HoxB8对结直肠癌细胞生长迁移的影响

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摘要目的：通过构建过表达HoxB8基因的慢病毒表达载体，研究HoxB8基因对结直肠癌细胞生长迁移的影响。方法：采用PCR技术扩增出人HoxB8基因全长，通过限制性内切酶EcoR I和SAC II双酶切，T4 DNA连接酶连接，克隆至慢病毒载体，构建Lenti-HoxB8-GFP重组载体。重组阳性克隆进行PCR和酶切鉴定，测序验证序列正确。将重组载体质粒以及3个辅助质粒共转染人肾上皮细胞（293T）进行慢病毒包装，收集病毒颗粒。然后将病毒颗粒感染结直肠癌细胞RKO，并将RKO细胞分为转染组（转染慢病毒重组质粒Lenti-HoxB8-GFP）、对照组（转染空白质粒Lenti-GFP）和空白对照组。RT-PCR和Western blot法检测病毒感染后 HoxB8在细胞中的表达情况。MTT、平板克隆形成实验和Transwell小孔试验检测过表达HoxB8基因对结直肠癌细胞生长迁移的影响。结果：经鉴定显示重组载体质粒构建成功，测序正确。293T细胞高效包装Lenti-HoxB8-GFP慢病毒，携带HoxB8基因的慢病毒感染RKO细胞后可看到大量绿色荧光，RT-PCR和Western blot法检测分别证实HoxB8基因和蛋白在转染组细胞中呈高表达。实验组细胞的增殖和平板克隆形成能力高于空白组及对照组。Transwell小孔试验结果显示HoxB8高表达能够促进结直肠癌细胞的转移。结论：成功构建HoxB8慢病毒表达载体，过表达HoxB8能够促进结直肠癌细胞的增殖、克隆形成以及迁移。

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	林飞燕1
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	李绍堂2

关键词 ： HoxB8, 慢病毒载体, 结直肠肿瘤, 实时荧光定量PCR, 蛋白质印记法

Abstract：Objective: To construct a lentiviral vector Lenti-HoxB8-GFP that containing human HoxB8 gene and investigate its role in colon cancer cells. Methods: The HoxB8 gene was amplified and cloned into a lentiviral vector by restriction endonuclease EcoR I/SAC II digestion and T4 DNA ligase ligation to form recombinant Lenti-HoxB8-GFP. The positive clones were identified by PCR and sequencing, after transformation into E. coli cells. Lentivirus were produced by co-transfection of lentiviral vector and the three packaging vector into 293T cells. The RKO cells were transduced with Lenti-HoxB8-GFP and the expression of HoxB8 was detected by Real-time PCR and western blotting Cell proliferation and colony forming ability were detected by MTT and plate clone assay. Cell migration was measured by Transwell assay. Results: The lentiviral vector Lenti-HoxB8-GFP containing HoxB8 gene was successfully constructed. Most of the cells had strong green fluorescence after transfected with the collected virus for 72 h. Real-time PCR and western blotting verified the overexpression of HoxB8 in the transfected RKO cells. Overexpression of HoxB8 promoted RKO cell proliferation and cell colony formation. Moreover, overexpression of HoxB8 increased colorectal cancer cell migration. Conclusion: HoxB8 gene promoted proliferation, cell colony formation and cell migration in colorectal cancer RKO cells.

Key words： HoxB8 lentiviral vector colorectal neoplasms Real-time PCR Western blot

收稿日期: 2015-04-03

基金资助:浙江省教育厅科研基金资助项目（Y201326738）；浙江省医药卫生科技计划项目（2013KYA129）；温州市科技计划项目（Y20140667）；浙江省自然科学基金资助项目（LY15H160057）。

通讯作者: 李绍堂，副主任医师，Email：lishaotang163@163.com。

作者简介: 林飞燕（1987-），女，浙江温州人，助理实验员。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2015/V45/I11/785

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