Construction Of HBax And HHGF Double Gene Co-expression Lentivirus And Its Effects On Vascular Endothelial Cells And Fibroblasts

(?)jective To construct and identify enhanced green fluorecence protein(EGFP) labeled lentiviral vector carrying hBax and hHGF Genes.To sutdy the effects of the lentiviral vector on the Endothelial cells and fibroblasts.Methods The PCR method and gateway technology were used to construct the positive plasmid and negative plasmid. They were packaged through co-transfected into human embryonic kidney cell line-293T with helper plasmids.Then the virus titer was examined by fluorescence microscope.This experiment choose human umbilical vein endothelial cells(HUVEC), human embryo lung fibroblasts(HELF) and NIH3T3cells as the purpose cells infected by the lentiviral vector to research,the cultured cells were divied into experimental group(treated with EGFP labeled lentiviral vector carrying hBax and hHGF genes),negative control group(lentiviral vector just carrying EGFP) and blank group(carrying nothing).The expression of Bax and HGF were detected by RT-PCR and Western blot.Morphological changes of the three groups cells were observed by inverted microscope.The proliferation of cells were measered by MTT while the impact of lentiviral vector on the migration of cells was measured by scarification test.Results The recombinant lentiviral transfer vector plasmids were constructed correctly,the titer of lentiviral-hBAX-eGFP-hHGF was7.8×10'TU/ml,and lentiviral-eGFP was9×107TU/ml.The results of RT-PCR and western blot showed that in the experimental group,the HUVEC expressed HGF and the level of Bax which was much higher than that of the other two groups after HUVEC infected was up-regulated significantly (P<0.05).Inverted microscope was used to observe that the cells are long spindle or irregular polygons and there were more bumps on the surface of cells.Compared with the other two groups, the proliferation and migration capacity ability of the experimental cells enhanced obviously (P<0.05).The expression levels of Bax and HGF in the experimental group was up-regulated significantly, it was much higher than that of the other two groups after NIH3T3cells and HELF infected(P<0.05).Inverted microscope was used to observe that cells of negative control group and blank group growed normally. At the time of72h after cells of experimental group infected, some cells occured shrinks, reduced in size;after96h,a portion of adherent cells fell off.Compared with the other two groups,MTT assay showed that the proliferation of NIH3T3cells and HELF of experimental group was obviously suppressed(P<0.05).Conclusion The lentiviral vector was successfully constructed and efficient lentivirus particles with high titer were packaged.The EGFP labeled lentiviral vector carrying hBax and hHGF genes could successfully infect purpose cells,interest gene and protein could be expressed after infeced. the proliferation and migration capacity ability of the experimental cells enhanced obviously.The proliferation of NIH3T3cells and HELF of experimental group was obviously suppressed.