上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (6): 744-.doi: 10.3969/j.issn.1674-8115.2011.06.014

• 论著（基础研究） • 上一篇    下一篇

p16基因克隆及其对结肠癌细胞SW480生长的抑制作用

孟庆凯, 张菁茹, 王 辉   

  1. 辽宁省肿瘤医院肠外科, 沈阳 110042
  • 出版日期:2011-06-28 发布日期:2011-06-27
  • 作者简介:孟庆凯（1971—）, 男, 副主任医师, 博士；电子信箱: mqk1971@126.com。
  • 基金资助:

    辽宁省自然科学基金（20092071）

Cloning of p16 gene and its role in inhibition of colon cancer SW480 cell growth

MENG Qing-kai, ZHANG Jing-ru, WANG Hui   

  1. Department of Enteric Surgery, Liaoning Cancer Hospital &|Institute, Shenyang 110042, China
  • Online:2011-06-28 Published:2011-06-27
  • Supported by:

    Natural Science Foundation of Liaoning Province, 20092071

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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CDK4 mRNA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。

关键词: p16基因, 克隆, 结肠癌细胞, D型细胞周期蛋白, 周期蛋白依赖性蛋白激酶4

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.ResultsCompared 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.

Key words: p16 gene, clone, colon cancer cells, cyclin D1, cyclin dependent kinase 4 p16