目的 探讨血管紧张素(1-7) (Ang(1-7))对胆管结扎所致肝纤维化大鼠肝窦血管生成的抑制作用及相应的分子机制. 方法 将18只Wistar雄性大鼠随机分为假手术(SHAM)组,胆总管结扎(BDL)组和Ang (1-7)治疗组.假手术组:行开腹术再关腹,在腹腔埋注射泵,以25μ g·kg-1.h1的速度持续注射等渗盐水;BDL组:开腹结扎胆总管建立肝纤维化模型,同时在腹腔埋注射泵,以25 μg· kg1.h-1的速度持续注射等渗盐水;Ang(1 7)治疗组:BDL建立肝纤维化模型,同时在腹腔埋注射泵,以25μg.kg11.h-1的速度持续注射Ang(1-7).4周后宰杀动物,取肝组织,行苏木素-伊红染色,进行肝纤维化评分;Masson胶原染色,测定胶原的表达;利用免疫组织化学,免疫印迹(Wetern blot),组织免疫荧光检测血管生成的标志:血管性血友病因子(vWF),血管内皮细胞生长因子A(VEGFA),血小板-内皮细胞黏附分子CD31的表达.根据资料不同采用One-way ANOVA检验、LSD法、Welch法、Dunnett’sT3法、t检验或相关性分析.结果 研究结果显示:在BDL组中,与SHAM组相比,肝纤维化评分(4.83±0.75对比0.00±0.00,p=0.000)、胶原面积(87.27±5.33对比0.98±0.11,P=0.000)、免疫组织化学检测vWF的表达(灰度值3.11±0.3对比1.00±0.07,p=0.000)、VEGFA的表达(灰度值8.38±1.64对比1.00±0.08,P=0.003)、CD31的表达(灰度值3.05±0.44对比1.00±0.12,P=0.000),Western blot检测vWF的表达(灰度值0.380±0.008对比0.270±0.007,P=0.000)、VEGFA的表达(灰度值0.270±0.005对比0.120±0.002,P=0.007)、CD31的表达(灰度值0.350±0.008对比0.060±0.004,P=0.000)均明显增高,差异有统计学意义(P值均＜0.01);免疫荧光检测BDL组肝组织vVWF、VEGFA、CD31阳性表达细胞较SHAM组明显增多.与BDL组相比,Ang(1-7)治疗组中肝纤维化评分(2.50±0.55对比4.83±0.75,P=0.000)、胶原面积(24.46±3.45对比87.27±5.33,P=0.000)、免疫组织化学检测vWF的表达(灰度值1.60±0.06对比3.11±0.30,P=0.000)、VEGFA的表达(灰度值4.68±0.58对比8.38±1.64,P=0.037)、CD31的表达(灰度值1.42±0.12对比3.05±0.44,P=0.009),Westem blot检测vWF的表达(灰度值0.190±0.007对比0.380±0.008,P=0.000)、VEGFA的表达(灰度值0.120±0.006对比0.270±0.005,P=0.000)、CD31的表达(灰度值0.130±0.006对比0.350±0.008,P=0.000)均显著下降,差异有统计学意义(P值均＜0.01);免疫荧光检测Ang(1-7)治疗组vWF、VEGFA、CD31阳性表达的细胞较BDL组明显减少;各分组中VEGFA的表达与vWF表达呈现显著正相关关系(r=0.956,P=0.000).结论 Ang(1-7)可抑制BDL诱导肝纤维化大鼠肝窦血管生成.
目的 探討血管緊張素(1-7) (Ang(1-7))對膽管結扎所緻肝纖維化大鼠肝竇血管生成的抑製作用及相應的分子機製. 方法 將18隻Wistar雄性大鼠隨機分為假手術(SHAM)組,膽總管結扎(BDL)組和Ang (1-7)治療組.假手術組:行開腹術再關腹,在腹腔埋註射泵,以25μ g·kg-1.h1的速度持續註射等滲鹽水;BDL組:開腹結扎膽總管建立肝纖維化模型,同時在腹腔埋註射泵,以25 μg· kg1.h-1的速度持續註射等滲鹽水;Ang(1 7)治療組:BDL建立肝纖維化模型,同時在腹腔埋註射泵,以25μg.kg11.h-1的速度持續註射Ang(1-7).4週後宰殺動物,取肝組織,行囌木素-伊紅染色,進行肝纖維化評分;Masson膠原染色,測定膠原的錶達;利用免疫組織化學,免疫印跡(Wetern blot),組織免疫熒光檢測血管生成的標誌:血管性血友病因子(vWF),血管內皮細胞生長因子A(VEGFA),血小闆-內皮細胞黏附分子CD31的錶達.根據資料不同採用One-way ANOVA檢驗、LSD法、Welch法、Dunnett’sT3法、t檢驗或相關性分析.結果 研究結果顯示:在BDL組中,與SHAM組相比,肝纖維化評分(4.83±0.75對比0.00±0.00,p=0.000)、膠原麵積(87.27±5.33對比0.98±0.11,P=0.000)、免疫組織化學檢測vWF的錶達(灰度值3.11±0.3對比1.00±0.07,p=0.000)、VEGFA的錶達(灰度值8.38±1.64對比1.00±0.08,P=0.003)、CD31的錶達(灰度值3.05±0.44對比1.00±0.12,P=0.000),Western blot檢測vWF的錶達(灰度值0.380±0.008對比0.270±0.007,P=0.000)、VEGFA的錶達(灰度值0.270±0.005對比0.120±0.002,P=0.007)、CD31的錶達(灰度值0.350±0.008對比0.060±0.004,P=0.000)均明顯增高,差異有統計學意義(P值均＜0.01);免疫熒光檢測BDL組肝組織vVWF、VEGFA、CD31暘性錶達細胞較SHAM組明顯增多.與BDL組相比,Ang(1-7)治療組中肝纖維化評分(2.50±0.55對比4.83±0.75,P=0.000)、膠原麵積(24.46±3.45對比87.27±5.33,P=0.000)、免疫組織化學檢測vWF的錶達(灰度值1.60±0.06對比3.11±0.30,P=0.000)、VEGFA的錶達(灰度值4.68±0.58對比8.38±1.64,P=0.037)、CD31的錶達(灰度值1.42±0.12對比3.05±0.44,P=0.009),Westem blot檢測vWF的錶達(灰度值0.190±0.007對比0.380±0.008,P=0.000)、VEGFA的錶達(灰度值0.120±0.006對比0.270±0.005,P=0.000)、CD31的錶達(灰度值0.130±0.006對比0.350±0.008,P=0.000)均顯著下降,差異有統計學意義(P值均＜0.01);免疫熒光檢測Ang(1-7)治療組vWF、VEGFA、CD31暘性錶達的細胞較BDL組明顯減少;各分組中VEGFA的錶達與vWF錶達呈現顯著正相關關繫(r=0.956,P=0.000).結論 Ang(1-7)可抑製BDL誘導肝纖維化大鼠肝竇血管生成.
목적 탐토혈관긴장소(1-7) (Ang(1-7))대담관결찰소치간섬유화대서간두혈관생성적억제작용급상응적분자궤제. 방법 장18지Wistar웅성대서수궤분위가수술(SHAM)조,담총관결찰(BDL)조화Ang (1-7)치료조.가수술조:행개복술재관복,재복강매주사빙,이25μ g·kg-1.h1적속도지속주사등삼염수;BDL조:개복결찰담총관건립간섬유화모형,동시재복강매주사빙,이25 μg· kg1.h-1적속도지속주사등삼염수;Ang(1 7)치료조:BDL건립간섬유화모형,동시재복강매주사빙,이25μg.kg11.h-1적속도지속주사Ang(1-7).4주후재살동물,취간조직,행소목소-이홍염색,진행간섬유화평분;Masson효원염색,측정효원적표체;이용면역조직화학,면역인적(Wetern blot),조직면역형광검측혈관생성적표지:혈관성혈우병인자(vWF),혈관내피세포생장인자A(VEGFA),혈소판-내피세포점부분자CD31적표체.근거자료불동채용One-way ANOVA검험、LSD법、Welch법、Dunnett’sT3법、t검험혹상관성분석.결과 연구결과현시:재BDL조중,여SHAM조상비,간섬유화평분(4.83±0.75대비0.00±0.00,p=0.000)、효원면적(87.27±5.33대비0.98±0.11,P=0.000)、면역조직화학검측vWF적표체(회도치3.11±0.3대비1.00±0.07,p=0.000)、VEGFA적표체(회도치8.38±1.64대비1.00±0.08,P=0.003)、CD31적표체(회도치3.05±0.44대비1.00±0.12,P=0.000),Western blot검측vWF적표체(회도치0.380±0.008대비0.270±0.007,P=0.000)、VEGFA적표체(회도치0.270±0.005대비0.120±0.002,P=0.007)、CD31적표체(회도치0.350±0.008대비0.060±0.004,P=0.000)균명현증고,차이유통계학의의(P치균＜0.01);면역형광검측BDL조간조직vVWF、VEGFA、CD31양성표체세포교SHAM조명현증다.여BDL조상비,Ang(1-7)치료조중간섬유화평분(2.50±0.55대비4.83±0.75,P=0.000)、효원면적(24.46±3.45대비87.27±5.33,P=0.000)、면역조직화학검측vWF적표체(회도치1.60±0.06대비3.11±0.30,P=0.000)、VEGFA적표체(회도치4.68±0.58대비8.38±1.64,P=0.037)、CD31적표체(회도치1.42±0.12대비3.05±0.44,P=0.009),Westem blot검측vWF적표체(회도치0.190±0.007대비0.380±0.008,P=0.000)、VEGFA적표체(회도치0.120±0.006대비0.270±0.005,P=0.000)、CD31적표체(회도치0.130±0.006대비0.350±0.008,P=0.000)균현저하강,차이유통계학의의(P치균＜0.01);면역형광검측Ang(1-7)치료조vWF、VEGFA、CD31양성표체적세포교BDL조명현감소;각분조중VEGFA적표체여vWF표체정현현저정상관관계(r=0.956,P=0.000).결론 Ang(1-7)가억제BDL유도간섬유화대서간두혈관생성.
Objective To explore the inhibitory effect of angiotensin (1-7) on hepatic sinusoid angiogenesis using a rat model of hepatic fibrosis.Methods Eighteen male Wistar rats were randomly divided into three equal groups for sham operation (untreated/uninduced control group),bile duct ligation (BDL) (untreated model group),or BDL with angiotensin (1-7) treatment (treated model group).Histological analysis was used to assess the liver fibrosis score,by hematoxylin-eosin staining,and the level of fibrosis,by Masson's trichrome staining.Immunohistochemistry,western blotting,and immunofluorescence were used to assess the expression of the angiogenesis markers vWF,VEGFA,and CD31.Results Compared with the untreated/uninduced control group,the untreated BDL model group showed remarkably higher fibrosis score,area of the type I collagen expression,and expression levels of vWF,VEGFA,and CD31.However,the angiotensin (1-7)-treatment protected against the BLD-related changes,as evidenced by decreased robustness and down-regulation of the corresponding indicators.Moreover,the expression level of VEGFA was highly correlated to the expression level of vWF (r =0.956,P =0.000).Conclusion BDL-induced hepatic fibrosis is accompanied by significant increases in angiogenesis-related factors,but angiotensin (1-7) treatment may inhibit hepatic sinusoid angiogenesis during the liver fibrosis process.
