新型组蛋白去乙酰化酶抑制剂对缺氧损伤心肌细胞的保护作用研究

发布时间：2018-04-12 00:29

本文选题：组蛋白去乙酰化酶抑制剂 + JZ　；参考：《军事医学》2015年01期

【摘要】：目的利用化学发光方法和细胞筛选模型,检测新型组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor,HDACi)JZ005的抑制组蛋白去乙酰化酶(histone deacetylases,HDACs)的活性;建立氯化钴损伤的心肌细胞缺氧模型,初步探讨JZ005对缺氧损伤细胞的保护作用。方法采用脂质体转染法将含有p21启动子元件的荧光素酶报告基因真核表达载体p CI-p21-Luc转入到人胚肾细胞293中,用G418筛选获得稳定转染荧光素酶报告基因的单克隆细胞系;采用已报道的HDACi曲古抑菌素A(trichostatina A,TSA)为阳性对照,检测细胞筛选模型的稳定性;用HDACi化学发光检测试剂盒及上述细胞筛选模型测定JZ005抑制HDACs的活性;用不同浓度的JZ005处理氯化钴缺氧损伤的大鼠胚胎心肌细胞(H9c2),MTT法检测JZ005对缺氧损伤细胞的保护作用。免疫印迹法检测JZ005处理后正常及缺氧损伤心肌细胞组蛋白H3的乙酰化水平变化。流式细胞术检测JZ005对H9c2细胞缺氧损伤后凋亡的影响。结果建立含p21启动子元件荧光素酶报告基因的HDACi细胞筛选模型;JZ005能够显著抑制HDACs的活性,浓度50~400μmol/L,抑制率50%。对缺氧损伤的心肌细胞具有明显保护作用,与对照组相比,细胞存活率提高38.33%、56.00%和35.20%,同时能够上调缺氧损伤心肌细胞组蛋白H3的乙酰化水平,拮抗缺氧损伤心肌细胞的凋亡,细胞凋亡数目从对照组的12.89%分别下降到给药组(25,50和100μmol/L)的6.63%、10.56%和8.89%。结论成功建立了HDACi的细胞筛选模型;JZ005作为一种新型的HDACi,具有明显的保护心肌细胞拮抗缺氧损伤的作用,提示JZ005有可能开发成一种治疗缺氧损伤的药物。
[Abstract]:Objective to detect the activity of histone deacetylase (histone deacetylase), a new inhibitor of histone deacetylase, HDA Ciorus JZ005, by chemiluminescence assay and cell screening model, and to establish an hypoxic model of cardiomyocytes injured by cobalt chloride, and to detect the activity of histone deacetylasesserine HDACs1, a new inhibitor of histone deacetylase (HDA), a new inhibitor of histone deacetylase.To investigate the protective effect of JZ005 on hypoxic injury cells.Methods the eukaryotic expression vector p CI-p21-Luc of luciferase reporter gene containing p21 promoter element was transfected into human embryonic kidney cell line 293 by liposome transfection, and a monoclonal cell line stably transfected with luciferase reporter gene was obtained by G418 screening.The stability of the cell screening model was detected by using the reported HDACi trigostatin A(trichostatina A(trichostatina as positive control, and the activity of JZ005 inhibiting HDACs was determined by HDACi chemiluminescence assay kit and the above cell screening model.The protective effect of JZ005 on hypoxic injury of rat embryonic cardiomyocytes was detected by MTT assay with different concentrations of JZ005.The acetylation level of histone H 3 in normal and hypoxic myocardial cells after JZ005 treatment was detected by Western blot.The effect of JZ005 on apoptosis of H9c2 cells after hypoxia injury was detected by flow cytometry.Results the screening model of HDACi cells containing luciferase reporter gene of p21 promoter element was established. JZ005 could significantly inhibit the activity of HDACs at a concentration of 50 渭 mol / L and inhibition rate of 50 渭 mol / L.Compared with the control group, the cell survival rate was increased by 56.00% and 35.20%, and the acetylation level of histone H3 was up-regulated and the apoptosis of hypoxic myocardial cells was antagonized.The number of apoptotic cells decreased from 12.89% in the control group to 6.63% and 8.89% in the drug administration group (25 渭 mol / L and 100 渭 mol / L), respectively.Conclusion as a new type of HDA Cii, JZ005 has been successfully established as a cell screening model of HDACi, which can protect cardiomyocytes from hypoxic injury, suggesting that JZ005 may be developed as a drug for the treatment of hypoxic injury.
【作者单位】：北华大学化学与生物学院吉林省中药生物技术科技创新中心;军事医学科学院野战输血研究所;神舟生物科技有限责任公司;
【基金】：国家传染病重大专项资助项目(2012ZX10001003)
【分类号】：R969

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