【作者】 王刚；

【导师】 吴小兵；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2011， 博士

【摘要】 肝细胞癌(简称肝癌)是最为常见的肝脏原发性恶性病变。由于传统的治疗手段如手术切除、肝移植和化疗的治疗效果不理想,迫切需要研究和发展新的治疗方法。腺病毒(Ad)介导的胸苷激酶/更昔洛韦(TK/GCV)自杀基因治疗是有临床应用前景的肝癌治疗方法,已经进入到Ⅱ期临床试验。研究发现,随着腺病毒治疗剂量的增加,抑瘤效果加强,但是TK/GCV系统对癌旁正常肝细胞的杀伤和腺病毒自身的肝毒性限制了其应用。microRNA-122(miR-122)是一种肝脏特异性microRNA (miRNA),在肝细胞中高表达,而在肝细胞癌中表达下降或缺失。因此,利用miR-122调控自杀基因表达有可能解决TK/GCV对正常肝细胞杀伤的问题。肝癌自杀基因治疗的另一个关键问题是需要研究安全高效的基因导入系统。重组8型腺相关病毒(rAAV8)是新发现的高度肝嗜性载体,具有无肝毒性和介导外源基因长期高效表达的特点。最近,静脉注射rAAV8至诱发型小鼠肝癌模型,成功地在肝癌细胞中表达了治疗基因。因此,rAAV8可能克服腺病毒由于自身肝毒性不能高剂量使用的缺点,成为肝癌自杀基因治疗的高效病毒载体。本研究有如下两个研究目的：一是利用miR-122调控机制在肝细胞癌中特异性表达外源基因,避免自杀基因治疗的肝毒性,提高安全剂量；二是探索rAAV8载体是否可以体内高效转导肝细胞癌并用于肝癌的自杀基因治疗。为此,进行了以下研究：首先,通过在分泌型荧光素酶Gluc基因的3’非翻译区(3’UTR)插入不同miRNA的靶序列构建了一系列miRNA传感器质粒,并将其通过水动力方法导入小鼠肝脏,研究miR-122等肝脏不同丰度miRNA对转基因的调控效率。其次,通过在报告基因和TK基因的3’UTR插入miR-122靶序列构建miR-122调控的报告基因和自杀基因表达质粒,将其通过水动力方法导入小鼠肝脏,研究miR-122调控的报告基因在肝脏的表达和TK/GCV系统的肝毒性。然后,选择低表达miR-122的肝癌细胞株建立移植型小鼠原位肝癌模型,研究小鼠静脉注射和瘤内注射rAAV8对肝脏组织和肝癌组织的转导效率,并与腺病毒载体进行比较,评价其用于肝癌自杀基因治疗的可能性。最后,根据前期结果选择Ad和／或rAAV8为载体,通过静脉注射和/或瘤内注射的途径转导miR-122调控的TK基因至上述小鼠肝细胞癌模型,并给予GCV处理,进行TK/GCV自杀基因治疗,评价其治疗效果和安全性,为肝癌的基因治疗提供新思路。主要研究结果如下：1.利用以Gluc为报告基因的miRNA传感器结合外周血Gluc检测建立了体外长期监测小鼠肝脏miRNA活性的方法,并成功监测肝脏内源性和外源性miRNA的活性。利用miRNA传感器研究了不同miRNA靶序列的插入对肝脏转基因表达的调控,发现miR-122靶序列对转基因的抑制显著高于miR-142和miR-34a。Gluc为报告基因的miRNA传感器为研究体内miRNA活性和体内miRNA对转基因的调控作用带来了便利。2.通过水动力法将miR-122调控的报告基因和TK基因转染小鼠肝脏,发现4个拷贝的miR-122靶序列的插入可以显著抑制报告基因在肝脏的表达,并有效避免了TK/GCV自杀基因治疗系统的肝毒性。证实了miR-122靶序列是一个高效的基因表达负调控元件,在基因治疗中可以利用miR-122强大的负调控机制避免自杀基因治疗的肝毒性。3.利用低表达miR-122的Hepa 1-6细胞建立了小鼠原位移植型肝细胞癌模型。研究了静脉注射rAAV8对正常小鼠和肝癌模型小鼠的转导特性,结果发现虽然静脉注射rAAV8介导了转基因在肝脏长期稳定的表达,但难以有效转导Hepa1-6移植瘤。又比较了瘤内注射rAAV8和Ad对肝癌模型小鼠的转导,结果发现rAAV8瘤内注射也难以转导Hepa 1-6移植瘤；而Ad瘤内注射则可以有效的转导Hepa 1-6肝癌细胞。鉴于Ad瘤内注射转导Hepa1-6移植瘤效率显著高于rAAV8,下一步miR-122调控的TK/GCV系统治疗小鼠原位肝癌的研究采用了Ad为载体经瘤内注射给药。4.小鼠Hepa1-6肝癌模型瘤内注射腺病毒介导的不含miR-122靶序列的报告基因发现,肿瘤和肝脏均有转基因的表达,并且肝脏中的表达甚至高于肿瘤；而瘤内注射腺病毒介导的miR-122调控的报告基因发现,报告基因在肿瘤中的表达无抑制,但在肝脏中的表达受到了显著抑制,从而造成外源基因在肿瘤中的表达显著高于肝脏,实现了外源基因在肿瘤中的特异性表达。5.小鼠Hepa 1-6肝癌模型瘤内注射腺病毒介导的miR-122调控的TK基因(Ad-TK-122T)和对照病毒Ad-TK并给予GCV处理发现,Ad-TK-122T和Ad-TK的抑瘤作用均随注射剂量的增加而加强,miR-122靶序列的插入不影响TK/GCV系统的抑瘤效果；与Ad-Fluc (Fluc,萤火虫荧光素酶)对照相比,1×1010 vp的Ad-TK-122T和Ad-TK均出现了显著的抑瘤作用。肝毒性评价发现,与Ad-Fluc相比,Ad-TK的肝毒性随剂量增加也显著增强,当达到1×1010vp剂量时出现显著肝毒性并造成小鼠死亡；与Ad-Fluc相比,1×1010 vp的Ad-TK-122T注射小鼠则未出现肝毒性。因此,利用miR-122调控的TK基因的表达可以避免TK/GCV系统的肝损伤,提高Ad瘤内注射安全剂量,从而获得更好的肝癌治疗效果。上述研究结果表明,miR-122靶序列是一个高效的肝脏基因表达负调控元件；利用miR-122调控机制可以实现外源基因在肝细胞癌中的特异性表达；利用miR-122调控机制可以提高肝癌自杀基因治疗中腺病毒瘤内注射的安全剂量,提高肿瘤的治疗效果。同时表明, rAAV8具有强大的肝嗜性,但并不意味着具有肝细胞癌嗜性,至少对于移植性Hepa1-6肝癌的转导效率很低；提示在肝癌基因治疗中需要选择对rAAV8易感的肿瘤模型进行自杀基因治疗,或者利用rAAV8高度嗜肝性特点在肝脏表达分泌型的抗肿瘤基因。更多还原

【Abstract】 Suicide gene therapy for hepatocellular carcinoma (HCC) using the adenovirus (Ad) vectors expressing herpes simplex virus thymidine kinase (TK) gene and ganciclovir (GCV) has shown promise in preclinical and clinical studies. However, this strategy was hampered by hepatotoxicity of Ad vectors and severe hepatic damage resulting from TK/GCV killing normal hepatocytes around the tumor. microRNA-122 (miR-122) is an abundant, liver-specific microRNA (miRNA) and found down-regulated or no expression in HCC. Using endogenous miR-122 to regulate TK expression may avoid killing the liver cells surrounding tumors by GCV treatment. Adeno-associated virus 8 (AAV8) is a new serotype and allows 100% transduction of hepatocytes and long-term gene expression without liver toxicity, but there is few data about the ability for recombinant AAV8 (rAAV8) vectors to transduce hepatocellular carcinoma in vivo.One aim of this research was to evaluate the regulation on transgene expression mediated by miR-122 and its ability of restricting the expression of therapeutic genes specifically in HCC rather than in normal liver cells to improve efficiency and safety of liver cancer suicide gene therapy; another aim was to investigate the ability for rAAV8 vectors to transduce HCC in vivo and the feasibility of applying to liver cancer gene therapy. First, sensor plasmids for detecting miRNA activities were constructed by adding miRNA target sequences to the 3’-untranslated region (3’UTR) of Gaussia princeps luciferase (Gluc) expression cassette. Sensor plasmids were delivered to mice by hydrodynamic tail vein (HTV) injection and the abilities for miR-122 and other miRNAs to regulate transgene expression in the liver were investigated. Then, the miR-122 regulated TK expressing plasmid was constructed by inserting miR-122 target sequences into the 3’UTR of TK gene and delivered to mice by HTV injection. The hepatotoxicity of TK/GCV system was evaluated by serum ALT, body weight and histopathological examination of liver. Then, Hepal-6 cells, a HCC cell line with down-regulated miR-122 expression were inoculated in liver to establish murine liver cancer models. rAAV8 or Ad vectors were given to tumor-bearing mice by intravenous or intratumoral injection and their transduction efficiencies to the liver and HCC were evaluated. Then, tumor-bearing mice were treated with intratumoral injection of different doses of Ad vectors expressing miR-122 regulated TK gene followed by treatment with GCV, and antitumor effects and liver toxicity were estimated.Our study showed delivering Gluc-based miRNA sensors to mice by HTV injection and then assaying Gluc activity in blood offered a sensitive and quantitative method for ex vivo monitoring of in vivo microRNAs in mouse liver. Results suggested CAG promoter-driven miRNA sensor was suitable for the long-term monitoring of endogenous miRNA activities such as miR-122, miR142 and miR-34a, as well as exogenously delivered miRNA. The Gluc expression in liver was inhibited significantly by insertion of the miR-122 target sequences (502.9-766.4-fold) rather than insertion of the miR-142 or miR-34a target sequences (6.8-10.8-fold). Moreover, the insertion of the miR-122 target sequences in the TK expression cassette avoided hepatotoxicity of GCV treatment.In our study, rAAV8 vectors demonstrated high efficiency of transgene expression in liver. However, rAAV8 vectors showed inefficiency of gene transfer to inoculated Hepal-6 HCC tissues either by intravenous or by intratumoral injection. Relative high transgene expression in HCC cells was acquired after intratumoral injection with Ad vectors, but there was higher tansgene expression in liver than tumor.Our study showed intratumoral injection with Ad vectors containing the miR-122 target sequences led to much higher tansgene expression in the tumor than the liver. Tumor-bearing animals received different doses of Ad vectors expressing TK by intratumoral injection and then treated with GCV. Both Ad vector bearing TK gene alone (Ad-TK) or with miR-122 target sequences (Ad-TK-122T) acquired viral dose-dependent antitumor effects. The liver toxicity was significantly elevated for Ad-TK, but no significant liver toxicity had been found for Ad-TK-122T, compared with the control vector Ad-Fluc (Fluc, Firefly luciferase). Intratumoral injection of Ad-TK-122T at dose of 1×1010 virus particles per mouse acquired significant antitumor effect without evident hepatotoxicity, but injection of Ad-TK at the same dose resulted in heavy hepatotoxicity and even death of mice.The research demonstrates that the strong regulation ability of miR-122 for transgene expression in the liver and the feasibility of utilizing miR-122 as a negative regulation element to prevent hepatotoxicity in liver-directed suicide gene therapy. miR-122 regulation can be used in intratumoral injection of Ad vector mediated TK/GCV system to acquire higher tolerable dose of vectors and enhance the safety of liver cancer suicide gene therapy. The research also demonstrates that rAAV8 is a robust vector for gene transfer to the liver. However, rAAV8 vectors show ineffective transduction for the experimentally inoculated HCC such as Hepa1-6 cells.更多还原