【作者】 金平；

【导师】 孔北华；

【作者基本信息】 山东大学 ， 妇产科学， 2005， 博士

【摘要】 随着肿瘤基因治疗研究的迅速发展,卵巢癌的基因治疗也成为近年来的研究热点。目前肿瘤基因治疗领域存在的关键技术问题是如何提高基因转导系统的高效性和靶向性。多聚乙烯基亚胺(polyethylenimine, PEI)作为近年来一种新型的非病毒载体,具有较高的转导效率和较低的细胞毒性,为基因转移提供了一条有效的新途径。以肿瘤特异性启动子hTERT和卵巢组织特异性启动子OSP-1调控下的自杀基因系统是卵巢癌基因治疗中的靶向性策略之一。其次在肿瘤的基因研究中,建立动物模型来研究肿瘤的发生、发展以及各种治疗手段的效果,是临床前肿瘤研究的必经之路。而在现有的肿瘤研究中,与临床相关的动物模型却存在着诸多问题。近年来生物荧光成像系统(bioluminescent imaging, BLI)作为一种新开发的动物模型,具有快速、灵敏、无创和高分辨率的特点,为肿瘤研究提供了良好的技术平台。本课题以多聚乙烯基亚胺为载体,介导靶向性的自杀基因系统转导人卵巢癌细胞,并利用生物荧光成像系统建立动物模型,研究其转导效率和靶向抗瘤效应。全文共分为两部分,现分部叙述如下。 第一部分 多聚乙烯基亚胺介导的卵巢特异性启动子调控下的自杀基因抑制卵巢癌生长的实验研究 目的:应用多聚乙烯基亚胺将卵巢特异性启动子OSP-1调控下的自杀基因HSV-tk导入人卵巢癌细胞,探讨其体外、体内的抗瘤效应和靶向性。 方法:(1)将pGL3-Luc质粒分别介导多聚乙烯基亚胺PEI、脂质体DOTAP和裸DNA转染人卵巢癌细胞SKOV3,检测荧光素酶表达RLU(relative luciferase unit);(2)利用PEI将卵巢特异性启动子调控下的真核表达质粒pOSP1-HSVtk更多还原

【Abstract】 Recently with the rapid development of tumor gene therapy, the reseach on ovarian cancer has also become a hotspot. To improve the transfection efficiency and targeted delivery of gene vector have become two critical problems in the field of gene therapy. Many approaches have been developed to solve these problems. Polycationic polyethylenimine (PEI), as a new kind of nonviral gene vector, has provided an effective solution with its higher transfection efficiency and lower toxicity. The suicide gene therapy with a tumor specific promoter (hTERT) or an ovarian specific promoter (OSP1) is one of these targeted delivery strategies for ovarian cancer. In addition, establishing some relevant animal models is a necessary method in preclinical oncology research in order to observate tumor growth and effect before or after some treatment in the field of tumor gene therapy. However, the relevant animal models remain many question and challenge at present. Fortunately, bioluminescent imaging (BLI), as a new kind of animal model with its sensitive, rapid, less invasive and greater accurate, has provided a excellent technical solution. This subject aimed to investigate the transfection efficiency and targeted killing activity using specific suicide gene therapy system and polyethylenimine mediated transfection by establishing animal model of BLI for ovarian cancer. This subject was divided into two parts and the relevant abstract was as follows.PART ⅠANTI-TUMOR STUDY OF SUICIDE GENE THERAPY WITH AN OVARIAN-SPECIFIC PROMOTER AND POLYETHYLENIMINE MEDIATED TRANSFECTION FOR OVARIAN CANCEROBJECTIVE: To investigate the anti-tumor and targeted delivery effects of HSV-tk suicide gene therapy adopting an ovarian-specific promoter (OSPl) for human ovarian carcinoma by polyethylenimine-mediated transfection in vitro and in vivo.METHODS: (1) Human ovarian carcinoma SKOV3 cells were transfected by pGL3-Luc plasmid mediated with polyethylenimine (PEI), DOTAP liposome and naked DNA respectively. The transfection efficiency was measured by RLU (relative luciferase unit); (2) The pOSPl-HSVtk plasmid containing an ovarian-specific promoter was transfected into the human ovarian carcinoma cell SKOV3 by PEI, human lung carcinoma cell NCI-H460 and human heptocellular carcinoma cell HepG2 and SMMC7721. The cytotoxicities resulted from GCV were evaluated using the MTT assay. The concentrations of GCV in SKOV3 cells were detected by high performance liquid chromatography (HPLC). The apoptosis of SKOV3 cells were observed by flow cytometry (FCM) and TdT-mediated dUTP nick end labeling (TUNEL) technique; (3) SKOV3 ovarian cancer xenograft model was established in BALB/C nude mice. The tumors were transfected with pOSPl-HSVtk using PEI as the transfection agent. The expressed TK activities were estimated by monitoring the GCV concentration change using a HPLC assay; (4) The weight of the nude mice, the tumor volumes and tumor weights were all recorded to calculate the tumor inhibition rates by weight and by volume. Histopathological analysis and TUNEL method were also performed; (5) SKOV3 cells were implanted intraperitoneally to BALB/C nude mice, then the survival period and survival prolong rate of BALB/C nude mice were evaluated the efficacy of PEI mediated pOSP1-HSVtk gene delivery followed by GCV treatment in vivo.RESULTS: (1)The polyethylenimine group had the highest luciferase activity(187.35 ± 6.48) compared with other two groups (45.74±5.98, 0.03 ± 0.00) with significant statistical difference(P<0.01); (2) GCV was shown to be toxicity only in SKOV3 cells, not in NCI-H460 cells, SMMC7721and HepG2 cells. The concentrations of GCV in SKOV3 cells detected by HPLC gradually decreased with increase of time. FCM showed the apoptotic rate of cells significantly increased in SKOV3 exposed to GCV 24h, 48h and 72h (8.42 ±0.76%, 18.50±1.78%, 34.80+ 3.46%). The substantial cell apoptosis in SKOV3 was confirmed by TUNEL; (3) Compared with the control group in vivo, GCV concentration in tumor tissues transfected pOSP1-HSVtk were shown to be significantly lower by HPLC (0.05>P>0.01); (4) The tumor volume and the tumor weight in the treated group were also significantly decreased (P<0.01). The tumor volume inhibition rate and the tumor weight inhibition rate were estimated to be 63.66% and 58.98% respectively. Histological examination revealed heavy haemorrhage and necrosis in the tumor tissues, and TUNEL confirmed substantial cell apoptosis in the treated group; (5) Compared with these control groups, the survival period of the treated group was remarkably longer in the ovarian cancer survival model (P<0.01). The survival prolong rate of treated group was 63.80%.CONCLUSION: The suicide gene therapy system using an ovarian-specific promoter by polyethylenimine mediated transfection had a tissue-specific killing activity for human ovarian cancer in vitro and in vivo, which indicated its potential to improve the efficiency and the targeting in the field of gene therapy.PART ⅡANTI-TUMOR EFFICACY STUDY OF DOUBLE SUICIDE GENETHERAPY WITH A hTERT PROMOTER FOR OVARIANCANCER BY POLYETHYLENIMINE MEDIATEDTRANSFECTIONOBJECTIVE: To evaluate the killing activity of double suicide gene therapy adopting a human telomerase reverse transcriptase (hTERT) promoter and polyethylenimine mediated transfection for human ovarian carcinoma in vivo by establishing traditional SKOV3 intraperitoneal implantation animal model as well as SKOV3-Luciferase intraperitoneal animal mode of bioluminescent imaging (BLI) respectively.METHODS: (1) SKOV3 cells were implanted intraperitoneally to BALB/C nude mice and the ovarian cancer survival model was established. Diverse dose (5 μ g, 30 μ g, 50 μ g) of pBTdel-279-CD-TK were given by intraperitoneal injection using PEI as the transfection agent. Diverse treatments were started by intraperitoneal injection PEI/pBTdel-279-CD-TK on the 1st, 10th and 20th day after tumor implantation. In addition, the two kinds of plasmid (PEI/pBTdel-279-TK, PEI/pBTdel-279-CD-TK) treatment were also observed. Then 5-fluorocytosine (5-FC) & ganciclovir (GCV) were injected into the cavity of the peritoneum. The survival period and the survival prolong rate were calculated; (2) Human ovarian carcinoma SKOV3 cells were transfected by luciferase and the bioluminescent stability was evaluated in vitro. SKOV3-Luciferase stable cell line was implanted intraperitoneally to nude mice to establish the ovarian cancer animal model expressing luciferase. According to whether PEI/pBTdel-279-CD-TK therapy were given, the animals were divided into the treated model and the control model respectively. BLI monitoring of tumors growth and anticancer efficacy of suicide gene therapy in vivo were also applied timely.RESULTS: (1) The survival rate of tree different dose groups(5 μ g,30 μ g,50 μ g) were 25%,75% and 50% respectively. The average survival period of the treated group which pBTdel-279-CD-TK was injected on the 1st day after tumorimplantation was longer than other two groups (p<0.05). There were no significantly difference between the 10th day treated group and the 20th day treated group (P>0.05). The average survival period of the pBTdel-279-CD-TK group were prior to the pBTdel-279-TK group (P<0.05), and the survival prolong rate of the two groups were 146.44% and 73.41% respectively; (2) BLI showed that SKOV3-Luciferase cell line and its ovarian cancer intraperitoneal animal model expressing luciferase were established successfully. With the time passed, the luciferase signal gradually proliferated in the control model whereas it hardly could be detected in the treated model in vivo.CONCLUSION: The suicide gene therapy system using a human telomerase reverse transcnptase (hTERT) promoter by polyethylenimine mediated transfection had a significantly killing activity on human ovarian cancer in vivo; especially it had remarkably anti-tumor effects in the early period of the tumor development; Bioluminescent imaging could be used to monitor and evaluate ovarian cancer development and suicide gene therapy efficacy of PEI/hTERT-CD-TK more accurately and more sensitively, which indicated its potential to improve and refine traditional animal models.更多还原