中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2014年
9期
671-675
,共5页
肝硬化%肝功能衰竭,急性%线粒体融合蛋白-2%三磷酸腺苷%活性氧
肝硬化%肝功能衰竭,急性%線粒體融閤蛋白-2%三燐痠腺苷%活性氧
간경화%간공능쇠갈,급성%선립체융합단백-2%삼린산선감%활성양
Liver cirrhosis%Liver failure,acute%Mitochondria fusion protein-2%Adenosine triphosphate%Reactive oxygen species
目的 比较肝硬化、慢加急性肝衰竭和正常大鼠肝组织线粒体融合蛋白2(Mfn2) mRNA和蛋白的表达差异,检测大鼠肝组织中三磷酸腺苷(ATP)、活性氧(ROS)的含量,探讨Mfn2表达差异在肝硬化及肝衰竭发生和发展过程中的意义.方法 通过腹腔注射四氯化碳植物油溶液建立大鼠肝硬化模型,在肝硬化基础上给予LPS/D-Ga急性攻击建立慢加急性肝衰竭大鼠模型,腹腔注射植物油建立正常对照组.正常对照组(NC组,n=10),肝硬化组(LC组,n=15),慢加急性肝衰竭组(LF组,n=15).Western blot法及免疫荧光法观察不同模型组大鼠肝组织中Mfn2的表达,实时荧光定量法检测不同模型肝组织中Mfn2 mRNA的表达,荧光分光光度法测定各组肝组织中ROS含量;生物发光法测定肝组织中ATP含量.数据统计计量资料以均数±标准差(-x±s)表示,样本均数比较用单因素方差分析,变量间相关性应用Pearson相关分析,回归分析采用直线回归方法.结果 Mfn2 mRNA在LC组、LF组肝组织中的的表达显著减低,相对表达量分别为(2.25±0.1)× 106、(0.95±0.5) ×106,与NC组的(2.67±0.4)×106比较,f值分别为-51.75,-6.73,P值分别为0.88、0.001,LC组与NC组比较,差异无统计学意义,LF组与NC组间差异有统计学意义;3组比较,F=451.30,P＜0.05,差异有统计学意义.Mfn2蛋白在LC组、LF组肝组织中的的表达显著减低,相对表达量分别为0.051±0.004、0.037±0.007,与NC组的0.254±0.008比较,t值分别为-19.41、-6.064,P值均＜0.05,差异有统计学意义;3组比较,F=444.98,P＜0.05,差异有统计学意义.与NC组比较,LC组、LS组ATP含量减少,ROS含量明显增加.Mfn2蛋白的表达与ATP含量呈正相关(r=0.982),Mfn2蛋白表达与ROS含量呈负相关(r=-0.803).结论 肝硬化、慢加急性肝衰竭大鼠模型中,肝组织中Mfn2表达降低引起ATP的合成减少,ROS产生增多,Mfn2表达减少与肝硬化、肝衰竭发生过程中能量代谢障碍有关.Mfn2蛋白的表达量与肝损伤程度相关.
目的 比較肝硬化、慢加急性肝衰竭和正常大鼠肝組織線粒體融閤蛋白2(Mfn2) mRNA和蛋白的錶達差異,檢測大鼠肝組織中三燐痠腺苷(ATP)、活性氧(ROS)的含量,探討Mfn2錶達差異在肝硬化及肝衰竭髮生和髮展過程中的意義.方法 通過腹腔註射四氯化碳植物油溶液建立大鼠肝硬化模型,在肝硬化基礎上給予LPS/D-Ga急性攻擊建立慢加急性肝衰竭大鼠模型,腹腔註射植物油建立正常對照組.正常對照組(NC組,n=10),肝硬化組(LC組,n=15),慢加急性肝衰竭組(LF組,n=15).Western blot法及免疫熒光法觀察不同模型組大鼠肝組織中Mfn2的錶達,實時熒光定量法檢測不同模型肝組織中Mfn2 mRNA的錶達,熒光分光光度法測定各組肝組織中ROS含量;生物髮光法測定肝組織中ATP含量.數據統計計量資料以均數±標準差(-x±s)錶示,樣本均數比較用單因素方差分析,變量間相關性應用Pearson相關分析,迴歸分析採用直線迴歸方法.結果 Mfn2 mRNA在LC組、LF組肝組織中的的錶達顯著減低,相對錶達量分彆為(2.25±0.1)× 106、(0.95±0.5) ×106,與NC組的(2.67±0.4)×106比較,f值分彆為-51.75,-6.73,P值分彆為0.88、0.001,LC組與NC組比較,差異無統計學意義,LF組與NC組間差異有統計學意義;3組比較,F=451.30,P＜0.05,差異有統計學意義.Mfn2蛋白在LC組、LF組肝組織中的的錶達顯著減低,相對錶達量分彆為0.051±0.004、0.037±0.007,與NC組的0.254±0.008比較,t值分彆為-19.41、-6.064,P值均＜0.05,差異有統計學意義;3組比較,F=444.98,P＜0.05,差異有統計學意義.與NC組比較,LC組、LS組ATP含量減少,ROS含量明顯增加.Mfn2蛋白的錶達與ATP含量呈正相關(r=0.982),Mfn2蛋白錶達與ROS含量呈負相關(r=-0.803).結論 肝硬化、慢加急性肝衰竭大鼠模型中,肝組織中Mfn2錶達降低引起ATP的閤成減少,ROS產生增多,Mfn2錶達減少與肝硬化、肝衰竭髮生過程中能量代謝障礙有關.Mfn2蛋白的錶達量與肝損傷程度相關.
목적 비교간경화、만가급성간쇠갈화정상대서간조직선립체융합단백2(Mfn2) mRNA화단백적표체차이,검측대서간조직중삼린산선감(ATP)、활성양(ROS)적함량,탐토Mfn2표체차이재간경화급간쇠갈발생화발전과정중적의의.방법 통과복강주사사록화탄식물유용액건립대서간경화모형,재간경화기출상급여LPS/D-Ga급성공격건립만가급성간쇠갈대서모형,복강주사식물유건립정상대조조.정상대조조(NC조,n=10),간경화조(LC조,n=15),만가급성간쇠갈조(LF조,n=15).Western blot법급면역형광법관찰불동모형조대서간조직중Mfn2적표체,실시형광정량법검측불동모형간조직중Mfn2 mRNA적표체,형광분광광도법측정각조간조직중ROS함량;생물발광법측정간조직중ATP함량.수거통계계량자료이균수±표준차(-x±s)표시,양본균수비교용단인소방차분석,변량간상관성응용Pearson상관분석,회귀분석채용직선회귀방법.결과 Mfn2 mRNA재LC조、LF조간조직중적적표체현저감저,상대표체량분별위(2.25±0.1)× 106、(0.95±0.5) ×106,여NC조적(2.67±0.4)×106비교,f치분별위-51.75,-6.73,P치분별위0.88、0.001,LC조여NC조비교,차이무통계학의의,LF조여NC조간차이유통계학의의;3조비교,F=451.30,P＜0.05,차이유통계학의의.Mfn2단백재LC조、LF조간조직중적적표체현저감저,상대표체량분별위0.051±0.004、0.037±0.007,여NC조적0.254±0.008비교,t치분별위-19.41、-6.064,P치균＜0.05,차이유통계학의의;3조비교,F=444.98,P＜0.05,차이유통계학의의.여NC조비교,LC조、LS조ATP함량감소,ROS함량명현증가.Mfn2단백적표체여ATP함량정정상관(r=0.982),Mfn2단백표체여ROS함량정부상관(r=-0.803).결론 간경화、만가급성간쇠갈대서모형중,간조직중Mfn2표체강저인기ATP적합성감소,ROS산생증다,Mfn2표체감소여간경화、간쇠갈발생과정중능량대사장애유관.Mfn2단백적표체량여간손상정도상관.
Objective To determine the differential protein and mRNA expressions of mitochondria fusion protein-2 (Mfn2) in hepatic tissues in conditions of cirrhosis and acute on chronic liver failure using rat model systems,and to determine the correlative effects on production of adenosine triphosphate (ATP) and reactive oxygen species (ROS).Methods A liver cirrhotic rat model (LC rats) was established by intraperitoneal injection of carbon tetrachloride (CCl4,in vegetable oil),and these mice were subsequently used (10 weeks later) to establish the acute on chronic liver failure rat model (LF rats) by injecting lipopolysaccharide and D-amino-galactose.Control groups (normal controls,NC rats) were established for each model by intraperitoneal injection of vegetable oil only.Protein expression of Mfn2 in liver was quantified by western blotting with fluorescence densitometry and immunofluorescence staining,and mRNA expression was measured by real-time fluorescence quantitative PCR.ROS levels in liver were measured by fluorescence spectrophotometry,and ATP content was measured by bioluminescence assay.Significance of inter-group differences was assessed by one-way ANOVA,and correlations were determined using bivariate statistical modeling.Results Mfn2 protein expression was significantly lower in the liver tissues from modeled rats than that from the control rats (LC:0.051±0.004 and LF:0.037±0.007 vs.NC:0.254±0.008;F=444.98,P ＜ 0.05).The mRNA expression followed the same trend of lower expression (LC:21.21±0.93 and LF:24.35±0.85 vs.NC:19.09±0.69; F=66.941,P ＜ 0.05).The ATP content in liver tissues was also significantly lower in the modeled rats (LC:2.07±0.05 mol/L and LF:1.81±0.11 mol/L vs.NC:3.24±0.08 mol/L; F =574.21,P ＜ 0.05).Lower Mfn2 expression was correlated with lower ATP content (r =0.982) and higher ROS content (r =0.803).Conclusion Reduced Mfn2 expression in liver tissue may cause a decrease in ATP synthesis and increase in ROS generation,thereby disrupting metabolism and increasing oxidative stress in the liver under conditions of cirrhosis and liver failure.
