乳腺肿瘤,PARP-1,RNA干扰,慢病毒感染 ," /> 乳腺肿瘤,PARP-1,RNA干扰,慢病毒感染 ,"/> Breast neoplasm,PARP-1,RNA interference,Lentivirus infections,"/> 慢病毒介导的靶向干扰PARP-1基因乳腺癌稳定细胞株的构建

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中国癌症防治杂志

中国癌症防治杂志 ›› 2017, Vol. 9 ›› Issue (2): 129-133.doi: 10.3969/j.issn.1674-5671.2017.02.09

• 基础研究 • 上一篇    下一篇

慢病毒介导的靶向干扰PARP-1基因乳腺癌稳定细胞株的构建

  

  1. 广西医科大学附属肿瘤医院放疗科；广西医科大学研究生院
  • 出版日期:2017-04-25 发布日期:2017-06-12
  • 通讯作者: 朱小东 zhuxiaodong@gxmu.edu.cn；赵伟 zwei1112@126.com
  • 基金资助:

    国家自然科学基金资助项目（81260346）；广西自然科学基金资助项目（2013GXNSFBA019199）；广西壮族自治区卫生厅自筹经费科研课题（z2012346）

Construction of a stable breast cancer cell line for lentivirus-mediated RNA interference to silence PARP-1

  • Online:2017-04-25 Published:2017-06-12

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1

摘要：

目的 构建慢病毒介导的人PARP-1基因短发夹RNA (shRNA）载体，并建立PARP-1基因稳定干扰的MDA-MB-231乳腺癌细胞株?方法 根据GenBank提供的PARP-1 cDNA序列，构建靶向PARP-1基因慢病毒载体，将慢病毒载体与病毒包装辅助质粒共同转染293T细胞，产生重组慢病毒并进行滴度测定?将重组慢病毒转染乳腺癌MDA-MB-231细胞株 (PARP-1组），同时设立阴性对照组和空白对照组?采用实时荧光定量PCR及Western blot检测细胞PARP-1基因mRNA及蛋白的表达?结果 成功构建了人PARP-1-RNAi重组慢病毒并转染至乳腺癌MDA-MB-231细胞株?空白对照组?阴性对照组和PARP-1组的PARP-1 mRNA相对表达量分别为1.001±0.056?1.022±0.021和0.166±0.041;PARP-1蛋白的相对表达量分别为0.963±0.006?0.943±0.008和0.573±0.011;与空白对照组和阴性对照组相比，PARP-1组PARP-1 mRNA和蛋白的表达均显著降低 (P<0.05）?结论 成功构建了PARP-1基因稳定干扰的乳腺癌MDA-MB-231细胞株?

关键词:

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Abstract:

Objective To construct a PARP-1 lentivirus interference vector and establish a stably infected human breast cancer MDA-MB-231 cell line. Methods According to genetic information of PARP-1 cDNA sequence provided by GenBank，a targeted PARP-1 lentiviral vector was constructed. Both the lentiviral vector and virus packaging plasmids were transfected into 293T cells to produce recombinant lentivirus，which was infected into the human breast cancer MDA-MB-231 cell line（PARP-1 group）. Negative control and blank control groups were also prepared. The mRNA and protein expression of PARP-1 was measured using real-time quantitative PCR and Western blot. Results PARP-1-RNAi lentiviral vector was constructed and infected into MDA-MB-231 cells. The mRNA expression levels of PARP-1 in blank control group，negative control group and PARP-1 group were，respectively，1.001±0.056，1.022±0.021 and 0.166±0.041； the relative expression levels of PARP-1 protein were 0.963±0.006，0.943±0.008 and 0.573±0.011. Compared with the blank control group and negative control group，the expression of PARP-1 mRNA and protein was significantly lower in the PARP-1 group（P<0.05）. Conclusion A breast cancer cell line stably expressing low levels of PARP-1 has been established.

Key words: Breast neoplasm')">