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目的:对小鼠凋亡相关新基因PNAS-4(mPNAS-4)进行克隆,构建其真核表达载体,并在小鼠Lewis肺癌细胞系LL2中转染表达;探讨mPNAS-4基因转染LL2细胞过表达所诱导的体外肿瘤细胞凋亡情况。方法:用RT-PCR从小鼠肝脏组织中克隆mPNAS-4编码区cDNA,构建重组真核表达载体pcDNA3.1(+)-mPNAS-4;用脂质体将pcDNA3.1(+)-mPNAS-4转染小鼠Lewis肺癌LL2细胞;用RT-PCR检测转染细胞中mPNAS-4的过表达情况;通过MTT、流式细胞术(FCM)及DNALadder分别检测转染细胞的增殖与凋亡情况。结果:从小鼠肝脏组织中克隆到mPNAS-4全长cDNA并成功构建其真核表达载体pcDNA3.1(+)-mPNAS-4,转染小鼠Lewis肺癌LL2细胞可使其mRNA表达明显上调。mPNAS-4过表达能抑制LL2细胞增殖并诱导其凋亡。结论:mPNAS-4过表达对小鼠Lewis肺癌LL2细胞的生长有明显的抑制和诱导凋亡作用。
OBJECTIVE: To clone the new gene of mouse PNAS-4 (mPNAS-4) and construct its eukaryotic expression vector and transfect it in Lewis lung carcinoma cell line LL2. To investigate the effect of mPNAS-4 gene transfection on LL2 In vitro tumor cell apoptosis induced by overexpression of cells. METHODS: cDNA of mPNAS-4 was cloned from mouse liver tissue by RT-PCR to construct recombinant eukaryotic expression vector pcDNA3.1 (+) - mPNAS-4. The pcDNA3.1 (+) - mPNAS- 4 transfected mouse Lewis lung carcinoma LL2 cells; RT-PCR detection mPNAS-4 over-expression in transfected cells; MTT, flow cytometry (FCM) and DNALadder were detected transfected cells proliferation and apoptosis . Results: The mPNAS-4 full-length cDNA was cloned from mouse liver tissue and its eukaryotic expression vector pcDNA3.1 (+) - mPNAS-4 was successfully constructed. The transfection of Lewis lung carcinoma LL2 cells significantly up-regulated the mRNA expression of mPNAS-4. Overexpression of mPNAS-4 can inhibit LL2 cell proliferation and induce its apoptosis. Conclusion: mPNAS-4 overexpression significantly inhibits the growth of Lewis lung carcinoma LL2 cells and induces apoptosis.