军事医学
軍事醫學
군사의학
BULLETIN OF THE ACADEMY OF MILITARY MEDICAL SCIENCES
2015年
1期
30-35,70
,共7页
王艳冰%任素萍%王庆军%乔志新%王春燕%郐启源%王钰%王璇琳%贺敏%李伟静%孙立伟%于群
王豔冰%任素萍%王慶軍%喬誌新%王春燕%鄶啟源%王鈺%王璇琳%賀敏%李偉靜%孫立偉%于群
왕염빙%임소평%왕경군%교지신%왕춘연%회계원%왕옥%왕선림%하민%리위정%손립위%우군
组蛋白去乙酰化酶抑制剂%JZ005%缺氧损伤%肌细胞,心脏%细胞保护%化学发光测定法
組蛋白去乙酰化酶抑製劑%JZ005%缺氧損傷%肌細胞,心髒%細胞保護%化學髮光測定法
조단백거을선화매억제제%JZ005%결양손상%기세포,심장%세포보호%화학발광측정법
histone deacetylase inhibitor%JZ005%hypoxia%myocytes,cardiac%cytoprotection%chemiluminescent measurements
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pCI-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。
目的：利用化學髮光方法和細胞篩選模型，檢測新型組蛋白去乙酰化酶抑製劑（ histone deacetylase inhibitor， HDACi）JZ005的抑製組蛋白去乙酰化酶（histone deacetylases，HDACs）的活性；建立氯化鈷損傷的心肌細胞缺氧模型，初步探討JZ005對缺氧損傷細胞的保護作用。方法採用脂質體轉染法將含有p21啟動子元件的熒光素酶報告基因真覈錶達載體pCI-p21-Luc轉入到人胚腎細胞293中，用G418篩選穫得穩定轉染熒光素酶報告基因的單剋隆細胞繫；採用已報道的HDACi麯古抑菌素A（ trichostatina A，TSA）為暘性對照，檢測細胞篩選模型的穩定性；用HDACi化學髮光檢測試劑盒及上述細胞篩選模型測定JZ005抑製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胞（H9c2），MTT法檢測JZ005對缺氧損傷細胞的保護作用。免疫印跡法檢測JZ005處理後正常及缺氧損傷心肌細胞組蛋白H3的乙酰化水平變化。流式細胞術檢測JZ005對H9c2細胞缺氧損傷後凋亡的影響。結果建立含p21啟動子元件熒光素酶報告基因的HDACi細胞篩選模型；JZ005能夠顯著抑製HDACs的活性，濃度50～400μmol／L，抑製率＞50％。對缺氧損傷的心肌細胞具有明顯保護作用，與對照組相比，細胞存活率提高38．33％、56．00％和35．20％，同時能夠上調缺氧損傷心肌細胞組蛋白H3的乙酰化水平，拮抗缺氧損傷心肌細胞的凋亡，細胞凋亡數目從對照組的12．89％分彆下降到給藥組（25，50和100μmol／L）的6．63％、10．56％和8．89％。結論成功建立瞭HDACi的細胞篩選模型；JZ005作為一種新型的HDACi ，具有明顯的保護心肌細胞拮抗缺氧損傷的作用，提示JZ005有可能開髮成一種治療缺氧損傷的藥物。
목적：이용화학발광방법화세포사선모형，검측신형조단백거을선화매억제제（ histone deacetylase inhibitor， HDACi）JZ005적억제조단백거을선화매（histone deacetylases，HDACs）적활성；건립록화고손상적심기세포결양모형，초보탐토JZ005대결양손상세포적보호작용。방법채용지질체전염법장함유p21계동자원건적형광소매보고기인진핵표체재체pCI-p21-Luc전입도인배신세포293중，용G418사선획득은정전염형광소매보고기인적단극륭세포계；채용이보도적HDACi곡고억균소A（ trichostatina A，TSA）위양성대조，검측세포사선모형적은정성；용HDACi화학발광검측시제합급상술세포사선모형측정JZ005억제HDACs적활성；용불동농도적JZ005처리록화고결양손상적대서배태심기세포（H9c2），MTT법검측JZ005대결양손상세포적보호작용。면역인적법검측JZ005처리후정상급결양손상심기세포조단백H3적을선화수평변화。류식세포술검측JZ005대H9c2세포결양손상후조망적영향。결과건립함p21계동자원건형광소매보고기인적HDACi세포사선모형；JZ005능구현저억제HDACs적활성，농도50～400μmol／L，억제솔＞50％。대결양손상적심기세포구유명현보호작용，여대조조상비，세포존활솔제고38．33％、56．00％화35．20％，동시능구상조결양손상심기세포조단백H3적을선화수평，길항결양손상심기세포적조망，세포조망수목종대조조적12．89％분별하강도급약조（25，50화100μmol／L）적6．63％、10．56％화8．89％。결론성공건립료HDACi적세포사선모형；JZ005작위일충신형적HDACi ，구유명현적보호심기세포길항결양손상적작용，제시JZ005유가능개발성일충치료결양손상적약물。
Objective To verify enzyme activity inhibition of a novel histone deacetylase inhibitor ( HDACi ) JZ005 using an HDACi chemiluminescence detection kit and a cell-based screening model .Methods The plasmid with p21 gene promoter elements and luciferase reporter gene was transfected into human embryonic kidney cells 293 , and the stable transfectants were established by G418 screening.Enzyme activity inhibition of JZ005 on histone deacetylases (HDACs) was verified by the HDACi chemiluminescence detection kit and the cell-based screening model .A well-known HDACi , tri-chostatin A ( TSA) was used as the positive control .MTT assay was used to detect the protection of rat H 9c2 myocardial cells suffering from CoCl 2-induced hypoxia and treated with different concentrations of JZ 005 .The expression of acetylated histone H3 protein of normal and CoCl 2-induced hypoxia H9c2 cells before and after JZ005 treatment was assayed by West-ern blotting while the effect of drug administration on apoptosis was detected by flow cytometry ( FCM) .Results An HDA-Ci cell-based screening system targeting the p21 gene promoter was ranging established .The JZ005, a HDACi, markedly suppressed the activity of HDACs by more than 50%with the concentration ranging from 50 to 400 μmol/L.JZ005 signifi-cantly protected H9c2 cells from hypoxia injury .Cell viability was increased by 38.33%,56.00% and 35.20% compared with control,accompanied by an enhanced acetylation level of histone H 3.JZ005(25,50 and 100 μmol/L) treatment sig-nificantly decreased the number of apoptotic cells (6.63%,10.56% and 8.89%) compared to control group (12.89%). Conclusion An HDACi cell-based screening system is successfully established .JZ005 effectively protects myocardial cells against hypoxia injury while enhancing the acetylation level of histone H 3.Our results indicate that JZ005 might be developed as a potential drug for hypoxia treatment .
