本文選題：卵巢癌 + 卵巢癌細(xì)胞株　； 參考：《山東醫(yī)藥》2017年39期

【摘要】：目的構(gòu)建核糖核苷酸還原酶亞基M2(RRM2)基因過表達(dá)的卵巢癌細(xì)胞株SKOV3,并觀察RRM2基因過表達(dá)的SKOV3細(xì)胞對(duì)順鉑的敏感性。方法提取SKOV3細(xì)胞總RNA,反轉(zhuǎn)錄得到c DNA,以其為模版進(jìn)行PCR反應(yīng),獲得RRM2基因。通過酶切(XhoⅠ、Bam HⅠ)連接的方法,將RRM2基因插入慢病毒表達(dá)載體p LVX-IRESZs Green1中,得到重組質(zhì)粒p LVX-IRES-RRM2。利用脂質(zhì)體將重組質(zhì)粒轉(zhuǎn)染293FT細(xì)胞,包埋病毒。將SKOV3細(xì)胞分為實(shí)驗(yàn)組和對(duì)照組,實(shí)驗(yàn)組轉(zhuǎn)染p LVX-IRES-RRM2慢病毒,96 h后測算慢病毒感染效率。對(duì)照組不進(jìn)行轉(zhuǎn)染。采用Western blotting法檢測兩組細(xì)胞中的RRM2蛋白;流式細(xì)胞儀檢測兩組凋亡細(xì)胞與死亡細(xì)胞,反映細(xì)胞對(duì)順鉑的敏感性。結(jié)果重組質(zhì)粒p LVX-IRES-RRM2經(jīng)限制性內(nèi)切酶XhoⅠ和Bam HⅠ雙酶切驗(yàn)證,各片段大小符合預(yù)期,測序結(jié)果顯示RRM2基因序列正確。實(shí)驗(yàn)組、對(duì)照組細(xì)胞中RRM2 mRNA相對(duì)表達(dá)量分別為0.280±0.055、0.102±0.022,RRM2蛋白相對(duì)表達(dá)量分別為0.582±0.049、0.228±0.032,實(shí)驗(yàn)組高于對(duì)照組,說明RRM2基因過表達(dá)的SKOV3細(xì)胞構(gòu)建成功。實(shí)驗(yàn)組、對(duì)照組凋亡細(xì)胞比例分別為0.39%±0.08%、9.60%±1.20%,死亡細(xì)胞比例分別為26.65%±2.20%、44.96%±2.80%,實(shí)驗(yàn)組凋亡細(xì)胞比例和死亡細(xì)胞比例均低于對(duì)照組(P均0.01)。結(jié)論成功構(gòu)建了RRM2基因過表達(dá)的SKOV3細(xì)胞,RRM2基因過表達(dá)的SKOV3細(xì)胞對(duì)順鉑的敏感性降低。
[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，

本文編號(hào)：1759025