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LipofectmineTM 2000 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pSilencer3.1-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 of drug-resistant MG-63 cells, and thus increase its sensitivity to chemotherapy drugs.

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）