【作者】 蒋祥龙；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 生殖工程， 2008， 博士

【摘要】 恶性肿瘤是严重危害人类健康的疾病之一,是继心、脑血管疾病之后引起人类死亡最常见的致死性疾病。传统的治疗以放疗、化疗结合手术治疗为主,但在过去的二十年里,肿瘤患者的生存率未有明显提高。其主要原因是因为对放疗、化疗药物的耐受及术后肿瘤转移和复发。肿瘤干细胞是一类具干细胞特性,极小数量细胞就能致瘤的细胞,是肿瘤发生、转移及复发的根源。人恶性畸胎瘤是一类发病率较低,但术后复发致死率较高的恶性肿瘤,其术后复发的5年生存率仅3%。人胚胎癌细胞系(NTERA-2)来源于人类睾丸畸胎瘤,具有体外形成三个胚层—外胚层、内胚层及中胚层的能力,同时表达一系列的胚胎干细胞标志,并具拟胚体,细胞单克隆形成能力,体内也具三胚层分化能力,因此,NTERA-2细胞被用作胚胎干细胞的研究模型并被认为是胚胎干细胞的恶性对照物——肿瘤干细胞。所以,NTERA-2细胞及畸胎瘤被认为是肿瘤干细胞基因治疗研究的最理想的模型。肿瘤坏死因子相关凋亡诱导配体是一个Ⅱ型膜蛋白,属于肿瘤坏死因子家族的典型成员,具有引起大多数肿瘤细胞凋亡,而对正常组织细胞无细胞毒的特性,因此,被认为是最有希望的抗肿瘤分子武器。其膜外部分即可溶性TRAIL (sTRAIL),氨基酸编码114-281,是引起肿瘤细胞凋亡的功能部分。大鼠progression-elevated gene-3 (PEG3)基因启动子是已被鉴定在癌症细胞中特异性高表达的启动子,萤光素双报告基因系统研究表明,PEG3启动子在大多肿瘤细胞系中活性高,而在正常的组织细胞中活性低或检测不到,我们的研究表明,PEG3启动子在胚胎干细胞中的活性极低,因此,PEG3启动子可能是作为肿瘤基因治疗,特别是肿瘤干细胞基因治疗的理想启动子,可能在肿瘤基因治疗过程中有效地解决肿瘤抑制基因的靶向问题。在本课题中,我们用PEG3启动子替换腺相关病毒中的CMV启动子,构建了治疗性载体AAV-PEG-sTRAIL,将sTRAIL基因转入NTERA-2细胞中,流式细胞仪检测到23.1±2.3%早期凋亡细胞；荧光显微镜下可观察到Hoechst 33258染色浓缩的细胞核,细胞中期凋亡的标志。荧光定量PCR检测sTRAIL的四个配体,诱导配体1(DcR1)、诱导配体2(DcR 2)、死亡受体(DR4)和死亡受体(DR5),结果表明DcR1、DcR 2表达量变化极小,但DR4和DR5有3倍以上表达量上升,Western blot检测了Caspase-3蛋白表达量,结果表明,转染sTRAIL基因的NTERA-2细胞可检测到19KD分离的Caspase-3,表明sTRAIL基因已经引起了NTERA-2细胞凋亡,转染sTRAIL基因与未转染sTRAIL基因的细胞致瘤研究表明,转染组4只NOD-SCID小鼠,60天仅一只小鼠致瘤,致瘤率为25%,而未转染组4只NOD-SCID小鼠,40天全部致瘤,致瘤率为100%,荷瘤小鼠治疗结果表明,sTRAIL基因治疗组的生存期比PBS及空载体治疗组明显延长,具有统计学意义(P=0.044),肿瘤体积较空载体及PBS对照组小；AAV-PEG-sTRAIL载体尾静脉注射后细胞毒研究表明,注射组小鼠的谷丙转氨酶和谷草转氯酶较对照组小鼠相比,未有明显的变化,且正常范围之内；HE染色表明,实验组小鼠的心、肝、脾、肺、肾均未出现可检测的组织坏死。sTRAIL基因对NTERA-2细胞及畸胎瘤的抑制作用,以及对正常组织缺乏可检测的细胞毒作用说明sTRAIL基因在PEG3启动子的驱动下,可对人恶性畸胎瘤进行有效的抑制。更多还原

【Abstract】 Carcinoma is one of a diseases of serious damage to human health and the leading causes of mortality except cardiovascular and cerebrovascular diseases. The survival rate of patients suffering postoperative recurrence has little progression despite the development of conventional therapeutic strategies including chemotherapy, radiation and surgical oncology over the past two decades. This failure of conventional therapy is due mainly to resistance to chemotherapy, radiation and the relapse and spreading after surgical treatment. Tumor stem cells are those that have the characters of adult stem cells and few tumor stem cells can cause the event of tumor. At the same time, tumor stem cells are the rootstock of development, metastasis and relapse in tumor occurring. Human teratocarcinomas is a disease that have higher mortality and lower incidence. The survival rate of patients suffering postoperative recurrence is only 3% in 5 years. Human embryonic carcinoma (EC) cell line (NTERA-2) cells derived from a metastasis of a human testicular teratocarcinoma have the ability to self-renew in vitro and maintain their ability to differentiate into all three lineages:ectoderm, mesoderm and endoderm. At the same time, EC cells express a set of important pluripotent markers that are exclusive to human ES cells but are not expressed in their differentiated derivatives. Hence, EC cells are considered to be the malignant counterparts of human embryonic stem cells and regarded as a mode for study of cancer stem cells and human embryonic stem cells.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a type II membrane protein belonging to tumor necrosis factor (TNF) family. TRAIL induces apoptosis in most neoplastic cell lines by binding to its specific functional receptor molecules on the surface of the target cells, while leaving intact most normal cells that can express non-functional decoy receptors. Hence, TRAIL is regarded as a promising molecular therapeutic weapon against malignancies. Membranous TRAIL is so called soluble TRAIL (sTRAIL114-281) forms a homotrimeric complex in serum with potent apoptosis-inducing activity.The promoter of rat progression-elevated gene-3 (PEG-3) is an available cancer-selective gene promoter. Many studies have shown that PEG-3 promoter can drive synthesis of apoptosis-inducing genes, which inhibits the growth of tumor, kills only cancer cells and spares normal cells. Our studies showed that the activity of PEG-3 promoter was very low in human embryonic stem cells and normal tissue cells. So, PEG-3 promoter is the perfect promoter in tumor gene therapy, especially in gene therapy of tumor stem cell.In our research, the CMV promoter of AAV vector was replaced by PEG-3 promoter, a therapeutic vector named AAV-PEG-sTRAIL was constructed. Transfected with AAV-PEG-sTRAIL in NTERA-2 cell line, sTRAIL was able to induce obvious apoptosis of NTERA-2 cells and apoptotic cell accounted for 23.1±2.3% of the population. In the absence of sTRAIL, the apoptotic rate was only 1.9±1.2%. Under microscopical examination, the condensed nuclei, a characteristic of apoptosis, could be found in NTERA-2 cells transfected with AAV-PEG-sTRAIL but not in the AAV-PEG transfected cells. NTERA-2 cells transfected with AAV-PEG-sTRAIL were subjected to quantitative reverse transcription-PCR (qPCR) and western blot analysis. DcRl, DcR2, DR4 and DR5 mRNA and cleaved caspase-3 protein were assessed in EC cells following treatment. Results of qPCR revealed that treatment with AAV-PEG-sTRAIL induced an (>3-fold) increase in DR5 and DR4 expression in NTERA-2 cell line. Interestingly, no significant change was found in the expression of either DcR1 or DcR2 following treatment. Western blot analysis indicated that activated caspase-3 (19kd) was also detected in EC cells treated with AAV-PEG-sTRAIL. These results indicate that the apoptosis of EC cells was caused by transfection with AAV-PEG-sTRAIL. Tumorigenicity experiment in vivo showed that 25%(one of four) was observed in 60 days when 1×106EC cell treated with AAV-PEG-sTRAIL group. In contrast,100%(four of four) tumor incidence was found in the untreated group at 40 day. Treatment with sTRAIL gene in models in nonobese diabetic/severe combined immunnodeficient (NOD/SCID) mice resulted in a suppression in tumor growth and statistically significant prolonged survival of the tumor-bearing mice (p=0.044). histopathologic studies showed no obvious lesions in liver, spleen, lung, or kidney. In addition, body weight, gross appearance, and behavior provided no signs of systemic toxicity. The successful inhibition of teratocarcinoma growth and the absence of detectable toxicty suggest a putative role for sTRAIL gene from PEG-3 promoter in treatment of tumor stem cells.更多还原