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目的:设计合成有效靶向Toll样受体4(TLR4)基因小干扰RNA(siRNA)表达载体,且挑选TLR4基因稳定沉默心肌细胞。方法:依据RNA干扰(RNAi)序列设计原则,以TLR4基因为靶基因,合成3对小发夹RNA(shRNA)寡核苷酸链,经退火、与线性化pSilence 2.1-U6neo质粒连接、酶切、测序并鉴定。脂质体法转染心肌细胞,新霉素(G418)加压筛选并收集稳定表达质粒的心肌细胞,并观察光镜下心肌细胞形态。RT-PCR及Western Blot法检测收集的心肌细胞TLR4mRNA和蛋白的表达,确定siRNA对TLR4的抑制效率。结果:测序鉴定插入发夹样序列正确,成功构建了TLR4基因siRNA表达载体;并获得了稳定沉默TLR4的心肌细胞。未见细胞毒性形态学改变。多种方法均证实siRNA作用于TLR4基因后,心肌细胞TLR4 mRNA和蛋白表达均被抑制,其中pSilence2.1-siTLR4-1抑制作用最强。结论:成功构建了靶向TLR4基因siRNA表达载体,并有效抑制心肌细胞TLR4表达。
OBJECTIVE: To design and synthesize small interfering RNA (siRNA) expression vector which can effectively target Toll-like receptor 4 (TLR4) gene and select stable TLR4 gene silenced cardiomyocytes. Methods: According to the principle of RNA interference (RNAi) sequence design, three pairs of small hairpin RNA (shRNA) oligonucleotides were synthesized by targeting TLR4 gene and annealed to the pSilence 2.1-U6neo plasmid. , Sequenced and identified. Myocardial cells were transfected by lipofectamine. Neonomycin (G418) was screened by pressure and the stable cardiomyocytes were harvested. The morphology of cardiomyocytes was observed under light microscope. The expression of TLR4 mRNA and protein in cardiomyocytes was detected by RT-PCR and Western Blot to determine the inhibitory efficiency of siRNA on TLR4. Results: The sequence of hairpin-like insert was identified by sequencing. The siRNA expression vector of TLR4 gene was successfully constructed and the cardiomyocytes stably silenced by TLR4 were obtained. No cytotoxic morphological changes were observed. A variety of methods confirmed that siRNA on TLR4 gene, myocardial cells TLR4 mRNA and protein expression were inhibited, of which pSilence2.1-siTLR4-1 the strongest inhibition. Conclusion: siRNA targeting TLR4 gene was constructed and the TLR4 expression was inhibited.