结缔组织生长因子在肝细胞上皮-间充质转分化及肝纤维化形成中的作用研究进展

纤维化是器官(肝、肾、肺等)慢性功能衰竭的一个共同机制,目前尚无有效防治方法.对上皮-间充质转分化(epithelial-to-mesenchymal transition,EMT)的研究使人们对器官纤维化的传统认识受到挑战,逆转分化已被认为是未来抗肝纤维化治疗的重要策略之一[l].肝细胞EMT被认为是肝纤维化时肝内肌成纤维细胞(myofibroblast)即活化肝星状细胞(HSC)的一种新的重要来源,对肝纤维化形成具有决定性影响[2-3].日益增加的证据表明,结缔组织生长因子(connective tissure growth factor,CTGF)可能介导了肝细胞EMT及肝纤维化形成[4-5].现就近年来CTGF在肝细胞EMT及肝纤维化形成中的作用研究进展作一综述.     

胆管上皮细胞增生在胆管结扎大鼠胆汁性肝纤维化形成中的作用

目的 研究胆管上皮细胞(biliary epithelia cells,BECs)增生在胆管结扎(bile duct ligation,BDL)大鼠胆汁性肝纤维化形成中的作用.方法 BDL的方法制作大鼠胆汁性肝纤维化模型,5周后杀鼠取材,观察内容包括大鼠血清总胆红素(TBlL)、总胆汁酸(TBA),肝组织羟脯氨酸(Hyp)含量,组织病理学,免疫组织化学观测Ⅰ型胶原(Col Ⅰ)、Ⅳ型胶原(Col Ⅳ)、层粘蛋白(LN)、纤连蛋白(FN)和α-平滑肌肌动蛋白(α-SMA);肝组织片激光显微切割(LCM)获取BECs,实时荧光定量PCR检测BECs表达Procollagen α1(Ⅳ)、血小板衍生生长因子B(PDGF-B)、结缔组织生长因子(CTGF)及转化生长因子β1(TGF-β1)mRNA.结果 1、胆管结扎模型大鼠血清TBiL和TBA含量均值分别为假手术组大鼠的22倍和3倍(P＜0.01);模型组大鼠肝组织Hyp含量是假手术组的4倍(P＜0.01);2、模型组大鼠BECs增生非常显著,肝实质细胞比例显著减少;3、模型组大鼠在增生胆管周围的LM表达显著增加;FN不但在肝窦内有阳性染色,还强烈地表达于增生的胆管周围;肝窦壁的Col Ⅰ阳性染色增强,增殖的BECs周围未见明显阳性染色;肝窦壁ColⅣ表达未见明显变化,但强烈表达于增殖的BECs周围的基底膜上;α-SMA阳性染色见于汇管区周围的肌成纤维细胞上,且这些肌成纤维细胞常常围绕在增生的胆管上皮细胞周围.4、模型组大鼠BECs的Procol α1(Ⅳ)、PDGF-B、TGF-β1及CTGF的mRNA表达量均显著增加(与假手术组比较,均为P＜0.001).结论 BECs增生是BDL大鼠胆汁性肝纤维化的启动因素和中心病理环节,抑制胆管上皮细胞的异常增生及其细胞生物学病理变化应是抗胆汁性肝纤维化治疗学研究的主要目标.     

淤胆型肝炎 不同配比黄芪汤干预大鼠胆汁淤积性肝硬化作用观察

目的：观察不同配比的黄芪汤（3：1、6：1、9：1）干预大鼠胆汁淤积性肝硬化的药理作用。方法：结扎大鼠胆总管制备大鼠胆汁淤积性肝硬化模型；结扎1周后，将模型大鼠随机分为模型组和药物干预组，药物干预分别给予不同配比的黄芪汤经口灌胃。模型组给予等量生理盐水；用药4周后杀鼠取材。     

内皮素-1诱导大鼠肾小管上皮细胞转分化的可能机制

目的:探讨内皮素-1(ET-1)是否可诱导大鼠肾小管上皮细胞转分化(TEMT)及可能的分子机制.方法:体外培养大鼠肾小管上皮细胞(NRK52E)并进行分组;倒置显微镜观察细胞的形态变化;Western印迹法检测细胞E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)、p38丝裂原活化蛋白激酶(p38MAPK)及磷酸化-p38MAPK(p-p38MAPK)蛋白的表达;逆转录-聚合酶链反应(RT-PCR)法检测细胞E-cadherin及α-SMA mRNA的表达. 结果:ET-1可诱导细胞由鹅卵石样变为梭形,下调E-cadherin表达,上调α-SMA、p38 MAPK及p-p38MAPK表达,增强p38MAPK活性(P＜0.05),而内皮素A受体拮抗剂BQ123能明显抑制这些变化(P＜0.05).p38MAPK特异性抑制剂SB203580可抑制ET-1诱导的细胞梭形性变,ET-1诱导的E-cadherin、α-SMA及p-p38MAPK表达改变及p38MAPK活性改变(P＜0.05),但对p38MAPK表达无明显影响(P＞0.05). 结论:ET-1可能通过激活肾小管上皮细胞p38 MAPK通路,下调E-cadherin的表达,同时上调d-SMA的表达,从而诱导肾小管上皮细胞转分化.     

血管内皮间质样转分化遗传示踪小鼠模型的构建及其在肝纤维化研究中的应用

目的 构建Cdh5-Cre^ERT/Acta2-tdTomato-STOP^floxed-eGFP knockin遗传示踪小鼠,并探究其在肝纤维化血管内皮细胞转化研究中的应用。方法 将Cdh5-Cre^ERT小鼠和Acta2-KI小鼠进行交配繁育,经PCR基因型鉴定得到Cdh5-Cre^ERT/Acta2-KI遗传示踪小鼠。通过分离、培养原代肝血窦内皮细胞（LSEC）和建立CCl₄肝纤维化模型,取LSEC和肝脏组织,分别进行免疫荧光染色,观察荧光蛋白tdTomato和eGFP的表达。结果 经他莫昔芬诱导后,Cdh5-Cre^ERT/Acta2-KI遗传示踪小鼠的LSEC和肝脏组织在体内外建立的内皮-间质样转分化条件下可表达eGFP,而未经诱导的对照组只表达tdTomato。结论 成功构建的Cdh5-Cre^ERT/Acta2-KI遗传示踪小鼠可实现对血管内皮-间质样转分化的有效标记,并为肝纤维化中肌纤维母细胞来源的多样性提供新的遗传示踪学依据。     

沙利度胺对肺泡上皮细胞上皮-间质转分化的作用

目的探讨沙利度胺对A549上皮-间质转分化的作用,阐明其抗肺纤维化的机理。方法 A549细胞分为3组,A:对照组;B:TGF-β1+DMSO组;C:TGF-β1+沙利度胺组。通过免疫印迹法检测A549细胞磷酸化p38 MAPK和JNK的蛋白,以及间质表型蛋白和上皮表型蛋白的表达;实时荧光定量PCR方法检测A549细胞p38 MAPK和JNK mRNA的表达。结果沙利度胺显著抑制(P0.05)TGF-β1诱导的A549细胞间质表型蛋白表达增加,可阻止(P0.05)A549细胞上皮表型蛋白表达的减少,显著减少活性MAPK蛋白及mRNA的表达(P0.05)。结论沙利度胺可能通过p38MAPK和JNK信号通道,从而抑制肺泡上皮细胞的上皮-间质转分化过程而发挥抗纤维化作用。     

胆汁淤积导致肝纤维化的机制及其阻断策略

胆汁淤积是指胆汁流形成、分泌和排泄障碍的一种病理状态,而肝纤维化是肝损伤引起的组织修复过程。胆汁淤积性肝病是由胆汁淤积、胆管渐进性破坏和肝内炎症持续存在所导致的一种慢性肝病,引起胆管细胞和肝细胞损伤,并逐步进展为肝纤维化。结合目前的研究进展,对胆汁淤积导致肝纤维化的发生机制和阻断策略作一综述。     

蛋白酶活化受体2在大鼠胆汁淤积性肝纤维化肝组织中的表达及其意义

目的 观察胆汁淤积性肝纤维化大鼠肝组织中蛋白酶活化受体2(PAR2)的动态变化,探讨其在肝纤维化形成中的意义. 方法 建立大鼠胆汁淤积性肝纤维化模型,同时设立假手术组和正常对照组,Western blot及RT-PCR检测术后2、4、6、8周肝组织中PAR2蛋白及mRNA的表达水平,并分析其与Ⅰ、Ⅲ型胶原表达水平的关系.对数据进行重复测量资料的方差分析.结果 肝纤维化组大鼠2周时即可见PAR2 mRNA及蛋白水平升高,随着肝纤维化时间的延长,4～6周时其表达水平进一步升高(P＜ 0.05),8周时达高峰(相对表达量分别为1.62±0.11、47.6±2.8,P＜ 0.01).Ⅰ、Ⅲ型胶原蛋白表达水平也随着肝纤维化程度的加重而升高,6～8周时达高峰(相对表达量均为65.6±2.7),且与PAR2的表达水平呈一致性.结论 PAR2在胆汁淤积性肝纤维化中的动态表达可能参与并促进肝纤维化的形成和发展.     

大鼠胆汁淤积性肝硬化的动态病理变化及其意义

目的：观察胆汁淤积性大鼠肝硬化形成过程中模型动态变化特点。方法：结扎大鼠胆总管制备大鼠胆汁淤积性肝硬化模型；结扎后3天、1周、2周、3用、4周、5周后杀鼠取材，观测大鼠一般状况，肝功能、肝组织羟脯氨酸（Hyp）含量测定，肝组织病理学及CK-19免疫组织化学观察。结果：与假手术组大鼠比较。模型组大鼠肝重、脾重、肝／体比、脾／体比均呈先增加后降低的动态变化；血清Alb含量持续下降，而TP含量呈先高后低的变化；血清ALT活性在结扎3天后最高，2周时最低；GGT活性及TBil含量自造模后呈持续增高；而ALP活性在2周时最高；肝组织Hyp含量从结扎后呈持续增加；结扎3天后汇管区已见胆管增生，至3周时增生逐渐加重。CK-19免疫组织化学染色观察，胆管上皮细胞第3天起开始增生，1周、2周时增生明显，3周后增生渐减。4周、5周时肝组织呈花环样改变，肝实质细胞显著减少。可见肝细胞去分化现象。结论：①胆汁淤积性大鼠肝硬化模型主要病理改变有胆汁淤积、胆管上皮细胞增生、肝细胞减少、胶原增生和沉积与新生的胆管上皮细胞关系密切。②血清ALT水平由高到低，5周末仅比假手术组稍高。AST活性一直维持在较高水平。③肝细胞的减少方式除了凋亡、坏死外，可见去分化现象。     

上皮细胞间质转换或分化与肝纤维化发生的研究进展

随着对肝纤维化发病机制研究的深入,上皮细胞间质转换或分化(epithelial to mesenchymal transition or transdifferentiation,EMT)在肝纤维化中的作用逐渐受到重视,现将EMT及其与肝纤维化相关的研究作一综述. 一、EMT的标志及其分类 EMT表现为细胞失去极性或失去细胞间的黏附等上皮特征,从而获得迁移特性.E钙黏蛋白是上皮细胞特有的表达分子.     

新型生长抑素类似物SOM230减轻胆总管结扎大鼠肝纤维化

目的 应用胆总管结扎大鼠肝纤维化模型,观察长效生长抑素类似物SOM230对胆管结扎所致大鼠肝纤维化的治疗作用.方法 雄性SD大鼠80只,随机分为模型组、高剂量预防组(80 mg/kg SOM230)、低剂量预防组(8 mg/kg SOM230)、假手术组及正常对照组.分别在术后第2周和第4周取心脏血及肝组织,观察肝组织...     

内质网应激参与蛋氨酸-胆碱缺乏饮食诱导大鼠脂肪性肝纤维化的发生与恢复

目的 探讨内质网应激在非酒精性脂肪性肝纤维化形成中的作用及饮食控制对脂肪性肝纤维化恢复的影响.方法 蛋氨酸-胆碱缺乏饮食 (MCDD) 喂养10周诱导非酒精性脂肪性肝纤维化大鼠模型,恢复组于第9周开始将MCDD转变为蛋氨酸-胆碱对照饮食 (MCCD) 喂养2周.纤维化和炎症程度采用组织病理染色方法检测,纤维化和炎症相关因子采用Western blot和实时定量聚合酶链反应 (RT-PCR) 方法检测,内质网应激标记物半胱氨酸天冬氨酸蛋白酶 (caspase)12、7和葡萄糖调节蛋白78 (GRP78) 采用免疫组织化学、Western blot和RT-PCR方法检测.计量资料采用统计分析软件SPSS13.0中的ANOVA程序进行单因素方差分析或q检验,并用LSD进行两两比较.结果 饮食由MCDD转变为MCCD后,组织病理学结果显示肝细胞脂肪沉积及炎症反应明显减轻,伴随着活化的肝星状细胞和枯否细胞减少,肝纤维化程度明显减轻.模型组大鼠肝细胞凋亡数为每5个高倍视野 (68.2±15.9) 个,明显高于正常组 (40.3±8.3) 个,P<0.05;肝细胞增殖/凋亡比值 (0.10±0.03) 明显低于正常组(0.19±0.03),P<0.01.恢复组大鼠肝细胞凋亡数为每5个高倍视野 (48.0±6.5)个,明显低于模型组 (68.2±15.9) 个,P<0.05;增殖数为每5个高倍视野 (17.2±4.4)个,明显高于模型组 (7.5±3.0) 个,P<0.01;肝细胞增殖/凋亡比值 (0.41±0.09) 也明显高于模型组 (0.10±0.03),P<0.01.模型组大鼠肝组织GRP78、caspase12、caspase7和cleaved caspase7蛋白质和mRNA表达均明显高于正常组(P<0.05或P<0.01),恢复组均明显低于模型组 (P<0.05或P<0.01).结论 大鼠饮食由MCDD转变为MCCD后,肝脏的炎症和纤维化程度迅速逆转,提示饮食摄取对于控制肝脏脂肪沉积和病理改变至关重要,且内质网应激参与了这一过程,阻断内质网应激尤其是Caspase12通路也许有助于非酒精性脂肪性肝纤维化的临床治疗.     

Effect of hepatic iron concentration reduction on hepatic fibrosis and damage in rats with cholestatic liver disease

AIM: To assess the effect of iron reduction after phlebotomy in rats with "normal" hepatic iron concentration (HIC) on the progression of hepatic fibrosis, as a result of bile duct ligation (BDL). METHODS: Rats underwent phlebotomy before or after sham operation or BDL. Animals undergone only BDL or sham operation served as controls. Two weeks after surgery, indices of hepatic damage and fibrosis were evaluated. RESULTS: Phlebotomy lowered HIC. Phlebotomy after BDL was associated with body weight increase, lower hepatic weight, less portal hypertension, less periportal necrosis, less portal inflammation, lower hepatic activity index score and higher albumin levels. On the other hand, phlebotomy before BDL was associated with body weight decrease and hepatic activity index score increase. Phlebotomy after sham operation was not associated with any hepatic or systemic adverse effects. CONCLUSION: Reduction of HIC after induction of liver damage may have beneficial effects in BDL rats. However, iron deficiency could induce impairment of liver function and may make the liver more susceptible to insults like BDL.     

A comparison of gene expression in mouse liver and kidney in obstructive cholestasis utilizing high-density oligonucleotide microarray technology

AIM: To assess the effects of obstructive cholestasis on a wider range of gene expression using microarray technology. METHODS: Male C57BL/6J mice underwent common bile duct ligation (BDL) and were matched with pair-fed sham-operated controls. After 7 d,the animals were sacrificed and total RNA was isolated from livers and kidneys. Equal amounts of RNA from each tissue were pooled for each group and hybridized to Affymetrix GeneChip(?)MG-U74Av2 containing a total of 12488 probe sets. Data analysis was performed using GeneSpring(?) 6.0 software. Northern analysis and immunofluorescence were used for validation. RESULTS: In sham-operated and BDL mice, 44 and 50% of 12488 genes were expressed in livers, whereas 49 and 51% were expressed in kidneys, respectively. Seven days after BDL, 265 liver and 112 kidney genes with GeneOntology annotation were up-regulated and 113 liver and 36 kidney genes were down-regulated in comparison with sham-operated controls. Many genes were commonly regulated in both tissues and metabolism-related genes represented the largest functional group. CONCLUSION: Following BDL, microarray analysis reveals a broad range of gene alterations in both liver and kidney.     

Dietary glycine blunts liver injury after bile duct ligation in rats

AIM： To investigate the effects of （dietary） glycine against oxidant-induced injury caused by bile duct ligation （BDL）.
METHODS： Either a diet containing 5% glycine or a standard diet was fed to male Sprague-Dawley （SD） rats. Three days later, BDL or sham-operation was performed. Rats were sacrificed 1 to 3 d after BDL. The influence of deoxycholic acid （DCA） in the presence or absence of glycine on liver cells was determined by measurement of calcium and chloride influx in cultivated Kupffer cells and lactate dehydrogenase （LDH） activity was determined in the supernatant of cultivated hepatocytes.
RESULTS： Serum alanine transaminase levels increased to about 600 U/L 1 d alter BDL. However, enzyme release was blunted by about two third in rats receiving glycine. Release of the alkaline phosphatase and aspartate aminotransferase was also blocked significantly in the group fed glycine. Focal necrosis was observed 2 d after BDL. Glycine partially blocked the histopathological changes. Incubation of Kupffer cells with DCA led to increased intracellular calcium that could be blocked by incubation with glycine. However, systemic blockage of Kupffer cells with gadolinium chloride had no effects on transaminase release. Incubation of isolated hepatocytes with DCA led to a significant release of LDH after 4 h. This release was largely blocked when incubation with glycine was performed.
CONCLUSION： These data indicate that glycine significantly decreased liver injury, most likely by a direct effect on hepatocytes. Kupffer cells do not appear to play an important role in the pathological changes caused by cholestasis.     

Fibrosis in autoimmune and cholestatic liver disease

Autoimmune and cholestatic liver disease account for a significant part of end-stage liver disease and are leading indications for liver transplantation. Especially cholestatic liver diseases (primary biliary cirrhosis and primary sclerosing cholangitis) appear to be different from other chronic liver diseases with regards to pathogenesis. Portal fibroblasts located in the connective tissue surrounding bile ducts appear to be different from hepatic stellate cells with regards to expression of marker proteins and response the profibrogenic and mitogenic stimuli. In addition there is increasing evidence for a cross talk between activated cholangiocytes and portal myofibroblasts. Several animal models have improved our understanding of the mechanisms underlying these chronic liver diseases. In the present review, we discuss the current concepts and ideas with regards to myofibroblastic cell populations, mechanisms of fibrosis, summarize characteristic histological findings and currently employed animal models of autoimmune and cholestatic liver disease.     

消黄方对二甲基亚硝胺致肝纤维化大鼠肺组织氧化应激及巨噬细胞激活的影响

目的 观察消黄方对二甲基亚硝胺( DMN)诱导的肝纤维化过程中大鼠肺组织氧化应激及巨噬细胞激活的干预作用.方法 腹腔注射DMN 4周制备大鼠肝纤维化模型,4周末处死全部大鼠.检测肺组织超氧化物歧化酶(SOD)活性,谷胱甘肽(GSH)及丙二醛( MDA)含量;免疫印迹、免疫组化及实时定量PCR检测CD68表达.结果 肺组织SOD活性及GSH含量在DMN染毒2周即降至较低水平,染毒4周维持低水平状态.MDA含量在DMN染毒2周到达较高水平,消黄方显著降低MDA含量并显著升高SOD活性及GSH含量.免疫组化显示正常组CD68在肺泡周呈弱阳性表达,DMN染毒过程中,CD68阳染进行性增强,消黄方组CD68阳染较弱.结论 DMN诱导肝纤维化过程中肺组织氧化应激和巨噬细胞激活紊乱.消黄方显著抑制肺组织氧化应激及巨噬细胞激活.     

Thy1.1阳性肝脏卵圆细胞在大鼠肝硬化形成与消减过程中的动态表达

目的 肝脏卵圆细胞（HOC）在二甲基亚硝胺（DMN）致大鼠肝硬化形成与消减过程中表达的动态变化，探讨其病理生理意义。方法 应用DMN腹腔注射（4周12次）制备大鼠肝硬化模型，分别于造模后3d、2、4周及终止造模后2、4周处死大鼠取肝组织，进行常规组织学观察，透射电镜下观察HOC超微结构，免疫组织化学和western blot方法检测Thy1．1的表达变化，应用图像分析法对所标记的HOC进行半定量测定以及光镜下计数细胞数量。结果 DMN造模4周大鼠已形成典型的肝硬化；终止造模后2周时肝组织病变较造模4周时有所减轻，终止造模后4周时炎症明显减轻并以不完全纤维间隔为主。Thy1．1阳性染色细胞正常大鼠肝组织未见到，造模后3d时可见微量表达，2周时呈散在分布，4周时明显增多，见于纤维间隔周围，终止造模后2周、即第6周时阳性染色显著强于造模4周，大量出现于汇管区，8周时较6周有所减少，表达量基本与4周时相等。阳性染色图像分析、光镜下阳染细胞计数及western blot三者的结果呈一致性。Western blot结果与免疫组织化学观察结果也具一致性。电镜下显示HOC具有形态小，核以卯圆型为主，高的核／浆比例等特点。结论 Thy1．1阳性的HOC可能与肝硬化的消减或逆转有关。