Research Of Effects Of Human Tumor Necrosis Factor-a Gene Transfection On Tongue Carcinoma Cells

Objective: ①To identify, extract and purify the pSV23SHTNF shuttle plasmid containing human tumor necrosis factor-α(hTNF-α) gene. ②To explore the optimum method to transfect Tca8113 tongue carcinoma cells with pSV23SHTNF shuttle plasmid mediated by cationic liposome. ③To study the effects of hTNF-αgene transfection on tongue carcinoma cells and its mechanism. ④To investigate the synergistic effects of hTNF-αgene transfection and interferon-γ(IFN-γ) on the growth of tongue carcinoma cells. ⑤To observe the expression level of hTNF-αafter human embryo myoblasts was transfected by hTNF-αgene. Methods: ①To detect whether cloned hTNF-αgene expressed in E. coli K12 MC 1061 bacteria with Western blot. ②To obtain highly purified shuttle plasmid containing hTNF-αgene with alkaline extraction and improved purification procedure. ③To observe the expression of hTNF-αgene with immunocytochemistry stain in Tca8113 tongue carcinoma cells after they were transfected by pSV23SHTNF shuttle plasmid mediated by DOSPER cationic liposome. ④pSV23SHTNF shuttle plasmid was transfected into Tca8113 tongue carainoma cells mediated by DOSPER cationic liposomes. The control group was only given equivalent liposomes, except plasmid. After culturing for 24, 48, 72 and 96 hours, the expression of hTNF-αgene in Tca8113 cells was analyzed by ELISA and the survival rate of transfected cells was assayed by MTT enzymatic labeling technique. The mechanism of hTNF-αeffect on Tca8113 cells was investigated through flow cytometric approach. ⑤The cultured Tca8113 tongue carcinoma cells was divided into 2 groups, one group was transfected with hTNF-αgene. Each one of the 2 groups was then divided into 5 subgroups, the subgroups were added IFN-γuntil the final IFN-γconcentrations respectively were 0, 10, 100, 1000 U/ml. After culturing for 48 hours, the survival rate of the all groups of cells was assayed by MTT enzymatic labeling technique. ⑥human embryo myoblasts were transfected with pSV23SHTNF shuttle plasmid mediated by DOSPER cationic liposomes. The control group was only given equivalent liposomes, except plasmid. After culturing for 24, 48, 72 and 96 hours, the expression of hTNF-αgene in Tca8113 cells was analyzed by ELISA and was also observed with immunocytochemistry stain. Result: ①Cloned hTNF-αgene expressed in E. coli K12 MC 1061 bacteria. The identified hTNF-αwas nondenatured oligomeric form, the molecular weight of which was about 45000. ②The concentration of the obtained plasmid was 649.5μg/ml and the ratio of OD260/ OD280 was 2.11. ③the transfected Tca8113 cells displayed remarkable overexprsssion of hTNF-α, compared with the nontransfected. ④The transfected Tca8113 cells displayed significantly overexprsssion of hTNF-α(P<0.05)in the culture medium. The survival rate of the transfected Tca8113 cells was decreased significantly (P<0.05), and the mechanism is that the overexpression of hTNF-αgene induced the apoptosis of the transfected Tca8113 cells. ⑤IFN-γalone did not affect the growth of Tca8113 cells (P>0.05). All the different concentrations of IFN-γand the transfection of hTNF-αsynergistically inhibited the growth of Tca8113 cells, the concentrations of IFN-γwere positively correlated with the inhibition effects (r =0.867,P<0.01). ⑥The transfected human embryo myoblasts displayed significantly overexprsssion of hTNF-α(P<0.05)in the culture medium. Conclusion: ①E. coli K12 MC 1061 bacteria were successfully transformed by shuttle plasmid containing hTNF-αgeng. ②The obtained plasmid through alkaline extraction and improved purification procedure fulfilled the demand of cellular transfection.③With the ratio of 4ul DOSPER / 1.5ugDNA, the transfection of plasmid mediated by DOSPER cationic liposomes result in high level of tranfection efficient. ④Transfection of hTNF-αgene in vitro mediated by cationic liposomes can induces the overexpression of hTNF-αand inhibit the growth of tongue carcinoma cells, the mechanism is that the overexpression of hTNF-αgene induced the apoptosis of the transfected Tca8113 cells. ⑤the combination with IFN-γcan enhance the inhibition effects of the hTNF-αtransfection on the tongue carcinoma cells. ⑥Transfection of hTNF-αgene to human myoblasts made myoblasts excrete high concentration of hTNF-α, implying it is feasible that transfecting muscle cells surrounding tongue carcinoma lesion with hTNF-αgene prevents tongue carcinoma from intruding into deeper muscle tissue.