Construction Of The Vector Of PhTERT-tumstatin And Its Antiangiogenic Effect

AbstractUnlimited proliferation of hepatoma cells and the continuous formation of new blood vessels are important reasons which are difficult to effectively control the Hepatic carcinoma. Hepatoma cell telomerase activation is the basis of their unlimited proliferation. Study confirmed that liver cancer cells with high expression of telomerase activity from the hTERT (human telomerase reverse transcriptase) gene promoter in the fully open. Angiogenesis is the key processes of tumor survival and metastasis. Targeted inhibition of tumor angiogenesis may indirectly inhibit tumor growth and metastasis. HCC is a highly vascularized tumor,therefore, using its specific hTERT promoter-driven anti-angiogenic genes to inhibit liver angiogenesis is a promising therapeutic strategy.ObjectiveConstruct the plasmid of phTERT-tumstatin gene vector driven by the hTERT promoter , To observe the specific expression and secretion of tumstatin transduced into HepG2 ,driven by the hTERT promoter and its anti-angiogenic effect in vitro.Methods1.Cells HepG2,L-02 and HUVEC were cultured in vitro cell culture. 2.Constructed phTERT-tumstatin plasmid and its control group. 3.We transfected HepG2 and L-02 with the plasmid of phTERT-tumstatin. 4.The expression of EGFP was examined with fluorescence microscope observations;The secretion of target protein tumstatin from HepG2 cells was detected by Western blotting. 5.MTS assay was used to measure cell proliferation of HepG2 cells after stably transfected by plasmids. 6.The effect of conditioned mediun (containing target protein or not) on the proliferation of HUVEC cells was detected by MTS assay. 7.The effect of target protein Tumstatin on the formation of HUVEC celluar tube structure can be observed through counting the number of tube branches.Results1.We successfully constructed phTERT-tumstatin, pCMV-tumstatin (positive control) and phTERT-EGFP(negative control) plasmids. 2.The specific expression and secretion of tumstatin was only observed in HepG2, but not in normal liver L-02 cells. 3.The target protein contained in CM-T inhibited the proliferation of HUVEC cells, inhibition rate reached (56.49±0.33)%. 4.The number of tube branches on Matrigel-coated plates supplemented with conditioned medium CM-T was (3.33±1.53)%. Compared with conditioned medium CM-N and CM-NT, supplement of CM-T significantly inhibited the cellular tube structure formation of HUVEC cells on Matrigel-coated plates【(3.33±1.53)% vs (24.44±3.11)%,(23.94±2.92)%,P<0.01)】。 ConclusionThe plasmid of hTERT-tumstatin was successfully constructed. The target protein selective expression and secretion in liver cancer cells can inhibit the proliferation of endothelial cells and vascular structure formation and almost have no influence on normal hepatic cells. The hTERT-tumstatin has a potential effect of targeted inhibition of angiogenesis .