Effect Of Human Visfatin MRNA Expression In 3T3-L1 Cells With Stable Transfection By TNF-a, Pioglitazone Treatment

Objective To construct the stable 3T3-L1 cell line transfected with pcDNA3.1(+)/hVisfatin and then to explore effect of human Visfatin mRNA expression by TNF-a, Pioglitaone treatment.Methods The recombinant eukaryotic expression plasmid pcDNA3.1(+)/hVisfatin was transformed into E coli JM109 and then transfected into 3T3-L1 cells by SuperFect Transfection Reagent after identification by digestion of restriction enzyme and sequencing. The stable transfected 3T3-L1 cell line was established after selection with G418, and human Visfatin mRNA expression was identified by RT-PCR. The stable transfected 3T3-L1 cells were treated with TNF-a(10 ng/ml), Pioglitaone(100 nmol/ml) for 24h respectively, compared with untreated control in the same period. The human Visfatin mRNA expression were measured by semi-quantitative RT-PCR.Resultsâ—‹1 5.4kb fragment and 1.5kb fragment were consistent with theoretical values after eukaryotic recombinant was digested by BamH I and Xho I, and identified by nucleotide sequence scaning. The RT-PCR result showed 3T3-L1 cells trasfected with pcDNA3.1(+)/hVisfatin had one 1.5kb fragment which was consistent with theoretical values, but negative control cells had no result above.â—‹2 Compared with undifferentiated 3T3-L1 cells trasfected with pcDNA3.1(+)/hVisfatin, human Visfatin mRNA expression in differentiated ones increased significantly (P<0.01) . In undifferentiated and differentiated 3T3-L1 cells transfected with pcDNA3.1(+)/hVisfatin, treatment with TNF-a significantly downregulated human Visfatin mRNA expression(P<0.01). However, Pioglitaone treatment did not significantly influence human Visfatin mRNA expression (P>0.05) . Conclusions The stable 3T3-L1 cell line transfected with pcDNA3.1(+)/hVisfatin is constructed successfully. In the stable transfected 3T3-L1 cells, the expression of human Visfatin mRNA seems to be correlated with differentiation of adipocytes. TNF-a treatment downregulates human Visfatin mRNA expression while Pioglitaone treatment has no significant effect.