SiRNA沉默HLA-A2表达的人骨髓间充质干细胞对人异体T淋巴细胞分泌功能及其诱导成骨能力的影响  

Effects of RNA silenced HLA-A2 in hBMSCs on secretory function of human allogeneic T lymphocytes and osteogenic ability of hBMSCs ＊

作　　者：单伟[1] 包翠芬[2] 郑仕杰[1] 刘雷 王岩 秦书俭[1] 

机构地区：[1]辽宁医学院解剖学教研室,锦州121001 [2]辽宁医学院科学实验中心,锦州121001 [3]锦州市妇婴医院不孕不育科,锦州121001 [4]辽宁辽河油田中心医院放射科,盘锦124000

出　　处：《解剖学杂志》2012年第2期145-148,F0002,共5页Chinese Journal of Anatomy

基　　金：辽宁省教育厅基金（LT2010061）;辽宁省自然科学基金（20102138）;辽宁医学院青年科技启动基金（Y2010z.04）

摘　　要：目的：探讨应用RNAi技术沉默HLA-A2表达的人骨髓间充质干细胞（hBMSCs）对人异体T淋巴细胞分泌功能的调节作用及诱导成骨能力的影响。方法：用siRNA转染至第3代hBMSCs将转染前、后各组的hBMSCs与PHA刺激的人异体T淋巴细胞共同培养，用ELISA检测各培养组上清液γ干扰素（IFN-γ）、白细胞介素2（IL-2）的水平；对2组细胞进行成骨诱导分化培养，观察细胞形态变化；碱性磷酸酶染色、计数和Von-Kossa染色、计数检测成骨能力。结果：hBMSCs表面抗原CD44、CDl66阳性。转染前hBMSCs的HLA-A2表达为阳性，转染后hBMSCs的HLA-A2表达为弱阳性；转染前hBMSCs的HLA-A2蛋白表达量高于转染后的HLA-A2蛋白表达量。转染前、后的hBMSCs均能抑制人T淋巴细胞分泌IFN-γ、IL-2，转染前、后比较有差异。ALP染色显示转染前、后的hBMSCs均出现胞质呈棕黑色的阳性反应，ALP活性检测结果显示2组之间无差异。Von-Kossa染色显示转染前、后的hBMSCs均有钙结节形成，统计2组之间无差异。结论：应用RNAi技术沉默HLA-A2表达的hBMSCs可以抑制PHA刺激人T淋巴细胞分泌因子IFN-γ、IL-2的水平，转染后的hBMSCs比未转染的hBMSCs抑制作用更强。应用RNAi技术沉默HLA-A2表达的hBMSCs，不影响其成骨能力。Objective： To explore the secretory function of human allogeneic T lymphocytes cocultured with human bone mar- row derived mesenchymal stem cells （hBMSCs）, in which the HLA-A2 gene was silenced using RNA interference, and, to observe the influence of this RNA interference on osteogenic ability of hBMSCs. Methods： The hBMSCs were harvested and identified. And, human allogeneic T lymphocytes stimulated by PHA were cocultured with 1st to 3rd passaged hBMSCs before and after transferred with siRNA targeting HLA-A2. 72 h later, the levels of IFN-＇/and IL-2 secreted from human allogeneic T lympho- cytes were detected using ELISA. Subsequently, the osteogenic induction of hBMSCs with and without RNA interference was per- formed in vitro. The osetogenic ability was judged by alkaline phosphatase （ALP） staining and Von-Kossa staining. Results： The cultured hBMSCs were CIN4 and CD166 positiv~ The RNA interference targeting HLA-A2 induced a significant down-regulation of HLA-A2 in hBMSCs. The levels of IFN-7 and IL-2 were decreased from human allogeneic T lymphocytes cocultured with trans- ferred or non-transferred hBM^s, especially with transferred ones. No difference was found both in ALP and Von-Kossa staining. Conclusion： The HLA-A2 silence in hBMSCs has stronger inhibition on IFN-7 and IL-2 secreted from human allogeneic T lympho- cytes than non-transferred hBM^s, but has no effects on its ostogenic ability.

关 键 词：骨髓间充质干细胞 RNAI技术 T淋巴细胞 分泌功能 成骨能力 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]