【作者】 赵礼金；

【导师】 王曙光；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 博士

【摘要】 原发性肝内胆管结石(primary Hepaticlithiasis),即肝胆管结石病,特指始发于肝内胆管系统的结石。是我国常见病、多发病,主要分布在华东、西南、中南地区。肝胆管结石病的病因目前还不完全清楚。已有临床和实验研究资料表明肝胆管结石的形成与胆道慢性炎症、细菌感染等因素有关。以往对原发性肝胆管结石病的基础研究多集中在胆石的成因方面,而从胆管上皮细胞去探讨原发性肝胆管结石的成因机制较少报道。胆管纤维化是肝胆管结石病的基本病理过程,其机制不清。EMT是具有极性的上皮细胞转换成具有活动能力、能够在细胞基质间自由移动的间质细胞过程。它以上皮细胞极性的丧失及间质特性的获得为重要特征,具体包括:细胞粘附分子(E-钙粘蛋白)表达减少;角蛋白为主的细胞骨架转变为波形蛋白(Vimentin)为主的细胞骨架,从而引起细胞形态的改变。TGF-β1是一种公认的的诱导EMT发生的细胞因子,它对细胞侵袭能力的促进作用往往伴随着细胞形态特征从上皮细胞向间叶细胞的转化。由于EMT与肿瘤的浸润及转移有关,因此关于EMT在肿瘤方面研究较多,也有关于EMT在良性病变的报道与研究,如在肝脏纤维化、肾小管纤维化等方面。Helen等人最近报告了在肝移植术后,肝内胆管上皮细胞存在着EMT现象,并且用相应的阻断剂能抑制EMT发生或逆转。Ho-Soon Choi等报告,用LPS剌激胆囊上皮细胞,能使胆囊上皮细胞中TGF-β1mRNA表达增加。这些研究说明在胆道系统中,胆管上皮细胞在特定的环境中(LPS)可能发生EMT。因此我们推测在原发性肝胆管结石形成机制中胆管上皮细胞可能发生EMT。为了检验我们的假想,本实验设计如下:首先,在肝胆管结石组织标本中,采用免疫组织化学的方法检测了上皮性标志物(E-cadherin、α-catenin)、间叶性标志物(Vimentin、α-SMA)在胆管上皮细胞中的的表达,并评价其临床意义;然后,在体外用人肝胆管上皮细胞(HIBEpiC)与LPS共培养,模仿胆道感染早期的变化,观察胆管上皮细胞能否发生EMT转化,分别用阻断及RNA干扰技术,来检测胆管上皮细胞发生EMT是否经过TGF-β1/Smads信号通路,以期探讨肝胆管上皮细胞发生EMT的可能机制。为了检验在活体内LPS能否使肝胆管上皮细胞发生EMT,我们选用SD大鼠,从胆总管注入一定剂量的LPS,在相应时段取胆管组织标本,用免疫组化方法检测上皮标志物和间叶标志物的表达情况。结果:1.31例原发性肝胆管结石胆管上皮细胞中E-cadherin、α-catenin表达阳性主要表现为胞膜呈棕黄色连续线性染色,也可见在胞浆中表达。呈正常阳性表达率分别为67.7%、74.2%,表达缺失率为32.3%、25.8%。Vimentin在中、大等胆管上皮细胞中11例表达阳性,阳性率为35.5%。α-SMA有9例表达阳性,阳性率为29.0%。TGF-β1阳性表达率为38.7%,Smad2/3阳性率为48.4%。Smad 2/3蛋白主要在小胆管上皮细胞核中表达。以上表明,在肝胆管结石胆管上皮细胞中上皮标志物表达缺失,间叶标志物表达阳性,提示原发性肝胆管结石形成过程中胆管上皮细胞可能发生了EMT,且可能是通过TGF-β1-Smad 2/3信号通路传导的。2.LPS刺激人肝内胆管上皮(HIBEpiC )24 h后可测到明显的TGF-β1表达,继续培养至48 h,TGF-β1 mRNA表达到高峰,72 h后开始下降,但仍维持较高水平。说明LPS可促使胆管上皮细胞TGF-β1的释放,且在48 h释放达高峰。3.PCR检测:上皮标志物E-cadherin mRNA的表达随培养时间的延长,其表达逐渐降低,在72 h时更明显。而S100A4和α-SMA的mRNA随培养时间的延长,其表达明显上调。Western blot结果显示,LPS刺激后,E-cadherin蛋白表达明显下调,S100A4和α-SMA蛋白表达明显上调与其mRNA表达水平一致。提示:LPS能诱导HIBEpiC发生上皮-间叶样表型转化。4.在加入LPS的细胞培养基中用紫杉醇(PT)预处理后,TGF-β1、Smad2、Smad3 mRNA表达明显下降,特别是在48 h最明显,与处理前相比,分别下降了60%、36%和28%。这说明,TGF-β1的特异阻断剂PT能阻断其信号通路TGF-β1- Smad2/3中TGF-β1的表达。表明:LPS诱导HIBEpiC发生EMT可能是通过TGF-β1- Smad2/3信号通路传导的。5.转染siRNA Smad2/3 DNA片断48 h后,荧光强度最强,72 h荧光强度逐渐减弱,细胞转染效率78%。荧光定量PCR发现,siRNA Smad2/3 DNA片断转染的胆管上皮细胞中的Smad2/3 mRNA的表达与对照组相比明显下降,而其上皮标志物E-cadherin在转染后表达明显上调,在48、72 h分别上调了64%和20%,间叶标志物S100A4和α-SMA表达明显下调。其中以Smad2/3基因沉默效果最好,在2时相点转染后,其表达下调90%以下。Western blot结果示:与干扰以前相比,48、72 h E-cadherin蛋白表达升高,分别升高了40%、20% ,S100A4和α-SMA表达则下降,在48 h分别下降了20%、35%,在72 h S100A4和α-SMA表达分别下降了21%、44%。48 h与72 h相比,72 h的S100A4和α-SMA沉默效果最好。进一步提示:LPS诱导HIBEpiC发生EMT可能是通过TGF-β1- Smad2/3传导的,而且,Smad2/3为该信号通路中的关健基因。6.LPS剌激SD大鼠胆管上皮细胞后,胆管上皮细胞中间叶标志物(S100A4、α-SMA)蛋白异常阳性表达,而上皮标志物表达缺失。提示:LPS在体内可以诱导胆管上皮细胞发生上皮-间叶样表型转化。结论:1.在原发性肝胆管结石胆管上皮细胞中存在上皮-间叶样表型转化。2.LPS可诱导人肝内胆管上皮细胞发生上皮-间叶样表型转化,并且可能是通过TGF-β1-Smad2/3信号传导的。3.在活体内,LPS可诱导胆管上皮细胞发生上皮-间叶样表型转化。更多还原

【Abstract】 Purpose: Primary hepatolithiasis (HL), a condition marked by the presence of calculus in intrahepatic ducts, is a common disease in China and is especially prevalent in the Eastern and Southern regions of China. Although HL is a benign disease, it is intractable and usually requires operative interventions due to frequent stone recurrences. Most importantly, this disease has a high association with cholangiocarcinoma. Clinical data and experimental studies indicate that hepatolithiasis is associated with chronic inflammation of bile ducts, biliary fibrosis, biliary stricture, and bacterial infections. However, the cellular and molecular etiology of hepatolithiasis has remained elusive. Many previous studies have focused on the formation of stones, while little attention has been paid to the role of bile duct epithelial cells (BEC). Epithelial-mesenchymal transition (EMT) is, by definition, a process whereby epithelial cells convert to migratory mesenchymal cells. This process is characterized by a loss of cell polarity and acquired expression of mesenchymal components. EMT results in a dramatic remodeling of the cytoskeleton, which includes reduced expression of cell adhesion molecules (i.e., E-cadherin) and the transition of keratin-based cytoskeleton into vimentin-based cytoskeleton, thus leading to changes in cell morphology. It has been reported that EMT contributes to both renal fibrogenesis and the fibrogenetic processes that occur in chronic liver diseases such as hepatocirrhosis.Members of the transforming growth factor-β(TGF-β) family are pleiotropic cytokines involved in multiple physiological and pathological processes, including cellular proliferation, adhesion, apoptosis, differentiation and immunoregulatory activities. TGF-β1 activates Smad signaling via its two cell surface receptors—TGF-βtype I receptor (TβRI) and TGF-βtype II receptor (TβRII)—leading to Smad-mediated transcriptional regulation of target genes.Therefore, we assumed that primary hepatolithiasis could be caused by EMT, wherein BECs lose differentiated functions and develop a fibroblast phenotype. This study explored whether intrahepatic biliary epithelial cells undergo EMT during hepatolithiasis by detecting changes in epithelial and mesenchymal markers via immunohistochemical assays. Results:1.Expressions of the epithelial markers E-cadherin andα-catenin were frequently lost in HL samples (32.3% and 25.9%, respectively), while the mesenchymal marker vimentin was found to be present in normal amounts in 35.5% of hepatolithiasis samples;α-SMA was also found to be present in 29.0% of samples. The positive rates of TGF-β1 and Smad 2/3 in the samples paralleled those of the mesenchymal markers, but was comparable between the HLBECs and the control cells (p>0.05).2.Twenty-four hours after the administration of LPS, marked TGF-β1 mRNA was detected. The expression of TGF-β1 mRNA reached the peak at 48 h, but the mRNA level decreased after 72 h. These results suggest that the expression of TGF-β1 was induced by LPS and reached a peak value at 48 h . Therefore, we detected the expression of proteins involved in the EMT process at 48 h and 72 h in the following experiments.3.Along with the exposure to LPS, the mRNA of epithelial markers E-cadherin decreased gradually, whereas the mesenchymal markers (S100A andα-SMA) increased significantly . However, the expression of markers S100A4 andα-SMA at different time points did not show any difference (p >0.05). Consistent with the results of :westblotting, the protein level of E-cadherin decreased , and S100A andα-SMA increased .4.Administration of paclitaxel alone did not change the expression of TGF-β1 mRNA. However, pretreatment with paclitaxel before administration of LPS significantly down-regulated the mRNA of TGF-β1, Smad2/3, especially at 48 h, when the mRNA of TGF-β1, Smad2 /3 decreased by 60%, 36% and 28%, respectively. These results indicated that PT was not a specific antagonist of Smad2/3 because the blockage of Smad2/3 by PT was not as efficient as that of TGF-β1 . We assumed that there were other proteins besides Smad 2/3 involved in the reduced transcription of TGF-β1 that resulted from treatment with PT.5.It was observed that 48 hours after transfection with siRNA Smad2/3, the fluorescence intensity reached its peak, but the fluorescence intensity gradually weakened after 72 hours. The transfection efficiency of HIBEpiCs was 78% (data not shown). The results of real-time PCR showed that the levels of Smad 2/3 mRNA in transfected cells were significantly decreased by even more than 90% at both 48 h and 72 h and that the mRNA of epithelial marker E-cadherin was significantly up-regulated. Both at 48 h and at 72 h, E-cadherin mRNA increased by 64% and 20%, respectively. Expression of mesenchymal markers S100A4 andα-SMA was significantly reduced . It was confirmed that Smad 2/3 played a key role in the TGF-β/Smad signaling pathway. Forty-eight to seventy-two hours after the transfection of siRNA to Smad2/3, the E-cadherin protein in HIBEpiCs increased by 40% and 20%, respectively . The level of S100A4 protein decreased by 21 % and 41%, andα-SMA protein decreased by 35 % and 44% . Inhibition of S100A4 andα-SMA at 72 h was more efficient than that at 48 h (p <0.01), but the expression of E-cadherin showed no difference between the two time points (p >0.05).6.In rat model, the mesenchymal marker vimentin was found to be present, while expressions of the epithelial markers E-cadherin andα-catenin were frequently lost in bile duct epithelial cells of rat with LPS administration, suggesting the development of EMT in bile duct epithelial cells.Conclusions:1.in BECs from patients with primary HL, epithelial markers were lost, the TGF-β1/Smad pathway was activated, and mesenchymal markers were upregulated. These data indicate that TGF-β1-mediated EMT plays a role in the formation of hepatolithiasis.2.LPS can induce the expression of TGF-?1 and a subsequent EMT in HIBEpiCs, and the inhibition of TGF-?1 or Smad 2/3 could reverse this EMT, suggesting that the TGF–?/Smad pathway may be a potential target for the prevention and treatment of biliary fibrosis.3.LPS could also induce the EMT of bile duct epithelial cells in rat model更多还原