Objective:To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant,laying a foundation for gene therapy research of malignant tumors.Methods:The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme.The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus;the products were cotransfered into HEK-293 cell line with helper plasmid PJM17.The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming.The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction(RT-PCR)procedure.The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Results:The p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing.High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells(1?10 11pfu/ml).The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR.Ad.NT4p53Ant had strong killing effect on HepG2 cells.Compared with Ad.GFP,Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells.Conclusion:The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques,laying a foundation for further research of gene therapy of cancer.

AIM: To investigate the effects of Astragalus polysacharin(APS) on human fibroblast and human umbilical vein endothelia cell (HUVEC) proliferation, as well as its acts on adhesion between white cells and HUVECs. METHODS: Human fibroblasts from distal and proximal skin away the ulcer were cultured as normal fibroblasts(NF) and wounded fibroblasts(WF). MTT assay was used for detecting cell proliferation, Rose Bengal staining and fluorescence immunohistology assay were used for examining the adhesion of human polymorpho-nuclear cell(PMN) and TPH-1 to HUVECs. RESULTS: 2 44-156 mg/L APS promoted WF proliferation, and 2 44-39 mg/L APS also promoted NF proliferation, but it did not show any proliferating effect on HUVECs. APS inhibited the adhesion of PMN or TPH-1 to HUVECs induced by tumor necrosis factor(TNF). At 25-100 mg/L, it also inhibited both VCAM-1 and ICAM-1 expression in HUVECs induced by TNF. Treatment with APS for 12 h also inhibited CD44 expression in HUVECs. CONCLUSION: APS shows mitogenic activity on both human normal and wounded fibroblasts. It also exerts anti-inflammation effects by inhibiting adhesion molecule expression and adhesion of white cells to HUVECs. [