【作者】 郑建伟；

【导师】 薛新波；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 原发性肝细胞癌(hepatocellularcarcinoma; HCC)是一种全球高发的恶性肿瘤之一,是我国及某些亚洲地区常见的恶性肿瘤。根据2009年WHO的统计,全世界男性恶性肿瘤发生率中,HCC发病率居于第4位。女性发生率略低于男性发生率,而为第7位。大约每年有近50万男女患者死于本病。在过去短短的20年中,我国HCC从恶性肿瘤死亡原因的第3位升至第2位。临床上肝癌患者的临床特点是：早期无特殊腹部症状,病情较为隐匿,不易被发现及诊断。大约70%-80%患者确诊时己属临床晚期(Ⅲ或Ⅳ期),无法手术治疗。肝癌浸润性生长并早期转移,是导致肝癌术后复发、疗效差最重要的原因之一,这也是导致癌症患者治疗效果不佳、死亡的最常见、最重要的原因。尽管近年来临床上应用许多新的治疗手段,但HCC的整体生存时间并未得到明显改善。肿瘤的基因治疗是近年来临床治疗研究的热点之一,尽管发现了许多与肝癌侵袭及转移相关的靶点分子,如p53、PTEN等,但是相关研究说明,它们只能在部分肝癌中起着抑制肿瘤细胞生长的作用且缺乏一定的特异性。因此,从生物学机制出发,研究和寻觅参与绝大多数肝癌发生及发展过程的关键性的特异的分子靶是现阶段国内外分子肿瘤学研究的热点。MDA-7/IL-24 (Melanoma Differentiation Associated gene-7)是本项目组成员Pual.B.Fisher教授利用亚减法技术于1993年从黑色素瘤细胞中经人IFN-p和MEZ(mezerin, PKC激活剂)诱生而获得的。最初的研究发现该基因在黑色素细胞中高表达而表达量与黑色素瘤细胞恶性度呈负相关,将该基因转染入恶性的黑色素瘤细胞后,发现它具有强烈的生长阻滞、促进分化和促凋亡作用,甚至可以逆转黑色素瘤的异型性,因此被命名为MDA-7,后来根据其生物学特性及染色体部位被命名为IL-24。随后的研究表明MDA-7基因不仅能引起黑色素瘤细胞生长抑制,而且能抑制其它多种肿瘤细胞的生长并促进其凋亡,包括黑色素瘤,肺癌,乳腺癌,胰腺癌,胶质瘤,卵巢癌和前列腺癌等。体外研究中发现,该基因具有强烈的旁路效应。更特别的是,即该基因在杀死肿瘤细胞的同时对正常分化的各种细胞均没有任何毒性作用,也就是说该基因具有强烈的特异选择性杀死肿瘤的特性。本文拟对MDA-7/IL-24对肝癌细胞的生物学作用机制入手,分析MDA-7/IL-24在缺氧及化疗药物作用下,对肝癌细胞侵袭及转移能力作用的变化。首先,我们成功构建携带MDA-7/IL-24基因的腺病毒真核表达载体。在缺氧条件下,观察肝癌细胞及正常肝细胞的变化,使用Ad.mda-7转染缺氧细胞以后分析相关因子在肝癌细胞缺氧情况下的调节机制。使用阿霉素作用肝癌细胞株HepG2后,肝癌细胞对阿霉素的细胞毒作用逐渐耐受,Ad.mda-7作用HepG2后可以明显增加阿霉素对肝癌细胞杀伤作用,并逆转肝癌细胞对阿霉素的多药耐药性。论文分为以下4个部分目的：本文通过构建携带MDA-7/IL-24基因的腺病毒真核表达载体,在正常肝细胞及肝癌细胞中异位表达MDA-7/IL-24基因,探讨MDA-7/IL-24对肝癌细胞的生物学作用。方法：将293细胞培养于6孔板中,使用穿梭质粒pSGCMV MDA-7/pSGCMV-GFP及腺病毒骨架质粒,以Polyfect介导转染293细胞,出现空斑后挑取2个空斑,分别转入500ul 5%DMEM培养液中。结果：阳性空斑可见293细胞呈CPE反应,细胞变大、变圆、漂浮,呈葡萄串聚集。取克隆1和克隆2的病毒DNA各1此为模板,通过PCR扩增出约630bp的特异性片段,其大小与阳性对照相符,而阴性对照腺病毒pSGCMV-GFP的病毒液无片段扩增。结论：成功构建携带DA-7/IL-24基因的腺病毒真核表达载体(Ad.mda-7)。该真核表达载体能转染MDA-7/IL-24基因,使其在正常肝细胞株LO2和肝癌细胞株HepG2细胞中高表达。目的：在缺氧条件下培养肝癌细胞,。比较正常及缺氧条件下,肝癌细胞迁移及转移能力的变化。方法：建立肝癌细胞MHCC-97H体外缺氧模型,将肝癌细胞分组培养正常培养对照组、低氧浓度培养48小时组、72小时组。采用光镜观察缺氧条件下,肝癌细胞的变化。MTT法、Transwell法研究缺氧条件下肝癌细胞生长及肿瘤细胞侵袭及转移能力的变化。荧光定量PCR、Western-blot法分析缺氧环境下肝癌细胞E-cadherin、Twist、Vimentin、FAK的mRNA及蛋白的表达。结论：肝癌细胞在缺氧状态下,细胞生长没有明显被抑制,细胞由间质细胞形态向上皮细胞形态过度,产生EMT,转移及侵袭能力增加。肿瘤细胞的E-cadherin表达进一步降低,而Twist、Vimentin、FAK却明显升高,促进肿瘤细胞侵袭及转移的能力增加。目的：使用Ad.mda-7作用于缺氧条件下的肝癌细胞MHCCC-97H,研究MDA-7逆转肝癌细胞EMT的作用机制。方法：以高转移潜能人肝癌细胞系MHCC-97H和肝正常细胞L02为实验对象,采用缺氧培养诱导肝癌细胞HepG2及MHCCC-97H细胞发生EMT,使用Transwell及MTT观察Ad.mda-7对缺氧肝癌细胞侵袭、转移及生长的作用。使用Real-timePCR及Western-blot观察MDA-7对EMT相关基因E-cadherin, Twist、Vimentin、FAK激酶的变化,阐明MDA-7基因对肝癌细胞上皮细胞间质转化(Epithelial-mesenchymal transition, EMT)的作用的影响。结果：MDA-7基因可以明显逆转肝癌细胞MHCCC-97H EMT的发生。Transwall表明MDA-7明显抑制抑制MHCC-97H侵袭及转移能力。Ad.mda-7明显增加细胞间质蛋白E-cadherinmRNA及蛋白的表达,Real-timePCR及Western-blot分析显示MDA-7能降低肝癌细胞Twist、Vimentin、FAK的mRNA及蛋白的表达(P<0.05)。结论Ad.mda-7通过增加E-cadherin蛋白的活性并抑制EMT相关因子的Twist、Vimentin、FAK的活性,抑制肝癌细胞MHCC-97H转移及侵袭的能力。目的：探讨黑色素瘤分化相关基因-7／白介素-24(mda-7/IL-24)基因联合阿霉素杀伤肝癌细胞HepG2及MHCC-97L并逆转肝癌细胞多药耐药的机制。方法以人肝癌细胞系HepG2及MHCC-97L为实验对象,使用MTT法和流式细胞仪比较Ad.mda-7联合阿霉素处理组与阿霉素组、Ad.mda-7组对肝癌细胞的作用差异。阐明MDA-7/IL-24对多药耐药的逆转作用的机制。实时定量PCR检测MDR-1,STAT-3、BCL-2、BAXmRNA的变化。Westernblot检测gp-170、stat3、P-stat-3、PKB、bcl-2、bax蛋白的表达的变化。结果低浓度的Ad.mda-7联合正常肝细胞的IC50浓度的阿霉素(1.5mg／L)使得细胞抑制率从阿霉素组的17.46%上升到79.5%,生长抑制逆转4.55倍(P<0.05)。联合化疗组MDR-1mRNA相对表达量从16.49±0.11下降至5.48±0.05。STAT-3 mRNA相对表达量从13.17±0.08上升至21.57±0.11。BCL-2及BAX表达与其他实验组相比较均有统计学意义(P<0.05)。联合实验组P-170蛋白的表达量较其他组明显降低,PKB蛋白表达量明显降低,磷酸化stat-3蛋白的表达量亦增加。结论Ad.mda-7具有逆转肝癌细胞HepG2多要耐药的作用,其下调MDR-1mRNA的表达的同时,通过抑制PKB蛋白和活化STAT-3信号通路的表达降低肝癌细胞HepG2对促进肝癌细胞凋亡。综上所述,MDA-7/IL-24基因可以明显逆转EMT,继而抑制肝癌细胞侵袭及转移能力的变化。在多药耐药中机制中,MDA-7也可以通过抑制内质网应激蛋白的作用及相关耐药蛋白的表达增加化疗药物对肿瘤细胞的杀伤作用。这也提示我们MDA-7不仅仅将是治疗肝癌的一个有效的分子靶标,而且是探索肝癌生物学机制的一个有效工具。更多还原

【Abstract】 Primary hepatocellular carcinoma (Hepatocellularcarcinoma; HCC) is a high incidence of malignant tumors in the world and also the common malignant tumors in China and some Asia era. According to WHO 2009 statistics, the incidence of HCC in male in the world, HCC incidence of living in the first four. Female incidence rate was slightly lower than male, but for the first seven. About 50 million men and women each year die from the disease patients. During the last 20 years, Chinese HCC patients causes of death from cancer No.3 to No.2. The liver cancer patients features in clinical are early non-specific abdominal symptoms, the condition is quite more hidden, difficult to detect and diagnose. About 70% to 80% patients had a clinical diagnosis they were late to do the surgery(III orⅣ).Liver cancer and the invasive growth and early metastasis were leaded to tumor recurrence and poor efficacy of one of the most important reason, which is leading to poor treatment of cancer patients, the death of the most common and most important reason. Although the clinical application in recent years, many new treatments, but the overall survival time of HCC has not been significantly improved. Cancer gene therapy clinical research in recent years one of the hot, Although found in many liver cancer invasion and metastasis-related target molecules, such as p53, PTEN, etc., but research shows that they are only part of the liver plays a role in inhibiting the growth of tumor cells and a certain lack of specificity. Therefore, starting from the biological mechanisms, research, and seeking participation in the development of vast majority of liver cancer and the critical molecular target specific molecular oncology at home and abroad at the present stage of researchPaper is divided into the following four parts.Objective:MDA-7 gene consists of seven exons and six introns, and IL-10, IL-19, IL-20 co-located on the 1st chromosome, also known as IL-24. We constructed the adenovirus vector carrying MDA-7/IL-24 gene. Using denovirus vector transfected the normal liver cells and liver cancer cells which were ectopic expression of MDA-7/IL-24 gene, studied on the biological effects of MDA-7/IL-24 on liver cancer cells. Methods: cultured the 293 cells in 6 well plate, using the PSGCMV MDA-7/pSGCMV-GFP shuttle plasmid and adenovirus backbone plasmid to Polyfect mediated transfection of 293 cells. appear plaque after plaque picked two were transferred to 500ul 5%DMEM culture medium. Results:the Plaque visible CPE in 6 well plate. the cells become large, rounded, floating, grapes were gathered. Clone 1 and clone 2 get a virus DNA in 1 of this as a template, PCR amplification of specific fragments of about 630bp, its size in line with the positive control, while the negative control adenovirus pSGCMV-GFP virus solution without PCR amplification.Objective:Under hypoxia environment, cultured the hepatoma cells.Comparison the ability of cells migration and metastasis changing in the different condition. Methods: established the hypoxia model of hepatoma cell line MHCC-97H in vitro. cultured the hepatoma cell in the normal and hypoxia group, each groups were cultured for 48 h,72 h. MTT and Transwell found the cell growth of MHCC-97H under the hypoxia and the change of tumor cell invasion and metastasis. The expression of E-cadherin, Twist, Vimentin, FAK of mRNA and protein were analysised by quantitative PCR and Western-blot. Results:the Hepatoma cells cultured in the hypoxia that no significant differentance the tumor cell growth was inhibited. The hepatric cells were change from the stromal cells form over to the epithelial cells, finally resulting in EMT. The cellls metastasis and invasion ability were increased. Tumor cells were further reduced the expression of E-cadherin but the Twist, Vimentin, FAK were significantly increased and then it promoted tumor cells to invasion and metastasis.Objective To explore the mechanism of the Melanoma differentiation associated gene-7(MDA-7/IL-24) inhibit hepatocellular carcinoma cell line MHCC-97H invasion and metastasis. Methods Based on the human hepatoma carcinoma cell(MHCC-97L) and hepatic cell line LO2, Using FCM and Transwall to observe the ability of invasion and metastasis after Ad.mda-7 treated MHCC-97H and LO2. Using immunofluorescence staining to indicate the expressing of E-cadherin in the heptaic cell lines. The expression levels of TWIST、Vimentin and E-cadherin was using Real-time PCR. Western blot perform to observe the change of protein twist, Vimentin,E-cadherin and FAK among teams. Result After transfection with 1000VP/cell Ad.mda-7, the growth of MHCC-97H had been arrested but no to LO2. After Ad.mda.7 transfected MHCC-97H, the EMT was reversed in the tumor cells. MHCC-97H marked CFSE and then FCM showed that MDA-7 selectively arrested MHCC-97H cell proliferation (P＜0.05). Then, transwall showed that MDA-7 could inhibited the tumor cell invasion and metastasis (P＜0.05) Finally, Real-timePCR and Western-blot showed that mda-7 could block the expressing of Twist, Vimentin and FAK were decreased.Objective To explore the mechanism of the Melanoma differentiation associated gene-7(HepG2) enhance adriamycin(ADM) kill the hepatoma carcinoma cell and reverses multidrug resistance(MDR). Methods Based on the human hepatoma carcinoma cell(HepG2), Using MTT assay and FCM to determine the difference among the 100VP/cell Ad.mda-7 add ADM, ADM, Ad.mda-7.To interpret the MDA-7/IL-24 reverses multidrug resistance. The expression levels of MDR-1,STAT-3,BCL-2,BAXmRNA was using real-time PCR. Westernblot was perform to observe the change of protein gp-170, stat-3, P-stat3, PKB, bcl-2, bax among those teams. Result After transfection with 100VP/cell Ad.mda-7, the growth suppression rate of HepG2 which was treated by ADM(1.5mg/L) rising from 17.46% to 79.5%.According to the change, killed cell of HepG2 was increase 4.55 time. It is important that real-time PCR showed MDR-1 mRNA was decreased from(16.49±0.11) to (5.48±0.05) and STAT-3 mRNA increased from (13.17±0.08) to (21.57±0.11) (P＜0.05).WB analyses also showed that P-170 and PKB was decreased after and the phosphorylation-stat-3 was increased. Conclusion Ad.mda-7 can reverse the multidrug resistance of ADM to HepG2. It inhibit the expression of MDR-1 mRNA, then arrest PKB protein active the signing of stat-3 to induce hepatoma carcinoma cell apoptosis.After all, MDA-7/IL-24 gene can significantly reverse the EMT, the inhibition of liver cancer cell invasion and metastasis of change. In the multi-drug resistance mechanisms, MDA-7 can also inhibit the endoplasmic reticulum stress the role of proteins and related proteins increase resistance of chemotherapeutic drugs on tumor cells in vitro. It also suggested that not only will we MDA-7 is an effective treatment of liver cancer molecular target, but also to explore the biological mechanisms of liver cancer is an effective tool.更多还原