摘要目的 观察补体对自体骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)的作用,并检测膜补体调节蛋白对这种补体作用的调节.方法 获取10例临床骨折患者的骨髓,分离、鉴定及培养BMSCs,并进行体外实验.Europium细胞活性实验,观察自体血清中补体对BMSCs的杀伤作用.每例患者BMSCs均分为BMSCs组、BMSCs+自体血清组和BMSCs+补体灭活自体血清组.采用流式细胞术检测骨髓间质干细胞膜上的补体膜攻击复合物(membrane attack complex,MAC)沉积;转染膜补体调节蛋白(membrane complement regulatory proteins,mCRPs),观察补体对BMSCs作用的变化.每例患者BMSCs均分为mCRPs未转染组以及各mCRPs转染组.结果 骨髓获取并分离培养的细胞经流式细胞仪检测表面标记物,其中＞95％为间质干细胞.10例患者的BMSCs与自体血清孵育后,BMSCs均有不同程度的损伤.BM-SCs组的细胞杀伤率为0.41％±1.48％,BMSCs+自体血清组为32.59％±2.73％,两者比较差异有统计学意义;而BMSCs+补体灭活自体血清组的细胞杀伤率为2.59％±3.08％,与BMSCs组相似.流式细胞仪检测发现在自体血清孵育组细胞表面可检测到补体激活产生的MAC.在转染mCRPs基因后,自体血清对于BMSCs的杀伤作用较未转染组下降,未转染组细胞杀伤率(41.70％±4.47％)分别与CD55转染组(21.87％±2.19％)、CD59转染组(18.67％±1.42％)、同时转染CD46+CD55+CD59组(28.43％±2.14％)比较,差异均有统计学意义.CD55、CD59和CD46+CD55 +CD59转染组均能有效阻止补体对细胞的损伤,而CD46的作用较差(杀伤率为39.30％±3.96％),与未转染组比较,差异无统计学意义.结论 自体血清中的补体能识别并杀伤来源于同一个体的BMSCs;外源性转染mCRPs能有效保护BMSCs免受补体的攻击.

abstractsObjective To explore the effect of complement activation on bone marrow mesenchymal stem cells (BMSCs)and evaluate the effect after transfection of complement regulatory proteins.Methods Bone marrow aspirate was harvested from 10 cases of patients suffered from fractures.Mesenchymal stem ceils were isolated,indentified cultured and then experimented in vitro.The complement cytotoxicity on the mesenchymal stem cells in autologous serum was measured by Europium cytotoxicity assay.The samples were divided into BMSCs group,BMSCs+ autologous human serum (AHS) group and BMSCs+ inactivated autologous human serum (iAHS) group.The complement membrane attack complex (MAC) deposited on the membranes was detected by flow cytometry.Finally,the cytotoxicity on BMSCs was measured after transfected with membrane complement regulatory proteins (mCRPs).All samples were divided into BMSCs with mCRPs untransfected group and BMSCs with mCRPs transfected group.Results More than 95％ of cells derived from bone marrow were identified to be mesenchymal stem cells through detection of cell surface markers by flow cytometry.The cytotoxicity of untreated cells was 0.41％± 1.48％.BMSCs harvested from the 10 patients all had cytotoxicity after incubated with autologous serum,and the cytotoxicity was 32.59％±2.73％,while cytotoxicity after incubated with complement inactivated autologous serum was 2.59％±3.08％,which was similar to control group.Complement attack complex (MAC) could be detected on the BMSCs incubated with autologous serum,which implied the complement activation.After transfection of mCRPs,the cytotoxicity of autologous serum on transfected cells was decreased.The cytotoxicity of untransfected cells (41.70％±4.47％) had significant difference compared to the cells transfected with CD55 (21.87％±2.19％),the cells transfected with CD59 (18.67％± 1.42％),and the cells transfected with CD46+CD55+CD59 (28.43％±2.14％).CD55,CD59 and CD46+CD55 +CD59 transfected groups could impair effectively the cytotoxicity from complement.However,the cytotoxicity impairment was less effective in CD46 transfected cells (39.30％±3.96％),which had no significant difference compared to untransfected cells.Conclusion Membrane complement regulatory proteins could effectively protect bone marrow mesenchymal stem cells from attacks by complement.