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目的构建转化生长因子-β1(Transforming growth factor-β1,TGF-β1)基因短发夹RNA(Short hairpin RNA,shRNA)表达质粒,并观察其对结肠癌细胞SW480侵袭能力的影响。方法设计并合成2对靶向TGF-β1基因的RNA干扰序列和1对阴性对照序列,并构建重组表达质粒pshRNA-TGF-β1a、pshRNA-TGF-β1b和pshRNA-TGF-β1c(阴性对照),以脂质体介导转染SW480细胞,荧光显微镜观察转染情况;RT-PCR和Western blot检测转染细胞中TGF-β1基因mRNA的转录水平和蛋白的表达水平;Transwell侵袭试验检测沉默TGF-β1基因表达对细胞侵袭能力的影响。结果 TGF-β1基因shRNA重组表达质粒经酶切鉴定和测序分析证明构建正确。与空白对照组相比,3种质粒转染的细胞均有较强的荧光表达;pshRNA-TGF-β1a和pshRNA-TGF-β1b组细胞TGF-β1基因mRNA的转录水平和蛋白的表达水平均明显减低(P<0.05);SW480细胞的侵袭能力也明显降低(P<0.05)。结论成功构建了TGF-β1基因shRNA重组表达质粒,其能有效抑制结肠癌细胞SW480中TGF-β1基因的表达,并可显著降低细胞的侵袭能力,为结肠癌的基因治疗提供了新的思路。
Objective To construct short hairpin RNA (shRNA) expression plasmid of Transforming growth factor-β1 (TGF-β1) gene and observe its effect on invasiveness of colon cancer cell line SW480. Methods Two pairs of RNA interfering sequences and one pair of negative control sequences targeting TGF-β1 gene were designed and synthesized. Recombinant plasmids pshRNA-TGF-β1a, pshRNA-TGF-β1b and pshRNA-TGF-β1c (negative control) Transfection of SW480 cells was performed by lipofectamine 2000. Transfection of SW480 cells was observed by fluorescence microscopy. Transcription levels of TGF-β1 mRNA and protein were detected by RT-PCR and Western blot. Transwell invasion assay was used to detect the expression of TGF- Effect of β1 Gene Expression on Cell Invasion. Results The shRNA recombinant expression plasmid of TGF-β1 gene was confirmed by restriction enzyme analysis and sequencing analysis. Compared with the blank control group, the transfected cells of the three plasmids all had strong fluorescence expression. The mRNA and protein expression levels of TGF-β1 mRNA in both pshRNA-TGF-β1a and pshRNA-TGF-β1b groups were significantly higher (P <0.05). The invasive ability of SW480 cells was also significantly decreased (P <0.05). Conclusion The shRNA recombinant expression plasmid of TGF-β1 gene was successfully constructed, which can effectively inhibit the expression of TGF-β1 gene in colon cancer cells SW480 and significantly reduce the invasion ability of cells, providing a new idea for the gene therapy of colon cancer.