磷酸钙法将MMP-2,MMP-3双基因干扰载体转入HEK293T细胞的效率观察

The Transfection Efficiency for Introducing MMP-2,MMP-3 Double Gene-silencing Vector into HEK293T Cells by Calcium Phosphate Method

检测用磷酸钙法能否将修饰后用于沉默MMP-2,MMP-3基因的慢病毒载体有效地转入到HEK293T细胞。利用磷酸钙法,MMP-2,MMP-3双基因干扰载体(MMP组)与对照质粒(对照组)被分别转染HEK293T细胞,在转染后的第24,48及72 h,通过计算绿色荧光蛋白(GFP)阳性细胞数的百分比来判断转染效率。转染24 h后,两组细胞均生长状况良好,荧光显微镜下观察,已有大量绿色荧光出现,计算MMP组转染效率为(91.5±6.2)%,对照组转染效率为(80.3±4.7)%;转染后48-72 h,两组感染效率均未见明显下降。与对照相比,插入了MMP-2,MMP-3shRNA模板序列的慢病毒载体没有降低磷酸钙法转染HEK293T细胞效率,反而有所增高。

Objective To evaluate whether modified lentiviral vector for silencing the MMP-2, MMP-3 double genes can be effectively introduced into HEK293T ceils by calcium phosphate method. Method MMP-2, MMP-3 double gene-sileneing vectors（ MMP group） and control vectors（ control group） were transfected into HEK293T cells using calcium phosphate method, respectively. 24 h,48 h and 72 h after transfection, the transfection efficiency were determined by calculating the percentage of GFP positive cells using fluorescent microscopy. Results 24 hours after transfection, two groups of cells grew well, bright green fluorescence can be observed under the fluouescene microscope, the transfection efficiency of MMP group was （91.5 ± 6.2）%, and control group was （ 80. 3 ± 4.7 ） %. During 48 - 72 h after transfection, there were not significant changes in transfection efficiency. Conclusions The modification of lentiviral vector into which the MMP-2 and MMP-3 double shRNAs templates were inserted does not decrease the calcium phosphate transfection efficiency, but increases it compared with the control vector.