CAJ | 학술논문

目的探讨miR-21调控肝卵圆细胞活化和增殖过程的可能机制.方法采用2-乙酰氨基芴(2-AAF)/部分肝切除法诱导SD大鼠肝卵圆细胞活化模型,分别于0、6、12、24、72、168 h处死动物,同时以单纯肝切除的相应时间点作为对照,分别提取各标本RNA逆转录后行SYBR Green实时荧光定量PCR,检测各组miR-21的表达,行两样本t检验,并用生物信息学方法分析其调控的miRNA转录分子和靶基因.实时荧光定量PCR检测其靶基因表达,Westem blot法检测其靶基因蛋白表达,进行两样本均值的t检验,以P＜ 0.05为差异有统计学意义. 结果成功建立大鼠肝卵圆细胞活化模型,检测miR-21的扩增信号,miR-21在实验组表达12h开始升高,24 h达到峰值,随后开始降低,168 h再次升高;而对照组6h开始升高,24h后开始下降后基本恢复原水平.实验组与对照组表达比较,实验组在6 h miR-21的表达低于对照组,t=3.029,P=0.039,差异有统计学意义;而在24 h和168 h的表达高于对照组,t值分别为-3.433、-5.105,P值均＜0.05,差异有统计学意义.根据三个数据库的综合评分,结合相关文献,我们选取Smad7为研究的靶基因.检测两组Smad7mRNA表达,对照组中在6h略有下降,后开始升高,在24 h达到峰值后开始下降恢复原水平;在实验组中,6h升高后至24 h达到峰值,随后开始降低,168 h又略有升高.对照组Smad7蛋白表达从6h开始降低,到24 h达到最低后开始升高;实验组6h升高,随后下降,168 h达到最低.Smad7 mRNA表达量变化趋势与miR-21表达变化趋势基本一致,Smad7蛋白表达和miR-21表达变化趋势呈负相关. 结论成功建立SYBR Green实时荧光定量PCR检测大鼠miR-21的方法,证实miR-21在肝卵圆细胞活化与增殖中起重要作用,Smad7作为miR-21的靶基因可能参与这一过程.
목적 탐토miR-21조공간란원세포활화화증식과정적가능궤제.방법 채용2-을선안기물(2-AAF)/부분간절제법유도SD대서간란원세포활화모형,분별우0、6、12、24、72、168 h처사동물,동시이단순간절제적상응시간점작위대조,분별제취각표본RNA역전록후행SYBR Green실시형광정량PCR,검측각조miR-21적표체,행량양본t검험,병용생물신식학방법분석기조공적miRNA전록분자화파기인.실시형광정량PCR검측기파기인표체,Westem blot법검측기파기인단백표체,진행량양본균치적t검험,이P＜ 0.05위차이유통계학의의. 결과 성공건립대서간란원세포활화모형,검측miR-21적확증신호,miR-21재실험조표체12h개시승고,24 h체도봉치,수후개시강저,168 h재차승고;이대조조6h개시승고,24h후개시하강후기본회복원수평.실험조여대조조표체비교,실험조재6 h miR-21적표체저우대조조,t=3.029,P=0.039,차이유통계학의의;이재24 h화168 h적표체고우대조조,t치분별위-3.433、-5.105,P치균＜0.05,차이유통계학의의.근거삼개수거고적종합평분,결합상관문헌,아문선취Smad7위연구적파기인.검측량조Smad7mRNA표체,대조조중재6h략유하강,후개시승고,재24 h체도봉치후개시하강회복원수평;재실험조중,6h승고후지24 h체도봉치,수후개시강저,168 h우략유승고.대조조Smad7단백표체종6h개시강저,도24 h체도최저후개시승고;실험조6h승고,수후하강,168 h체도최저.Smad7 mRNA표체량변화추세여miR-21표체변화추세기본일치,Smad7단백표체화miR-21표체변화추세정부상관. 결론 성공건립SYBR Green실시형광정량PCR검측대서miR-21적방법,증실miR-21재간란원세포활화여증식중기중요작용,Smad7작위miR-21적파기인가능삼여저일과정.
Objective To investigate the possible mechanisms of miR-21-mediated regulation of proliferation and activation of hepatic oval cells.Methods The 2-acetamidofluorene/partial hepatectomy (2-AAF/PH) method was applied to generate hepatic oval cell activation model in male Sprague-Dawley rats; after the 7 days of 2-AAF/PH or PH alone (control),the rats were sacrificed at 0 h,6 h,12 h,24 h,72 h and 168 h.Expression of miR-21 was detected by real-time PCR and differences between groups were evaluated using the two-sample t-test.Differential transcription of miR-21 target genes was assessed bioinformatically,and with western blotting to detect changes in protein expression of the target gene.Results The rat hepatic oval cell activation model was successfully established.The 2-AAF/PH rats showed miR-21 expression beginning to increase at 12 h,peaking at 24 h,and decreasing thereafter until an increase at 168 h.For the control group,the miR-21 expression began to increase at 6 h,until 24 h when expression began steadily declining to reach the original level.Compared to the control group,the experimental group showed expression of miR-21 that was significantly less at 6 h (P =0.039,t =3.029) and significantly more at 24 h and 168 h (P =0.026,t =-3.433 andP =0.007,t =-5.105).Among the predicted target genes of miR-21 were WW domain containing E3 ubiquitin protein ligase 1 (WWD),Smad family member 7 (Smad7),and polybromo-1 (Pbrm1).Smad7 protein expression began to decrease at 6 h in the control group,until reaching its minimum at 24 h when it increased; in the experimental group,SMAD7 expression increased at 6 h,then began to decrease with the minimum detected at 168 hour.In the control group,the Smad7 mRNA expression decreased slightly at 6 h,then began to increase,reaching its peak at 24 h when the expression fell to the original level.In the experimental group,the Smad7 mRNA expression began to increase at 6 h and reached its peak at 24 h when it decreased; the expression was little more than its original level at 168 h.Smad7 protein expression was negatively correlated with miR-21,and Smad7 mRNA expression was positively correlated with miR-21 but negatively correlated with Smad7 protein expression.Conclusion miR-21 may play a vital role in the activation and proliferation of hepatic oval cells.As a target gene of miR-21,Smad7 might be involved in the process.