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%,P<0.05)。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)%,P<0.05]。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(P<0.05)。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。
Objective： To investigate the effect of Livin gene （an inhibitor of apoptosis protein） on reversing the drug-resistance of osteosarcoma. Methods： Drug-resistant MG-63 cell strain was established in vitro by continuous exposure to doxorubicin at gradually increased concentrations. The resistance index of drug-resistant MG-63 cells was examined by MTT method; Livin protein expressions in MG-63 cells and durg-resistant MG-63 cells were determined by Western blotting.Livin shRNA eukaryotic expression vector （pSilencer3.1-H1 neo-Livin si） was constructed and then transfected into drug-resistant MG-63 cells by using Lipofectmine 2000. Expression change of Livin mRNA and protein in drug-resistant MG-63 cells before and after the transfection was respectively measured by Real-time PCR and Western blotting. The distribution of cell cycle and apoptosis were determined by flow cytometry.The analysis of chemotherapeutic sensitivity of drug-resistant MG-63 cell to doxorubicin was performed by MTT. Results： The recombinant eukaryotic expression vector Silencer3.1-H1 neo-Livin si was successfully constructed.Resistance index to doxorubicin of drug-resistant MG-63 cells （MG-63/R） was 81.32±5.33. Livin shRNA could significantly inhibit the mRNA and protein expression of Livin in MG-63/R cells compared with untransfect-ed group and non-specific transfected group（down-regulated by 72% and 69% at mRNA and protein level respectively,all P〈0.05）. The flow cytometry analysis showed there was significantly higher apoptosis rate in Livin shRNA transfected group than that of untransfected group and non- specific transfected group （[22.4±3.2]% vs [4.2±1.1]%, [4.7±0.6]%,P〈0.05）. After adding doxorubicin, the proliferation of three groups of cells was all inhibited at different levels in time-dependent manner; However, the cell survival rate in Livin shRNA transfected group was significantly lower than that in other two groups （P〈0.05）. Conclusion：Livin shRNA could efficiently promote the ,apoptosis
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2017年第12期1375-1380,共6页
Chinese Journal of Cancer Biotherapy
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)~~
