IFN-γ/sPD-1联合过表达的小鼠骨髓间充质干细胞具有较高生物活性和遗传稳定性  

Mouse bone marrow mesenchymal stem cells co-overexpressing IFN-γ/sPD-1 have high biological activity and genetic stability

作　　者：颉亚辉 吕珍 吴建军 魏兴民 郑贵森 XIE Yahui;LYU Zhen;WU Jianjun;WEI Xingmin;ZHENG Guisen(Department of Mathematics and Biostatistics,School of Public Health,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学公共卫生学院数学与生物统计学教研室,甘肃兰州730000

出　　处：《细胞与分子免疫学杂志》2024年第9期806-812,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：甘肃省教育厅2023年高校教师创新基金(2023B-104);甘肃省自然科学基金(21JR11RA139);甘肃省青年科技基金(21JR11RA147)。

摘　　要：目的研究慢病毒介导的γ干扰素(IFN-γ)及可溶性程序性细胞死亡蛋白1(sPD-1)过表达对骨髓间充质干细胞(BMSC)生物活性及遗传稳定性的影响。方法全骨髓贴壁法分离培养BMSC;流式细胞术鉴定BMSC表面标志物的表达;扩增分别带有绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)的IFN-γ及sPD-1基因序列,通过慢病毒转染分别构建IFN-γ、sPD-1和IFN-γ/sPD-1双基因过表达的BMSC,采用倒置显微镜观察细胞形态,实时荧光定量PCR和Western blot法分别检测IFN-γ和sPD-1的mRNA和蛋白表达;CCK-8法、BMSC端粒酶活性检测法分析IFN-γ和sPD-1过表达BMSC的生物活性和遗传稳定性。结果成功分离培养了CD44、CD105阳性,CD34、CD11b、CD45阴性的BMSC。与空载体组相比,IFN-γ/sPD-1联合组的IFN-γ、sPD-1 mRNA表达水平显著提高,但各组细胞生长情况类似。与空载体组相比,IFN-γ组端粒酶活性显著降低,但sPD-1组和IFN-γ/sPD-1联合组端粒酶活性变化不明显。结论IFN-γ和sPD-1联合过表达的BMSC具有较高生物活性和遗传稳定性。Objective To investigate the effects of lentivirus-mediated overexpression of interferon gamma(IFN-γ)and soluble programmed death 1(sPD-1)on the biological activity and genetic stability of bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were isolated and cultured by whole bone marrow adhesion method.The expression of surface markers of BMSCs was identified by flow cytometry.IFN-γand sPD-1 gene sequences with green fluorescent protein(GFP)and red fluorescent protein(RFP)were amplified,and BMSCs overexpressing IFN-γ,sPD-1,and IFN-γ/sPD-1 were constructed by lentiviral transfection.The cell morphology was observed using an inverted microscope.The mRNA and protein expression of IFN-γand sPD-1 were detected by real-time fluorescence quantitative PCR and Western blot analysis,respectively.The biological activity of the IFN-γand sPD-1 overexpression BMSCs was assessed through functional assays,while their genetic stability was analysed using karyotyping.Results BMSCs positive for CD44 and CD105 and negative for CD34,CD11b and CD45 were successfully isolated and cultured.Compared with the empty group,IFN-γand sPD-1 mRNA expression levels were significantly higher in the combined IFN-γ/sPD-1 group,but the growth of cells was similar in all groups;the telomerase activity was significantly reduced in the IFN-γgroup,but the changes in telomerase activity were not obvious in the sPD-1 group and the combined IFN-γ/sPD-1 group.Conclusion BMSCs overexpressing IFN-γand sPD-1 in combination has high biological activity and genetic stability.

关 键 词：γ干扰素(IFN-γ) 可溶性程序性细胞死亡蛋白1(sPD-1) 骨髓间充质干细胞(BMSC) 遗传稳定性 

分 类 号：Q311[生物学—遗传学] Q344.13[医药卫生—肿瘤] R730.51[医药卫生—临床医学]