论文部分内容阅读
目的 构建葡萄糖糖基神经酰胺合成酶(GCS)小干扰RNA表达载体,观察其对乳腺癌耐药细胞(MCF- 7 /ADR)GCS基因表达和耐药性的影响。方法 设计合成2对GCS基因的特异性siRNA,分别定向克隆入真核表达载体pSUPER,构建pSUPER -GCS1和pSUPER -GCS2重组质粒,脂质体LipofectAMINE2000介导转染MCF -7 /ADR细胞,空载体pSUPER转染作为对照,逆转录聚合酶链反应(RT- PCR)分析GCSmRNA的表达,流式细胞仪观察GCS蛋白的表达,四氮唑盐法检测阿霉素对MCF -7 /ADR细胞的半数抑制浓度(IC50 ),流式细胞技术检测细胞凋亡率的改变。结果 酶切分析和测序证实成功构建了针对GCS的siRNA表达载体pSUPER -GCS1和pSUPER GCS2。两对重组质粒均可特异性抑制GCS基因表达,转染后48hGCSmRNA抑制率分别为89 .4%、88. 5%,GCS蛋白含量分别下降为8 .3%±1 .0%, 9. 2%±0 .8%,四氮唑盐法显示重组质粒对阿霉素耐药性相对逆转率分别为93. 7%、91. 6%,明显提高了乳腺癌细胞对于阿霉素的药物敏感性,流式细胞仪结果表明细胞凋亡率增加为15. 38±1 .16, 13. 92±1 .73,而空载体对照组无以上作用。结论 GCS特异性siRNA表达载体构建成功,且有效抑制了GCS基因表达,并通过诱导耐药细胞凋亡率增加,逆转乳腺癌细胞多药耐药。
Objective To construct small interfering RNA expression vector of glucose glycosylceramide synthase (GCS) and observe its effect on GCS gene expression and drug resistance of drug-resistant breast cancer cells (MCF-7 / ADR). Methods Two pairs of specific siRNAs for GCS gene were designed and synthesized. The recombinant plasmid pSUPER-GCS1 and pSUPER-GCS2 were cloned into the eukaryotic expression vector pSUPER respectively. LipofectAMINE2000 was transfected into MCF-7 / ADR cells. pSUPER transfection was used as a control, GCS mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), GCS protein expression was observed by flow cytometry, and half of doxorubicin on MCF-7 / ADR cells Inhibitory concentration (IC50), flow cytometry detection of apoptosis rate changes. Results Restriction analysis and sequencing confirmed the successful construction of siRNA expression vectors pSUPER-GCS1 and pSUPER GCS2 for GCS. Two pairs of recombinant plasmids could specifically inhibit the expression of GCS gene. The inhibition rates of GCS mRNA 48 h after transfection were 89.4% and 88.5%, respectively, and the GCS protein contents decreased to 8.3% ± 1.0% and 9.2% respectively % ± 0 .8%, the relative reversion rates of the recombinant plasmid to doxorubicin resistance by the tetrazolium salt method were 93.7% and 91.6%, respectively, which obviously enhanced the drug sensitivity of the breast cancer cells to doxorubicin The results of flow cytometry showed that the apoptosis rate increased to 15. 38 ± 1.16, 13. 92 ± 1.73, while the control group without the above effect. Conclusion The GCS-specific siRNA expression vector was constructed successfully and effectively inhibited the expression of GCS gene, and reversed the multi-drug resistance of breast cancer cells by inducing the increase of apoptosis rate of multi-drug resistant cells.