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目的构建人肺癌整合素连接激酶(Integrin-linked kinase,ILK)基因siRNA重组表达质粒,并检测其对人肺腺癌A549细胞增殖的影响。方法人工合成靶向ILK基因的siRNA干扰序列,克隆至载体pGenesil-1中,构建重组表达质粒pGenesil-1-ILK,利用脂质体转染A549细胞,经G418稳定筛选后,采用RT-PCR和Western blot检测细胞中ILK基因mRNA的转录水平及蛋白的表达水平,MTT法检测细胞的增殖活力。结果重组表达质粒pGenesil-1-ILK经SalⅠ单酶切及测序证明构建正确,其转染A549细胞后,细胞ILK基因mRNA的转录水平和蛋白的表达水平均明显降低(P<0.05),且细胞的增殖能力也显著降低(P<0.05)。结论成功构建了人ILK基因siRNA重组表达质粒,ILK基因沉默可显著抑制A549细胞增殖,为肺癌靶向基因治疗的研究提供了新的思路。
OBJECTIVE: To construct siRNA recombinant expression plasmid of integrin-linked kinase (ILK) gene in human lung cancer cell line A549 and to investigate its effect on the proliferation of human lung adenocarcinoma A549 cells. Methods siRNA interference targeting ILK gene was synthesized and cloned into pGenesil-1 vector. The recombinant plasmid pGenesil-1-ILK was constructed and transfected into A549 cells by lipofectamine 2000. After stable screening with G418, Western blot was used to detect the mRNA transcription level and protein expression level of ILK gene. The proliferation activity of the cells was detected by MTT assay. Results The recombinant plasmid pGenesil-1-ILK was digested with SalⅠ and sequenced to confirm that the recombinant plasmid pGenesil-1-ILK was constructed correctly. Transfection of A549 cells resulted in a significant decrease of mRNA and protein levels of ILK mRNA (P <0.05) The proliferation ability was also significantly lower (P <0.05). Conclusion The siRNA recombinant expression plasmid of human ILK gene was successfully constructed. Silencing ILK gene can significantly inhibit the proliferation of A549 cells, which provides a new idea for the targeted gene therapy of lung cancer.