系统性红斑狼疮患者骨髓间充质干细胞的体外扩增及鉴定

In vitro amplification and identification of bone marrow mesenchymal stem cells from patients with systemic lupus erythematosus

目的:探讨系统性红斑狼疮患者骨髓间充质干细胞的体外培养方法、生物学功能、表面标记表达的异常与否。方法:选择2004-12/2005-12于南京医科大学第一附属医院风湿科初诊为系统性红斑狼疮患者5例,均为女性,年龄20～35岁,诊断符合美国风湿病学院(ARA)1997年修订的诊断标准,所有患者知情同意后,获取骨髓标本。采用密度梯度离心法和贴壁筛选法分离培养5例系统性红斑狼疮患者骨髓间充质干细胞,观察其形态学改变,测定其生长曲线,采用流式细胞仪鉴定骨髓间充质干细胞表面标记物及分析细胞周期。结果:5例系统性红斑狼疮患者骨髓间充质干细胞均分离培养成功,其中1例因出现细菌污染而终止培养。①传代细胞生长较原代要快,细胞在8～10d即可达到90%融合。对P1、P2代细胞生长曲线进行观察,发现这2代细胞具有以下共同特征:传代培养的潜伏期为1～3d,第4天起呈对数增长,至第7天达到高峰,之后进入平台期。②流式细胞术检测间充质干细胞细胞表面抗原显示,细胞高表达CD105、CD13、CD90、CD73,而CD34、CD45、CD38、CD33阴性,3代以后的骨髓间充质干细胞纯度达到了95%以上,基本上排除了骨髓单个核细胞和巨噬细胞的污染。③间充质干细胞的周期分析显示,扩增后的间充质干细胞9.46%的细胞处于S/G2/M期,90.54%处于G0/G1期。结论:系统性红斑狼疮患者骨髓间充质干细胞体外可有效分离培养,所培养扩增的细胞成分单一,且保持干细胞特性,适于以后临床应用。

AIM： To explore the in vitro culture approaches, biological function and expression of surface marking of bone marrow mesenchymal stem cells （MSCs） from patients with systemic lupus erythematosus （SLE）. METHODS： Totally 5 female SLE patients aged 20-35 years were selected at Department of Rheumatology, First Affiliated Hospital, Nanjing Medical University from December 2004 to December 2005. The diagnosis was accorded with the diagnostic criteria of American Rheumatism Association （ARA） in 1997. MSCs were isolated from bone marrow of 5 SLE patients after their agreement by density centrifugation and adhesive culture in vitro. The morphological change and the proliferation growth curve of MSCs were observed in primary and passage cultures. The surface makers and cell cycle were detected by flow cytometry （FCM）. RESULTS： MSCs were successfully isolated from 5 SLE patients, and MSCs from one patient were stopped culturing, because of bacterial contamination.①Passage cells grew rapidly as compared with the primary cells. These cells were 90% confluence for 8-10 days. After observing the growth curve of P1 and P2 cells, it was found that the latency phase was for 1-3 days, and from the 4^th day these cells entered log phase of growth, reached the peak on the 7= day and then entered platform phase.②After flow cytometry, MSCs surface antigen verified that MSCs expressed CD105, CD13, CD90, CD73, but did not express CD34, CD45, CD38, CD33. The purity of the third generation was more than 95%, and bone marrow mononuclear cells and macrophagus contamination were excluded basically.③ MSCs cycle analysis displayed that 9.46% of the cells were in S/G2/M, and 90.54% of the cells were in G0/G1. CONCLUSION： The MSCs from SLE patients can be cultured purely and extensively proliferate and retain stem cell biological feature. The methods of MSCs culture can be used for clinical MSCs therapy.