【作者】 王利华；

【导师】 周平；

【作者基本信息】 中南大学 ， 影像医学与核医学， 2010， 硕士

【摘要】 研究背景：乳腺癌是女性最常见的恶性肿瘤之一,已严重威胁女性健康。随着对肿瘤发病机制认识的不断深入,以及分子生物学等学科的迅速发展,基因治疗已成为一种全新的肿瘤治疗模式,其中自杀基因治疗作为一种颇具临床应用潜力的治疗策略,是近年来肿瘤基因治疗的研究热点。自杀基因疗法主要通过直接杀伤作用和旁观者效应来杀伤肿瘤细胞,但各自杀基因体系又有不同特点,联合应用可取长补短,增强疗效。目前应用最广泛的是单纯疱疹病毒胸苷激酶/更昔洛韦(HSV-TK/GCV)和胞嘧啶脱氨基酶/5-氟胞嘧啶(CD/5-Fc)两大自杀基因体系,且两者有很强的互补性,联合应用不仅可以提高疗效、扩大肿瘤治疗谱,还能抑制继发性耐药的产生。基因治疗的靶向性是关系到治疗安全性的重要问题,自杀基因只能在肿瘤组织表达而不在正常组织表达,在自杀基因前端连接上肿瘤组织特异性启动子就能达到这一目的。研究表明,血管内皮细胞生长因子受体(KDR)高表达于肿瘤血管内皮细胞及多种肿瘤细胞,而在正常组织不表达或表达甚微。因此,以KDR启动子(KDRp)驱动自杀基因,可使自杀基因在肿瘤血管内皮细胞及肿瘤细胞特异性表达,达到双重靶向治疗作用。近年来,由于病毒载体存在的安全性问题,载体研究主要转向于非病毒载体。脂质体是最常用的非病毒载体,安全、操作简便,但是转染效率较低。微泡造影剂是一种新型的基因载体,在超声波的辐照下不仅能将裸质粒导入靶细胞,还能提高脂质体等载体的转染效率,被认为是今后非病毒载体发展的重要方向。目的：1.构建含KDRp及双自杀基因CD/TK的肿瘤特异性质粒载体pEGFP-KDRp-CD/TK。2.超声辐照微泡联合脂质体转染pEGFP-KDRp-CD/TK质粒于乳腺癌MCF-7细胞,观察转染效率及给予前药后对MCF-7细胞的体外杀伤作用。方法：1.采用PCR扩增出KDRp、CD及TK基因片段,对各PCR产物进行TA克隆,构建成重组T质粒T-KDRp、T-CD、T-TK,再逐个将重组T质粒上的目的基因亚克隆至表达载体pEGFP-C2质粒上。对PCR结果凝胶电泳检测,对各重组质粒进行酶切电泳检测及测序鉴定。2.采用本实验组最优化超声及微泡参数。将培养的MCF-7细胞分为5组：①裸质粒组；②脂质体组；③超声辐照微泡组；④超声辐照脂质体组；⑤超声辐照微泡联合脂质体组,转染pEGFP-KDRp-CD/TK质粒于MCF-7细胞,荧光显微镜下观察GFP表达情况,流式细胞仪定性检测转染效率。用超声辐照微泡联合脂质体法转染MCF-7细胞后(未转染的MCF-7细胞作为对照组),给予前药0.1mg/ml GCV或(和)2mg/ml 5-FC, MTT检测自杀基因体系对MCF-7细胞的体外杀伤作用。结果：1.PCR扩增出的KDRp、CD、TK基因片段凝胶电泳条带大小与设计相符；各重组T质粒酶切电泳条带大小与设计相符,测序结果与GenBank比对一致；重组质粒pEGFP-KDRP-CD/TK测序,各基因片段序列与前期测序结果一致；其中KDRp为563bp,CD为1284bp,TK为1132bp。2.荧光显微镜下观察超声辐照微泡联合脂质体组的GFP表达最多、最强。流式细胞仪检测结果显示超声辐照微泡联合脂质体组的转染率最高(39.59±1.19)%,与其他各组比较差异有统计学意义(P<0.05)；超声辐照脂质体组(27.72±1.21)%次之,与脂质体组(22.96±0.93)%比较差异有统计学意义(P<0.05),超声辐照微泡组(21.84±1.04)%和脂质体组比较,差异无统计学意义(P>0.05)。裸质粒组的转染率最低(0.78±0.03)%(P<0.001)。3.MTT检测结果显示转染组MCF-7细胞的抑制率明显高于未转染组(P<0.001)；转染组的MCF-7细胞在联合用药时的抑制率为(90.77±2.68)%,明显高于单一用药组,差异有统计学意义(P<0.05)。且CDI值<1,提示双自杀基因系统有协同作用。4.转染组各前药组细胞抑制率均显著高于超声辐照微泡联合脂质体对MCF-7细胞的转染率(39.59±1.19)%(P<0.05),表现出显著的旁观者效应。结论：1.成功构建了由KDRp驱动的CD/TK双自杀基因重组质粒载体pEGFP-KDRp-CD/TK。2.超声辐照微泡联合脂质体能明显提高基因转染效率,其转染率明显高于超声辐照脂质体组、超声辐照微泡组及单纯脂质体组。3.CD/5-Fc和TK/GCV双自杀基因体系间存在协同效应,转染双自杀基因后的MCF-7细胞,联合用药比单一用药有更显著的治疗效果。4.旁观者效应在一定程度上弥补了载体系统转染效率不高的缺陷,增加了超声辐照微泡联合脂质体转染法在自杀基因治疗应用中的可行性。5.超声辐照微泡联合脂质体法转染双自杀基因体系是较理想的乳腺癌基因治疗策略。更多还原

【Abstract】 Background:Breast cancer is one of the most common malignant tumor, threatening the health of women. With the knowledge improvement of tumorous pathogenesis, and the rapid development of molecular biology, gene therapy represents a novel treatment model in cancer therapy. And the suicide gene therapy as a potential strategy for clinical application, is the hotspot of tumor gene therapy in recent years.Suicide gene system kills tumor cells by direct cytotoxic effect and bystander effect, and every system has its characteristic, therefor combined using of different systems can make up for each other and enhance efficacy. Currently the most widely used systems are herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) and cytosine deaminas/5-fluorocytosine (CD/5-Fc), and they have highly complementary. Combined using of them has several advantages, such as improving anti-tumor effect, avoiding drug resistance, etc.Targeted expression of gene is related to the security of gene therapy, suicide gene should specifically express only in tumor than normal tissue, that could be realized by making use of specific promoter. Most of the studies demonstrated that kinase domain-containing receptor (KDR) over-expresses in the majority of the solid cancer cells and neogenetic vascular endothelial cells of neoplasma but not in normal cells, so that KDR promoter (KDRp) could make genes of interest over-express in tumor cells and its vascular endothelial cells.In recent years, the focuse of research shift to non-viral vectors, because of viral vectors’security risks. Liposome is the most common non-viral vector, safe and convenient, but its transfection efficiency is low. Microbubble contrast agent is a novel gene vector, with the ultrasound irradiation it can not only transfect naked plasmid into target cells, but also enhance the transfection efficiency of liposome, considering the important part of non-viral vectors research.Objective: 1.To construct the tumor specific vector containing KDRp and CD/TK suicide genes.2.To transfect the pEGFP-KDRp-CD/TK into MCF-7 cells in vitro by ultrasound mediated microbubble destruction combined with liposome, and then investigate the transfection efficiency and the killing effect after given the prodrugs.Methods:1.Fristly, the KDRp, CD and TK gene fragments were amplified with polymerase chain reaction (PCR) technique. Secondly, every PCR product was cloned to T-vector respectively, establishing recombinant T-plasmid T-KDRp, T-CD, and T-TK. Finally, the target gene fragments of recombinant T-plasmids were subcloned to expression vector pEGFP-C2 gradually. The fragments and plasmid vectors were identified by cataphorerising and sequence analysising.2.The optimized ultrasound and microbubble paramaters were used. MCF-7 cells were dividcd into 5 groups,①naked plasmid group,②liposome group,③ultrasound irradiated microbubble group,④ultrasound irradiated liposome group,⑤ultrasound irradiated microbubble+liposome group. The transfection efficieny of pEGFP-KDRp-CD/TK in MCF-7 cells was detected by fluorescence microscope and flow cytometry. The killing effects of double suicide genes on MCF-7 cells (transfected and untransfected) that treated with 0.1mg/ml GCV,2mg/ml 5-Fc or combination GCV and 5-FC were determined by the method MTT.Result:1.The gene fragments amplified with PCR were equal to the expection. The sequence of every recombinant plasmid was identical with the array published in GeneBank. And the length of KDRp, CD and TK gene fragments was 563bp,1184bp and 1131bp respectively.2.The GFP of fifth group was the most and strongest observed by fluorescence microscope. The results of flow cytometry showed that the transfection efficiency of the fifth group was the highest one (39.59±1.19)% compared with the other groups (P<0.05), the fourth group was the next one (27.72±1.21)%, the third group (21.84±1.04)% was as near as the second group (22.96±0.93)%(P>0.05), and the first group is the lowest one (0.78±0.03)%(P<0.001).3.The results of MTT showed that the inhibition ratios of transfected MCF-7 cells were significantly higher than untransfected groups (P< 0.001), the inhibition ratio of transfected MCF-7 cells treated with GCV and 5-Fc was (90.77±2.68)%, obviously higher than the single prodrug group GCV or 5-Fc (P<0.05), furthermore the value of CDI was less than 1, both of these prompt that it has the synergistic killing efficacy between the two suicide gene systems.4.Every inhibition ratio of transfected groups was obviously higher than the transfection efficiency of MCF-7 cells (39.59±1.19)%(P<0.05), showing a significant bystander effect.Conclusions:1.The recombinant plasmid vector pEGFP-KDRp-CD/TK containing KDRp and CD/TK suicide genes was constructed successfully.2.Ultrasound mediated microbubble destruction combined with liposome can enhance the efficiency of gene transfection. The transfection efficiency is much higher than ultrasound irradiated liposome group, ultrasound irradiated microbubble group and liposome group.3.It has the synergistic efficacy between CD/5-Fc and TK/GCV suicide gene systems. The combination of both prodrugs were more effective than either one on the MCF-7 cells transfected with double suicide genes.4.Bystander effect to some extent makes up the weakness of low transfection efficiency, enhancing the feasibility of the transfection method of ultrasound mediated microbubble destruction combined with liposome in suicide gene therapy.5.Ultrasound mediated microbubble destruction combined with liposome transfecting double suicide genes is an ideal strategy for breast cancer gene therapy.更多还原