CAJ | 학술논문

目的观察应用组蛋白脱乙酰酶抑制剂丙戊酸钠调节染色体组蛋白低乙酰化水平对肝癌细胞增殖的作用,并进一步检测Cyclin A、Cyclin D1、Cychn E、P21 Waf/cip1蛋白及mRNA表达变化,探讨其分子作用机制.方法应用0.75～4.0 mmol/L丙戊酸钠作用于肝癌细胞系HepG2细胞后,MTT法检测细胞生长抑制、细胞克隆形成试验观察克隆形成率、碘化丙啶标记流式细胞术检测细胞周期、间接免疫荧光法分析Cyclin A、Cyclin D1、Cyclin E、P21Waf/cip1蛋白表达,RT-PCR检测分析Cyclin A、Cyclin D1、Cyclin E、P21Waf/cip1mRNA表达.结果 0.75～4.0 mmol/L丙戊酸钠作用24、48、72、96 h,观察组出现了时间-剂量依赖趋势的生长抑制,同时细胞克隆形成率显著降低,对照组细胞增殖周期G1、S、M期所占比例未见明显改变,而观察组则随药物浓度、作用时间的不同而出现不同程度的细胞增殖周期G1期阻滞.Gl期比例由55.4%～82.8%不等,差异有统计学意义(P＜0.01).观察组Cyclin A、Cyclin D1蛋白及mRNA表达均被明显下调而LP21Waf/cip1蛋白、mRNA表达则被明显上调,与对照组比较差异均有统计学意义(均P＜0.01);而CyclinE蛋白和mRNA表达变化则未见明显差异(P＞0.05).结论通过应用特异性组蛋白脱乙酰酶抑制剂调节组蛋白乙酰化修饰可明显抑制肝癌细胞生长、抑制细胞克隆形成、阻滞细胞增殖周期于G1期;丙戊酸钠作为组蛋白脱乙酰酶抑制剂可明显抑制肝癌细胞增殖,其作用机制是通过上调P21Waf/cip1 mRNA蛋白表达,下调Cyclin D1、Cyclin A mRNA蛋白表达分别和/或协同实现.
목적 관찰응용조단백탈을선매억제제병무산납조절염색체조단백저을선화수평대간암세포증식적작용,병진일보검측Cyclin A、Cyclin D1、Cychn E、P21 Waf/cip1단백급mRNA표체변화,탐토기분자작용궤제.방법 응용0.75～4.0 mmol/L병무산납작용우간암세포계HepG2세포후,MTT법검측세포생장억제、세포극륭형성시험관찰극륭형성솔、전화병정표기류식세포술검측세포주기、간접면역형광법분석Cyclin A、Cyclin D1、Cyclin E、P21Waf/cip1단백표체,RT-PCR검측분석Cyclin A、Cyclin D1、Cyclin E、P21Waf/cip1mRNA표체.결과 0.75～4.0 mmol/L병무산납작용24、48、72、96 h,관찰조출현료시간-제량의뢰추세적생장억제,동시세포극륭형성솔현저강저,대조조세포증식주기G1、S、M기소점비례미견명현개변,이관찰조칙수약물농도、작용시간적불동이출현불동정도적세포증식주기G1기조체.Gl기비례유55.4%～82.8%불등,차이유통계학의의(P＜0.01).관찰조Cyclin A、Cyclin D1단백급mRNA표체균피명현하조이LP21Waf/cip1단백、mRNA표체칙피명현상조,여대조조비교차이균유통계학의의(균P＜0.01);이CyclinE단백화mRNA표체변화칙미견명현차이(P＞0.05).결론 통과응용특이성조단백탈을선매억제제조절조단백을선화수식가명현억제간암세포생장、억제세포극륭형성、조체세포증식주기우G1기;병무산납작위조단백탈을선매억제제가명현억제간암세포증식,기작용궤제시통과상조P21Waf/cip1 mRNA단백표체,하조Cyclin D1、Cyclin A mRNA단백표체분별화/혹협동실현.
Objective To investigate the suppressing effect on hepatocellular carcinoma cell proliferation by up-regulating histone acetylizad level with a selective inhibitar of HDACs-Valproate acid sodium(VPA).And the mechanism which involved was clarified by detecting the protein and mRNA expressions of Cyclin A、Cyclin D1、Cyclin E、P21Walf/cipl.Methods HepG2 cells wero cultured with 0.75-4.0 mmol/L valproic acid(VPA)for 24,48,72,96hrs in vitro.The inhibition rate was studied by MTT assay:clone forming inhibit rate was tested by clonay assay;cell cycle was analyzed by flow cytometry with PI assay,and the protein and mRNA expressions of Cyclin A、Cydin D1、Cychn E、P21Waf/cip1 of HepG2 cells after 1.5.3.0 mmol/L VPA treated wero analyzed by indirect immunofluorescence technique and RT-PCR respectively.Results The inhibition rate of experimental group increased significantly(P＜0.001)and a dose and acting time dependence was found.Clones in experimental groups decreased more than in control group.As for cell cycle,the percentage of G1,S,M phrase in control group remained the same.Compared with control group,0.75 and 4.0 mmol/L VPA induced a significant arrest in G1 phrase(P＜0.001)and a total of 55.4%～82.8% G1 phrase of cells which cultured with VPA.Cychn A.Cyclin D1 were down-regulated both at mRNA and protein level(P＜0.001)while P21Waf/cip1 was up-regulated both at mRNA and protein level.Conversely,neither mRNA nor protein expression of Cyclin E showed any difierence between experimental and control group(P＞0.05).Conclusion Up-regulating histone acetylizad level can inhibit hepatocellular carcinoma cell proliferation,inhibit cell clone formation,induce cell cycle arrest in G1 phrase.VPA.as a Ⅰ class of histone deacetylase inhibitor can be used as an option in the treatment of hepetoma.The mechanism includes upregulating P21Waf/cip1 mRNA and protein expression,down-regulating Cyclin A,Cyclin D1 mRNA and protein expression.