【摘要】 目的探讨p16基因对结肠癌细胞生长的抑制作用。方法采用亚克隆技术将p16基因克隆入真核表达载体pEGFP-N3中,Lipofectamine法将p16基因转染入结肠癌细胞株SW480。采用MTT法检测未转染细胞(SW480组)、转染空质粒细胞(SW480-GFP组)和转染p16细胞(SW480-p16组)培养24、48和72 h后细胞的增殖率;Real-Time PCR和Western blotting分别检测3组细胞的p16、CDK4、cyclin D1 mRNA和蛋白的表达情况。结果 p16基因克隆入真核表达载体,并成功转染结肠癌细胞后,在基因和蛋白质水平均有外源性p16基因表达,与SW480组比较,相对表达量均有显著增加(P<0.01)。MTT检测结果显示:培养48 h和72 h后,SW480-p16组的细胞相对增殖率均显著低于SW480组和SW480-GFP组(P<0.01)。Real-Time PCR检测结果显示:SW480-p16组的p16 mRNA相对表达量接近SW480-GFP组的2.38倍,接近SW480细胞的2.59倍,差异具有统计学意义(P<0.01);SW480-p16组的cyclin D1 mRNA相对表达量明显低于SW480组和SW480-GFP组细胞(P<0.05),而CDK4mRNA的相对表达量略低于SW480组和SW480-GFP组,差异无统计学意义(P>0.05)。Western blotting检测结果显示:与SW480-GFP组和SW480组比较,SW480-p16组的P16蛋白表达量明显升高;而CDK4和cyclin D1蛋白表达量明显降低,差异均有统计学意义(P<0.01)。结论 p16基因转染入结肠癌细胞,具有抑制癌细胞生长的功能。更多还原

【Abstract】 Objective To investigate the inhibitory effect of p16 gene on the growth of colon cancer cell line SW480.Methods p16 gene was cloned into eukaryotic express vector pEGFP-N3 using subclone technique,and the recombinant plasmid was transfected into SW480 cells using Lipofectamine method.MTT assay was performed to analyse the proliferation of cells without transfection(SW480 group),cells transfected with blank plasmid(SW480-GFP group) and cells transfected with p16(SW480-p16 group) after culture for 24,48 and 72 h.Real-Time PCR and Western blotting were employed to detect the expression of p16,CDK4 and cyclin D1 mRNA and protein respectively.Results Compared with SW480 group and SW480-GFP group,the expression of p16 gene in SW480-p16 group was significantly increased(P<0.01).MTT assay indicated that the cell proliferation in SW480-p16 group was significantly lower than that in SW480 group and SW480-GFP group after culture for 48 h and 72 h(P<0.01).Real-Time PCR analysis revealed the expression of p16 mRNA in SW480-p16 group which was 2.59 times of that in SW480,and 2.38 times in SW480-GFP(P<0.01).The expression of cyclin D1 mRNA in SW480-p16 group was significantly lower than that in SW480 group and SW480-GFP group(P<0.05),and the expression of CDK4 mRNA in SW480-p16 group was slightly lower than that in SW480 group and SW480-GFP group(P>0.05).Western blotting demonstrated that the expression of p16 protein in SW480-p16 group was significantly higher than that in SW480-GFP group and SW480 group(P<0.01),while the expression of CDK4 and cyclin D1 protein in SW480-p16 group was significantly lower than that in SW480-GFP group and SW480 group(P<0.01).Conclusion Colon cancer cells transfected with p16 gene can resume the function of inhibiting tumor growth.更多还原