外组装补体膜攻击复合物建立肾小管上皮细胞亚溶破模型

Establishment of complement sublytic tubular epithelial cells model by assembled membrane attack complex in vitro

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一2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sMAC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sMAC沉积，流式细胞仪检测sMAC对HK-2表面HLA-DR分子表达的影响。结果确定C56 1：480，NHS 1：20为HK-2最适亚溶破量；LSCM显示sMAC沉积于HK-2细胞表面；不同时相观察sMAC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 sublytic membrane attack complex （sMAC） model and to identify the sublytic effects on renal tubular epithelial cell （TEC）. Methods An activated complex of C56 was generated by treatment of acute phase serum and C7- C9 come from fresh normal human serum to assemble sMAC on TEC. The sublytic dose of sMAC was determined by LDH release assay, sMAC deposition was identified by laser confocal microscope（LSCM） ; the expressions of HLA-DR on TEC were detected by flow cytometry analysis. Results The optimization sublytic dose of MAC was determined as C56 1：480 and NHS 1：20. sMAC deposition TEC cells was seen under a LSCM; sMAC increased the expression of HLA-DR after 72h of stimulation（P〈0.01 vs controls）. Conclusion The sublytic membrane attack complex （sMAC） model of renal tubular epithelial cell is established successfully, sMAC can increase the expressions of HLA-DR on TEC, which suggests that sMAC may have proinflammatory effects on TECs.