大鼠胆汁淤积性肝内胆管损伤早期动态病理变化实验研究
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摘要
[目的]观察大鼠梗阻性黄疸后胆汁淤积性肝损伤过程中胆管上皮细胞早期动态病理变化及确定肝纤维化的时间点,并通过同时期细胞角蛋白(CK-19)、转化生长因子1(TGF-β1)、Ⅳ型胶原免疫组织化学变化规律来讨论肝损伤后肝纤维化病理变化过程中小胆管上皮细胞的作用。
[方法]结扎大鼠胆总管(ligating common bile duct,LCD)的大鼠为模型,使大鼠形成梗阻性黄疸(OJ),观察OJ 1、3、7、14 d大鼠一般状况,肝功能:TBil、ALP、GGT、Alb肝组织病理学及CK-19、TGF-β1、Ⅳ型胶原免疫组织化学观察。
[结果]①与对照组大鼠比较,模型组大鼠血清Alb水平呈持续下降趋势,GGT活性及TBil水平持续增高,ALP水平持续上升1周后略下降。②肝脏病理组织学变化:胆总管结扎后原有肝内胆管上皮细胞呈扁平形,管腔略宽大。第3天汇管区、小叶周边已可见到新生小胆管样上皮细胞,1周后可见新生小胆管样结构,2周时汇管区及小叶周边部分呈纤维化改变,但未形成完整纤维间隔。③肝组织CK-19、TGF-β1、Ⅳ型胶原免疫组织化学染色观察:胆总管结扎后3天大鼠的肝组织汇管区、小叶周边部新生小胆管样上皮细胞CK-19表达呈弱阳性,1周时新生小胆管样上皮细胞数量增多,CK-19表达增强,2周后汇管区小叶周边部小胆管样上皮细胞已形成新生小胆管,CK-19阳性细胞表达增多增强。Ⅳ型胶原免疫组织化学染色观察1周时已有胆管外周基底膜及胶原纤维层增厚,2周时自汇管区有细纤维隔延肝界板延伸但未形成完整纤维间隔。TGF-β1在1周时阳性表达增加,肝内分布均匀,胆管增生区和正常肝细胞区域均表达,2周时表达最强。
[结论]①结扎大鼠胆总管后第3天有新生小胆管上皮细胞生成,后期形成新生小胆管。②新生小胆管增生趋势与激活肝纤维化主要细胞因子TGF-β1表达相一致,肝内原有及新生胆管上皮细胞可能为胆汁性肝损伤引起的胆小管周围纤维化及肝纤维化启动的主要激活细胞。③术后1周时肝组织已有纤维化形成迹象,但胆汁性肝损伤后细纤维隔延肝界板延伸标志着肝纤维化病理过程开始的时间点可能为术后第2周。
【Objective】To observe the early dynamic pathological change in bile duct epithelial cells in obstructive jaundice and to determine the time point of liver fibrosis,and also to discuss the role of the bile duct epithelial cells in the development of liver fibrosis by the immunohistochemistry study of CK-19,TGF-β_1,typeⅣcollagen.
【Methods】We ligated the common bile duct in the rats to establish the liver injury model.Rats were divided into 5 groups:Ⅰ(control group),Ⅱ(lst day after the ligation),Ⅲ(3st day after the ligation),Ⅳ(7st day after the ligation),Ⅴ(14st day after the ligation).Samples were collected from the different groups.The levels of serum(Tbil,ALP,GGT,Alb)were tested,and HE stainning and Immunohistochemistry stainning of CK-19,TGF-β_1,typeⅣcollagen were performed in the rat's liver tissues.
【Results】①Serum Alb decreased compared with sham-operated group;GGT and TBil increased continuously after ligation of the common bile duct,ALP increased first and dropped a little bit after one week.②Histopathology in liver:Epithelial cells of Intrahepatic bile duct became flat after ligation and more epithelial cells appeared around acini hepatis after 3 days,the new bile ducts formed after 1 week,hepatic fibrosis was starting around acini hepatis after 2 weeks,but fiber intermission was not completely formed.③Immunohistochemistry study of CK-19,TGF-β_1 typeⅣcollagen in hepatic tissue:CK-19 in epithelial cells of small bile duct surrounding the Ministry of lobular showed weak positive after 3 days of ligation.The number of bile duct epithelial cells increased and CK-19 expression became stronger in one week. New small bile appeared after two weeks,in the mean time CK-19 expression became even more stronger.Base membrane of bile duct and collagen layer were thickened after one week.Small fiber compartment extended but fiber interval was not completely formed after 2 weeks.TGF-β_1 expression increased evenly in liver tissue after 1 week and reached the strongest expression after 2 weeks.
【Conclusion】①The new epithelial cells appeared after 3 days ligation of the common bile duct,and new bile duct formed later.②The formation of the new bile duct was bile duct,and new bile duct formed later.②The formation of the new bile duct was accorded with TGF-β_1 expression.And epithelialis cells of bile ducts were one of the chief activated cells in the development of hepatic fibrosis③Quasi- hepatic fibrosis was formed after 1 week ligation of the common bile duct.After 2 weeks small fiber compartment extended but fiber interval was not completely formed yet.That may mark the beginning of the hepatic fibrosis.
[方法]结扎大鼠胆总管(ligating common bile duct,LCD)的大鼠为模型,使大鼠形成梗阻性黄疸(OJ),观察OJ 1、3、7、14 d大鼠一般状况,肝功能:TBil、ALP、GGT、Alb肝组织病理学及CK-19、TGF-β1、Ⅳ型胶原免疫组织化学观察。
[结果]①与对照组大鼠比较,模型组大鼠血清Alb水平呈持续下降趋势,GGT活性及TBil水平持续增高,ALP水平持续上升1周后略下降。②肝脏病理组织学变化:胆总管结扎后原有肝内胆管上皮细胞呈扁平形,管腔略宽大。第3天汇管区、小叶周边已可见到新生小胆管样上皮细胞,1周后可见新生小胆管样结构,2周时汇管区及小叶周边部分呈纤维化改变,但未形成完整纤维间隔。③肝组织CK-19、TGF-β1、Ⅳ型胶原免疫组织化学染色观察:胆总管结扎后3天大鼠的肝组织汇管区、小叶周边部新生小胆管样上皮细胞CK-19表达呈弱阳性,1周时新生小胆管样上皮细胞数量增多,CK-19表达增强,2周后汇管区小叶周边部小胆管样上皮细胞已形成新生小胆管,CK-19阳性细胞表达增多增强。Ⅳ型胶原免疫组织化学染色观察1周时已有胆管外周基底膜及胶原纤维层增厚,2周时自汇管区有细纤维隔延肝界板延伸但未形成完整纤维间隔。TGF-β1在1周时阳性表达增加,肝内分布均匀,胆管增生区和正常肝细胞区域均表达,2周时表达最强。
[结论]①结扎大鼠胆总管后第3天有新生小胆管上皮细胞生成,后期形成新生小胆管。②新生小胆管增生趋势与激活肝纤维化主要细胞因子TGF-β1表达相一致,肝内原有及新生胆管上皮细胞可能为胆汁性肝损伤引起的胆小管周围纤维化及肝纤维化启动的主要激活细胞。③术后1周时肝组织已有纤维化形成迹象,但胆汁性肝损伤后细纤维隔延肝界板延伸标志着肝纤维化病理过程开始的时间点可能为术后第2周。
【Objective】To observe the early dynamic pathological change in bile duct epithelial cells in obstructive jaundice and to determine the time point of liver fibrosis,and also to discuss the role of the bile duct epithelial cells in the development of liver fibrosis by the immunohistochemistry study of CK-19,TGF-β_1,typeⅣcollagen.
【Methods】We ligated the common bile duct in the rats to establish the liver injury model.Rats were divided into 5 groups:Ⅰ(control group),Ⅱ(lst day after the ligation),Ⅲ(3st day after the ligation),Ⅳ(7st day after the ligation),Ⅴ(14st day after the ligation).Samples were collected from the different groups.The levels of serum(Tbil,ALP,GGT,Alb)were tested,and HE stainning and Immunohistochemistry stainning of CK-19,TGF-β_1,typeⅣcollagen were performed in the rat's liver tissues.
【Results】①Serum Alb decreased compared with sham-operated group;GGT and TBil increased continuously after ligation of the common bile duct,ALP increased first and dropped a little bit after one week.②Histopathology in liver:Epithelial cells of Intrahepatic bile duct became flat after ligation and more epithelial cells appeared around acini hepatis after 3 days,the new bile ducts formed after 1 week,hepatic fibrosis was starting around acini hepatis after 2 weeks,but fiber intermission was not completely formed.③Immunohistochemistry study of CK-19,TGF-β_1 typeⅣcollagen in hepatic tissue:CK-19 in epithelial cells of small bile duct surrounding the Ministry of lobular showed weak positive after 3 days of ligation.The number of bile duct epithelial cells increased and CK-19 expression became stronger in one week. New small bile appeared after two weeks,in the mean time CK-19 expression became even more stronger.Base membrane of bile duct and collagen layer were thickened after one week.Small fiber compartment extended but fiber interval was not completely formed after 2 weeks.TGF-β_1 expression increased evenly in liver tissue after 1 week and reached the strongest expression after 2 weeks.
【Conclusion】①The new epithelial cells appeared after 3 days ligation of the common bile duct,and new bile duct formed later.②The formation of the new bile duct was bile duct,and new bile duct formed later.②The formation of the new bile duct was accorded with TGF-β_1 expression.And epithelialis cells of bile ducts were one of the chief activated cells in the development of hepatic fibrosis③Quasi- hepatic fibrosis was formed after 1 week ligation of the common bile duct.After 2 weeks small fiber compartment extended but fiber interval was not completely formed yet.That may mark the beginning of the hepatic fibrosis.
引文
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[12]陶艳艳 刘成海 TGF-β_1刺激肝星状细胞活化与肝纤维化的分子病理机制研究进展中西医结合肝病杂志2006;16(2):125-129
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[2]姜军 普通外科实验动物模型的特点和基本要求 中国普外基础与临床杂志2001;11(8):87-90
[3]李德旭 杨镇 邱新光等 猪胆汁性肝纤维化的形成机制研究中华实验外科杂志2003;1(20):16-20
[4]Bucher NLR,Farmer S.Liver regeneration after partialhepatectomy:genes and metabolism [A].In:Strain AJ,Diehl AM.Liver Growth and Repair[M].London:pp.Chapman & Hall,1998;27-30.
[5]颜天华 肿瘤坏死因子肝损伤机理研究进展 国外医学药学分册 1998;12(25):330-335
[6]李春霞 戴立里 肝星状细胞与肝纤维化研究进展 国际消化病杂志 2007;4(27):108-115
[7]周斌 张培建 胆管上皮细胞的生理及其与胆管疾病的相关性 中国普通外科杂志2007;16(7):680-683
[8]葛明建 王梅 李良彬 等CK19 RT-PCR法检测淋巴结中肺癌微小转移灶 中国肺癌杂志2000;2(3)1:23-25
[9]苏勤 刘彦仿 龚民族 大鼠胚胎肝内胆管的发生机制-细胞角道白和层粘连蛋白免疫组织化学研究 第四军医大学学报1994;15(3):75-77
[10]谢明 周建华 文继舫 蒋海英 乳腺癌基质金属蛋白酶-9及前哨淋巴结CK19 mRNA表达的相关性 实用医学杂志 2007;(23)7:102-105
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[12]陶艳艳 刘成海 TGF-β_1刺激肝星状细胞活化与肝纤维化的分子病理机制研究进展中西医结合肝病杂志2006;16(2):125-129
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