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目的研究靶向人胰岛素样生长因子1类受体(IGFIR)基因miR30-shRNA慢病毒对肝癌细胞生长的影响。方法针对已经筛选确定的IGF1R基因干扰有效序列,合成靶序列的OligoDNA,退火形成双链DNA,与经XhoI和EcoRI酶切后的pPRIME载体连接产生pPRIME-IGF1R-miR30-shRNA。用后者,psPAX2及pMD2G3质粒共转染包装细胞293T细胞,包装产生慢病毒。慢病毒PRIME-IGF1R-miR30-shRNA大量扩增,氯化铯梯度离心纯化,测病毒滴度。RT-PCR及Western blot检测IGF1R表达,CCK-8法检测细胞生长。结果成功构建PRIME-IGF1R-miR30-shRNA,滴度为4.58×109PFU/mL;该慢病毒能抑制肝癌细胞IGF1R表达及生长。结论IGF1R基因miR30-shRNA慢病毒靶向转染可有效地封闭肝癌细胞的IGF1R表达,降低肝癌细胞的增殖能力,为以IGFIR基因为靶点的肝癌生物治疗的进一步研究奠定了实验基础。
Objective To investigate the effect of miR30-shRNA lentivirus targeting human insulin-like growth factor 1 receptor (IGFIR) gene on the growth of hepatoma cells. Methods Targeted IGF1R gene was screened for the effective sequence. Oligo DNA of target sequence was synthesized and annealed to form double-stranded DNA. The pPRIME-IGF1R-miR30-shRNA was ligated with pPRIME vector digested with XhoI and EcoRI. In the latter, psPAX2 and pMD2G3 plasmids were co-transfected into packaging cells 293T cells and packaged to produce lentivirus. The lentivirus PRIME-IGF1R-miR30-shRNA was extensively amplified and purified by cesium chloride gradient centrifugation to determine the virus titer. The expression of IGF1R was detected by RT-PCR and Western blot, and the cell growth was detected by CCK-8. Results The PRIME-IGF1R-miR30-shRNA was successfully constructed and its titer was 4.58 × 109 PFU / mL. The lentivirus could inhibit IGF1R expression and growth in hepatoma cells. Conclusion Targeting transfection of IGF1R gene miR30-shRNA lentivirus can effectively block the expression of IGF1R in hepatocellular carcinoma cells and reduce the proliferation of hepatocellular carcinoma cells. This study lays the foundation for the further study of biological treatment of hepatocellular carcinoma with IGFIR gene.