论文部分内容阅读
目的构建并筛选小鼠p300特异性siRNA载体,并观察p300表达降低对心脏特异性转录因子的调控作用,为进一步研究p300介导的组蛋白乙酰化失衡对心脏发育的影响奠定基础。方法针对小鼠p300基因的保守序列区域设计并构建3个重组质粒p300RNAi1,p300RNAi2和p300RNAi3。利用阳离子脂质体将其分别转染入体外培养的乳鼠心肌细胞,分别利用RT-PCR和免疫荧光从mRNA和蛋白水平检测p300表达,判断抑制效率,并检测心脏特异性转录因子GATA4的表达水平。结果 pSOS-HUS组和p300RNAi2组中p300mRNA水平表达较正常对照组无明显降低,而p300RNAi1组和p300RNAi3组中p300mRNA表达量较正常组有明显降低(分别为0.2208±0.0200,0.1708±0.0400,vs0.5095±0.0300),且差别有统计学意义(P<0.05),抑制率分别为56.7%和66.5%。转染p300RNAi1组和转染p300RNAi3组的GATA4mRNA表达水平(分别为0.4231±0.1100,0.2627±0.0800)明显低于正常组(0.8564±0.0700)和阴性对照组(0.8415±0.0400),差别有统计学意义(P<0.05)。结论 p300特异性siRNA质粒载体构建成功,p300RNAi1和p300RNAi3具有确切阻断p300蛋白表达的作用,并下调GATA4的表达。
Objective To construct and screen mouse p300-specific siRNA vector and to observe the effect of decreasing p300 expression on cardiac-specific transcription factors, so as to lay a foundation for further studies on the effect of p300-mediated histone acetylation imbalance on heart development. Methods Three recombinant plasmids p300RNAi1, p300RNAi2 and p300RNAi3 were designed and constructed according to the conserved sequence of mouse p300 gene. The recombinant plasmids were transfected into neonatal rat cardiomyocytes by cationic liposome respectively. The expression of p300 was detected by RT-PCR and immunofluorescence respectively at mRNA and protein levels to determine the inhibitory efficiency. The expression of cardiac-specific transcription factor GATA4 Level. Results The expression of p300 mRNA in pSOS-HUS group and p300RNAi2 group was lower than that in normal control group, but p300 mRNA expression in p300RNAi1 group and p300RNAi3 group was significantly lower than that in normal group (0.2208 ± 0.0200,0.1708 ± 0.0400 vs0.5095 ± 0.0300), and the difference was statistically significant (P <0.05), the inhibition rates were 56.7% and 66.5% respectively. The levels of GATA4 mRNA in transfected p300RNAi1 group and p300RNAi3 transfected group were 0.4231 ± 0.1100,0.2627 ± 0.0800 respectively, which were significantly lower than those in normal group (0.8564 ± 0.0700) and negative control group (0.8415 ± 0.0400) P <0.05). Conclusion p300 specific siRNA plasmid vector was successfully constructed. P300RNAi1 and p300RNAi3 could block the expression of p300 protein and down-regulate GATA4 expression.