【摘要】 目的利用慢病毒介导的RNA干扰技术下调CD133+HepG2肝癌干细胞中CD133的表达,探讨其对肝癌干细胞增殖及化疗敏感性的影响。方法利用流式分选技术筛选CD133+HepG2细胞并检测分选前后CD133表达量,体外成球及体内成瘤实验鉴定其"干性";随后以CD133 mRNA编码序列为干扰靶点,采用慢病毒介导的shRNA转染CD133+细胞。应用RT-PCR和Western Blot检测CD133 mRNA及蛋白表达,克隆形成实验检测转染前后细胞增殖情况,CCK-8法和流式细胞仪检测在化疗药物顺铂作用下转染前后细胞的生长抑制率及凋亡情况。结果分选前后CD133表达率分别为(3.36±1.80)%,(88.74±3.19)%;CD133+细胞相对于CD133-细胞有更强的体外成球和体内成瘤能力;转染CD133 shRNA后的细胞CD133 mRNA表达明显受到抑制(P<0.05),蛋白表达水平降低,增殖能力显著下降(P<0.01)。顺铂对转染CD133 shRNA后细胞生长抑制作用明显提高(P<0.01),且其在顺铂作用下的凋亡率也显著增加(P<0.05)。结论慢病毒介导的shRNA能显著下调CD133+肝癌干细胞CD133基因的表达,抑制其增殖并提高其对顺铂的敏感性。更多还原

【Abstract】 Objective To study the effects of CD133 suppression by lentivirus-mediated RNA interference(RNAi) on the proliferation and chemosensitivity of CD133+ cancer stem cells(CSCs) sorted from HepG2 cell line.Methods CD133+ and CD133-cells were sorted from HepG2 cell line by flow cytometry,and the expression of CD133 before and after cell sorting were detected.The stem cell property of sorted CD133+ cells were validated by sphere-forming assay in vitro and xenograft experiments in vivo.Lentivirus-mediated short haipin RNA(shRNA) targeting CD133 were transfected into CD133+ cells,and CD133 mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blotting,respectively.Before and after the transfection,the proliferative ability of CD133+ cells was evaluated by colony formation assay,and the cell growth inhibition rate and apoptosis following ciplatin exposure were detected using CCK-8 assay and flow cytometry.Results The sorted CD133+ cells showed a high purity of(88.74±3.19)%,as compared with the purity of(3.36±1.80)% before cell sorting.CD133+ cells showed a high tumor sphere formation ability and tumorigenesis capacity compared with CD133-cells.CD133 shRNA transfection significantly inhibited CD133 mRNA and protein expressions in CD133+ cells(P<0.01),resulting also in a significantly lowered cell proliferative ability(P<0.01) and an increased growth inhibition rate(P<0.01) and obviously increased cell apoptosis(P<0.05) after cisplatin exposure.Conclusions Lentivirus-mediated RNAi for CD133 suppression inhibits the proliferation of CD133+ liver cancer stem cells and increases their chemosensitivity to cisplatin.更多还原