【作者】 李天宇；

【导师】 王曙光；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 近年来,国内外大量流行病学和实验室研究发现,丙型肝炎病毒(hepatitis C virus,HCV)感染与胆管癌发生关系密切。HCV核心区基因编码的核心蛋白(hepatitis C virus core protein,HCVc),在细胞的信号转导、蛋白的相互作用、致癌及脂质代谢中起着重要作用,它可以通过激活NF-κB信号通路参与胆管癌的发生。上皮-间叶样表型转化(epithelial-mesenchymal transition,EMT)是上皮细胞来源的恶性肿瘤的重要生物学现象,其实质是上皮细胞获得了成纤维细胞样表型,特征是细胞标志物改变和细胞内骨架重排,结果是细胞间粘附减弱、细胞运动能力大大增强。鉴于NF-κB信号通路在诱导EMT过程中的重要作用以及EMT可以促进肿瘤细胞的侵袭和转移,我们研究了HCVc蛋白与胆管癌细胞EMT的关联性,旨在证明HCVc蛋白可以诱导胆管癌EMT发生从而促进其侵袭和转移的可能性。赖氨酰氧化酶样2基因(lysyl oxidase-like 2 enzyme gene,LOXL2)是EMT的关键因子之一,由于其在胆管癌组织中存在着较高表达,并与胆管癌组织的分化、侵袭和转移密切相关,我们同时探讨了LOXL2在HCVc蛋白诱导胆管癌细胞EMT中的作用,旨在初步阐明胆管癌EMT的发生机理。为了检验我们的假想,本实验设计如下:首先,在胆管癌组织标本上,采用免疫组织化学的方法检测了HCVc蛋白、上皮性标志物(E-cadherin、α-catenin、β-catenin)、间叶性标志物(N-cadherin、vimentin、fibronectin)在胆管癌癌组织中的表达,并评价其临床意义;然后,转染含有HCVc全长基因序列的重组质粒载体到胆管癌细胞QBC939中,观察转染后细胞形态的变化,并检测细胞上皮性标志物、间叶性标志物及LOXL2的表达情况;最后,通过共转染HCVc基因和LOXL2 shRNA基因到QBC939中,检测转染后细胞上皮性标志物、间叶性标志物的表达变化情况,以评价LOXL2在HCVc蛋白诱导胆管癌细胞EMT中的作用。结果:1.34例胆管癌组织中HCVc蛋白的阳性表达率是47.1%,上皮性标志物的缺失率分别是E-cadherin 55.9%,α-catenin 70.6%,β-catenin 55.9%,间叶性标志物的阳性表达率分别是N-cadherin 50%,vimentin 44.1%,fibronectin 55.9%,HCVc蛋白的阳性表达与E-cadherin和α-catenin的缺失及N-cadherin、vimentin和fibronectin的阳性表达相关(P值均<0.05),并与胆管癌组织的淋巴结及其它脏器转移具有相关性(P值均<0.05)。这些结果提示,HCVc蛋白可能通过诱导胆管癌组织上皮-间叶样表型转化的发生,促进了胆管癌的侵袭和转移。2.经过基因测序鉴定,PGST-HCVc195含有HCVc全长基因序列,质粒转染靶细胞后,RT-PCR及real-time PCR检测到HCVc mRNA的表达,免疫细胞化学和Western blotting检测到HCVc蛋白的表达。结果表明,HCVc基因成功地转染到胆管癌细胞QBC939中,目的基因得到高表达。3.重组质粒转染细胞5天后,空载体对照组细胞维持母细胞的上皮样表型,而表达HCVc基因的实验组细胞转化为梭形,变得离散;通过RT-PCR、real-time PCR、免疫细胞化学和Western blotting等方法进一步检测转染HCVc基因后胆管癌细胞标志物的表达变化,发现上皮性标志物E-cadherin在实验组细胞中的表达水平显著低于对照组细胞,同时间叶性标志物Vimentin、Fibronectin在实验组细胞中的表达明显高于对照组,这进一步提示,HCVc蛋白诱导胆管癌细胞发生了上皮-间叶样表型转化。4.细胞运动和侵袭实验结果显示,转染HCVc基因的实验组细胞运动和侵袭能力显著强于空载体对照组细胞,这提示,HCVc蛋白诱导胆管癌细胞发生上皮-间叶样表型转化后,提高了细胞的运动和侵袭能力。5.转染PGST-HCVc195重组质粒及PGEX-3ks空载体的实验组和对照组QBC939细胞48小时后,通过real-time PCR和Western blotting的方法检测LOXL2基因和蛋白的表达,结果显示:转染HCVc基因的实验组细胞LOXL2基因和蛋白的表达水平明显高于空载体对照组细胞,说明HCVc基因可能上调了LOXL2基因转录,增加了其蛋白的表达。6.经过酶切和基因测序鉴定,证明LOXL2 shRNA基因正确插入到所构建的重组质粒载体中,在其与HCVc基因共转染靶细胞后,荧光显微镜下实验组及对照组均可见绿色荧光蛋白的表达,通过real-time PCR和Western blotting的方法检测,发现两组细胞均有HCVc mRNA和蛋白的表达,同时,通过real-time PCR、免疫细胞化学和Western blotting的方法也检测到LOXL2 mRNA及蛋白的表达实验组明显低于空载体对照组,这说明,HCVc及LOXL2 shRNA基因成功地转染胆管癌细胞QBC939,目的基因得到较好的表达或沉默。7.重组质粒共转染细胞48小时后,通过real-time PCR和Western blotting的方法检测转染HCVc和LOXL2 shRNA基因后,胆管癌细胞标志物的表达变化,发现上皮标志物性E-cadherin在实验组细胞中的表达水平显著高于对照组细胞,而间叶性标志物Vimentin、Fibronectin在实验组细胞中的表达明显低于对照组,结果提示,LOXL2在HCVc蛋白诱导胆管癌细胞发生上皮-间叶样表型转化中发挥了重要作用。结论:1. HCVc蛋白可以诱导胆管癌细胞发生上皮-间叶样表型转化,其特征是胆管癌细胞形态由圆形、椭圆形、多角形,排列紧密,转化为梭形,离散;E-cadherin等上皮性标志物表达下调,Vimentin、Fibronectin等间叶性标志物表达增强,而且发生上皮-间叶样表型转化后,胆管癌细胞的运动和侵袭转移能力显著提高。2. LOXL2在HCVc蛋白诱导胆管癌细胞发生上皮-间叶样表型转化中发挥了重要作用。更多还原

【Abstract】 Purpose: Cholangiocarcinoma (CC) is associated with chronic hepatitis C virus(HCV) infection. Hepatitis C virus core protein(HCVc) is an important protein product encoded by the HCV genome, it’s function involved in interaction of cell protein, cell signal transduction, carcinogenesis and lipids metabolism. It perhaps participates carcinogenesis of cholangiocarcinoma by triggering NF-κB signaling pathways. Epithelial-mesenchymal transition(EMT) was defined by the formation of mesenchymal cells from epithelia in different embryonic territories. Recently, increasing evidences suggested that EMT was involved in cancer invasion and metastasis. In a view of significance of NF-κB signaling pathway in EMT, we studied the correlation between HCVc protein and CC EMT, intended to prove HCVc protein can induce CC EMT. The high positive expression of LOXL2 which is a key regulator of EMT was observed in CC tissues and the positive expression of LOXL2 was associated with CC tissues’invasion and metastasis, so we studied the effect of LOXL2 in the CC EMT induced by HCVc protein to preliminaryly reveal the mechanism of CC EMT.Experimental Design: In the first part of the experiment, we examined the expression of HCVc protein, epithelial markers:E-cadherin、β-catenin andα-catenin, mesenchymal markers:N-cadherin、vimentin and fibronectin by immunohistochemistry, and assessed their correlation and clinic pathological significance. In the second part of the experiment, we transfected a recombinant plasmid vector containing HCVc gene into QBC939 cell to observe the morphological change under microscope 5 days after transfection.The expression and localization of HCVc and LOXL2 proteins, epithelial and mesenchymal markers were determined by RT-PCR、Realtime PCR、immunocytochemistry and western blotting. In the third part, we co-transfected recombinant plasmid vector containing HCVc gene and LOXL2 shRNA to assesse the effect of LOXL2 in QBC939 EMT induced by HCVc protein.Results:1. In CC tissues, the positive expression rate was observed in 47.1% for HCVc protein, 50% for N-cadherin, 44.1% for Vimentin, 55.9% for Fibronectin and the decreased expression rate was E-cadherin for 55.9%,α-catenin for 70.6%,β-catenin for 55.9%, The positive expression of HCVc protein were associated with the decreased expression of E-cadherin、α-catenin and the positive expression of N-cadherin、Vimentin、Fibronectin(P<0.05), a positive-correlation between the expression of HCVc protein and metastasis of ganglia lymphatica and other organs were found(P<0.05). HCVc protein perhaps promote cholangiocarcinoma tissues’infiltration and metabasis by inducing it’s EMT.2. The HCVc gene was obtained from PGST-HCVc195 plasmid. After it transfected into QBC939 cell, the high expression of HCVc mRNA and protein were observed by RT-PCR and Realtime PCR, immunocytochemistry and western blotting. 3. The QBC939 cell transfected with HCVc gene underwent a morphological change from a classic epithelial morphology to a spindle-like shape 5 days after transfection. In addition to this, the HCVc protein significantly decreased E-cadherin expression, but increased vimentin and fibronectin expression in QBC939 cell.4. The cell migration and invasion assays indicated that transfection of HCVc gene drastically enhanced the migratory and invasive potentials of QBC939 cell.5. After the QBC939 cell transfected with HCVc gene 48 hours, the expression of LOXL2 was significantly increased by HCVc protein.6. The HCVc gene was obtained from PGST-HCVc195 plasmid and LOXL2 shRNA gene was obtained from pGenesil-shLOXL2, after they co-transfected into QBC939 cell, the high expression of HCVc and the decreased expression of LOXL2 mRNA and protein were observed by RT-PCR、Realtime PCR、immunocytochemistry and western blotting.7. After co-transfection 48 hours, the decreased expression of E-cadherin and increased expression of vimentin and fibronectin induced by HCVc protein were inhibited by LOXL2 gene interference in QBC939 cell.Conclusions: The clinic pathological data indicated that loss of E-cadherin and gain of vimentin, fibronectin may lead to a more aggressive tumor behavior in CC tissues, it may has relation with HCVc protein. HCVc gene transfection data suggested that it can induce QBC939 cell EMT. The result of HCVc gene and LOXL2 shRNA co-transfection indicated that LOXL2 can inhibit EMT induced by HCVc protein in QBC939 cell, so we presumed that QBC939 undergoing EMT maybe triggered by the key EMT-inducing factor LOXL2. Furthermore, HCVc protein enhances migratory and invasive potentials of QBC939 cell.更多还原