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目的构建Krüppel-like factor4(KLF4)过表达慢病毒载体,探讨其对胃癌细胞株BGC-823生物学行为的影响。方法检测BGC-823中KLF4 mRNA的表达水平;用真核表达质粒pcDNA3.1IE-KLF4-EGFP,将KLF4基因连入慢病毒载体pLv-UbC-IRES2-EGFP中,构建pLv-KLF4-IRES2-EGFP重组慢病毒表达载体。将酶切和测序鉴定后的重组质粒转染至BGC-823中,观察转染情况,RT-PCR检测KLF4 mRNA。慢病毒包装后转染BGC-823细胞,Western印迹检测KLF4蛋白。结果重组质粒经酶切和DNA测序证实目的基因插入正确;pcDNA3.1IE-KLF4-EGFP转染BGC-823细胞后KLF4 mRNA升高。慢病毒包装后转染BGC-823检测到目的蛋白KLF4。KLF4能够将细胞阻滞于G1/S,抑制其生长、促进细胞凋亡,减少细胞侵袭能力。结论转染BGC-823后KLF4蛋白检测证实慢病毒载体成功构建,KLF4能抑制胃癌细胞的恶性转化。
Objective To construct a lentiviral vector with over-expression of Krüppel-like factor 4 (KLF4) and investigate its effect on the biological behavior of gastric cancer cell line BGC-823. The expression of KLF4 mRNA in BGC-823 cells was detected by ELISA. The KLF4 gene was inserted into the lentiviral vector pLv-UbC-IRES2-EGFP using eukaryotic expression plasmid pcDNA3.1IE-KLF4-EGFP to construct pLv-KLF4-IRES2-EGFP Recombinant lentivirus expression vector. After restriction endonuclease digestion and sequencing, the recombinant plasmids were transfected into BGC-823 cells, and the transfection conditions were observed. The KLF4 mRNA was detected by RT-PCR. After lentivirus packaging, BGC-823 cells were transfected and KLF4 protein was detected by Western blotting. Results The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing. The insertion of pcDNA3.1IE-KLF4-EGFP increased the expression of KLF4 mRNA in BGC-823 cells. After the lentivirus was packaged, the target protein KLF4 was detected by transfection of BGC-823. KLF4 can block cells in G1 / S, inhibit its growth, promote apoptosis, reduce cell invasive ability. Conclusion The results of KLF4 protein detection after transfection of BGC-823 confirmed the successful construction of lentiviral vector. KLF4 could inhibit the malignant transformation of gastric cancer cells.