RNA干扰Livin基因表达增强骨肉瘤MG-63耐药细胞对多柔比星的化疗敏感性

发布时间：2018-06-16 09:19

本文选题：骨肉瘤 + Livin基因　；参考：《中国肿瘤生物治疗杂志》2017年12期

【摘要】：目的:研究凋亡抑制蛋白基因Livin在逆转骨肉瘤耐药中的作用。方法:应用多柔比星逐步诱导法诱导人骨肉瘤MG-63细胞建立耐药细胞株,MTT实验检测耐药细胞株的耐药指数,Western blotting法检测MG-63细胞和耐药细胞中Livin蛋白表达的差异。构建Livin shRNA真核表达载体,应用LipofectmineTM2000转染至耐药骨肉瘤细胞中,Real-time PCR和Western blotting分别检测Livin shRNA转染前后MG-63耐药细胞中Livin基因和蛋白表达的变化,流式细胞术检测细胞凋亡的变化,MTT法检测对多柔比星化疗敏感性的变化。结果:成功构建重组质粒p Silencer3.1-H1 neo-Livin si。诱导的耐药细胞MG-63/R对多柔比星的耐药指数为81.32±5.33。Livin shRNA可抑制MG-63细胞中Livin基因和蛋白表达,明显低于空白对照组和非特异性转染组(分别下调72%和69%,P0.05)。特异性转染Livin shRNA组MG-63/R细胞的凋亡率显著高于未转染组和非特异性转染组[(22.4±3.2)%vs(4.2±1.1)%、(4.7±0.6)%,P0.05]。3组细胞在加入多柔比星后增殖均受到不同程度的抑制,且呈明显的时间-效应关系,但特异性转染组细胞的存活率均显著低于其他两组(P0.05)。结论:应用RNA干扰技术下调Livin的基因表达可有效促进耐药MG-63骨肉瘤细胞的凋亡,从而增加其对化疗药物的敏感性。
[Abstract]:Aim: to study the role of apoptosis inhibitor gene Livin in reversing drug resistance in osteosarcoma. Methods: human osteosarcoma MG-63 cells were induced by doxorubicin step by step to establish a drug-resistant cell line. MTT assay was used to detect the difference of Livin protein expression between MG-63 cells and drug-resistant cells by Western blotting assay. Livin shRNA eukaryotic expression vector was constructed. Real-time PCR and Western blotting were used to detect the expression of Livin gene and protein before and after transfection into drug-resistant osteosarcoma cells by Lipofectmine TM2000. Apoptosis was detected by flow cytometry and the sensitivity to doxorubicin was detected by MTT assay. Results: the recombinant plasmid pSilencer3.1-H1 neo-Livin si. was successfully constructed. The resistance index of MG-63 / R to doxorubicin was 81.32 卤5.33. Livin shRNA inhibited the expression of Livin gene and protein in MG-63 cells, which was significantly lower than that in control group and non-specific transfection group. The apoptosis rate of MG-63 / R cells in specific transfection group was significantly higher than that in non-transfection group and non-specific transfection group [22.4 卤3.2)%vs(4.2 卤1.1] .3 cells were inhibited by doxorubicin. But the survival rate of the cells in the specific transfection group was significantly lower than that in the other two groups. Conclusion: down-regulation of Livin gene expression by RNA interference can effectively promote apoptosis of drug-resistant MG-63 osteosarcoma cells and increase its sensitivity to chemotherapeutic drugs.
【作者单位】：南昌大学第二附属医院骨科;江西省都昌县中医院骨科;
【基金】：国家自然科学基金资助项目(No.81660443) 江西省卫计委科技项目资助(No.20155278)~~
【分类号】：R738.1

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