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目的:构建小鼠NOMO1基因RNA干扰载体,比较其对P19细胞NOMO1基因的抑制效率,以研究NOMO1基因与P19细胞向心肌细胞方向分化的关系。方法:利用Designer3.0软件设计小鼠NOMO1基因干扰片段,合成shRNA序列,再行寡核苷酸双链的合成,退火后形成的寡核苷酸双链克隆到pGPU6/Hygro载体的黏性末端,连接产物转化到感受态细胞,增菌,质粒扩增,质粒DNA抽提,使用PstⅠ、BamHⅠ进行双酶切和DNA测序鉴定重组克隆。将小鼠NOMO1基因RNA干扰载体转染P19细胞,以RT-PCR技术验证NOMO1基因在P19细胞中的mRNA表达。以DMSO诱导分化方案诱导P19细胞向心肌细胞分化,采用定量RT-PCR技术检测P19细胞中α-MHC等基因mRNA的表达,评估NOMO1与P19干细胞向心肌细胞方向分化的关系。结果:双酶切证实shRNA正确插入质粒,测序结果表明插入的序列正确。载体转染的P19细胞筛选稳定表达株,经RT-PCR和Western blot验证4个位点设计的干扰质粒对NOMO1在P19细胞中的mRNA表达均具有较高的抑制效率。转染后P19细胞的α-MHC基因mRNA表达则显著下调(P<0.05)。结论:成功构建小鼠NOMO1基因RNA干扰载体,为研究NOMO1基因与P19细胞向心肌细胞方向分化的关系提供了稳定的转染细胞工具,NOMO1可能通过诱导α-MHC等基因表达,促进P19干细胞向中胚层的分化。
OBJECTIVE: To construct NOMO1 RNA interference vector and compare its inhibitory effect on NOMO1 gene in P19 cells, so as to study the relationship between NOMO1 gene and P19 cells toward cardiomyocytes. METHODS: The mouse NOMO1 gene fragment was designed by using Designer3.0 software to synthesize the shRNA sequence. After the oligonucleotide double strand was synthesized, the annealed oligonucleotide double strand was cloned into the cohesive end of pGPU6 / Hygro vector , Ligated into competent cells, amplified bacteria, plasmid amplification, plasmid DNA extraction, using Pst Ⅰ, BamH Ⅰ double digestion and DNA sequencing identified recombinant clones. The NOMO1 gene RNA interference vector was transfected into P19 cells, and the mRNA expression of NOMO1 gene in P19 cells was verified by RT-PCR. The differentiation of P19 cells into cardiomyocytes was induced by DMSO induction. The mRNA expression of α-MHC in P19 cells was detected by quantitative RT-PCR and the relationship between NOMO1 and P19 stem cells was analyzed. Results: Double enzyme digestion confirmed that shRNA was inserted into the plasmid correctly. Sequencing results showed that the inserted sequence was correct. The vector-transfected P19 cells were screened for stable expression. The four plasmids designed by RT-PCR and Western blot confirmed that NOMO1 had a higher inhibitory effect on the mRNA expression of NOMO1 in P19 cells. The expression of α-MHC mRNA in P19 cells was significantly down-regulated after transfection (P <0.05). Conclusion: The NOMO1 gene RNA interference vector was successfully constructed, which provided a stable transfected cell tool for the study of the relationship between NOMO1 gene and the differentiation of P19 cells into cardiomyocytes. NOMO1 may promote the expression of α-MHC and other genes, Mesodermal differentiation.