| With the incidence rate of atherosclerosis growing, it has been become the main causes of mortality and morbidity in our country. The treatment of symptomatic coronary stenosis by percutaneous transluminal coronary angioplasty (PTCA) is widely applied. However, their efficacy is limited by restenosis which occurs in 20% to 50% of cases. Catheter-based gene therapy has emerged as one of the most promising approaches for preventing coronary restenosis, because it appears capable of delivering therapeutic gene specifically to the location of the disease, at a precise site in the arterial wall. The choice of efficacious therapeutic gene and appropriate vector are the crux of the matter for a successful gene therapy. According to the pathogenesis mechanisms of restenosis, the therapeutic gene choice must be match the principles as follows: which can inhibit smooth muscle cell (SMC) proliferation and migration, prevent thrombosis, and enhance re-endothelialization. Among the viral vectors, adenoviral vectors and adeno-associated viral vectors are used for vascular gene delivery in animal models more frequently than the others. It is difficult for using a single therapeutic gene to meet all of the therapeutic needs simultaneously. So we focus on the strategy of co-expression of p21 and antisense thrombin receptor gene (ATR) in adeno-associated viral vector, or p21 and tissue inhibitor of matrix metalloproteinases-4 (TIMP-4) gene in adenoviral vector to inhibit SMC proliferation and migration simultaneously.Construction and Package of Recombinant Adeno-associated Virus Vectors (rAAV) Carrying p21, ATR and Both of ThemObjective To construct and package the rAAV vectors containingp21, or ATR, or both of them for the following gene therapy experiment in vitro and in vivo.Methods Vector plasmids, carrying GFP, ATR, p21 or both ATR and p21 (AP), were constructed and lipotransfected into BHK-21 cells respectively. Then the cell lines containing transgenes were established after G418 selection. In order to detect the transgenes in the cell lines , the genomic DNA of them were abstracted and digested by restriction enzymes, and then the Southern hybridization was performed. The expression of GFP was detected under fluorescence microscopy. The recombinant adeno-associated viruses (rAAV/GFP, rAAV/ATR, rAAV/p21 and rAAV/AP) were packaged in the cell lines by the infection of rHSV which carries rep-cap genes. The titer of the rAAV calculated by the results of dot blot.Results (1) The cell lines, integrated with the AAV vectors, were obtained by screening with G418 and confirmed by southern hybridization. They are BHK/p21, BHK/ATR, BHK/AP and BHK/GFP. (2) The green fluorescence was detected by microscope in the BFIK/GFP at the fifth day after transfection. It indicated that the expression of GFP was in the cells. (3) The rAAV vectors, rAAV/GFP, rAAV/p21, rAAV/ATR and rAAV/AP were generated in the cell lines by HSVl-rc / A UL2 infection. The recombinant viruses containing transgenes had been detected by dot hybridization. The titers of rAAV/p21, rAAV/ATR and rAAV/AP are 1.07 X-1012(particles/ml), 1.25 X 101 (particles/ml) and 9.88X 1014(particles/ml) respectively.Conclusion (1) The genes carried by AAV vector plasmid (pSNAV) can be transferred and integrated into the genomic DNA of BHK cells. (2) BHK/GFP cells can express GFP effectively. (3) The high titer rAAV (1 X 10n~14particles/ml) can be yielded by the system of HSVl-rc / A UL2.The Effect of p21 and ATR Co-expression on Human Aorta SmoothMuscle Cells (ASMC)Objective To study the effect of p21 and ATR co-expression on proliferation and apoptosis of human ASMC by a bicistronic rAAV vector.Methods The aorta was taken from fetus at the age of 7-8 month. The primary human ASMCs were isolated and cultured. When they grew to confluence, subcultured with the DMEM containing 10-15% FBS. The fifth to eighth passage cells were used to be infected with rAAV/p21, rAAV/ATR, rAAV/AP and rAAV/GFP respectively. The expression of transgenes was confirm...
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