siRNA沉默HLA-A2表达的hMSCs对人异体T淋巴细胞分泌IFN-γ和IL-2功能的影响  

siRNA silencing the expression of HLA-A2 gene in hMSCs decreases the secretion of IFN-γ and IL-2 in cocultured allogeneic human T lymphocytes

作　　者：郑仕杰[1] 于洋 单伟[1] 郑德宇[1] 秦书俭[1] 包翠芬[3] 

机构地区：[1]辽宁医学院解剖教研室 [2]河北省秦皇岛市91404部队后勤部医院 [3]辽宁医学院科学实验中心

出　　处：《解剖科学进展》2011年第2期147-150,153,共5页Progress of Anatomical Sciences

基　　金：辽宁省科学技术厅计划课题(N0.2006408002-5)

摘　　要：目的探讨应用siRNA技术下调HLA-A2表达的人骨髓间充质干细胞(human mesenchymal stemcells,hMSCs)对人异体T淋巴细胞分泌功能的调节作用。方法从成人骨髓中分离和培养hMSCs,并且通过免疫细胞化学检测其表面标志;利用人工合成HLA-A2靶向小分子干扰RNA转染至第3代hMSCs,然后将转染前后的hMSCs,与经植物血凝素(PHA)刺激的人异体T淋巴细胞共同培养。用ELISA分别检测各培养上清液中T淋巴细胞分泌IFN-γ和IL-2的水平。结果原代培养3d后,约70%hMSCs贴壁生长,7d后细胞迅速增殖,进入对数生长期,14d左右进入平台期,第3代细胞多呈梭形生长,形态比较均一,hMSCs表面抗原CD44、CD166阳性。HLA-A2靶向小分子干扰RNA转染3天后,可见少量细胞死亡,免疫荧光染色可见细胞核和部分胞浆呈阳性表达。转染HLA-A2靶向小分子干扰RNA的和未转染的hMSCs均能抑制人T淋巴细胞分泌IFN-γ和IL-2,转染后的hMSCs抑制作用强于未转染的hMSCs(<0.05)。结论体外沉默HLA-A2基因表达可增强hMSCs抑制PHA刺激人T淋巴细胞分泌因子。Objective To investigate the effect of small interfering RNA（siRNA） silencing the expression of HLA-A2 gene in human mesenchymal stem cells（hMSCs） on the secretion of IFN-γ and IL-2 in allogeneic human T lymphocytes cocultured with the hMSCs in vitro. Methods hMSCs from human marrow cells were isolated and expanded,then cultured and purified by sticking plastic bottle. Using synthetic HLA-A2 targeting siRNA transfected into the 3rd generation hMSCs,the levels of INF-γand IL-2 secretion in T lymphocytes in different culture conditions were determined by EL1SA after human allogeneic T lymphocyte proliferation stimulated by phytohemagglutinin（PHA） . Results 70% hMSCs were adhered to wall following 3 days primary culture,hMSCs were amplified at day 7 and entered into logarithmic phase and into platform at day 14. hMSCs at passage 3 grew fast with spindle-shape and showed CD166-and CD44-positive. Some hMSCs were died and positive immunofluorescence was found in cytoplasm and nucleus of hMSCs 3 days after HLA-A2 targeting siRNA transfection. Both HLA-A2 targeting siRNA transfected hMSCs or nontransfected hMSCs inhibits the secretiom of INF-γand IL-2 in T lymphocytes,but the inhibited effect of transfected hMSCs is stronger than that of nontransfected hMSCs. Conclusion Silencing the expression of HLA-A2 in hMSCs can increase the inhibited effect of hMSCs on the secretiom of INF-γand IL-2 in T lymphocytes stimulated by PHA.

关 键 词：骨髓间充质干细胞 HLA-A2基因 SIRNA干扰 人T淋巴细胞 

分 类 号：R331.22[医药卫生—人体生理学]