【作者】 钱滢；

【导师】 周平；

【作者基本信息】 中南大学 ， 影像医学与核医学， 2011， 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,基因治疗成已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。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,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。而利用肿瘤特异性调控元件(启动子或增强子)以驱动自杀基因选择性表达于特定的靶细胞中,能准确杀伤肿瘤细胞,保护正常组织,减少毒副作用,这也是自杀基因临床应用的主要优势。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,微泡造影剂作为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,还能提高转染效率,被认为是今后非病毒载体发展的重要方向。本项目拟应用VEGF启动子驱动时CD/TK双自杀基因系统对乳腺癌细胞MCF-7的作用研究,联合使用超声辐照微泡,观察该系统在乳腺癌中的应用效果,为进一步开展自杀基因靶向治疗乳腺癌的研究提供实验依据。目的1.构建乳腺癌移植瘤裸鼠模型(MCF-7体内模型)。2.超声辐照微泡介导pEGFP-KDRp-CD/TK质粒转染乳腺癌裸鼠模型,观察该治疗系统对乳腺癌的体内抑瘤作用。方法建立乳腺癌移植瘤裸鼠模型,将20只荷瘤裸鼠随机分成4组,每组5只。I组：空白对照组,仅给予前药5-FC与GCV；II组：仅注射裸质粒与前药5-FC与GCV;Ⅲ组：注射裸质粒与前药5-FC与GCV,并予超声辐照；Ⅳ组：注射靶向超声造影剂与前药5-FC与GCV,并予超声辐照。从处理开始当天起,每3天测量肿瘤大小,计算肿瘤的体积,观察不同处理组的肿瘤生长情况；处理结束后5天处死裸鼠,剥除肿瘤并称重,计算各处理组与空白对照组相比较的抑瘤率；使用倒置荧光显微镜观察目的基因体内转染情况,并据此计算基因转染率；RT-PCR法检测目的基因的表达；免疫组化法计数肿瘤的微血管密度。根据以上指标了解肿瘤的生长情况和目的基因的体内转染情况,从而评估超声辐照微泡介导双自杀基因转染对裸鼠乳腺癌移植瘤的体内抑瘤作用。结果1.Ⅱ、Ⅲ、Ⅳ组处理组的抑瘤率分别为3.72%、21.40%、47.13%,同空白对照组(Ⅰ组)比较,裸质粒组(Ⅱ组)的肿瘤大小较之I组没有明显的差异(P>0.05),而超声辐照组(Ⅲ组)和超声微泡联合辐照组(Ⅳ组)的肿瘤大小较之Ⅰ组均有明显的差异(P<0.05),尤其以超声微泡联合辐照组的肿瘤的体积抑制最为显著。2.Ⅳ组在超声微泡的联合作用下,其基因转染的效率要明显高于Ⅲ组超声组,两者比较有显著的统计学差异(P<0.001)；超声组与裸质粒组比较,基因转染效率稍高,但其差异没有统计学意义(P>0.05)。3.Ⅲ组(超声组)、Ⅳ组(超声微泡组)的肿瘤组织中均出现了2415bp的CD/TK基因阳性片段,而在Ⅰ组(空白对照组)、II组(裸质粒组)的肿瘤组织中未出现该阳性片段。4.Ⅳ组在超声微泡的联合作用下,其对肿瘤血管的抑制作用要明显高于Ⅲ组超声组,两者比较有明显的统计学差异(P<0.05)；Ⅲ组(超声组)、Ⅳ组(超声微泡组)与空白对照组相比较,其差异均有统计学意义(P<0.05)；裸质粒组与空白对照组比较,MVD计数稍低,但其差异没有统计学意义(P>0.05)。结论：1.皮下接种法建立裸鼠乳腺癌移植瘤动物模型具有操作简单、模型稳定、成功率高等优点,并且该模型能保持人乳腺癌肿瘤细胞的部分病理学特点及生物学特性；2.应用MCF-7能成功的建立乳腺癌移植瘤裸鼠动物模型,并能方便地观测移植瘤的生长情况；3.超声辐照可提高裸鼠移植瘤组织的微血管通透性,促进基因转染物质进入细胞,超声辐照微泡则能更有效的介导目的基因转染,提高基因转染率；4.重组质粒转基因,荷瘤动物肿瘤组织内可以表达CD/TK融合基因的产物,联合超声辐照微泡,可以更有效的介导目的基因在肿瘤组织中表达,使该治疗系统具有满意的体内抑瘤作用,表现为对肿瘤生长的抑制和肿瘤微血管密度的下降；5.超声辐照微泡介导双自杀基因系统是较理想的乳腺癌基因治疗策略。更多还原

【Abstract】 BackgroundBreast 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. Currently the most widely used systems are herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) and cytosine deaminas/5-fluorocytosine (CD/5-Fc). Combined using of them has several advantages, such as improving anti-tumor effect, avoiding drug resistance, etc.The specific expression of suicide genes in the tumor cells so as to selectively damage tumor cells could be realized by taking advantage of some tumor specific transcription modulating elements(promoter or enhanser). So, the anti-tumor effect is quite satisfied with mild side-effects. It is the main dominance of suicide gene therapy in clinic application. So that KDR promoter (KDRp) could make genes of interest over-express in tumor cells and its vascular endothelial cells.In recent years, 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.The project planed to apply CD/TK fusion suicide gene system driven by VEGF promoter to MCF-7 tumor cells, transfecting by ultrasound mediated microbubble destruction. Then, the effect of the system therapy for breast cancer was observed. So, the study Was carried out to provide further experimental evidences to treat the breast cancer with double suicide genes.Objective 1.Construction of nude mice model bearing human breast cancer (MCF-7 in vivo).2.To transfect the pEGFP-KDRp-CD/TK into breast cancer model in nude mice by ultrasound mediated microbubble, and then investigate the anti-tumor effect of the treatment system on breast cancer in vivo.MethodsConstruction of nude mice model bearing human breast cancer,20 nude mice were randomly divided into 4 groups (n=5). GroupⅠ(control group):only injection of 5-FC and GCV. GroupⅡ:injection of naked plasmid,5-FC and GCV. GroupⅢ:injection of naked plasmid,5-FC and GCV, ultrasonic irradiation was given at the same time. GroupⅣ: injection of targeted ultrasound contrast agents,5-FC and GCV, ultrasonic irradiation was given at the same time. Measurement of tumor size from the day of treatment every 3 days to calculate the tumor’ s size and observe the tumor growth of different groups. The nide mice were killed after end of the treatment, the tumor were removed and weighted to calculate the inhibition rate compared with the control group. The target gene transfer situation in vivo was observed by inverted fluorescence microscope to calculate the gene transfer rate. Target gene expression was detected by RT-PCR. Microvessel density was counted by immunohistochemistry. The growth of tumor and target gene transfection in vivo were observed by indicators above to assess the tumor inhibitory effection of the tumor in nude mice by ultrasound microbubble mediated double suicide gene transfer.Results1.The inhibition rate of groupⅡ, groupⅢ, groupⅣis 3.72%, 21.40%,47.13%, the tumor size between groupⅡand groupⅠshowed no statistically significant difference(P>0.05). The tumor size of groupⅢand groupⅣshowed statistically significant difference compared with groupⅠ(P<0.05). The inhibition of tumor size of groupⅣwas most significant.2. The gene transfection rate of group IV mediated microbubble was higher than that of groupⅢ, and there was statistically significant difference the two group (P＜0.001). The gene transfection rate of group Ⅲwas higher than that of groupⅡ, but there was no statistically significant difference the two group (P>0.05)3. CD/TK expressed in the tumor of groupⅢand groupⅣ, but not expressed in the tumor of groupⅠand groupⅡ.4.The tumor angiogenesis inhibition of group IV mediated microbubble was higher than that of groupⅢ, and there was statistically significant difference the two group (P<0.05). That of groupⅢand groupⅣcompared with control group, there were statistically difference (P<0.05). The MVD count of groupⅡwas lower than that of control group, but there was no statistically significant difference the two group (P>0.05).Conclusions1. Construction of nude mice model bearing human breast cancer with subcutaneous inoculation has the advantages of simply operation, stability, high success rate. The model is able to maintain some of the human breast tumor’ s biological characteristics and pathological features.2. Construction of nude mice model bearing human breast cancer with MCF-7 cells, the growth of transplanted tumor could be observed conveniently.3.Ultrasonic irradiation can increase the microvascular permeability of transplanted tumor and promot transfection of genetic material into cells. Ultrasound mediated microbubble can transfer target gene more efficiently to enhance gene transfer rate.4. Ultrasound mediated microbubble can transfer target gene expressed in tumor tissue, which making a satisfactory tumor inhibition by the treatment system showed the inhibition of tumor growth and decline in tumor MVD.5.Ultrasound irradiate microbubble mediated double suicide gene system is a perfect strategy of gene therapy for breast cancer.更多还原