Change of TNF? level after the transfection of ?CREB eukaryotic expressive vector in neonatal rat cardiomyocytes / 中国病理生理杂志

ABSTRACT

AIM: To construct an eukaryotic expressive vector of cAMP response element binding protein (?CREB ) and observe the regulation effects of ?CREB transfection on TNF? transcription in cardiomyocytes. METHODS: ?CREB gene was obtained after PCR amplification from human heart cDNA library with CREB specific primers. After digestion and ligation, the complete cDNA was inserted into pAdTrack, a shuttle plasmid. Neonatal rat ventricular myocytes were dissociated and cultured. The myocytes were transfected with the vector by liposome-lipofectamine. The expression of transfected ?CREB was confirmed and evaluated with green fluorescent protein (GFP), competitive RT-PCR, and immunocytochemistry. Concentrations of TNF? in cultured supernatants of cardiomyocytes were measured with radioimmunoassay from control, forskolin (10 ?mol/L), transfected, and transfected with adding forskolin (10 ?mol/L) groups. RESULTS: The expression levels of ?CREB in transfected group (mRNA ratio to ?-actin, 1.00?0.05; positive protein 28.88%?9.05%) was significantly higher than that in control group (mRNA 0.76?0.04, P