Experimental Study Of Gene Therapy Witn HAG(K1-3) In Gastric Carcinoma

Objective:To detect the expression of recombinant hAG(K1～3) in eukaryotic cells, and to investigate the growth inhibitory effect of hAG(K1~3) to SGC-7901 cells and HUVEC in vitro. To lay the foundation for the tumorous gene therapy with multiple gene by investigate the inhibitory effect of hAG(K1~3) combinated with TRAIL.Methods: (1) Total mRNA was extracted from human hepatic tissue and then the hAG cDNA was obtained by RT-PCR. The hAG(K1~3) gene was cloned into pEGFP-N1 vector and the pEGFP-N1-hAG vector was constructed;(2)The recombinant plamid was transfected into COS-7 cells,and the expression of hAG–GFP fusion protein in COS-7 cells was analyzed by RT-PCR and Western blot;(3) hAG(K1~3) strongly inhibited the proliferation of HUVEC(P<0.05)in vitro ,while it had no inhibition effect on SGC-7901 cells;(4) The plasmid of pEGFP-N1-hAG was transfected into SGC-7901 cells and HUVECs to detect the inhibition ability of angiostatin by MTT colorimetric assay;(4) SGC-7901 cells were injected subcutaneously into nude mice to form transplanted tumor,than the recombinant plamid of pBud-hAG and pBud-hAG-TRAIL constructed by subclone were injected into the tumor to investigate their inhibitory effect on tumor growth and the microvessel density (MVD) of the tumor. Results: (1)The length of hAG(K1~3) gene obtained was 836bp,which was correspond with the target gene;(2)The recombinant plamid of pEGFP-N1-hAG was constructed successfully,and the fusion protein of hAG(K1～3)-GFP was expressed in COS-7 cells;(3) hAG(K1～3) could inhibit tumor angiogenesis in vivo,and hAG(K1～3) conbinated with TRAIL could inhibit tumor growth more significantly.Conclusion: Angiostatin can inhibit tumor angiogenesis through inhabiting the growth of vascular endothelial cells, thereby inhibiting tumor growth.And angiostatin has no apparent effect on the tumor cells themselves.hAG(K1～3) conbinated with TRAIL could inhabit tumor growth more efficiently.