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RIP3基因重组质粒构建及其表达对MCF7细胞死亡方式的影响

路灿,徐惠君,贾勇圣,佟仲生   

  1. 天津医科大学附属肿瘤医院
  • 收稿日期:2013-04-15 修回日期:2013-10-21 出版日期:2014-02-15 发布日期:2014-02-15
  • 通讯作者: 佟仲生

Construction of Recombinant Plasmid of Human RIP3 Gene and Its Expression effects on the death of Breast Cancer MCF7 Cells

  • Received:2013-04-15 Revised:2013-10-21 Published:2014-02-15 Online:2014-02-15

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摘要： 【摘要】 目的 构建RIP3基因过表达重组质粒，建立稳定过表达RIP3基因的乳腺癌细胞株，并证实融合蛋白在细胞内的表达、定位及对MCF7细胞死亡方式的影响。方法 逆转录聚合酶链反应(RT-PCR)检测4种乳腺癌细胞及正常乳腺上皮细胞中RIP3 mRNA的表达。以正常乳腺上皮细胞MCF10A cDNA为模板，PCR扩增RIP3基因cDNA全长，将合成的RIP3编码区序列，克隆入mCherry载体的N末端，构建重组质粒mCherry-RIP3，对重组质粒进行酶切鉴定及DNA测序。钙法转染293T细胞，收集病毒感染MCF7细胞，basticidin (4 mg/L)维持筛选，构建稳定表达细胞株。Western blot、荧光显微镜等检测目的基因表达效率及蛋白定位。显微镜下观察TNF-α及zVAD处理下mCherry-RIP3-MCF7细胞的死亡形态及比例。结果 RIP3 mRNA 在乳腺癌细胞中普遍低表达。RIP3基因成功克隆入载体。过表达mCherry-RIP3基因的MCF7细胞可见红色荧光蛋白表达，定位于胞质。Western检测到目的基因RIP3在转染细胞中为过表达。mCherry-RIP3转染后增强MCF7细胞对TNF-α联合z-VAD 诱导的细胞坏死的敏感性。结论 成功构建RIP3基因过表达重组质粒，获得外源性RIP3稳定过表达的乳腺癌MCF7细胞株，mCherry-RIP3定位于胞质，并在TNF-α介导的程序性坏死中起作用。

关键词: 受体相互作用蛋白3, 乳腺癌, 质粒, 程序性坏死, 肿瘤坏死因子α

Abstract: Abstract: Objective: Construct the recombinant RIP3 overexpressed plasmids and transfect them in breast cancer MCF7 cells. Identify the expression and localization of fusion protein, as well as its effect on the death way of MCF7 cells. Methods: The expression of RIP3 mRNA in four breast cancer cell lines and one mammary epithelial cell was detected by reverse transcription polymerase chain reaction (RT-PCR). The RIP3 coding sequence was amplified by polymerase chain reaction and subcloned into mCherry vector to construct recombinant plasmids. The plasmids were transfected into MCF7 cells by lentivirus after DNA sequencing, then screened by basticidin (4 mg/L) for 1 week. The efficiencies of RIP3 expression were validated by Western blotting. The death way of mCherry-RIP3-MCF7 cells was observed under the treatment of TNF-αand zVAD. Results: The lowest expression of RIP3 mRNA was seen in MCF7 cells. The sequencing results validated the well recombinant plasmids. The expression of mCherry-RIP3 fusion protein with a molecular weight of 85 ku was detected by Western blot. mCherry-RIP3 expression enhance the sensitivity of MCF7 cells to TNF-α and z-VAD induced cell death. Conclusion：The recombinant RIP3 overexpressed plasmids were successfully constructed, and the stable MCF7 cells with ectopic RIP3 transfection were obtained. The mCherry-RIP3 fusion protein was expressed in the cytoplasm and was conformed to mediate TNF-α induced necroptosis.

Key words: RIPK3 protein, human, Breast neoplasms, Plasmids, Necroptosis, TNF-α