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目的构建在哺乳动物细胞中表达 SMN1 shRNA 的质粒,并初步探讨其对人骨髓间充质干细胞全长 SMN mRNA 及蛋白的抑制作用,为进一步建立脊髓性肌萎缩症细胞模型提供实验依据。方法根据 GenBank 中 SMN1 cDNA 序列设计5条 SMN1靶向 shRNA 并将其克隆至 pRNAT-U6.1/Neo 载体中 U6启动子的下游,形成能在细胞内表达 SMN1特异性短发夹状 RNA 的重组质粒pshRNA-SMN1;在脂质体介导下将重组质粒 pshRNA-SMN1转染人骨髓间充质干细胞(hMSC),RT-PCR、Western 印迹分别检测 fl-SMN mRNA 及 fl-SMN 蛋白的表达。结果琼脂糖凝胶电泳及 DNA 测序证实重组质粒 pshRNA-SMN1构建成功,其中 pshRNA-SMN1-1组和 pshRNA-SMN1-4组转染后明显抑制了 hMSCs fl-SMN mRNA 及蛋白的表达(均 P<0.05),在 mRNA 水平上干扰效率分别为64.05%、61.04%,蛋白水平上干扰效率分别为52.97%和61.57%,而未影响△7-SMN mRNA 的表达。结论构建的 pshRNA-SMN1重组质粒能有效地抑制人骨髓间充质干细胞 fl-SMN mRNA 及蛋白的表达。
Objective To construct plasmids expressing SMN1 shRNA in mammalian cells and to investigate the inhibitory effect on SMN mRNA and protein of human bone marrow mesenchymal stem cells in vitro and to provide experimental evidence for establishing a cell model of spinal muscular atrophy. Methods According to the sequence of SMN1 in GenBank, five SMN1-targeting shRNAs were designed and cloned into the downstream of U6 promoter in pRNAT-U6.1 / Neo vector to form recombinant SMN1-specific short hairpin RNA Plasmid pshRNA-SMN1 was transfected into human bone marrow mesenchymal stem cells (hMSC) by liposome. The expression of fl-SMN mRNA and fl-SMN protein were detected by RT-PCR and Western blot respectively. RESULTS: The recombinant plasmid pshRNA-SMN1 was successfully constructed by agarose gel electrophoresis and DNA sequencing. The expression of fl-SMN mRNA and protein in hMSCs was significantly inhibited by pshRNA-SMN1-1 group and pshRNA-SMN1-4 group (all P <0.05). The interference efficiency at mRNA level was 64.05% and 61.04% respectively, and the interference efficiency at protein level was 52.97% and 61.57% respectively, without affecting the expression of △ 7-SMN mRNA. Conclusion The constructed pshRNA-SMN1 recombinant plasmid can effectively inhibit the expression of fl-SMN mRNA and protein in human bone marrow mesenchymal stem cells.