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目的:构建组蛋白去乙酰化酶(histone deacetylase 1,HDAC1)过表达胶质瘤U87细胞系,探讨HDAC1过表达对胶质瘤细胞化疗药耐药性的影响。方法:分别用HDAC1重组过表达慢病毒载体p CDH-CMV-MCS-HDAC1-EF1-Puro和阴性空载体病毒p CDH-CMV-MCS-EF1-Puro转染U87MG细胞,通过嘌呤梯度浓度筛选出HDAC1稳定过表达细胞系U87MGHDAC1和空载体对照细胞系U87MG-Control,经Western Blotting鉴定,用不同浓度替尼泊苷(VM-26)和顺铂(cisplatin,DDP)对两种细胞进行处理,MTT法检测存活率,Hoechst/PI双染法检测细胞凋亡,Western Blotting检测Bcl-2、Bax及Caspase-3蛋白表达。结果:成功构建U87MG-HDAC1稳定过表达细胞系。与U87MG-Control组相比,U87MG-HDAC1组中U87MG-HDAC1蛋白的表达显著升高[(1.148±0.024)vs(0.580±0.003),P<0.01];药物处理后,U87MG-HDAC1细胞存活率显著升高[0.1μg/ml VM-26:(95.57±0.45)%vs(68.8±1.49)%,P<0.01],[0.08μg/ml DDP:(99.20±7.4)%vs(72.48±2.03)%,P<0.01];凋亡细胞个数比例明显下降(均P<0.05),Caspase-3蛋白表达量显著降低(P<0.01),Bcl-2/Bax蛋白表达量比值明显升高(P<0.01)。结论:胶质瘤U87MG细胞中HDAC1过表达明显增强细胞对化疗药耐药性与Caspase-3和Bcl-2/Bax表达有关。
OBJECTIVE: To construct a histone deacetylase 1 (HDAC1) overexpressing glioma U87 cell line to investigate the effect of HDAC1 overexpression on chemoresistance of glioma cells. Methods: U87MG cells were transfected with HDAC1 recombinant lentiviral vector pCDH-CMV-MCS-HDAC1-EF1-Puro and negative vector pCDH-CMV-MCS-EF1-Puro respectively. The HDAC1 Stably overexpressing cell line U87MGHDAC1 and empty vector control cell line U87MG-Control, identified by Western Blotting, were treated with different concentrations of teniposide (VM-26) and cisplatin (DDP) Survival rate was detected. Hoechst / PI double staining was used to detect apoptosis. Western Blotting was used to detect the protein expressions of Bcl-2, Bax and Caspase-3. Results: U87MG-HDAC1 stable over-expression cell line was successfully constructed. The expression of U87MG-HDAC1 protein in U87MG-HDAC1 group was significantly higher than that in U87MG-Control group [(1.148 ± 0.024) vs (0.580 ± 0.003), P <0.01] (P <0.01), [0.08μg / ml DDP: (99.20 ± 7.4)% vs (72.48 ± 2.03), P <0.01] %, P <0.01]. The percentage of apoptotic cells decreased significantly (all P <0.05), the expression of Caspase-3 protein decreased significantly (P <0.01), and the ratio of Bcl- <0.01). Conclusion: Overexpression of HDAC1 in glioma U87MG cells significantly enhances the chemoresistance of cells to chemotherapeutic drugs and the expression of Caspase-3 and Bcl-2 / Bax.