ApoE修饰的Lipofectamine 2000介导HSV-tk基因对肝癌杀伤作用的研究

发布时间：2018-03-28 22:03

本文选题：载脂蛋白E　切入点：基因治疗　出处：《山西医科大学》2017年硕士论文

【摘要】：目的:1.将ApoE(载脂蛋白E)与阳离子脂质体Lipofectamine 2000偶联,构建能特异转染肝(癌)细胞的基因运载体。2.检测在体外细胞实验中,ApoE-Lipofectamine 2000的转染特异性及转染效率,并检测其对细胞的毒性作用。3.通过ApoE-Lipofectamine 2000介导的pcDNA3.1-pSurvivin-TK联合更昔洛韦(GCV)系统的体内抑制试验,观察该系统对裸鼠肝癌模型的靶向杀伤作用。方法:1.将ApoE与Lipofectamine 2000偶联,制备ApoE修饰的Lipofectamine2000。2.在体外细胞实验中,质粒包裹实验分析脂质体与质粒的结合比;脂质体携带pGenesil-1质粒转染肝细胞HL-7702、肝癌细胞HepG2、人胚肾细胞HEK293T、大肠癌细胞SW480,并通过荧光显微镜及流式细胞仪检测转染效率;MTT法分析脂质体对细胞的毒性作用。3.在体内试验中,将HepG2细胞接种于裸鼠右腋皮下,建立肝癌裸鼠移植瘤模型;待肿瘤长至约为200 mm3时,将荷瘤裸鼠随机分为三组;A组:肿瘤空白对照组;B组:Lipofectamine 2000+pcDNA3.1-pSurvivin-TK+GCV治疗组;C组:ApoE-Lipofectamine 2000+pcDNA3.1-pSurvivin-TK+GCV治疗组。观察各组治疗后瘤体积及瘤重的变化,计算抑瘤率;RT-PCR法与Western blotting法观察脂质体介导的HSV-tk质粒在瘤组织中的表达情况;HE染色与TUNEL法观察治疗后移植瘤的凋亡坏死情况。结果:1.制备ApoE修饰的Lipofectamine 2000运载体,质粒包裹实验显示质粒与Lipofectamine 2000和ApoE修饰的Lipofectamine 2000的质量体积比分别为1:2,1:2.5时,可完全包裹质粒。2.流式结果表明,ApoE-Lipofectamine 2000对HL-7702和HepG2细胞的转染效率明显高于Lipofectamine 2000,转染效率有显著性差异,具有统计学意义(P0.05,P=0.03,P=0.001)。在HEK293T、SW480细胞中,两种脂质体转染效率无明显差异,无统计学意义(P0.05,P=0.93,P=0.55)。3.MTT实验表明ApoE修饰后的Lipofectamine 2000没有增加Lipofectamine2000对HepG2细胞的毒性作用(P0.05)。4.成功建立裸鼠肝癌移植瘤模型;治疗后,与对照组相比,瘤体积和瘤重均减小,B、C组抑瘤率分别为(29.46±2.51)%,(46.18±1.79)%,二者相比差异有统计学意义(P0.05,P=0.007)。5.Western blotting与RT-PCR结果显示,在A组未见明显条带,B、C两组均检测到HSV-tk质粒成功表达。6.HE染色与TUNEL法显示,两种脂质体介导的HSV-tk质粒联合GCV治疗对肝癌移植瘤均具有一定的杀伤作用,但ApoE修饰后的Lipofectamine 2000介导的抑制作用更明显。结论:1.成功构建了一种肝癌靶向基因递送载体,ApoE-Lipofectamine 2000。2.ApoE修饰的Lipofectamine 2000对肝(癌)细胞具有较高的亲和力,能够靶向转染肝(癌)细胞。3.ApoE-Lipofectamine 2000介导的pcDNA3.1-pSurvivin-TK联合更昔洛韦(GCV)系统对裸鼠肝癌模型具有靶向杀伤作用。
[Abstract]:Objective 1. To couple apolipoprotein E (ApoE) with cationic liposome Lipofectamine 2000 to construct gene carrier .2. to detect the transfection specificity and transfection efficiency of ApoE-Lipofectamine 2000 in vitro. In vivo inhibition test of ApoE-Lipofectamine 2000 mediated pcDNA3.1-pSurvivin-TK combined with ganciclovir (GCV) system was used to observe the targeted killing effect of the system on nude mice liver cancer model. Methods: 1. ApoE was coupled with Lipofectamine 2000. ApoE modified Lipofectamine 2000.2 was prepared. In vitro cell experiment, plasmid encapsulation assay was used to analyze the binding ratio of liposomes to plasmids. Liposomes carrying pGenesil-1 plasmid were transfected into hepatocytes HL-7702, HepG2, HEK293T, SW480. the transfection efficiency was detected by fluorescence microscope and flow cytometry. HepG2 cells were inoculated subcutaneously in the right axilla of nude mice to establish the transplanted tumor model of hepatoma in nude mice, and when the tumor reached about 200 mm3, The nude mice were randomly divided into three groups: control group B (control group) and control group (group B) treated with 2000 pcDNA3.1-pSurvivin-TK GCV. The changes of tumor volume and tumor weight after treatment were observed in group C (group C) and group C (group C) treated with ApoE-Lipofectamine 2000 pcDNA3.1-pSurvivin-TK GCV. The expression of HSV-tk plasmid mediated by liposome in tumor tissue was observed by RT-PCR and Western blotting. The apoptosis and necrosis of transplanted tumor were observed by HE staining and TUNEL method. Results 1. The ApoE modified Lipofectamine 2000 carrier was prepared. Plasmid encapsulation assay showed that when the mass / volume ratio of the plasmid to Lipofectamine 2000 modified by Lipofectamine 2000 and ApoE was 1: 2 / 1: 2.5, the plasmid could be completely encapsulated. Flow cytometry showed that the transfection efficiency of ApoE-Lipofectamine 2000 on HL-7702 and HepG2 cells was significantly higher than that of Lipofectamine 2000, and there was significant difference in transfection efficiency between ApoE-Lipofectamine 2000 and Lipofectamine 2000. There was no significant difference in transfection efficiency between the two liposomes in HEK293TnSW480 cells. The results showed that Lipofectamine 2000 modified with ApoE did not increase the toxicity of Lipofectamine2000 to HepG2 cells. Compared with the control group, the tumor volume and tumor weight of the control group were significantly lower than that of the control group. The tumor inhibition rates in the group C were 29.46 卤2.51g and 46.18 卤1.79g, respectively. The difference between the two groups was statistically significant. The results of Western blotting and RT-PCR showed that the tumor volume and weight of the two groups were significantly lower than those in the control group. The successful expression of HSV-tk plasmid was detected in group A and group C. 6. He staining and TUNEL staining showed that both liposome-mediated HSV-tk plasmids combined with GCV therapy had a certain killing effect on liver cancer transplanted tumor. But the inhibitory effect of ApoE modified Lipofectamine 2000 was more obvious. Conclusion: 1. A hepatoma targeting gene delivery vector, Lipofectamine 2000 modified by 2000.2.ApoE, was successfully constructed, and Lipofectamine 2000 had high affinity to liver (cancer) cells. ApoE-Lipofectamine 2000 mediated pcDNA3.1-pSurvivin-TK combined with ganciclovir has targeted killing effect on nude mice liver cancer model. 3.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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