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1375~1380.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[J].孙廓,袁初平,李怀强,廖琦.中国肿瘤生物治疗杂志,2017,24(12)
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    点此下载全文    点此浏览HTML全文
孙廓  袁初平  李怀强  廖琦
1.南昌大学第二附属医院 骨科,江西 南昌 330006; 2.江西省都昌县中医院骨科,江西都昌332600,1.南昌大学第二附属医院 骨科,江西 南昌 330006; 2.江西省都昌县中医院骨科,江西都昌332600,1.南昌大学第二附属医院 骨科,江西 南昌 330006; 2.江西省都昌县中医院骨科,江西都昌332600,1.南昌大学第二附属医院 骨科,江西 南昌 330006; 2.江西省都昌县中医院骨科,江西都昌332600
基金项目: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)
DOI:10.3872/j.issn.1007-385X.2017.12.006
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。
关键词:骨肉瘤  Livin基因  RNA干扰  多柔比星  耐药
Livin gene silence by RNA interference enhances the chemotherapeutic sensitivity of drug resistant MG-63 osteosarcoma cells to doxorubicin    Download Fulltext
SUN Kuo  YUAN Chuping  LI Huaiqiang  LIAO Qi
1.Department of Orthopedics, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China; 2. Department of Orthopedics, Duchang County Hospital of Traditional Chinese Medicine, Duchang 332600, Jiangxi, China,1.Department of Orthopedics, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China; 2. Department of Orthopedics, Duchang County Hospital of Traditional Chinese Medicine, Duchang 332600, Jiangxi, China,1.Department of Orthopedics, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China; 2. Department of Orthopedics, Duchang County Hospital of Traditional Chinese Medicine, Duchang 332600, Jiangxi, China and 1.Department of Orthopedics, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China; 2. Department of Orthopedics, Duchang County Hospital of Traditional Chinese Medicine, Duchang 332600, Jiangxi, China
Fund Project:Project supported by the National Natural Science Foundation of China(No.81660443), and the Health and Family Planning Commision of Jiangxi Province(No. 20155278)
Abstract:
      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.
Keywords:osteosarcoma  Livin gene  RNA interference  doxorubicin  drug resistance
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