目的比较阳离子聚合物Xfect与PDS-1000基因枪系统转染人骨髓间充质干细胞(hBMSCs)的转染效率及两种方法对细胞增殖能力的影响。方法应用贴壁法原代培养hBMSCs,取第3代细胞以流式细胞术鉴定细胞表面抗原CD29、CD34、CD45、CD90,并分别用Xfect与PDS-1000基因枪系统将pcDNA-胰岛素样生长因子1(IGF-1)质粒转导入hBMSCs中。转染后在不同时间点使用倒置荧光显微镜观察绿色荧光,计数阳性细胞,计算细胞转染率及细胞漂浮率;以酶联免疫吸附试验(ELISA)方法检测IGF-1分泌量。结果 hBMSCs培养成功后,高表达CD29、CD90,低表达CD34、CD45。在转染后1、2d,两种方法细胞转染率差异有统计学意义(t=4.734、5.324,P〈0.05)。两种方法转染后细胞IGF-1分泌量均较转染前增加,但两种方法间比较差异有统计学意义(t=2.220-10.678,P〈0.05)。两种方法转染均有部分细胞脱落。结论两种方法均可将pcDNA-IGF-1基因转染入hBMSCs内,转染后细胞能分泌IGF-1。Xfect转染试剂转染效率较高,PDS-1000基因枪系统转染效率较低,两者对细胞都造成一定损伤。
Objective To compare the efficiency and cell reproductive activity after transfection of Xfect and PDS-1000 to hBMSCs. Methods Using attachment method,hBMSCs were primarily cultured.The third generations of cells were taken for detection of cell surface antigens CD29,CD34,CD45 and CD90using flow cytometer.Xfect and PDS-1000 were respectively used to deliver pcDNA3.1/Neo vector containing IGF-1to hBMSCs.After transfection,the green fluorescence was observed by inversion fluorescent microscope at different time points.The fluorescence-positive cells,cell transfection efficiency and cell flotation rate were calculated.The secretory volume of IGF-1was detected with ELISA. Results After hBMSCs were successfully cultured,high expressions of CD29 and CD90,low expressions of CD34 and CD45were noted.After 1and 2days of transfection,the difference in cell transfection efficiency between the two methods was statistically significant(t=4.734,5.324;P〈0.05).After transfection using the two methods,though the secretory volume of IGF-1was all increased compared with before,the difference in between Xfect and PDS-1000 was significant(t=2.220-10.678,P〈0.05).Detachment of some cells could be seen in both transfection methods. Conclusion Both Xfect and PDS-1000 can transfer the pcDNA-IGF-1gene into hBMSCs and secrete IGF-1after the transfection.The transfection efficiency of Xfect is higher than that of PDS-1000,but both of the methods cause certain damage to the cells.