Background SMAD proteins have recently been identified as the first family of putative transforming growth factor-β_1(TGF-β_1) signal transducers. This study was to investigate the effects of TGF-β_1 and signal pr...Background SMAD proteins have recently been identified as the first family of putative transforming growth factor-β_1(TGF-β_1) signal transducers. This study was to investigate the effects of TGF-β_1 and signal protein Smad3 on rat cardiac hypertrophy.Methods The incorporation of [3H]-leucine was measured to determine the hypertrophy of cardiomyocyte incubated with different doses of TGF-β_1 in cultured neonatal cardiomyocytes. The model of rat cardiac hypertrophy was produced with constriction of the abdominal aorta. At different times after the operation, rats were killed, and their left ventricular mass index (LVMI) determined. The mRNA expression of TGF-β_1 and Smad3 of cultured cells and hypertrophic left ventricles were assessed by RT-PCR. The protein expression of Smad3 was assessed by Western blot.Results In cultured neonatal cardiomyocytes, TGF-β_1 significantly promoted incorporation of [3H]-leucine. With the concentration of 3 pg/L, it increased the expression of Smad3 in mRNA and protein levels after 15 minutes, and continued for up to 8 hours of cultured cardiomyocytes. The LVMI and the expression of TGF-β_1 (mRNA) and Smad3 (mRNA and protein) of hypertrophic left ventricle were increased by day 3 after the operation and continued to the 4th week. The peak expression of these was in the second week after operation.Conclusion TGF-β_1 has positive effects on rat cardiomyocyte hypertrophy. Signal protein Smad3 could be related to the pathologic progression of rat cardiac hypertrophy.展开更多
文摘Background SMAD proteins have recently been identified as the first family of putative transforming growth factor-β_1(TGF-β_1) signal transducers. This study was to investigate the effects of TGF-β_1 and signal protein Smad3 on rat cardiac hypertrophy.Methods The incorporation of [3H]-leucine was measured to determine the hypertrophy of cardiomyocyte incubated with different doses of TGF-β_1 in cultured neonatal cardiomyocytes. The model of rat cardiac hypertrophy was produced with constriction of the abdominal aorta. At different times after the operation, rats were killed, and their left ventricular mass index (LVMI) determined. The mRNA expression of TGF-β_1 and Smad3 of cultured cells and hypertrophic left ventricles were assessed by RT-PCR. The protein expression of Smad3 was assessed by Western blot.Results In cultured neonatal cardiomyocytes, TGF-β_1 significantly promoted incorporation of [3H]-leucine. With the concentration of 3 pg/L, it increased the expression of Smad3 in mRNA and protein levels after 15 minutes, and continued for up to 8 hours of cultured cardiomyocytes. The LVMI and the expression of TGF-β_1 (mRNA) and Smad3 (mRNA and protein) of hypertrophic left ventricle were increased by day 3 after the operation and continued to the 4th week. The peak expression of these was in the second week after operation.Conclusion TGF-β_1 has positive effects on rat cardiomyocyte hypertrophy. Signal protein Smad3 could be related to the pathologic progression of rat cardiac hypertrophy.
文摘目的设计并筛选针对大鼠Smad3基因的siRNA,构建靶向Smad3基因的shRNA重组慢病毒。方法针对大鼠Smad3基因设计并合成6对siRNA(siRNA001～006)及1对无关对照siRNA,转染大鼠肝细胞株BRL-3A,应用Western Blot检测各siRNA对SMAD3蛋白表达的抑制作用,挑选抑制效率高的siRNA。依据所得的序列合成并克隆到pLL3.7载体中,与包装质粒pRSV-rev、pMDLg-pRRE和VSV-G共转染293FT细胞获得靶向Smad3的慢病毒。通过流式细胞仪绿色荧光蛋白(GFP)荧光计数来检测病毒滴度。结果 Western Blot检测证实siRNA001、siRNA005、siRNA006对SMAD3蛋白表达的抑制作用明显,抑制率分别可达83.36%、86.99%及64.88%,而对照siRNA无明显作用。酶切和测序结果显示Smad3 shRNA及对照shRNA重组载体质粒pLL3.7-shRNA构建成功,将构建的质粒进行慢病毒包装可产生有感染活性的慢病毒颗粒。结论筛选出针对大鼠Smad3基因有明显抑制作用的3对siRNA,并成功构建表达相应shRNA的4种重组慢病毒,为研究调控Smad3的表达对肝再生或肝纤维化的影响提供了实验条件。
