泡球蚴感染中期小鼠纤维化肝组织中TGF-β1/Smad信号通路的表达和意义
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摘要
目的：探讨泡球蚴(Em)感染中期BALB/c小鼠致纤维化肝组织中α-SMA、I型胶原、Ⅲ型胶原、Ⅳ型胶原、TGF-β1、TβRI、Smad2、Smad3、Smad4和Smad7的表达状况及泡球蚴致肝纤维化的发病机制。方法：建立Em感染疾病动物模型,将BALB/c小鼠随机分为两组：实验组,以泡状棘球蚴原头蚴混悬液0.1ml/只经开腹肉眼直视下肝叶内注射接种小鼠。对照组,以同样方法注入0.1ml 0.9%生理盐水／只。于造模后12wk处死小鼠,取肝组织和病灶组织。部分肝组织10%福尔马林固定、石蜡包埋,4μm连续切片,HE和Masson染色,光镜下泡球蚴感染病灶周围病理改变和纤维化程度。一部分装入干净Ep管冻存-80℃,提取RNA,进行实时荧光定量检测。采用实时荧光定量PCR (qRT-PCR)检测肝组织α-SMA、Ⅰ型胶原、Ⅲ型胶原和Ⅳ型胶原以及TGF-β1和Smad7mRNA表达水平；免疫组织化学技术检测肝组织上述各指标在肝组织内的表达及定位；HE和Masson染色,光镜下观察肝组织病理改变和肝组织纤维化程度。结果：实验组小鼠病灶周围以形成典型的大小和形状各异的囊泡为主要特征,囊泡外周纤维结缔组织增生明显,存在不同程度的纤维化。免疫组化结果显示：α-SMA、Ⅰ型胶原、Ⅲ型胶原、Ⅳ型胶原、TGF-β1、TβR1、Smad2、Smad3、Smad4和Smad7在囊泡外围肉芽肿和对照组的显色指数分别为(6.43±0.59 VS 1.48±0.33)、(4.43±0.57 VS1.12±0.28)、(4.83±0.42 VS 0.84±0.26)、(3.95±0.33 VS 0.72±0.18)、(3.90±1.39VS0.28±0.17)、(3.98±0.95 VS 0.32±0.18)、(3.60±±0.93 VS 0.20±0.14)、(3.95±0.40 VS0.32±0.18)、(4.23±0.35 VS 0.36±0.17),差异均有显著地统计学意义(P<0.01)。在mRNA水平,Ⅰ型胶原、Ⅲ型胶原、TGF-(β1、Smad2、Smad3、Smad4在实验组的表达明显高于对照组,分别为：(3.60±2.51 VS 1.0±0.43)、(14.07±6.73 VS 1.00±0.34)、(2.19±0.13 VS 1.00±0.25)、(4.43±1.32 VS 1.00±0.28)、(1.92±0.38 VS 1.00±0.18),差异具有明显统计学意义(P<0.05)。结论：泡球蚴感染中期小鼠肝组织纤维化的主要胶原成分为Ⅰ型胶原、Ⅲ型胶原。在囊泡外围组织纤维化严重的区域,TGF-β1、TβRI、Smad2、Smad3、Smad4和Smad7的表达明显,提示TGF-β1/Smad信号通路参与泡球蚴感染小鼠病灶纤维化过程。
Objective:To investigate the expression of a-SMA, collagen type I, collagen type III, collagen type IV, transforming growth factor-β1, TPRI, p-Smad2/3, Smad4 and Smad7in BALB/c mouse's fibrotic liver infected by Echinococcus multilocularis (Em) during the middle stage of infection (12wk), and to explore the Echinococcus multilocularis-fibrogenic molecular mechanism. Methods:Building Animal models, BALB/c mouse were randomly allocated into two groups:a model group, and a normal group.The E.multilocularis infected mice were injected 0.1mL pooled metacestode lesions into the left liver lobe under opening abdomen, and control mice were injected 0.1mL saline using the same surgical procedure. The mouse of experiment group were killed and to get the tissue of liver the at the end of 12weeks. Some liver pieces were fixed in 10%buffered formalin for histology assay to detect the degree of liver fibrosis by the immunohistochemistry assay. Other liver pieces were snap-frozen in fluid nitrogen immediately, and then conserved in the-80℃refrigerator. The mRNA of liver's a-SMA, collagen typeⅠ, collagen typeⅢ, TGF-β1 TβRI Smad2、Smad3、Smad4、Smad7 were analysed by Quantitative Real-time Reverse Transcription PCR (qRT-PCR). The protein expression and location of a-SMA, collagen type I, collagen type III, collagen type IV, TGF-β1, TβRI, Smad2, Smad3, Smad4, Smad7 were analysed by immuhistochemistry. The variable pathology and the level of liver fibrosis were analysed by Heand Massion and observed by lightmicroscope. Results:The experimental group had lesions characterized by many metacestode vesicles with different sizes and shapes. Hyperplasia of fibro connective tissue with different degrees of fibrosis was apparent around metacestode vesicles.The immunohistochemically result showed that:the expression ofα-SMA, collagen typeⅠ, collagen typeⅢ, collagen typeⅣ, TGF-β1, TβRI, Smad2, Smad3, Smad4 and Smad7 (6.43±0.59 VS 1.48±0.33), (4.43±0.57 VS 1.12±0.28), (4.83±0.42 VS 0.84±0.26), (3.95±0.33 VS 0.72±0.18), (3.90±1.39 VS 0.28±0.17), (3.98±0.95 VS 0.32±0.18),(3.60±0.93 VS 0.20±0.14), (3.95±0.40 VS 0.32±0.18), (4.23±0.35 VS 0.36±0.17). was higher in periparastic granulomas in comparison to the control group. The Quantitative Real-time Reverse Transcription PCR result showed as follows:the mRNA level expression of collagen typeⅠ, collagen typeⅢ, TGF-(31, Smad2, Smad3, Smad4 was (3.60±2.51 VS 1.00±0.43), (14.07±6.73 VS 1.00±0.34), (2.19±0.13 VS 1.00±0.25), (4.43±1.32 VS 1.00±0.28), (1.92±0.38 VS 1.00±0.18), was higher than than in control group. Conclusion:The major collagen in hepatic fibrosis of mice during the middle stage of infection in mice infected by Echinococcus multilocularis were collagen typeⅠand collagen typeⅢ. Pathological injury, fibrosis, and upregulation of TGF-β1, TβRI, Smad2, Smad3, Smad4, Smad7 expression occurred in the mouse live as a result of E. multilocularis infection, which may indicate that TGF-β/Smad pathway was involved in parasite induced hepatic fibrosis in mice.
Objective:To investigate the expression of a-SMA, collagen type I, collagen type III, collagen type IV, transforming growth factor-β1, TPRI, p-Smad2/3, Smad4 and Smad7in BALB/c mouse's fibrotic liver infected by Echinococcus multilocularis (Em) during the middle stage of infection (12wk), and to explore the Echinococcus multilocularis-fibrogenic molecular mechanism. Methods:Building Animal models, BALB/c mouse were randomly allocated into two groups:a model group, and a normal group.The E.multilocularis infected mice were injected 0.1mL pooled metacestode lesions into the left liver lobe under opening abdomen, and control mice were injected 0.1mL saline using the same surgical procedure. The mouse of experiment group were killed and to get the tissue of liver the at the end of 12weeks. Some liver pieces were fixed in 10%buffered formalin for histology assay to detect the degree of liver fibrosis by the immunohistochemistry assay. Other liver pieces were snap-frozen in fluid nitrogen immediately, and then conserved in the-80℃refrigerator. The mRNA of liver's a-SMA, collagen typeⅠ, collagen typeⅢ, TGF-β1 TβRI Smad2、Smad3、Smad4、Smad7 were analysed by Quantitative Real-time Reverse Transcription PCR (qRT-PCR). The protein expression and location of a-SMA, collagen type I, collagen type III, collagen type IV, TGF-β1, TβRI, Smad2, Smad3, Smad4, Smad7 were analysed by immuhistochemistry. The variable pathology and the level of liver fibrosis were analysed by Heand Massion and observed by lightmicroscope. Results:The experimental group had lesions characterized by many metacestode vesicles with different sizes and shapes. Hyperplasia of fibro connective tissue with different degrees of fibrosis was apparent around metacestode vesicles.The immunohistochemically result showed that:the expression ofα-SMA, collagen typeⅠ, collagen typeⅢ, collagen typeⅣ, TGF-β1, TβRI, Smad2, Smad3, Smad4 and Smad7 (6.43±0.59 VS 1.48±0.33), (4.43±0.57 VS 1.12±0.28), (4.83±0.42 VS 0.84±0.26), (3.95±0.33 VS 0.72±0.18), (3.90±1.39 VS 0.28±0.17), (3.98±0.95 VS 0.32±0.18),(3.60±0.93 VS 0.20±0.14), (3.95±0.40 VS 0.32±0.18), (4.23±0.35 VS 0.36±0.17). was higher in periparastic granulomas in comparison to the control group. The Quantitative Real-time Reverse Transcription PCR result showed as follows:the mRNA level expression of collagen typeⅠ, collagen typeⅢ, TGF-(31, Smad2, Smad3, Smad4 was (3.60±2.51 VS 1.00±0.43), (14.07±6.73 VS 1.00±0.34), (2.19±0.13 VS 1.00±0.25), (4.43±1.32 VS 1.00±0.28), (1.92±0.38 VS 1.00±0.18), was higher than than in control group. Conclusion:The major collagen in hepatic fibrosis of mice during the middle stage of infection in mice infected by Echinococcus multilocularis were collagen typeⅠand collagen typeⅢ. Pathological injury, fibrosis, and upregulation of TGF-β1, TβRI, Smad2, Smad3, Smad4, Smad7 expression occurred in the mouse live as a result of E. multilocularis infection, which may indicate that TGF-β/Smad pathway was involved in parasite induced hepatic fibrosis in mice.
引文
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