中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
3期
207-212
,共6页
孔今波%任伟光%米红梅%赵素贤%张玉果%南月敏
孔今波%任偉光%米紅梅%趙素賢%張玉果%南月敏
공금파%임위광%미홍매%조소현%장옥과%남월민
肝硬化%骨桥蛋白%转化生长因子β1%小鼠%模型,动物%酒精性肝纤维化
肝硬化%骨橋蛋白%轉化生長因子β1%小鼠%模型,動物%酒精性肝纖維化
간경화%골교단백%전화생장인자β1%소서%모형,동물%주정성간섬유화
Liver cirrhosis%Osteopontin%Transforming growth factor-β1%Mouse%Model,animal%Alcoholic liver fibrosis
目的 快速建立小鼠酒精陛肝纤维化模型,为酒精性肝纤维化发病机制及防治策略的研究奠定基础.方法 64只C57BL/6J小鼠随机分为对照组、四氯化碳组、乙醇组、溶剂对照组及乙醇+四氯化碳组.乙醇+四氯化碳组小鼠,造模前4周以含4％乙醇Lieber-DeCarli液体饲料喂养,第5周起联合5％四氯化碳腹腔注射,于造模第0、4、5、6、7、8周各处死6只.其余各组小鼠于造模第8周处死.采用酶法检测小鼠血清ALT及AST水平;HE及Masson染色观察肝组织病理学变化,并对肝脂肪变、炎症活动及纤维化程度评分.免疫组织化学染色观察肝组织α-平滑肌肌动蛋白(α-SMA)表达变化;实时定量RT-PCR及Western blot方法检测肝组织骨桥蛋白(OPN)及转化生长因子β 1 (TGFβ1)mRNA及蛋白表达动态变化.结果 乙醇+四氯化碳组小鼠于造模第4周出现轻～中度肝脂肪变,第5周出现炎细胞浸润,窦周出现纤维组织沉积,第6～7周肝小叶内可见点、灶状肝细胞坏死,炎细胞浸润、窦周纤维组织沉积呈进行性加重,第8周肝细胞片状坏死,桥接纤维化形成.免疫组织化学染色显示:α-SMA主要表达于活化肝星状细胞及纤维组织沉积区域,随造模时间延长表达逐渐增强.乙醇+四氯化碳组小鼠肝组织OPN表达随造模时间延长逐渐增强,第0、4、5、6、7、8周mRNA相对表达量依次为1.01±0.13、0.80±0.20、1.83±0.25、2.94±0.19、3.45±0.31及5.99±0.17,各组比较,F=476.270,P＜ 0.01;蛋白相对表达量依次为0.19±0.06、0.48±0.05、0.52±0.06、1.02±0.10、1.52±0.11及1.50±0.08,各组比较,F=298.027,P＜0.01.TGF β 1表达自第5周明显上调,并呈持续高表达,mRNA相对表达量依次为1.03±0.18、1.07±0.23、3.19±0.40、3.31±0.28、1.58±0.18及2.08±0.26,各组比较,F=85.546,P＜0.01;蛋白相对表达量依次为0.24±0.08、0.28±0.12、1.26±0.16、0.96±0.12、1.09±0.25、1.10±0.20,各组比较,F=43.639,P＜0.01.结论 Lieber-DeCarli乙醇液体饲料喂养联合微量四氯化碳腹腔注射可成功建立小鼠酒精性肝纤维化模型,符合酒精性肝纤维化病理特点及病变过程.肝组织OPN及TGFβl表达在酒精性肝纤维化发生及进展中发挥重要作用.
目的 快速建立小鼠酒精陛肝纖維化模型,為酒精性肝纖維化髮病機製及防治策略的研究奠定基礎.方法 64隻C57BL/6J小鼠隨機分為對照組、四氯化碳組、乙醇組、溶劑對照組及乙醇+四氯化碳組.乙醇+四氯化碳組小鼠,造模前4週以含4％乙醇Lieber-DeCarli液體飼料餵養,第5週起聯閤5％四氯化碳腹腔註射,于造模第0、4、5、6、7、8週各處死6隻.其餘各組小鼠于造模第8週處死.採用酶法檢測小鼠血清ALT及AST水平;HE及Masson染色觀察肝組織病理學變化,併對肝脂肪變、炎癥活動及纖維化程度評分.免疫組織化學染色觀察肝組織α-平滑肌肌動蛋白(α-SMA)錶達變化;實時定量RT-PCR及Western blot方法檢測肝組織骨橋蛋白(OPN)及轉化生長因子β 1 (TGFβ1)mRNA及蛋白錶達動態變化.結果 乙醇+四氯化碳組小鼠于造模第4週齣現輕～中度肝脂肪變,第5週齣現炎細胞浸潤,竇週齣現纖維組織沉積,第6～7週肝小葉內可見點、竈狀肝細胞壞死,炎細胞浸潤、竇週纖維組織沉積呈進行性加重,第8週肝細胞片狀壞死,橋接纖維化形成.免疫組織化學染色顯示:α-SMA主要錶達于活化肝星狀細胞及纖維組織沉積區域,隨造模時間延長錶達逐漸增彊.乙醇+四氯化碳組小鼠肝組織OPN錶達隨造模時間延長逐漸增彊,第0、4、5、6、7、8週mRNA相對錶達量依次為1.01±0.13、0.80±0.20、1.83±0.25、2.94±0.19、3.45±0.31及5.99±0.17,各組比較,F=476.270,P＜ 0.01;蛋白相對錶達量依次為0.19±0.06、0.48±0.05、0.52±0.06、1.02±0.10、1.52±0.11及1.50±0.08,各組比較,F=298.027,P＜0.01.TGF β 1錶達自第5週明顯上調,併呈持續高錶達,mRNA相對錶達量依次為1.03±0.18、1.07±0.23、3.19±0.40、3.31±0.28、1.58±0.18及2.08±0.26,各組比較,F=85.546,P＜0.01;蛋白相對錶達量依次為0.24±0.08、0.28±0.12、1.26±0.16、0.96±0.12、1.09±0.25、1.10±0.20,各組比較,F=43.639,P＜0.01.結論 Lieber-DeCarli乙醇液體飼料餵養聯閤微量四氯化碳腹腔註射可成功建立小鼠酒精性肝纖維化模型,符閤酒精性肝纖維化病理特點及病變過程.肝組織OPN及TGFβl錶達在酒精性肝纖維化髮生及進展中髮揮重要作用.
목적 쾌속건립소서주정폐간섬유화모형,위주정성간섬유화발병궤제급방치책략적연구전정기출.방법 64지C57BL/6J소서수궤분위대조조、사록화탄조、을순조、용제대조조급을순+사록화탄조.을순+사록화탄조소서,조모전4주이함4％을순Lieber-DeCarli액체사료위양,제5주기연합5％사록화탄복강주사,우조모제0、4、5、6、7、8주각처사6지.기여각조소서우조모제8주처사.채용매법검측소서혈청ALT급AST수평;HE급Masson염색관찰간조직병이학변화,병대간지방변、염증활동급섬유화정도평분.면역조직화학염색관찰간조직α-평활기기동단백(α-SMA)표체변화;실시정량RT-PCR급Western blot방법검측간조직골교단백(OPN)급전화생장인자β 1 (TGFβ1)mRNA급단백표체동태변화.결과 을순+사록화탄조소서우조모제4주출현경～중도간지방변,제5주출현염세포침윤,두주출현섬유조직침적,제6～7주간소협내가견점、조상간세포배사,염세포침윤、두주섬유조직침적정진행성가중,제8주간세포편상배사,교접섬유화형성.면역조직화학염색현시:α-SMA주요표체우활화간성상세포급섬유조직침적구역,수조모시간연장표체축점증강.을순+사록화탄조소서간조직OPN표체수조모시간연장축점증강,제0、4、5、6、7、8주mRNA상대표체량의차위1.01±0.13、0.80±0.20、1.83±0.25、2.94±0.19、3.45±0.31급5.99±0.17,각조비교,F=476.270,P＜ 0.01;단백상대표체량의차위0.19±0.06、0.48±0.05、0.52±0.06、1.02±0.10、1.52±0.11급1.50±0.08,각조비교,F=298.027,P＜0.01.TGF β 1표체자제5주명현상조,병정지속고표체,mRNA상대표체량의차위1.03±0.18、1.07±0.23、3.19±0.40、3.31±0.28、1.58±0.18급2.08±0.26,각조비교,F=85.546,P＜0.01;단백상대표체량의차위0.24±0.08、0.28±0.12、1.26±0.16、0.96±0.12、1.09±0.25、1.10±0.20,각조비교,F=43.639,P＜0.01.결론 Lieber-DeCarli을순액체사료위양연합미량사록화탄복강주사가성공건립소서주정성간섬유화모형,부합주정성간섬유화병리특점급병변과정.간조직OPN급TGFβl표체재주정성간섬유화발생급진전중발휘중요작용.
Objective To create a convenient method to establish an alcoholic liver fibrosis model in mice and use it to explore the putative pathogenic mechanisms involving the immunomodulatory proteins osteopontin (OPN) and transforming growth faetor-β1 (TGF-β1).Methods Forty C57BL/6J mice were fed the Lieber-DeCarli 4％ ethanol-containing liquid diet for four weeks,followed by an additional four weeks of the 4％ ethanol diet combined with intraperitoneal injection of carbon tetrachloride (CCl4 5％ solution in olive oil；2ml/kg body weight,2 times/week) to induce alcoholic liver fibrosis.Control groups (n =6 each) included:normal diet；normal diet plus CCl4 injections；ethanol diet alone；ethanol diet plus solvent (olive oil) injections.Model establishment was monitored by sacrificing six mice at model inception (week 0),and weeks 4,5,6,7,and 8 of modeling to collect liver tissues and blood for histological and biochemical analyses.Extent of hepatic steatosis,inflammation,and fibrosis was assessed by hematoxylin-eosin and Masson staining.Liver function markers,serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels,were tested by automated enzymatic assays.Alpha-smooth muscle actin (α-SMA) expression was detected by immunohistochemistry.The mRNA and protein expression of OPN and TGF-β1 was detected by real-time quantitative reverse transeriptionPCR and western blotting,respectively.Significance of differences between multiple groups was assessed by one-way ANOVA analysis followed by least significant difference t-test or Kruskal-Wallis H test followed by the Mann-Whitney U test.Results Compared to the control groups,the group of mice administrated ethanol and CCl4 developed mild to moderate hepatic steatosis at week 4 of modeling,progressive necroinflammation and perisinusoidal and portal fibrosis from weeks 5-8,and irregular necrosis and bridging fibrosis at week 8.In addition,the model group showed progressive up-regulation of α-SMA expression in the activated hepatic stellate cells (HSCs) and fibrotic areas from weeks 5-8.Both hepatic OPN and TGF-β1 showed signifieantly increasing trends in mRNA and protein expressions from weeks 5-8 (OPN mRNA:1.83 ± 0.25,2.94 ± 0.19,3.45 ± 0.31,and 5.99 ± 0.17 (F =476.27,P ＜ 0.001)；OPN protein:0.52 ± 0.06,1.02 ± 0.10,1.52 ± 0.11 and 1.50 ± 0.08 (F =298.03,P ＜ 0.001)；TGF-β1 mRNA:13.19 ± 0.40,3.31 ± 0.28,1.58 ± 0.18 and 2.08± 0.26 (F =85.55,P＜ 0.001)；TGF-β1 protein:1.26 ± 0.16,0.96 ± 0.12,1.09 ± 0.25 and 1.10 ± 0.20 (F=43.64,P ＜ 0.001).Conclusion Feeding C57BL/6J mice the Lieber-DeCarli ethanol-containing liquid diet combined with CCl4 intraperitoneal injection is a convenient method to establish a model of alcoholic liver fibrosis within a relatively short amount of time (eight weeks).Progression of alcoholic liver fibrosis is accompanied by increased hepatic expression of OPN and TGF-β1,which may contribute to the pathogenic mechanism of this disease and may be targets of future molecular therapies.
