血管生成相关因子在大鼠布加综合征肝纤维化模型中的表达及意义

Expression and significance of angiogenesis-related factors in liver fibrosis model of Budd-Chiari syndrome in rats

目的探讨血管内皮生长因子(VEGF)、诱导型一氧化氮合酶(iNOS)、血管生成素(Angpt)-1及内皮素(ET)-1在大鼠布加综合征(BCS)肝纤维化模型中的表达及意义。方法结扎雄性SD大鼠肝后段下腔静脉建立BCS动物模型,按数字表法分为对照组(20只)、实验组(80只)和假手术组(80只),实验组及假手术组又各分4个亚组(实验1、3、6、12周,各20只)。各组行免疫组织化学、苏木精-伊红(HE)及马松(Masson)染色,以实时定量聚合酶链反应(Real-time PCR)及蛋白质印迹法(Western blot)检测ET-1、VEGF、iNOS及Angpt-1的表达量。各因子水平在各组间差异采用方差分析,亚组间两两比较采用LSD检验,以皮尔逊(Pearson)及斯皮尔曼(Spearman)法做相关性分析。结果实验组1、3、6、12周VEGF(1.41±0.18、6.54±0.78、4.19±0.41、2.16±0.25)、iNOS(2.06±0.21、8.88±0.95、4.85±0.51、2.79±0.28)及Angpt-1(9.57±0.91、6.20±1.39、4.53±0.49、2.55±0.33)的mRNA水平均高于对照组(VEGF:0.97±0.08;iNOS:1.01±0.11;Angpt-1:1.01±0.10)及假手术组(VEGF:F=366.124、412.266;iNOS:F=443.326、1170.864;Angpt-1:F=213.868、503.210,P<0.05),差异有统计学意义。实验组内的VEGF(1.41±0.18、6.54±0.78、4.19±0.41、2.16±0.25)、iNOS(2.06±0.21、8.88±0.95、4.85±0.51、2.79±0.28)及Angpt-1(9.57±0.91、6.20±1.39、4.53±0.49、2.55±0.33)的mRNA表达差异均有统计学意义(F=293.799、346.260、137.121,P<0.05),亚组间两两比较差异均有统计学意义(P<0.05)。ET-1、VEGF、iNOS及Angpt-1在各组间及组内的蛋白表达变化规律与相应mRNA表达一致。VEGF(1.41±0.18、6.54±0.78、4.19±0.41、2.16±0.25)与iNOS(2.06±0.21、8.88±0.95、4.85±0.51、2.79±0.28)、Angpt-1(9.57±0.91、6.20±1.39、4.53±0.49、2.55±0.33)正相关(r=0.983、0.364,P<0.05),iNOS(2.06±0.21、8.88±0.95、4.85±0.51、2.79±0.28)与Angpt-1(9.57±0.91、6.20±1.39、4.53±0.49、2.55±0.33)正相关(r=0.377,P<0.05)。结论BCS模型中的iNOS、Angpt-1及VEGF高表达且互为正相关,三者参与调控BCS肝内外血管生成及肝纤维化进程。

Objective To explore the expression and significance of angiogenesis-related factors in liver fibrosis model of budd-chiari syndrome(BCS)in rats.Methods The animal model of BCS was established by partially ligating the inferior vena cava in rats.All animals were divided into control group(20 rats),experimental group(80 rats)and sham group(80 rats).The experimental group and sham group were divided into four subgroups(1,3,6,12 weeks)of 20 rats each.After the experiment,liver tissues in each group were collected for immunohistochemistry,hematoxylin and eosin(HE),and Masson staining.The levels of endothelin(ET)-1,vascular endothelial growth factor(VEGF),inducible nitric oxide synthase(iNOS)and angiopoietin(Angpt)-1 were examined respectively.Variance analysis was used to analyze the differences of each factor level among groups.LSD test was used for multiple comparisons among subgroups.Pearson method and Spearman method were used for correlation analysis.Results The mRNA expression of VEGF(1.41±0.18,6.54±0.78,4.19±0.41,2.16±0.25),iNOS(2.06±0.21,8.88±0.95,4.85±0.51,2.79±0.28)and Angpt-1(9.57±0.91,6.20±1.39,4.53±0.49,2.55±0.33)were compared between control group and sham group respectively,and the overall difference was not statistically significant(VEGF:F=0.487,P>0.05;iNOS:F=0.651,P>0.05;Angpt-1:F=0.969,P>0.05);the overall difference was statistically significant in the experimental group compared with the control group and the sham group(VEGF:F=366.124,412.266;iNOS:F=443.326,1170.864;Angpt-1:F=213.868,503.210,P<0.01);the expression of each factor in the subgroups of experimental group was higher than that of control group(VEGF:0.97±0.08;iNOS:1.01±0.11;Angpt-1:1.01±0.10)and sham subgroups at the same time(P<0.05).The overall differences of mRNA expression of VEGF(1.41±0.18,6.54±0.78,4.19±0.41,2.16±0.25),iNOS(2.06±0.21,8.88±0.95,4.85±0.51,2.79±0.28)and Angpt-1(9.57±0.91,6.20±1.39,4.53±0.49,2.55±0.33)in the experimental group at each time point after the operation were significant(F=293.799,346.260,137.121,P<0.01),and the comparison between each subgroup were also statistically significant(P<0.05).VEGF(1.41±0.18,6.54±0.78,4.19±0.41,2.16±0.25)was positively correlated with iNOS(2.06±0.21,8.88±0.95,4.85±0.51,2.79±0.28)and angpt-1(9.57±0.91,6.20±1.39,4.53±0.49,2.55±0.33,r=0.983,0.364,P<0.05),and iNOS(2.06±0.21,8.88±0.95,4.85±0.51,2.79±0.28)was positively correlated with angpt-1(9.57±0.91,6.20±1.39,4.53±0.49,2.55±0.33,r=0.377,P<0.05).Conclusion The expression of iNOS,Angpt-1 and VEGF in BCS model is increased and positively correlated each other.They are involved in the regulation of intrahepatic and extrahepatic angiogenesis and liver fibrosis in BCS,but the specific mechanism needs further study.