【作者】 李玉姬；

【导师】 张桂英；

【作者基本信息】 中南大学 ， 消化内科， 2010， 硕士

【摘要】 背景与目的：结肠癌是常见多发的恶性肿瘤之一,在全球男性、女性常见的恶性肿瘤中分别排第四位和第三位。在我国,结肠癌的发病率居全部恶性肿瘤的第四位。目前结肠癌的治疗以手术治疗为主,辅以化疗、放疗等。由于发病隐匿,大部分患者就诊时已属中晚期,失去手术机会,而化疗、放疗有严重的全身毒副作用,特异性差。因此,基因靶向治疗结肠癌是目前研究的热点。基因治疗的载体包括病毒载体和非病毒载体两类。病毒载体有免疫原性,可诱发机体产生免疫反应,在安全性方面存在潜在的危险。而非病毒载体无毒性,安全性高,故在研究中应用更多。磷酸钙纳米颗粒(CPNP)作为一种非病毒载体,具有转染效率高、对人体无毒副作用、使用安全性高等特点。自杀基因如CD/5-FC、HSV-TK/GCV系统对结肠癌有杀伤作用,且CD、TK两者联合之后的协同作用较之单独作用能更好地发挥对肿瘤的杀伤作用。肿瘤血管生成对肿瘤的生长、转移起到至关重要的作用,VEGF是促进肿瘤血管生成的重要因子,使用RNA干扰技术高度特异性地沉默VEGF表达,可有效抑制肿瘤血管生成,从而达到抑制肿瘤生长的目的。因此,本实验使用非病毒载体CPNP作为基因转染载体,应用CMV增强子、CEA启动子与融合自杀基因yCDglyTK整合,联合应用针对VEGF的RNA干扰技术,研究其对人结肠癌Lovo细胞的体外靶向性杀伤作用。方法：将结肠癌Lovo细胞分五组,空白对照组(L1组),其它四组分别为用CPNP为载体瞬时转染pcDNA3.1(-) Null (L2组)、pcDNA3.1(-)CVyCDglyTK (L3组)、pGenesil-shVEGF(L4组)、pcDNA3.1(-) shVEGF/yCDglyTK (L5组)等,用RT-PCR、免疫荧光检测yCDglyTK、VEGF基因的表达,用MTT法和流式细胞仪检测5-FC对各组细胞的杀伤作用和凋亡效率。实验数据采用SPSS 13.0和Excel2003进行统计分析,P＜0.05认为有统计学意义。结果：1、yCDglyTK及VEGF基因表达：(1)L3组、L5组有yCDglyTK基因mRNA及蛋白表达的增强；(2)各组细胞均有VEGF基因mRNA及蛋白的表达,其中L4组、L5组的表达较其它三组减弱。以上说明CPNP能成功介导四种质粒转染Lovo细胞。2、转染CPNP-DNA对各组Lovo细胞的增殖抑制作用：MTT结果显示L5组在5-FC的作用下生存率低于其他各组(P<O.05),说明转染联合基因治疗体系pcDNA3.1(-)shVEGF/yCDglyTK对Lovo细胞的增殖抑制作用较单独转染自杀基因yCDglyTK或shVEGF强。3、转染CPNP-DNA对各组Lovo细胞的凋亡作用：流式细胞仪结果显示各组细胞凋亡率分别为：L1组1.92%,L2组5.57%,L3组43.8%,L4 20.2%,L5组67.8%,其中L5凋亡率最高,说明转染联合基因治疗体系pcDNA3.1(-)shVEGF/yCDglyTK对Lovo细胞的凋亡作用较单独转染自杀基因yCDglyTK或shVEGF强。结论：1、磷酸钙纳米颗粒能成功介导pcDNA3.1(-)Null. pcDNA3.1(-)CVyCDglyTK、pGenesil-shVEGF、pcDNA3.1(-)shVEGF /yCDglyTK等四种质粒转染结肠癌Lovo细胞。2、yCDglyTK基因能有效抑制Lovo细胞的增殖,VEGF-shRNA能有效抑制VEGF的表达。3、联合基因治疗体系pcDNA3.1(-)shVEGF/yCDglyTK较单独的自杀基因治疗及RNA干扰更有效地杀伤结肠癌Lovo细胞。更多还原

【Abstract】 Background and Aim:Colon carcinoma is one of most common malignancies, which is the fourth most common cancer in men and the third in women worldwide. In China, the incidence is in the fourth place of all malignancies. Nowadays the chief therapy of colon carcinoma is surgery, coupled with radiotherapy and chemotherapy. A majority of patients has been found in the advanced stage and loses the chance of surgery because the symptoms are occult. Furthermore, radiotherapy and chemotherapy have a severe toxic and side-effect and weak specificity. Consequently, the gene targeted therapy of colon carcinoma has become the focus of researches. The vectors of gene therapy consist of two kinds, viral vectors and non-viral vectors. Because of immunogenicity, viral vectors can induce immunological reaction of body and have potential risk of safety, while non-viral vectors are non-toxic and safe, which are applied more in researches. As a kind of non-viral vectors, the calcium phosphate nanoparticle (CPNP) has many nice characteristics like high transfection efficiency, nonpoisonous side effect to human body, and high safety and so forth. The suicide genes such as CD/5-FC and HSV-TK/GCV systems have a lethal effect on colon carcinoma cells. Furthermore, the synergistic action of the unity of CD and TK has a more powerful lethal effect on carcinoma cells than each of them. Tumor angiogenesis plays a vital role in tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is one of most important substances to promote tumor angiogenesis. RNA interference can efficiently inhibit tumor angiogenesis by highly specific silence of VEGF gene expression, and eventually lead to the purpose of tumor growth inhibition. Hence, in this experiment, the non-virus vector CPNP is used as a gene transfection vector, and the integration of cytomegalovirus (CMV) enhancer, carcinoembryonic antigen (CEA) promoter and fusion suicide gene yCDglyTK combined with RNA interference of VEGF has a targeted lethal effect on human colon cancer cells Lovo in vitro. Method:The Lovo cells were divided into five group, one of which was the blank controller (Group L1), and the rest were separately transiently transfected into pcDNA3.1 (-) Null (Group L2),pcDNA3.1 (-) CVyCDglyTK (Group L3),pGenesil-shVEGF (Group L4),pcDNA3.1(-)-shVEGF/yCDglyTK(Group L5) using CPNP as a vector. The expression of the yCDglyTK and VEGF gene was detected by RT-PCR and immunofluorescence. MTT assay and flow cytometry (FCM) were used to detect the cytotoxic effects and apoptosis rates of the yCDglyTK/ 5-FC system. SPSS 13.0 and Excel 2003 were used in data analysis. There was a statistical significance when P＜0.05.Results:1. The expression of yCDglyTK and VEGF genes:(1) The expression of yCDglyTK mRNA and protein was enhanced in Group L3 and L5; (2) All groups expressed VEGF mRNA and protein, but the expression was weakened in Group L4 and L5. It showed that CPNP can successfully induce the transfection of four plasmids into Lovo cells.2. The anti-proliferative effect of transfection of CPNP-DNA in Lovo cells:MTT analysis showed that Group L5 had the lowest survival rate of all on the effect of 5-FC (P<0.05). It showed that transfection of the united gene therapic system pcDNA3.1(-)shVEGF/yCDglyTK had a more potential anti-proliferative effect on Lovo cells than yCDglyTK or shVEGF separately.3. The apoptosis of transfection of CPNP-DNA in Lovo cells:The apoptosis rates of FCM were as follows:Group L1 1.92%, Group L2 5.57%, Group L3 43.8%, Group L4 20.2%, Group L5 67.8%. Group L5 had the highest apoptoticrate. It showed that the transfection of the united gene therapic system pcDNA3.1(-)shVEGF/yCDglyTK led to higher apoptosis in Lovo cells than yCDglyTK or shVEGF separately.Conclusions:1.CPNP can successfully induce the transfection of pcDNA3.1(-)Null, pcDNA3.1(-)CVyCDglyTK, pGenesil-shVEGF and pcDNA3.1(-)shVEGF/yCDglyTK into colon cancer Lovo cells.2. The yCDglyTK gene can effeciently inhibit the proliferation of Lovo cells, and VEGF-shRNA can efficiently inhibit the expression of VEGF.3. The united gene therapic system of pcDNA3.1(-)shVEGF/yCD -glyTK is much more efficienct than suicide gene therapy or RNA interference separately in killing colon cancer Lovo cells.更多还原