RRM2基因过表达卵巢癌细胞株SKOV3的构建及对顺铂敏感性观察

发布时间：2018-04-16 13:06

  本文选题：卵巢癌 + 卵巢癌细胞株　； 参考：《山东医药》2017年39期

【摘要】：目的构建核糖核苷酸还原酶亚基M2(RRM2)基因过表达的卵巢癌细胞株SKOV3,并观察RRM2基因过表达的SKOV3细胞对顺铂的敏感性。方法提取SKOV3细胞总RNA,反转录得到c DNA,以其为模版进行PCR反应,获得RRM2基因。通过酶切(XhoⅠ、Bam HⅠ)连接的方法,将RRM2基因插入慢病毒表达载体p LVX-IRESZs Green1中,得到重组质粒p LVX-IRES-RRM2。利用脂质体将重组质粒转染293FT细胞,包埋病毒。将SKOV3细胞分为实验组和对照组,实验组转染p LVX-IRES-RRM2慢病毒,96 h后测算慢病毒感染效率。对照组不进行转染。采用Western blotting法检测两组细胞中的RRM2蛋白;流式细胞仪检测两组凋亡细胞与死亡细胞,反映细胞对顺铂的敏感性。结果重组质粒p LVX-IRES-RRM2经限制性内切酶XhoⅠ和Bam HⅠ双酶切验证,各片段大小符合预期,测序结果显示RRM2基因序列正确。实验组、对照组细胞中RRM2 mRNA相对表达量分别为0.280±0.055、0.102±0.022,RRM2蛋白相对表达量分别为0.582±0.049、0.228±0.032,实验组高于对照组,说明RRM2基因过表达的SKOV3细胞构建成功。实验组、对照组凋亡细胞比例分别为0.39%±0.08%、9.60%±1.20%,死亡细胞比例分别为26.65%±2.20%、44.96%±2.80%,实验组凋亡细胞比例和死亡细胞比例均低于对照组(P均0.01)。结论成功构建了RRM2基因过表达的SKOV3细胞,RRM2基因过表达的SKOV3细胞对顺铂的敏感性降低。
[Abstract]:Objective to construct a ovarian cancer cell line SKOV3 with overexpression of ribonucleotide reductase subunit (RRM2) gene, and to observe the sensitivity of SKOV3 cells with overexpression of RRM2 gene to cisplatin.Methods the total RNAs of SKOV3 cells were extracted, the c DNA was obtained by reverse transcription, and the RRM2 gene was obtained by PCR reaction using it as template.The RRM2 gene was inserted into the lentivirus expression vector p LVX-IRESZs Green1 and the recombinant plasmid pLVX-IRES-RRM2 was obtained.The recombinant plasmid was transfected into 293FT cells by liposome and the virus was encapsulated.SKOV3 cells were divided into experimental group and control group. The infection efficiency of lentivirus was measured after 96 h transfection of p LVX-IRES-RRM2 lentivirus in experimental group.The control group was not transfected.Western blotting assay was used to detect the RRM2 protein in the two groups, and flow cytometry was used to detect the apoptotic cells and the dead cells to reflect the sensitivity of the cells to cisplatin.Results the recombinant plasmid p LVX-IRES-RRM2 was confirmed by restriction endonuclease Xho 鈪，

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