【作者】 陈选民；

【导师】 张桂英；

【作者基本信息】 中南大学 ， 消化内科， 2010， 硕士

【摘要】 背景及目的：胃癌是人类常见的恶性肿瘤之一,在全球范围内,发病率居恶性肿瘤的第4位,病死率居恶性肿瘤的第2位。在我国,胃癌的发生率与病死率均居恶性肿瘤前2位。胃癌的预后差,手术、放射治疗、化学治疗效果有限,迫切需要新的有效的治疗方法。从肿瘤的恶性转化和肿瘤发展的分子遗传学来看,胃癌是一种获得性的多阶段的基因病,它的生长、侵袭、凋亡与基因相关。目前基因治疗肿瘤的方式主要为自杀基因治疗、抑癌基因治疗、反义基因治疗、免疫基因治疗,以及在单一基因治疗策略基础上发展起来的联合基因治疗。胃癌的基因治疗中,自杀基因是研究较多的一类抗肿瘤基因,如自杀基因CD/5-FC、HSV-TK/GCV系统具有强大的杀伤肿瘤细胞,抑制肿瘤生长的作用。RNA干扰,是近几年来发展起来的一门新兴的在转录水平上的基因阻断技术,是一种双链RNA (double-stranded RNA,dsRNA)分子在mRNA水平上关闭相应序列基因的表达或使其沉默的过程,也就是序列特异性的转录后基因沉默(post transcriptional gene silencing, PTGS)。研究表明,VEGF的RNA干扰技术是一种有效的抑制肿瘤细胞增殖、迁移、侵袭和血管生成的策略。肿瘤的发生是复杂的多因素、多步骤、涉及多基因事件发生的过程,针对单一环节的转基因治疗常常不能达到满意的治疗效果,近十年来,人们开始把目光转向联合基因治疗。所谓肿瘤联合基因治疗是指几种不同目的基因之间、目的基因与化疗药物、放射线及生物反应修饰剂之间联合应用以求获得更佳的抗肿瘤效果,同时尽可能地降低对机体产生的毒副作用。研究显示,联合基因治疗存在协同效应,能够增强对肿瘤的杀伤作用。为了探索更有效的胃癌基因治疗体系,我们在pcDNA3.1(-)-CV-yCDglyTK骨架基础上,将来自pGenesil-shVEGF的U6-shRNA表达框亚克隆至该骨架载体上,旨在通过联合两种作用机制的不同基因治疗体系,获得更有效的杀伤胃癌细胞的方法,为胃癌的基因治疗提供新策略。方法：以磷酸钙纳米颗粒为载体,介导pcDNA3.1(-) Null, pGenesil-shRNA, pcDNA3.1(-)-CV-yCDglyTK, pcDNA3.1(-)-shVEGF /yCDglyTK转染胃癌细胞SGC7901,用荧光显微镜观察报告基因GFP表达情况,结合流式细胞技术测定转染效率；RT-PCR检测细胞中yCDglyTK及VEGFmRNA的表达情况；Western-blot分析yCDglyTK及VEGF蛋白的改变；MTT法检测5-FC对转染细胞的增殖的影响；流式细胞术检测转染细胞的凋亡情况。结果：1、磷酸钙纳米颗粒成功介导4种质粒转染SGC7901细胞,转染效率约54%；2、转染pcDNA3.1(-)-CV-yCDglyTK组及pCDNA3.1(-)-shVEGF/yCDglyTK组SGC7901细胞有yCDglyTK mRNA及相关蛋白表达,对照组及其余两组细胞无该基因的表达；转染pGenesil-shVEGF组及pcDNA3.1(-)-shVEGF/yCDglyTK质粒组SGC7901细胞VEGF表达受抑制,相对于内参P-actin的表达量,前者为0.36±0.04,后者为0.41±0.04,与对照组的0.66±0.05相比,有统计学差异(P<0.05)；3、MTT法检测结果显示pcDNA3.1(-)-CV-yCDglyTK组及pcDNA3.1(-)-shVEGF/yCDglyTK组细胞生长均明显受抑,相对于转染pcDNA3.1(-)-CV-yCDglyTK质粒组,pcDNA3.1(-)-shVEGF/ yCDglyTK组对5-FC有更高的敏感性(P<0.05)。4、流式细胞仪检测结果显示SGC7901组、pcDNA3.1(-) Null组,pGenesil-shVEGF组,pcDNA3.1(-)-CV-yCDglyTK组,pcDNA3.1(-)-shVEGF/yCDglyTK组凋亡率分别为：(3.24±0.40)%、(4.56±0.71)%、(21.6±1.11)%、(56.2±2.59)%、(67.9±4.78)%,联合质粒组凋亡率显著高于其余4组(P<0.05)。结论：1、磷酸钙纳米颗粒可成功介导pcDNA3.1(-) Null,pGenesil-shVEGF, pcDNA3.1(-)-CV-yCDglyTK,pcDNA3.1(-)-shVEGF/yCDglyTK等四种质粒转染SGC7901细胞；2、联合基因治疗体系pcDNA3.1(-)-shVEGF/yCDglyTK可有效抑制SGC7901细胞VEGF的表达。3、联合基因治疗体系pcDNA3.1(-)-shVEGF/yCDglyTK可有效地抑制SGC7901细胞的增殖,其作用要强于单独的RAN干扰或自杀基因,提示融合自杀基因与VEGF干扰质粒间存在协同效应。更多还原

【Abstract】 Background and Purposes:Gastric cancer is one of the common malignant tumors in humans,whose incidence ranks the first in four malignant tumors over the worldwide and No.2 on cancer mortality rate. In China, the incidence and mortality of gastric cancer tops the first. Prognosis of gastric cancer is poor, for the limited effect of surgery, radiation therapy, chemotherapy. So the need for new effective treatment is urgent. From malignant transformation and tumor development in terms of molecular genetics, cancer is a multi-stage acquired genetic disease, its growth, invasion and apoptosis are all associated with genes. Currently the main ways for gene therapy are suicide gene therapy, tumor suppressor gene therapy, antisense gene therapy, immune gene therapy, and combined gene therapy,a gene therapy strategie developed on the basis of a single.For gastric cancer gene therapy, suicide gene is more studied in all anti-tumor genes, such as suicide gene CD/5-FC, HSV-TK/GCV system, have powerful effect of anti-tumor and inhibit growth of tumor. RNA interference, discovered and developed in recent years, a new gene technology blocking genes on the level of transcription, is a double-stranded RNA (double-stranded RNA, dsRNA) molecules to close the corresponding gene sequence or silence the process of the expression on the mRNA level, that is sequence-specific post-transcriptional gene silencing (post transcriptional gene silencing, PTGS).Researches shows that, RNA interference towards VEGF is an effective strategy to inhibit proliferation, migration, invasion and vascular generation of tumor cells. Tumorigenesis is processes involved complex multi-factors, multi-steps and multi-genetic events. For a single link gene therapy treatment often can not achieve satisfactory results over the past decade, people began to turn their attentions to combined gene therapy. Combined gene therapy is the combination among several target genes, gene and chemotherapy drugs, radiation and biological response modifiers, in order to get a better combination between the anti-tumor effect, while reducing as much as possible on the body produced side effects. Most of these researches showed that combined gene therapy embraced synergistic effects, which can enhance anti-tumor effect. In order to explore more powerful gene treatment for system gantric cancer, we will clone the U6-shRNA expression cassette from pGenesil-shVEGF into the vector backbone of pcDNA3.1 (-)-CV-yCDglyTK frame basis.Through two mechanisms of gene therapy systems,we hope obtain a more effective method to killi cancer cells to provide a new strategies for clinical practice.Methods:Gastric cancer cell SGC7901 were transfected by plasmids of pcDNA3.1 (-) Null, pGenesil-shVEGF, pcDNA3.1 (-)-CV-yCDglyTK and pcDNA3.1 (-)-shVEGF/yCDglyTK mediated by carriers of calcium phosphate nanoparticles. Expressions of GFP reporter gene were observed by the fluorescence microscopy, combined with flow cytometry to determine transfection efficiency; Expressions of yCDglyTK and VEGFmRNA were detected by RT-PCR; Changes of protein of VEGF and yCDglyTK were analyzed by Western-blot; The inhibition effects of 5-FC on proliferation of transfected cells were analyzed by MTT; Apoptosis of transfected cells were observed by flow cytometry. Results:1. Four kinds of plasmids could successfully transfect SGC7901 cells mediated by calcium phosphate nanoparticles, transfection efficiency measured was about 54%; 2. The groups transfected with pcDNA3.1 (-)-CV-yCDglyTK and pCDNA3.1 (-)-shVEGF/yCDglyTK expressed yCDglyTK mRNA and protein,while the control group and the remaining two cells had no expression; The expression of VEGF of groups transfected with pGenesil-shVEGF and pcDNA3.1 (-)-shVEGF/yCDglyTK plasmid was inhibited, compared to internal reference P-actin expression level, the former was 0.36±0.04, the latter was 0.41±0.04, compared with the control group,which was 0.66±0.05, there was a significantly difference (P<0.05);3. MTT tested 5 groups of cells treated by 5-FC. The survival rate showed that growth of pcDNA3.1 (-)-CV-yCDglyTK group and pcDNA3.1 (-)-shVEGF/ yCDglyTK group were significantly inhibited, compared to pcDNA3.1 (-)-CV-yCDglyTK group pcDNA3.1 (-)-shVEGF/yCDglyTK group had a higher sensitivity to 5-FC (P<0.05).4. Results of flow cytometry also confirmed pcDNA3.1 (-)-shVEGF/yCDglyTK group had a higher apoptosis rate of (67.9±4.78)%, higher than the other 4 groups(P<0.05), which were (3.24±0.40)%、(4.56±0.71)%、(21.6±1.11)%、(56.2±2.59) %.Conclusions:1. Four kinds of plasmids, pCDNA3.1 (-) Null, pGenesil-l-hVEGF4-shRNA, pCDNA3.1(-)-CV-yCDglyTK, pCDNA3.1 (-)-shVEGF /yCDglyTK could successfully transfect SGC7901 cells mediated by calcium phosphate nanoparticles.2. Combined gene therapy system pcDNA3.1 (-)-shVEGF/yCDglyTK can inhibit the expression of VEGF in gastric cancer cells.3. Combined gene therapy system pCDNA3.1 (-)-shVEGF/yCDglyTK can effectively inhibit the proliferation of gastric cancer cells, whose effect is stronger than separate RAN interference plasmid or suicide gene. There is a synergistic effect between fusion suicide gene and the VEGF interference plasmid.更多还原