SOCS1在人骨髓间充质干细胞中的表达以及稳定表达SOCS1间充质干细胞株的建立

The Expression Pattern of SOCS1 in Bone Marrow MSCs and the Construction of MSCs with the Over-expression of SOCS1

目的探讨不同炎性因子刺激下细胞因子信号转导抑制分子-1(suppressor of cytokine signaling1,SOCS1)在人骨髓间充质干细胞(mesenchymal stem cells,MSCs)中的表达情况,并研究稳定表达SOCS1的MSCs细胞株的建立方法。方法用SOCS1相关炎性因子刺激剂处理24小时后裂解MSCs,Westen Blot检测各组细胞SOCS1的表达。通过Gateway技术,构建出表达SOCS1基因的载体质粒p Final/PGK-puro-EF1α-SOCS1-IRES-EGFP,将其与包装质粒共转染293FT细胞产生慢病毒,通过多次感染将载体导入人骨髓间充质干细胞,流式细胞术筛选出稳定表达SOCS1的人骨髓间充质干细胞,并用Westen Blotting、流式细胞术、成骨成脂肪诱导分化来鉴定。结果 SOCS1蛋白在IFN-γ、Pam3CSK4、Poly(I∶C)、LPS和ODN 2006刺激的MSCs中表达升高(P<0.01)。表达载体包装出的病毒感染后的MSCs中SOCS1蛋白水平提高,流式细胞检测表达(>95%)CD29、CD44、CD73、CD90、CD105、CD166,不表达(<2%)CD34和CD45,在相应诱导剂诱导下可分化为成骨细胞和脂肪细胞。结论多种炎性因子能诱导MSCs内SOCS1表达升高,说明SOCS1可能参与了MSCs的免疫调节功能;成功建立了稳定表达SOCS1的MSCs细胞株。

Objective To investigate the expression patterns of SOCS1 induced by inflammatory molecules in human bone marrow MSCs and to construct MSCs with the over- expression of SOCS1. Methods Inflammatory molecules were used to stimulate MSCs, their total proteins were extracted 24 hours later and were assay by Western Blotting for detecting SOCS1 and GAPDH proteins. Using Gateway technology, we construct SOCS1 gene expression vector pFinal/PGK- puro- EF1α- SOCS1- IRES- EGFP. This vector was mixed with Lentiviral Packaging plasmid to transfect into 293FF cells to package the Lentivirus with SOCS1 gene. MSCs with the over-expression of SOCS1 was established by the Lentivirus multi-infection, then sorted by flow eytometry to purify transgenic MSCs, and assay by Western Blotting,flow cytometry,osteogenic and adipogenic differentiation potential test. Results After stimulated with IFN-γ, Pam3CSK4,Poly （I： C）,LPS and ODN 2006,the protein expression of SOCS1 was relatively increased（P 〈0.01 ）. The protein expression of SOCS1 was increased in the transgenic MSCs. The overexpression SOCS1 had osteogenic and adipogenie differentiation potential and expressed CD29, CD44, CD73, CD90, CD105, CD166, did not express CD34, CD45. Conclusion SOCS1 possibly take part in interaction of immuneregulation of MSCs. The overexpression SOCS1 transgenic MSCs was successfully established.