ΔCREB真核表达载体的构建及对心肌细胞TNFα含量的影响

Change of TNFα level after the transfection of ΔCREB eukaryotic expressive vector in neonatal rat cardiomyocytes

目的 :构建ΔCREB的真核表达载体 ,观察其对TNFα等细胞因子的影响 ,探索心衰发生发展的始动因素。方法 :利用穿梭质粒pAdTrack构建核转录因子ΔCREB的真核表达载体pAdTrack -ΔCREB ,然后转染培养的乳鼠心肌细胞。将心肌细胞随机分为空白对照组、forskolin(10 μmol/L)组、转染组和转染后加forskolin组 ,观察各组心肌细胞ΔCREB的表达以及TNFα含量的变化。结果 :①定量逆转录多聚酶链反应 (RT -PCR)显示 ,ΔCREB/β-actin定量比值 ,转染组 (1 0 0 10 16± 0 0 5 35 2 0 )明显高于对照组 (0 76 132 2± 0 0 4 4 0 90 ) ,(P <0 0 1) ;免疫细胞化学检测心肌细胞ΔCREB蛋白标记阳性率 ,转染组 (2 8 88% +9 0 5 % )亦明显高于对照组 (6 6 3% +3 38% ) ,(P <0 0 1)。②细胞上清TNFα含量 ,转染后加forskolin组 (34 36pmol/L± 12 17pmol/L)明显高于对照组 (16 91pmol/L± 5 16pmol/L) ,(P <0 0 1)。结论 :转染ΔCREB可显著升高心肌细胞TNFα含量。cAMP介导的CREB转录激活作用促进了TNFα基因表达 ,可能在心力衰竭的发展中起重要作用。

AIM: To construct an eukaryotic expressive vector of cAMP response element binding protein (ΔCREB ) and observe the regulation effects of ΔCREB transfection on TNFα transcription in cardiomyocytes. METHODS: ΔCREB 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 ΔCREB was confirmed and evaluated with green fluorescent protein (GFP), competitive RT-PCR, and immunocytochemistry. Concentrations of TNFα in cultured supernatants of cardiomyocytes were measured with radioimmunoassay from control, forskolin (10 μmol/L), transfected, and transfected with adding forskolin (10 μmol/L) groups. RESULTS: The expression levels of ΔCREB in transfected group (mRNA ratio to β-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<0.01; protein 6.63%±3.38%, P<0.01). Also, concentrations of TNFα in transfected with adding forskolin group(34.36 pmol/L±12.17 pmol/L) were higher than that in control group (16.91 pmol/L±5.16 pmol/L,P<0.01). CONCLUSION: Overexpression of ΔCREB activated the transcription of TNFα gene in cardiomyocytes, and this may play a key role in the pathophysiology of heart failure.