Inhibitory effect of mouse angiostatin cDNA gene transduction on human liver cancer cell / 中华消化杂志

ABSTRACT

Objective To study the effect of the mouse angiostatin cDNA gene transfecting into human liver cancer cell line HCC7721 on cell growth, cell cycle phase distribution, cell morphology in vitro and tumorigenesis in vivo , and the mechanisms. Methods The gene fragment of mouse angiostatin cDNA was directly cloned into an eukaryotic expression plasmid pcDNA3.1(+) of the promoter CMV between the multicloning sites HindⅢ and XbaⅠ and confirmed the correct recombinant plasmid pcDNA3.1(+) angio through enzymatic digestion and gene sequencing. Then it was transfected into human liver cancer cell line HCC7721 with liposome, pcDNA3.1(+) as vecter control and liposome as mock control. After 30 day selection by neomycin G418, angiostatin expression at the levels of mRNA and protein in vitro were tested by RNA dot blot and FACS respectively. Cell morphology under was observed light microscope, made growth curves were made, and cell cycle distribution checked in FACS. Animal model was set up to study tumorigenesis in vivo of those cells transplanted subcutaneously in the right hind legs of nude mice BALBc. Microvessel density (MVD) was analyzed and angiostatin expression in the tumor tissues by in situ immunohistochemistry. Results The recombinant eukaryotic expression plasmid pcDNA3.1(+) angio was constructed Auccessfully. After selection of G418 for about 4 weeks there were macroscopic cell clones in the experimental group and vector control group, but no survival cells in the mock control. In vitro angiostatin were expressed in the experimental group, but not in the vector control. There weren't significant changes in cell morphology, cell growth curves and cell cycle distribution between the experimental and the control group. However, the nude mice experiment showed that tumorigenic capability of the experimental cells had been reduced greatly. Immunochemistry study showed that there were much less microvessels and conversely stronger angiostatin positive staining in the tumor tissues than in the control group. Conclusions Mouse angiostatin has no direct inhibition on human liver cancer cell line HCC7721 in vitro , but it inhibits effectively the tumorigenesis of HCC7721 in vivo , which is probably due to its inhibition on tumor angiogenesis in a paracrine path way.