CAJ | 학술논문

目的通过监测急性肝功能衰竭小鼠体内miR-122的表达,探讨miR-122与小鼠急性肝衰竭疾病程度和进展之间的关系,为肝功能衰竭的早期诊断提供新的生物学标志物. 方法将BALB/C小鼠随机分为4组,实验组用D-氨基半乳糖(D-GalN,900 mg/kg)联合脂多糖(LPS,10 μg/kg)腹腔注射建立肝衰竭模型,对照组3组,分别予以D-GalN(900 mg/kg),LPS(10 μg/kg)和等渗盐水腹腔注射,在不同时间点观察小鼠病死率、肝脏组织学变化,给药后0、1、3、5、7、9 h分别留取血清、肝脏组织标本,实时定量逆转录多聚酶链反应检测小鼠体内miRNA-122和炎症因子的表达,LNA(锁核酸)-Northern blot验证miRNA-122的表达,生化分析仪检测血清中ALT、AST水平,酶联免疫吸附法检测血清中炎症因子水平.组间均数比较用two-WayANOVA方差分析,相关性分析采用Pearson和Spearman相关分析.结果 D-GalN/LPS给药24 h,小鼠病死率率达80%以上,而3个对照组则无一只小鼠死亡;肝脏特异性miR-122在正常小鼠肝脏内含量丰富(ct≈14),D-GalN/LPS诱导后1 h,miR-122即发生了明显的变化(P=0.013),表现为上调,之后随疾病的进展,miR-122表达进行性下降,9 h下调最为明显(ct≈15,P=0.002);ALT/AST于给药1 h无明显变化,3 h后呈明显上升趋势,7 h达高峰,之后ALT/AST急剧下降;对miR-122和ALT的表达对比,发现在该模型中miR-122比ALT变化快,且持久;肝衰竭相关炎症因子肿瘤坏死因子(TNF)α和白细胞介素(IL)-6在肝组织和血清中的变化一致,均上调(P<0.05); miR-122和ALT、TNFα和IL-6的相关性分析显示miR-122与以上三项指标均呈良好的相关性(相关系数分别为-0.505、0.493和0.674、).结论肝衰竭小鼠体内肝脏特异性miR-122和ALT呈负相关关系,但又较ALT更敏感,更持久地反映肝细胞损伤程度,且miR-122表达变化与肝脏炎症损伤相关因素TNF α、IL-6均具良好的相关性,推测miR-122有望成为判断急性肝衰竭肝细胞损伤程度的一个新的分子生物学标志物. 目的通過鑑測急性肝功能衰竭小鼠體內miR-122的錶達,探討miR-122與小鼠急性肝衰竭疾病程度和進展之間的關繫,為肝功能衰竭的早期診斷提供新的生物學標誌物. 方法將BALB/C小鼠隨機分為4組,實驗組用D-氨基半乳糖(D-GalN,900 mg/kg)聯閤脂多糖(LPS,10 μg/kg)腹腔註射建立肝衰竭模型,對照組3組,分彆予以D-GalN(900 mg/kg),LPS(10 μg/kg)和等滲鹽水腹腔註射,在不同時間點觀察小鼠病死率、肝髒組織學變化,給藥後0、1、3、5、7、9 h分彆留取血清、肝髒組織標本,實時定量逆轉錄多聚酶鏈反應檢測小鼠體內miRNA-122和炎癥因子的錶達,LNA(鎖覈痠)-Northern blot驗證miRNA-122的錶達,生化分析儀檢測血清中ALT、AST水平,酶聯免疫吸附法檢測血清中炎癥因子水平.組間均數比較用two-WayANOVA方差分析,相關性分析採用Pearson和Spearman相關分析.結果 D-GalN/LPS給藥24 h,小鼠病死率率達80%以上,而3箇對照組則無一隻小鼠死亡;肝髒特異性miR-122在正常小鼠肝髒內含量豐富(ct≈14),D-GalN/LPS誘導後1 h,miR-122即髮生瞭明顯的變化(P=0.013),錶現為上調,之後隨疾病的進展,miR-122錶達進行性下降,9 h下調最為明顯(ct≈15,P=0.002);ALT/AST于給藥1 h無明顯變化,3 h後呈明顯上升趨勢,7 h達高峰,之後ALT/AST急劇下降;對miR-122和ALT的錶達對比,髮現在該模型中miR-122比ALT變化快,且持久;肝衰竭相關炎癥因子腫瘤壞死因子(TNF)α和白細胞介素(IL)-6在肝組織和血清中的變化一緻,均上調(P<0.05); miR-122和ALT、TNFα和IL-6的相關性分析顯示miR-122與以上三項指標均呈良好的相關性(相關繫數分彆為-0.505、0.493和0.674、).結論肝衰竭小鼠體內肝髒特異性miR-122和ALT呈負相關關繫,但又較ALT更敏感,更持久地反映肝細胞損傷程度,且miR-122錶達變化與肝髒炎癥損傷相關因素TNF α、IL-6均具良好的相關性,推測miR-122有望成為判斷急性肝衰竭肝細胞損傷程度的一箇新的分子生物學標誌物.
목적 통과감측급성간공능쇠갈소서체내miR-122적표체,탐토miR-122여소서급성간쇠갈질병정도화진전지간적관계,위간공능쇠갈적조기진단제공신적생물학표지물. 방법장BALB/C소서수궤분위4조,실험조용D-안기반유당(D-GalN,900 mg/kg)연합지다당(LPS,10 μg/kg)복강주사건립간쇠갈모형,대조조3조,분별여이D-GalN(900 mg/kg),LPS(10 μg/kg)화등삼염수복강주사,재불동시간점관찰소서병사솔、간장조직학변화,급약후0、1、3、5、7、9 h분별류취혈청、간장조직표본,실시정량역전록다취매련반응검측소서체내miRNA-122화염증인자적표체,LNA(쇄핵산)-Northern blot험증miRNA-122적표체,생화분석의검측혈청중ALT、AST수평,매련면역흡부법검측혈청중염증인자수평.조간균수비교용two-WayANOVA방차분석,상관성분석채용Pearson화Spearman상관분석.결과 D-GalN/LPS급약24 h,소서병사솔솔체80%이상,이3개대조조칙무일지소서사망;간장특이성miR-122재정상소서간장내함량봉부(ct≈14),D-GalN/LPS유도후1 h,miR-122즉발생료명현적변화(P=0.013),표현위상조,지후수질병적진전,miR-122표체진행성하강,9 h하조최위명현(ct≈15,P=0.002);ALT/AST우급약1 h무명현변화,3 h후정명현상승추세,7 h체고봉,지후ALT/AST급극하강;대miR-122화ALT적표체대비,발현재해모형중miR-122비ALT변화쾌,차지구;간쇠갈상관염증인자종류배사인자(TNF)α화백세포개소(IL)-6재간조직화혈청중적변화일치,균상조(P<0.05); miR-122화ALT、TNFα화IL-6적상관성분석현시miR-122여이상삼항지표균정량호적상관성(상관계수분별위-0.505、0.493화0.674、).결론 간쇠갈소서체내간장특이성miR-122화ALT정부상관관계,단우교ALT경민감,경지구지반영간세포손상정도,차miR-122표체변화여간장염증손상상관인소TNF α、IL-6균구량호적상관성,추측miR-122유망성위판단급성간쇠갈간세포손상정도적일개신적분자생물학표지물.
Objective To investigate the expression of miR-122 and its relationship with progression and development of acute liver failure in mice induced by D-GalN/LPS,and to explore new biomarker(s)for early diagnosis of acute liver failure.Methods BALB/C mice were randomly divided into four groups:the mice were given D-GalN(900 mg/kg body weight)and LPS(10 μg/kg body weight)intraperitoneally(i.P.)to construct the acute liver model;whereas the control groups were given D-GalN(900 mg/kg),LPS (10 μg/kg)and normal saline respectively.All biochemical and histological indexes were determined at 0,1,3,5,7 and 9h respectively after administration.Real-time RT-PCR were used to detect the expression of miR-122 and pro-inflammatory cytokines,furthermore,the expression of miR-122 was verified by LNA (lock nucleic acid)-Northern-blot.ALT and AST levels were tested by biochemistry analyzer.Serum proinflammatory cytokine levels were tested by ELISA.Results The mortality rate was about 80% at 24h after D-GalN/LPS treatment,but no mortality was observed in the other three control groups.Liver special miRNA miR-122 was highly expressed in liver tissue of normal mice(ct≈14),it was up-regulated significantly(P=0.013)at first hour after treatment then down-regulated according to the development of acute liver failure,the change was more obvious at 9h(ct≈15,P=0.002).ALT and AST levels increased obviously at 3h after treatment and reached peak at 7 hours then they were declined sharply.It was found that the expression of miR-122 was faster and more durable than ALT.Pro-inflammatory cytokines related to acute liver failure including TNFα and IL-6 were all up-regulated in serum as well as liver tissue(P<0.05).Correlation analysis showed that miR-122 had anegative correlation with ALT (correlation coefficients-0.505)and positive correlations with TNF α and IL-6(correlation coefficients were 0.493 and 0.674 respectively).Conclusions Liver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.