【作者】 祝贺；

【导师】 崔满华；

【作者基本信息】 吉林大学 ， 妇产科学， 2011， 博士

【摘要】 卵巢癌在女性生殖系统恶性肿瘤中发病率居第三位,死亡率居首位。由于卵巢癌发病具有隐匿性,加上缺乏有效的预防措施和筛选方法,75-80%的患者明确诊断时已是晚期。尽管大多数患者在接受初次手术和化疗后能得到缓解,但复发率仍较高,使得卵巢癌患者的5年存活率始终徘徊于30%左右,因此寻找新的治疗方案迫在眉睫。近些年,多种基因治疗策略应用于卵巢癌治疗,包括分子化疗、基因突变补偿、免疫增强、溶瘤病毒、RNAi和多基因联合治疗等,其中有一些已进入临床实验阶段,但有效性和安全性始终限制着基因治疗在临床的广泛应用。为提高基因治疗的安全性,人们开始了基因的靶向治疗研究,即将目的基因通过载体系统高效转入肿瘤细胞,并只在肿瘤细胞表达而发挥杀伤作用,不影响机体的正常组织。同时为了提高基因治疗的有效性,人们不断寻找有效的肿瘤杀伤基因,并联合化学疗法以提高基因治疗的效果。近年来,陆续报道了一些卵巢组织的特异性启动子,例如细胞色素P450芳香化酶(P450c19/CYP19)启动子、抑制素α(Inhibinα)启动子和苗勒管抑制物质Ⅱ型受体(MISⅡR)启动子等,但后续研究又发现这些启动子在非卵巢组织上也有表达,缺乏卵巢组织的高度特异性,限制了其在科研和临床上的应用。卵巢特异性启动子(ovarian-specific promoter-1,OSP-1)是在小鼠卵巢组织中特异性表达的逆转录病毒样元件,在多种卵巢肿瘤细胞和正常卵巢细胞中均有较高的活性,而在非卵巢细胞中几乎无活性。一些体内外研究证实该启动子可以启动肿瘤杀伤基因在卵巢癌细胞内特异性表达。所以应用OSP-1为启动子,可实现肿瘤治疗基因在卵巢肿瘤组织的靶向表达,最大限度地降低对其他组织的毒副作用,增强卵巢癌基因治疗的安全性。理想的治疗基因应该对肿瘤细胞有选择性的生长抑制和凋亡作用,同时有抗肿瘤血管生成作用和强烈的旁观者效应,即转导细胞对非转导细胞的细胞毒作用,从而扩大了肿瘤的杀伤效果及抑制肿瘤的转移和扩散,和其他治疗措施(放疗、化疗)联合应用还能产生协同效应,从而能够从多方面补偿由于基因转染效率低而带来的肿瘤基因治疗效果低下的缺陷。白介素24(interleukin-24, IL-24)就是集以上优点于一身的多功能肿瘤抑制基因,被喻为肿瘤的“魔法子弹”。基于该基因治疗肿瘤的良好的前期实验基础,本研究拟应用该基因进行卵巢癌基因治疗。但目前IL-24的生理功能还不清楚,为获得良好治疗效果而应用大剂量IL-24究竟会对全身系统造成什么样的副反应更是个未知数,如何在巩固和加强IL-24抗肿瘤能力的同时又最大限度地降低临床副反应,提高安全性,是IL-24能否在临床得到广泛应用的关键,因此本研究在拟应用IL-24进行卵巢癌基因治疗时,采用基因靶向卵巢癌细胞表达的基因靶向治疗策略,使IL-24基因只在卵巢癌细胞内表达,从而使该基因集中在肿瘤组织的局部表达,避免该基因对全身系统可能造成的副作用。同时检测IL-24能否增强卵巢癌细胞对化疗药紫杉醇、顺铂的敏感性并探讨其机制,为IL-24安全有效地用于卵巢癌临床治疗提供实验基础。本实验研究内容如下：1构建靶向卵巢癌细胞特异性表达的真核基因表达载体方法：应用基因合成方法合成OSP-1启动子,通过基因重组方法将该启动子片段插入到真核基因表达载体pcDNA3.0内,替换该载体内的巨细胞病毒启动子与增强子序列,从而构建成由OSP-1启动基因表达的pcDNA3.0-OSP-1载体。在此基础上将报告基因β半乳糖苷酶基因(β-gal)克隆入pcDNA3.0-OSP-1载体的OSP-1启动子下游,构建成由OSP-1启动报告基因表达的pcDNA3.0-OSP-1--β-gal载体。将该报告基因载体应用脂质体介导的基因转染方法分别转入人卵巢癌细胞株SKOV3细胞、人肝癌细胞株HepG2细胞及人成纤维细胞株BJ细胞中,72h后应用β半乳糖苷酶细胞原位染色法观察β半乳糖苷酶基因在三组细胞内的表达。结果：pcDNA3.0-OSP-1-β-gal载体转染的三组细胞,只有SKOV3细胞表达β半乳糖苷酶,而其他两组细胞没有表达。结论：本研究构建的靶向卵巢癌细胞特异性表达的真核基因表达载体具有启动外源基因在卵巢癌细胞内特异性表达的功能。2 IL-24基因在卵巢癌细胞内的特异性表达方法：应用RT-PCR方法从外周血单个核细胞内调取人IL-24基因cDNA片段,将获得PCR产物克隆入T载体,该载体经测序鉴定获得的IL-24基因序列与核酸数据库序列一致后,应用基因重组技术将IL-24基因片段分别克隆入pcDNA3.0及pcDNA3.0-OSP-1载体,构建成pcDNA3.0-IL-24表达载体和pcDNA3.0-OSP-1-IL-24表达载体。将上述载体应用脂质体介导的基因转染技术分别转入人卵巢癌细胞株SKOV3细胞、人肝癌细胞株HepG2细胞及人成纤维细胞株BJ细胞中,并应用G418进行基因转染的稳定筛选。对基因稳定表达的细胞应用RT-PCR检测IL-24基因在细胞内的InRNA表达水平,应用ELISA方法检测细胞上清中IL-24基因蛋白含量。结果：pcDNA3.0-IL-24表达载体转染的三组细胞中,IL-24基因mRNA表达水平与细胞上清中IL-24基因蛋白含量明显高于各组的未转染细胞；pcDNA3.0-OSP-1-IL-24表达载体转染的三组细胞中,只有SKOV3细胞的IL-24基因mRNA表达水平与细胞上清中IL-24基因蛋白含量明显高于本组的未转染细胞,其他两组细胞无明显差异。结论：本研究构建的pcDNA3.0-OSP-1-IL-24表达载体可实现IL-24基因在卵巢癌细胞内的特异性表达。3 IL-24基因靶向抑制卵巢癌细胞增殖的研究方法：将基因转染的各组细胞以1×104每孔接种于24孔培养板,通过细胞计数方法计数生长8天的细胞数量,绘制生长曲线,从而判断IL-24对细胞生长的影响；通过流式细胞术检测细胞周期分布情况。取pcDNA3.0-OSP-1-IL-24质粒稳定转染的SKOV3细胞培养第6天收集的细胞上清以不同稀释浓度作用于SKOV3细胞,分别于24h,48h,72h应用MTT法进行细胞活性检测。结果：pcDNA3.0-IL-24表达载体转染的三组细胞中：BJ细胞生长曲线与本组未转染细胞相比无差异。而SKOV3细胞与HepG2细胞的生长在第5天开始明显受到抑制,与本组未转染细胞相比均有明显差异。pcDNA3.0-OSP-1-IL-24表达载体转染的三组细胞中：只有SKOV3细胞的生长在第6天开始受到明显抑制,与本组未转染细胞相比有明显差异,其他两组细胞无明显差异。流式细胞术检测的细胞周期显示,在SKOV3细胞组：转染pcDNA3.0-IL-24与pcDNA3.0-OSP-1-IL-24质粒载体的细胞与基因未转染的细胞相比G1期细胞数明显增多,S期细胞明显减少。MTT检测结果分析显示pcDNA3.0-OSP-1-IL-24质粒载体稳定转染SKOV3细胞的培养上清对于SKOV3细胞的生长有抑制能力,随着浓度的减少,抑制能力下降。结论：通过本研究构建的pcDNA3.0-OSP-1-IL-24表达载体获得的IL-24基因表达产物可特异性地抑制卵巢癌细胞的增殖,并使G1期细胞增多。其分泌表达的IL-24可有效抑制基因未转染的SKOV3细胞生长,具有抗肿瘤的旁观者效应。4 IL-24基因对卵巢癌细胞化疗药物敏感性的影响方法：将紫杉醇与顺铂两种化疗药物分别作用于SKOV3细胞,稳定转染pcDNA3.0-IL-24的SKOV3细胞及稳定转染pcDNA3.0-OSP-1-IL-24的SKOV3细胞,72h后应用MTT检测细胞活性,根据检测的吸光值计算化疗药物对细胞生长的抑制率,并计算化疗药物对各组细胞抑制的IC50%值。应用实时定量PCR检测各组细胞ERCC1、TUBB3基因的表达。结果：转染IL-24的SKOV3细胞对紫杉醇和顺铂的敏感性与未转染的SKOV3细胞相比有显著提高,其IC50%值分别降低了6倍与10倍。荧光定量PCR结果显示转染IL-24的SKOV3细胞的ERCCl基因与TUBB3基因表达与未转染的SKOV3细胞相比,分别下降10倍与4倍。结论：转染IL-24基因可以有效提高SKOV3细胞对于紫杉醇、顺铂的敏感性,其机制可能与IL-24降低卵巢癌细胞内TUBB3基因与ERCC1基因的表达有关。通过上述研究,本实验成功构建了IL-24基因靶向卵巢癌细胞特异性表达的真核表达载体pcDNA3.0-OSP-1-IL-24,体外实验实现了IL-24基因在卵巢癌细胞内的特异性表达与肿瘤杀伤作用,并确定转染IL-24基因能有效提高卵巢癌细胞对紫杉醇与顺铂敏感性。本研究取得的研究成果将为提高卵巢癌基因治疗的安全性与有效性以及将IL-24基因联合化疗应用于卵巢癌临床治疗提供了有力的体外实验依据。更多还原

【Abstract】 Ovarian cancer has a third morbidity in the female reproductive system malignancies, and the mortality rate is the first. Due to lacking of effective preventive measures and screening methods to Ovarian cancer, which invades hidingly,75%-80% of patients present with advanced stage disease while diagnosed. Although the majority of patients who received initial surgery and chemotherapy could be eased, the recurrence rate is still high, which makes 5-year survival rate is always at around 30%. Therefore, finding new therapy strategies of ovarian cancer is urgent for the truth of lacking of an effective treatment.Recent years, several gene therapy strategies have been used in the treatment of ovarian cancer, such as molecular chemotherapy, mutation compensation, immune enhancement, oncolytic virus, RNAi and multi-gene therapy, etc, some of which have been applied in clinical trials, but the efficacy and safety of gene therapy has always been the limites of clinical applications in a wide range. In order to improve the safety of gene therapy, people began gene targeted therapy, the aim gene is highly transfered into tumor cells by vector system, and only kill the tumor cells without affecting the normal tissue. Meanwhile, in order to improve the efficacy of gene therapy, people are constantly looking for effective tumor-killing genes, as well as which is combined with chemotherapeutics to enhance the effectiveness of gene therapy.These years, some ovarian tissue specific promoters have been reported one after another, such as cytochrome P450 aromatase (P450c19/CYP19) promoter, inhibin a promoter and Mullerian inhibiting substance typeⅡreceptor (MISⅡR) promoter, etc. On the following study, researchers found that these promoters also express in non-ovarian tissues, and their non-highly specific to ovarian tissue limits the application in researches and clinical trials. Ovarian-specific promoter-1(OSP-1) is a retro virus-like elements, which expresses specifically in the mouse ovary tissue, acts highly in majority ovarian cancer cells and normal ovarian cells but none in non-ovarian cells. Some vivo and vitro studies confirmed that the promoter can start tumor-killing genes expressing specifically in ovarian cancer cells. Therefore, specific expression of tumor therapy gene in ovarian cancer tissue can be achieved by OSP-1 promoter, which can minimize side effects to other organizations and enhance the safety of gene therapy for ovarian cancer.The ideal therapy gene should inhibit the growth of tumor cells selectively as well as result in cell apoptosis, while it has anti-angiogenic effect and strong bystander effect, which means transduced cells have cytotoxicity on non-transduced cells, so that expanding the anti-tumor effect and inhibiting metastasis and diffusion of tumor. Combinating with other treatments (radiotherapy or chemotherapy), the gene can create synergy, which can improve the efficacy of gene transfer in many ways, so that change the low effect of tumor gene therapy. Interleukin-24 (IL-24) is a multi-functional tumor suppressor gene, which has these advantages mentioned above. It has been hailed as the tumor’s "magic bullet", based on the good preliminary experiments of IL-24 gene therapy for Cancer, In this study, IL-24 gene therapy is to be applied for ovarian cancer At present, the physiological function of IL-24 is not clear, what will happen to human system after receiving high dose IL-24 therapy is unknown. How to consolidate and strengthen the anti-tumor capacity of IL-24 and minimize the side effects to improve security at the same time is the key to widely clinical application of IL-24. Therefore, our study plans to adopt gene-targeting therapy in the application of IL-24 gene therapy for ovarian cancer, so that IL-24 gene only expresses in the ovarian cancer cells, concentrating on tumor tissue to avoid the potential side effects to human system. Meanwhile, we want to detect whether IL-24 gene can enhance the sensitivity of ovarian cancer cells to chemotherapeutic drugs or not, such as paclitaxel and cisplatin, as well as explore the mechanism, so that we can try to provide experimental basis for IL-24 gene therapy for ovarian cancer safely and efficiently in clinical. This study was as follows:1. Construct eukaryotic expression vector targeting ovarian cancer cellMethods:Compose OSP-1 promoter through gene synthesis method, insert the promoter fragment into the eukaryotic expression vector named pcDNA3.0 by genetic recombination method, which replaces the cytomegalovirus promoter and enhancer sequences in the vector, so that pcDNA3.0-OSP-1 drived by OSP-1 was constructed. On this basis, clone the report gene namedβ-galactosidase gene (β-gal) into the vector, ensure it is in the downstream of OSP-1 promoter to construct pcDNA3.0-OSP-1--β-gal vector, of which report gene is drived by OSP-1. Then we transfer pcDNA3.0-OSP-1--β-gal vector into ovarian cancer cell line SKOV3, human hepatoma cell line HepG2 and human fibroblast cell line BJ cells by Liposomes, and obeserve the expression of (3-gal in the three groups of cells byβ-galactosidase staining cells in situ afer 72h.Result:In these three groups of cells, only SKOV3 cells expressβ-galactosidase, while the other two groups not.Conclusion:The pcDNA3.0-OSP-1--β-gal vector can drive foreign gene expressing specifically in ovarian cancer cells.2. Specific expression of IL-24 gene in ovarian cancer cellsMethod:Human IL-24 cDNA fragments was amplified from peripheral blood mononuclear cells by RT-PCR, clone the products of RT-PCR into T-vector, then identified the results by DNA sequencing. We clone the IL-24 gene into pcDNA3.0 and pcDNA3.0-OSP-1 vector by genetic recombination method respectively, construct pcDNA3.0-IL-24 and pcDNA3.0-OSP-1-IL-24 expression vectors. These two expression vectors are transferred into ovarian cancer cell line SKOV3, human hepatoma cell line HepG2 and human fibroblast cell line BJ cells by Liposomes, after the stable selection of G418, we detect the expressing level of IL-24 gene mRNA in cells by RT-PCR and the protein product of IL-24 gene in cell supernatant by ELISA.Result:In the three groups of cells transfected with pcDNA3.0-IL-24 expression vector, the expressing levels of IL-24 gene mRNA in cells and the protein product of IL-24 gene in cell supernatant are highly more than nontransfected cells, as the same as the SKOV3 cells transfected with pcDNA3.0-OSP-1-IL-24 expression vectors, but the other two groups had no differences between transfected cells and not.Conclusion:The pcDNA3.0-OSP-1-IL-24 expression vectors constructed in this study can achieve specific expression in ovarian cancer cells.3. Inhibition of IL-24 gene targeting of ovarian cancer cellsMethod:Plate the transfected cells in 24-well culture plates with the dose of 1×104 cells per well, count the number of cells in 8 days by cell counting method. Then describe the growth curve to identify the influence of IL-24 gene on cells, and detect the cell cycle distribution by flow cytometry. Dilute the supernatant collected from the SKOV3 cells to different concentrations, which were stably transfected with pcDNA3.0-OSP-1-IL-24 plasmids and cultured for 6 days,then deal SKOV3 cells with the supernatant。After 24 hours,detect the cells activity by MTT, as well as 48 hours and 72 hours later.Result:In the three groups of cells transfected with pcDNA3.0-IL-24 expression vector, there is no difference between transfected cells and nontransfected cells in the growth curve of BJ cells. While, the growth of SKOV3 and HepG2 cells were significantly inhibited since the 5th day, compared with nontransfected cells, there are distinct differences. In the three groups transfected with pcDNA3.0-OSP-1-IL-24 expression vector, only the growth of SKOV3 cells was significantly inhibited since the 6th day compared to nontransfected cells, and the other two groups had no differences. The result of flow cytometry showed the G1 phase cells of SKOV3 increased mostly and the S phase cells of SKOV3 decreased mostly in the two groups transfected with pcDNA3.0-IL-24 and pcDN A3.0-OSP-1-IL-24 expression vectors comparing to nontransfected cells. The result of MTT showed that the supernatant of the SKOV3 cells stably transferred with pcDNA3.0-OSP-1-IL-24 plasmids could inhibit the growth of SKOV3 cells.The inhibition capability depended on the concentration of the supernatant.Conclusion:The expression production of pcDNA3.0-OSP-1-IL-24 can specifically inhibits the proliferation of ovarian cancer cells, and increases the G1 phase cells. The IL-24 product expressed by pcDNA3.0-OSP-1-IL-24 plasmids can effectively inhibit the growth of non-transfected SKOV3 cells, which play the role of anti-tumot bystander effect.4. Influence of IL-24 gene on the sensitivity of ovarian cancer cells to chemotherapeutic drugsMethod:We use paclitaxel and cisplatin respectively to deal with SKOV3 cells, SKOV3 cells stably transfected with pcDNA3.0-IL-24 and pcDNA3.0-OSP-1-IL-24 respectively, then detect the cells activity by MTT after 72 hours and calulate the cell inhibition rate by chemotherapeutics according to the detected result, as well as the IC50% value of cells inhibiton. Detected the expression of ERCC1and TUBB 3 gene by fluorescence quantitative PCR.Result:The sensitivity of SKOV3 cells transfected with IL-24 gene to paclitaxel and cisplatin were increased evidently comparing to nontransfected cells, and their IC50% values were reduced by 6 times and 10 times respectively, Comparing to nontransfected cells, the expression of ERCC1 and TUBB3 gene mRNA of transfecred SKOV3 cells reduced by 10 times and 4 times respectively.Conclusion:Transfecting IL-24 gene can enhance the senstivity of SKOV3 cells to paclitaxel and cisplatin, and the machanism maybe down-regulating the expression of TUBB3 gene and ERCCl gene.Above all, we successfully constructed eukaryotic expression vector pcDNA3.0-OSP-1-IL-24 targeting ovarian cancer cells, and achieved the specific expression and anti-tumor function of IL-24 gene in ovarian cancer in vitro, and confirmed that transfecting IL-24 gene to ovarian cancer cells can effectively increase their sensitivity of paclitaxel and cisplatin. This study provid strong experimental evidences in viro for improving the safety and efficacy of ovarian cancer gene therapy and applying IL-24 gene trerapy combinating with chemotherapy in Clinical treatment of ovarian cancer.更多还原