本文選題：組蛋白去乙�；敢种苿� + JZ　； 參考：《軍事醫(yī)學(xué)》2015年01期

【摘要】：目的利用化學(xué)發(fā)光方法和細(xì)胞篩選模型,檢測(cè)新型組蛋白去乙酰化酶抑制劑(histone deacetylase inhibitor,HDACi)JZ005的抑制組蛋白去乙�；�(histone deacetylases,HDACs)的活性;建立氯化鈷損傷的心肌細(xì)胞缺氧模型,初步探討JZ005對(duì)缺氧損傷細(xì)胞的保護(hù)作用。方法采用脂質(zhì)體轉(zhuǎn)染法將含有p21啟動(dòng)子元件的熒光素酶報(bào)告基因真核表達(dá)載體p CI-p21-Luc轉(zhuǎn)入到人胚腎細(xì)胞293中,用G418篩選獲得穩(wěn)定轉(zhuǎn)染熒光素酶報(bào)告基因的單克隆細(xì)胞系;采用已報(bào)道的HDACi曲古抑菌素A(trichostatina A,TSA)為陽性對(duì)照,檢測(cè)細(xì)胞篩選模型的穩(wěn)定性;用HDACi化學(xué)發(fā)光檢測(cè)試劑盒及上述細(xì)胞篩選模型測(cè)定JZ005抑制HDACs的活性;用不同濃度的JZ005處理氯化鈷缺氧損傷的大鼠胚胎心肌細(xì)胞(H9c2),MTT法檢測(cè)JZ005對(duì)缺氧損傷細(xì)胞的保護(hù)作用。免疫印跡法檢測(cè)JZ005處理后正常及缺氧損傷心肌細(xì)胞組蛋白H3的乙酰化水平變化。流式細(xì)胞術(shù)檢測(cè)JZ005對(duì)H9c2細(xì)胞缺氧損傷后凋亡的影響。結(jié)果建立含p21啟動(dòng)子元件熒光素酶報(bào)告基因的HDACi細(xì)胞篩選模型;JZ005能夠顯著抑制HDACs的活性,濃度50~400μmol/L,抑制率50%。對(duì)缺氧損傷的心肌細(xì)胞具有明顯保護(hù)作用,與對(duì)照組相比,細(xì)胞存活率提高38.33%、56.00%和35.20%,同時(shí)能夠上調(diào)缺氧損傷心肌細(xì)胞組蛋白H3的乙酰化水平,拮抗缺氧損傷心肌細(xì)胞的凋亡,細(xì)胞凋亡數(shù)目從對(duì)照組的12.89%分別下降到給藥組(25,50和100μmol/L)的6.63%、10.56%和8.89%。結(jié)論成功建立了HDACi的細(xì)胞篩選模型;JZ005作為一種新型的HDACi,具有明顯的保護(hù)心肌細(xì)胞拮抗缺氧損傷的作用,提示JZ005有可能開發(fā)成一種治療缺氧損傷的藥物。
[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.
【作者單位】： 北華大學(xué)化學(xué)與生物學(xué)院吉林省中藥生物技術(shù)科技創(chuàng)新中心;軍事醫(yī)學(xué)科學(xué)院野戰(zhàn)輸血研究所;神舟生物科技有限責(zé)任公司;
【基金】：國(guó)家傳染病重大專項(xiàng)資助項(xiàng)目(2012ZX10001003)
【分類號(hào)】：R969

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本文編號(hào)：1738379