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.
Department of Mathematics and Biostatistics, School of Public Health, Gansu University of Traditional Chinese Medicine, Lanzhou 730000, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2024 Sep;40(9):806-812.
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-γ and 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-γ and sPD-1 were detected by real-time fluorescence quantitative PCR and Western blot analysis, respectively. The biological activity of the IFN-γ and 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-γ and 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-γ group, 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-γ and sPD-1 in combination has high biological activity and genetic stability.
目的 探讨慢病毒介导的干扰素γ（IFN-γ）和可溶性程序性死亡蛋白1（sPD-1）过表达对骨髓间充质干细胞（BMSCs）生物学活性和遗传稳定性的影响。方法 采用全骨髓贴壁法分离培养BMSCs。通过流式细胞术鉴定BMSCs表面标志物的表达。扩增带有绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）的IFN-γ和sPD-1基因序列，通过慢病毒转染构建过表达IFN-γ、sPD-1以及IFN-γ/sPD-1的BMSCs。使用倒置显微镜观察细胞形态。分别通过实时荧光定量PCR和蛋白质免疫印迹分析检测IFN-γ和sPD-1的mRNA和蛋白表达。通过功能测定评估过表达IFN-γ和sPD-1的BMSCs的生物学活性，同时使用核型分析来分析其遗传稳定性。结果 成功分离培养出CD44和CD105阳性、CD34、CD11b和CD45阴性的BMSCs。与空载体组相比，IFN-γ/sPD-1联合组中IFN-γ和sPD-1的mRNA表达水平显著更高，但所有组的细胞生长情况相似；IFN-γ组的端粒酶活性显著降低，但sPD-1组和IFN-γ/sPD-1联合组中端粒酶活性变化不明显。结论 联合过表达IFN-γ和sPD-1的BMSCs具有较高的生物学活性和遗传稳定性。
