重组腺病毒介导反义C-myc基因对人肝癌细胞治疗的有效性及安全性研究

【作者】 余昌中；

【导师】 应大君；

【作者基本信息】 第三军医大学 ， 人体解剖学， 2001， 博士

【摘要】 肝癌的发病率和死亡率高，是我国第二位常见恶性肿瘤，传统的手术、化疗及放射治疗效果均不理想，为此，国内外都在寻找和开展肝癌治疗新方法—基因治疗的研究。对肝癌相关基因的研究，使人们清楚的认识到肝癌的发生发展是一个多种因素、多种机制、多个阶段的复杂过程，其中涉及到多种癌基因的激活和抑癌基因的功能丧失。阻断或抑制癌基因的高表达是肿瘤基因治疗的策略之一，为此反义技术得以发展应用，并在一些肿瘤的基因治疗中取得良好的效果而具有广泛应用的前景。 c-myc属于细胞原癌基因，其编码的62KD核蛋白通过与核蛋白Max形成二聚体结合特异的DNA序列（CACTGT）激活许多靶基因的转录，其中很多基因产物与细胞周期调控密切相关。myc癌基因的异常表达是形成肿瘤的步骤之一，同时myc基因又与其它癌基因在肿瘤形成的复杂过程中具有协同作用。从细胞癌变到肿瘤形成再到转移侵润的启动、促癌、演进及转移各个阶段，c-myc均与其它一些原癌基因被激活参与作用。c-myc癌基因的高表达参与了肝癌的形成和生长。通过反义RNA抑制c-myc基因的过表达，使依赖其基因产物的肿瘤细胞生长周期改变、发生凋亡，有报道在前列腺癌及转移乳腺癌的基因治疗中有效。 随着基因转移技术的不断完善和分子生物学的飞速发展，基因治疗已从实验室走向临床。理想的基因转移载体是基因治疗能够进入临床的关键之一。在众多的基因转移方法中，腺病毒载体介导的基因转移，以其高转染率和良好的靶向性而成为肿瘤基因治疗中广为使用的方法。腺病毒载体具有很好的嗜肝向性，体内外研究均证明了腺病毒载体介导的基 因在肝脏细胞的高效转移。但是，腺病毒载体介导的肝癌基因治疗的真 正应用于临床，还必需解决临床安全性问题，即其临床使用对机体的毒 副作用。因此临床应用前对其安全性进行评价至关重要。 本研究采用兔疫组化法，以检测C－myc、hcf毛癌基因在肝癌组织中 表达情况，探讨其在肝癌发生发展中的作用及其在判断预后中的价值。 采用构建成功的Ad－ASmyc，利用反义基因在翻译水平上阻断c－p的过 表达，抑制肝癌细胞的生长并促使其凋亡，以寻找肝癌基因治疗新的有 效方法。经Beagle犬肝动脉、门静脉途径注射重组腺病毒介导反义c－D叹c 基因 （Ad－ASm）载体，观测其在体内是否能有效转导入正常肝细胞、持 续时间、体内器官分布及毒副作用，以评价AdAS侧治疗肝癌的临床 前期安全性。 本研究获得以下主要结果： 1．原发性肝癌组织中C侧C蛋白阳性表达率较高，B＊K蛋白阳性表 达率虽然较低，但其阳性表达往往伴随着C 4r1V4r1VC蛋白阳性表达，二者联合 检测有助于判断肝癌的恶性程度及预后。 2．重组腺病毒介导的反义 C－pC基因（A－ASmyC）滴度较高，可达到 5 X 10” PFMl，并可高效转导入斗种人肝癌细胞系①ELj402、QSG－ 7701、SMMCJ、HCCq204X人胚肺H倍体细胞ZBS及正常人肝细 胞LOZ细胞。 3．Ad－AS啊c自够引起人肝癌细胞c－p、hcf－2 RNA及 c－myC、hcf－2 蛋白表达下调，抑制肝癌细胞生长和导致肝癌细胞凋亡。 4．c－p c表达水平不同的肝癌细胞系，Ad·ASmyc的作用效果不同， 与细胞c－msc表达水平高低有关，c－msc表达高者治疗效果更明显。说明 反义C－my C基因疗法对不同的细胞类型治疗效果有差异性。 5．通过两种肝癌细胞系①ELJ402、S删CJ）裸鼠皮下移植瘤的 体内实验证实，瘤内直接注射Ad－AS呷治疗裸鼠皮下移植肝癌有效。 6．经犬肝动脉或门静脉途径注射AdASIny均可持续转导至肝细胞 达3周，病毒可播散至脾脏、肾脏、胃、心脏及皮肤，但对组织器官无明 ·Vll· X》显毒副作用，仅在较大剂量时肝脏组织镜下可见Ad－ASmrc剂量依赖性的轻微炎症反应。 7．注射Ad－ASmyc后，血清产生针对腺病毒载体的中和抗体，大剂量Ad叭Smyc犬较小剂量A小ASmyC犬抗体滴度高，但平均抗体滴度皆较低。临床应用AdASmyc进行肝癌的基因治疗是安全可行的。 本研究结果表明，原癌基因C－IT［yC在肝癌组织及人肝癌细胞系中均为高表达；AdAS侧对体外培养的人肝癌细胞系及裸鼠体内皮下移植肝癌治疗有效；经犬肝动脉或门静脉途径注射AdAS啊C对机体无明显毒副作用，为临床应用Ad－ASmyc基因治疗肝癌提供了有力的实验基础。更多还原

【Abstract】 Hepatocellular carcinoma ( HCC ) is one the commonest cancer in the world and one the most difficult tumors to treat. The morbidity and mortality of this disease is high. The most effective therapy is surgical resection, but still limited. The results of other approaches, such as chemotherapy or radiotherapy are also disappointing. Therefore, it is urgent to explore a new and effective treatment method. Gene therapy, although in its infancy at the present time, may have a significant role to play in the future management of HCC. Proto-oncogene c-myc encodes a nuclear 62KD phosphoprotein, which acts as a key regulator of cell growth and differentiation, when dimerized with its partner protein Max, c-myc functions as transcription factor, capable of both activating and repressing transcription. Increased expression of c-myc is seen in many cancers such as breast, prostate, lung, and others. Importantly, elevated expression of c-myc combined with other oncogene has a synergistic effect in the development of tumor. Overexpression of c-myc exists in all stage from the tumorigenesis to growth of tumor, metastasis in many tumors and so does in the development of HCC. Thus, using antisense RINA blocks the overexpression of c-myc gene in HCC cells, the decreased expression of c-myc gene can contribute to change the cells growth cycle and induce apoptosis. This kind of antisense gene therapy may be a potential clinical utility for HCC. With the improvement of gene transferring system and the progression of molecular biology, gene therapy had performed human clinical trial. First, efficient gene transfer is essential for successful gene therapy. A number of gene transfer vectors have been developed. Adenoviral vectors transduce dividing and non-dividing cells of tissues with efficiency. These vectors have been used to transduce foreign genes into a variety of tumor cells. Adenoviral vectors can efficiently infect normal hepatocytes in vitro and in vivo because of ?III their natural tropism for hepatocytes. Second, the safety required to be evaluated before adenovirus mediated gene transfer vector really becomes a feasibility therapy for clinical liver cancer. In this study, immunohistochemical stainning technique (S-P) was used to evaluate the effect of the expression of c-myc, Bcl-2 protein in the carcinogenesis and tumor development of hepatocellular carcinoma and their clinical values. The observation of morphological changes, growth curve, DNA ladder, FCM, RT-PCR and Western blot analysis after hepatocellular cells infected Adenovirus-mediated antisense c-myc gene (Ad-ASmyc) were used in this study to explore the inhibitory effect of Ad-ASmyc on growth of hepatocellular cells in vifro and Three kinds dose of Ad-ASmyc, 1O7pfu~ lO2pfii and ~o~pftj, for treatment of hetero-transplanted tumor in nude mice was studied in vivo. Ad-ASmyc was infused into the hepatic artery or portal vein in Beagle dogs to observe its transduction efficiency, duration, distribution in organs, toxicity and to assess the preclinical safety of Ad-ASmyc. Conclusion presented in this study can be summarized as follows: 1. The higher positive expression of c-myc was found in HCC tissues. The positive expression of Bcl-2 protein was lower and associated with the positive expression of c-myc protein. c-myc and Bcl-2 were associated with the occurrence and development of I-ICC and both com更多还原