中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2012年
9期
671-676
,共6页
申春燕%陈永平%阳韬%陆小蒟%李春艳%林镯%宋梅
申春燕%陳永平%暘韜%陸小蒟%李春豔%林鐲%宋梅
신춘연%진영평%양도%륙소구%리춘염%림탁%송매
肝硬化%骨形成蛋白-7%转化生子因子β1%Arkadia%模型,动物
肝硬化%骨形成蛋白-7%轉化生子因子β1%Arkadia%模型,動物
간경화%골형성단백-7%전화생자인자β1%Arkadia%모형,동물
Liver cirrhosis%Bone morphogenetic protein-7%Transforming growth factor β1%Arkadia%Models,animal
目的 探索E3泛素连接酶Arkadia在CCl4所致肝纤维化小鼠肝组织中的动态表达,以及骨形成蛋白7 (BMP-7)对其的阻断作用. 方法 30只健康雄性ICR小鼠,随机分为正常对照组(6只)、模型组(18只)和BMP-7干预组(6只);模型组再按不同的时间点分为4周、8周和12周3个亚组.在小鼠双后肢皮下交替注射60％ CCl4/花生油溶液(5ml/kg),每周2次,共持续12周,制备肝纤维化模型；BMP-7干预组在皮下注射CCl4的同时,从第9周开始腹腔注射BMP-7 (300Pg/g)溶液,隔天1次,共4周.苏木素-伊红(HE)和Masson染色观察肝组织病理变化;RT-PCR、免疫组织化学和Western blot法检测肝组织中Arkadia的mRNA和蛋白质表达量.样本均数比较用单因素方差分析和(或)配对t检验,方差齐时采用LSD -t检验;相关性分析用Pearson直线相关分析法. 结果 成功建立了肝纤维化小鼠模型.模型组Arkadia、Smad7和TGFβ 1的mRNA表达量在纤维化过程中逐渐升高,12周时达高峰(相对表达量分别为1.3434±0.0149、1.2200±0.0093和1.1258±0.0065),BMP-7干预组表达量降低(相对表达量分别为0.9867±0.0169、0.9517±0.0120和0.9029±0.0085),与对照组(相对表达量分别为0.5398±0.0025、0.7467±0.0072和0.6318±0.0041)相比,差异有统计学意义(F值分别为812.801、451.462和998.957,P值均＜0.01);BMP-7干预组Arkadia、Smad7和TGF β1的mRNA表达明显低于12周模型组(t值分别为12.108、18.737和16.364,P值均＜0.01).正常肝组织中Arkadia、Smad7、TGF β1蛋白仅在汇管区少量表达(相对表达量分别为2.1702±0.0518、2.0798±0.0547和2.4515±0.0487),模型组中其表达量逐渐增加(12周的相对表达量分别为3.4198±0.0279、5.4480±0.0565和5.2619±0.0530),主要在汇管区和肝细胞胞质内表达,BMP-7干预后表达量降低(分别为3.1457±0.0424、3.5616±0.0491和3.5282±0.0195),与对照组相比,差异有统计学意义(F值分别为8.399、609.690和900.561,P值均＜0.01);与12周模型组相比,BMP-7干预组Arkadia、Smad7、TGF β1的蛋白质表达量明显降低(t值分别为23.438、11.667和42.889,P值均＜0.01).结论 Arkadia在肝纤维化进展过程中呈上升趋势,BMP-7具有抗肝纤维化作用并可以抑制纤维化过程中Arkadia的表达.
目的 探索E3汎素連接酶Arkadia在CCl4所緻肝纖維化小鼠肝組織中的動態錶達,以及骨形成蛋白7 (BMP-7)對其的阻斷作用. 方法 30隻健康雄性ICR小鼠,隨機分為正常對照組(6隻)、模型組(18隻)和BMP-7榦預組(6隻);模型組再按不同的時間點分為4週、8週和12週3箇亞組.在小鼠雙後肢皮下交替註射60％ CCl4/花生油溶液(5ml/kg),每週2次,共持續12週,製備肝纖維化模型；BMP-7榦預組在皮下註射CCl4的同時,從第9週開始腹腔註射BMP-7 (300Pg/g)溶液,隔天1次,共4週.囌木素-伊紅(HE)和Masson染色觀察肝組織病理變化;RT-PCR、免疫組織化學和Western blot法檢測肝組織中Arkadia的mRNA和蛋白質錶達量.樣本均數比較用單因素方差分析和(或)配對t檢驗,方差齊時採用LSD -t檢驗;相關性分析用Pearson直線相關分析法. 結果 成功建立瞭肝纖維化小鼠模型.模型組Arkadia、Smad7和TGFβ 1的mRNA錶達量在纖維化過程中逐漸升高,12週時達高峰(相對錶達量分彆為1.3434±0.0149、1.2200±0.0093和1.1258±0.0065),BMP-7榦預組錶達量降低(相對錶達量分彆為0.9867±0.0169、0.9517±0.0120和0.9029±0.0085),與對照組(相對錶達量分彆為0.5398±0.0025、0.7467±0.0072和0.6318±0.0041)相比,差異有統計學意義(F值分彆為812.801、451.462和998.957,P值均＜0.01);BMP-7榦預組Arkadia、Smad7和TGF β1的mRNA錶達明顯低于12週模型組(t值分彆為12.108、18.737和16.364,P值均＜0.01).正常肝組織中Arkadia、Smad7、TGF β1蛋白僅在彙管區少量錶達(相對錶達量分彆為2.1702±0.0518、2.0798±0.0547和2.4515±0.0487),模型組中其錶達量逐漸增加(12週的相對錶達量分彆為3.4198±0.0279、5.4480±0.0565和5.2619±0.0530),主要在彙管區和肝細胞胞質內錶達,BMP-7榦預後錶達量降低(分彆為3.1457±0.0424、3.5616±0.0491和3.5282±0.0195),與對照組相比,差異有統計學意義(F值分彆為8.399、609.690和900.561,P值均＜0.01);與12週模型組相比,BMP-7榦預組Arkadia、Smad7、TGF β1的蛋白質錶達量明顯降低(t值分彆為23.438、11.667和42.889,P值均＜0.01).結論 Arkadia在肝纖維化進展過程中呈上升趨勢,BMP-7具有抗肝纖維化作用併可以抑製纖維化過程中Arkadia的錶達.
목적 탐색E3범소련접매Arkadia재CCl4소치간섬유화소서간조직중적동태표체,이급골형성단백7 (BMP-7)대기적조단작용. 방법 30지건강웅성ICR소서,수궤분위정상대조조(6지)、모형조(18지)화BMP-7간예조(6지);모형조재안불동적시간점분위4주、8주화12주3개아조.재소서쌍후지피하교체주사60％ CCl4/화생유용액(5ml/kg),매주2차,공지속12주,제비간섬유화모형；BMP-7간예조재피하주사CCl4적동시,종제9주개시복강주사BMP-7 (300Pg/g)용액,격천1차,공4주.소목소-이홍(HE)화Masson염색관찰간조직병리변화;RT-PCR、면역조직화학화Western blot법검측간조직중Arkadia적mRNA화단백질표체량.양본균수비교용단인소방차분석화(혹)배대t검험,방차제시채용LSD -t검험;상관성분석용Pearson직선상관분석법. 결과 성공건립료간섬유화소서모형.모형조Arkadia、Smad7화TGFβ 1적mRNA표체량재섬유화과정중축점승고,12주시체고봉(상대표체량분별위1.3434±0.0149、1.2200±0.0093화1.1258±0.0065),BMP-7간예조표체량강저(상대표체량분별위0.9867±0.0169、0.9517±0.0120화0.9029±0.0085),여대조조(상대표체량분별위0.5398±0.0025、0.7467±0.0072화0.6318±0.0041)상비,차이유통계학의의(F치분별위812.801、451.462화998.957,P치균＜0.01);BMP-7간예조Arkadia、Smad7화TGF β1적mRNA표체명현저우12주모형조(t치분별위12.108、18.737화16.364,P치균＜0.01).정상간조직중Arkadia、Smad7、TGF β1단백부재회관구소량표체(상대표체량분별위2.1702±0.0518、2.0798±0.0547화2.4515±0.0487),모형조중기표체량축점증가(12주적상대표체량분별위3.4198±0.0279、5.4480±0.0565화5.2619±0.0530),주요재회관구화간세포포질내표체,BMP-7간예후표체량강저(분별위3.1457±0.0424、3.5616±0.0491화3.5282±0.0195),여대조조상비,차이유통계학의의(F치분별위8.399、609.690화900.561,P치균＜0.01);여12주모형조상비,BMP-7간예조Arkadia、Smad7、TGF β1적단백질표체량명현강저(t치분별위23.438、11.667화42.889,P치균＜0.01).결론 Arkadia재간섬유화진전과정중정상승추세,BMP-7구유항간섬유화작용병가이억제섬유화과정중Arkadia적표체.
Objective This study explored the dynamic expression of the E3 ubiquitin-protein ligase gene,Arkadia,in response to carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model and investigated the differential expression that occurs following treatment with the anti-fibrotic bone morphogenetic protein-7 (BMP-7).Methods Thirty healthy male imprinting control region (ICR) mice were randomly assigned to three groups:normal (control; n =6),CCl4-induced model group (model; n =18),and CCl4-induced model with BMP-7 treatment group (treatment; n =6).The model group was further divided into three subgroups (n =6 each) for analysis at 4,8 and 12 weeks after fibrosis induction.Liver fibrosis was induced by hypodermic injections of 60％ CCl4/peanut oil (5 mL/kg)to the hind legs of mice two-times per week in alternating legs for a period of 12 weeks.At week 9,the treatment group of CC14-induced mice were given an intraperitoneal injection of BMP-7 (300 pg/g) simultaneously with that day's hypodermic injection of 60％ CCl4/peanut oil,and then every other day for a period of four weeks.The pathological changes in liver tissues were observed after staining with hematoxylin-eosin (HE) and Masson's trichrome.Messenger RNA (mRNA) and protein expression of Arkadia in liver were evaluated using reverse transcription-polymerase chain reaction and immunohistochemistry and Western blotting,respectively.Results Mouse models of liver fibrosis were successfully established by CCl4 exposure.Arkadia,Stmad7 and TGF-β1 mRNA levels were up-regulated in the model group in a time-dependent manner (vs.control group),and BMP-7 treatment led to significant down-regulation of the CCl4-induced expression of the three genes (vs.control group:F=812.80,451.46,and 998.96,respectively; P ＜ 0.01).At week 12,the mRNA levels ofArkadia,Smad7,and TGF-β1 were significantly lower in the BMP-7 treatment group than in the model group (t =12.108,18.737,and 16.364,respectively; P ＜ 0.01).Arkadia,Smad7,and TGF-β1 protein staining was weak in the portal area of control liver tissue.In contrast,the model group showed significantly stronger staining for all three proteins in the portal area and in the cytoplasm of liver cells.The staining of Arkadia,Smad7,and TGF-β1 proteins was significantly lower in the treatment group (vs.control group:F =8.399,609.690,and 900.561,respectively;P ＜ 0.01).At week 12,the protein levels ofArkadia,Smad7,and TGF-β1 were significantly lower in the treatment group than in the model group (t =23.438,11.667,and 42.889,respectively; P ＜ 0.01).Conclusion Arkadia expression gradually increased along with the development of liver fibrosis but was suppressed by treatment with the anti-fibrotic factor,BMP-7.
