Experimental Study Of Triton X-100Promoting Trasfection Of DNA Mediated With Liposomes And The Influence To Rat Marrow Mesenchymal Stem Cells

Objective:To study the effect of Triton X-100promoting gene transfection mediated with liposomes and the influence on rat marrow mesenchymal stem cells.Methods:1、Obtaining the marrow mesenchymal stem cells from wistat rat and culturing the stem cell.2、Amplification and construction of the plamid of pegfp-BMP2-N1and pegfp-VEGF165-N1by method of colibacillus in vitro.3、To determine the optimum concentration of TritonX-100for promoting liposome mediated gene transfection with ELISA meter by the way of MTT.4、In proper concentration of Triton X-100, Liposome mediated BMP2and VEGF165gene transfected to bone marrow mesenchymal stem cells. The group was divided into7according to the following types of trasfection agents:liposome+pegfp-BMP2-N1group,liposome+pegfp-VEGF165-N1group,TritonX-100+liposome+pegfp-BMP2-N1group,TritonX-100+liposome+pegfp-VEGF165-N1group, Triton X-100+pegfp-BMP2-N1group, Triton X-100+pegfp-VEGF165-N1group and the supernatant only with no agent group.Rat marrow mesenchymal stem cells was treated with the previously list agents.5、After48hours of transfecting,detect the GFP in cells through inverted fluoresrence microscope and take the picture.6、 After transfection of72h, the relative expression level of BMP2and VEGF165was assayed by real-time fluorescence quantitative PCR in transfection. we use the way of Delta-delta Ct to detect the relative mRNA expression in cells.7、The experimental data were homogeneity of variance test and normality test. The t test was used to compare the two groups of single variable data, single factor analysis of variance comparing multiple groups of data, nonparametric test to compare the variance not neat, count data using chi-square test. It was considered statistically significant when the P value was below0.05. The statistical analyses were performed with the software of SPSS12.0.Results:1、The appropriate concentration of Triton X-100used to promot gene trasfection of rat marrow mesenchymal stem cells mediated with liposomes is0.01%. The OD value is0.127by the method of MTT and the blank cells was0.151. It is no statistical significance(P<0.05). In this concentration, Triton X-100was not effect on the growth of rat marrow mesenchymal stem cell.2、After trasfecting for48h,GFP was detected through inverted fluoresrence microscope in cells treated with the agent liposome+pegfp-BMP2-N1、the agent liposome+pegfp-VEGF165-N1、the agent Triton X-100+liposome+pegfp-BMP2-N1and the agent Triton X-100+liposome+pegfp- VEGF165-N1,while the Triton X-100agent alone and the no agent cells not exist. The amout of the GFP in cells treated with the agent Triton X-100+liposome+pegfp-BMP2-N1and the agent Triton X-100+liposome+pegfp-VEGF165-N1is more than the agent liposome+pegfp-BMP2-N1and the agent liposome+pegfp-VEGF165-N1by gross observation.3、After trasfecting for72h, the relative expression in cells treated with the agent Triton X-100+liposome+pegfp-BMP2-N1is5.94,while the agent liposome+pegfp-BMP2-N1is4.99. The transfection efficiency was improved by19%. The relative expression in cells treated with the agent Triton X-100+liposome+pegfp-VEGF165-N1is3.48,while the agent liposome+pegfp-VEGF165-N1is2.77. The transfection efficiency was improved by25.6%. There were significant statistic differences in BMP2and VEGF165in experimental group and conventional transfection group expression (P<0.05).Conclusions:1、The optimal concentration of Triton X-100for the promotion of liposome mediated gene transfection is0.01%. In this concentration, Triton X-100has no effect on the growth of rat marrow mesenchymal stem cells.2、Triton X-100can promote the liposome mediated gene transfection of rat bone marrow mesenchymal stem cells. The trasfection rate could get up19%-25.6%.3、the reason of Triton X-100improving the transfection efficiency of gene transfection mediated with liposomes to rat marrow mesenchymal stem cells could be that it has the ability of dissolving the Lipid material between liposomes and cell membrane, thus promoting the lipo-DNA complex trasfection into cells by the endocytosis of cells or the fusion between the cell membrane.