GP73抗体修饰阳离子脂质体介导HSV-tk基因对肝癌细胞的选择性杀伤作用研究

发布时间：2018-04-18 20:04

本文选题：GP73抗体 + 阳离子脂质体　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:1.筛选肝癌细胞和其他非肝癌细胞表面高尔基体膜蛋白73(GP73)阳性表达率,确定GP73在肝癌细胞表面表达的特异性;2.制备普通阳离子脂质体;3.将普通阳离子脂质体(lipoplexes)与GP73抗体偶联,构建一种新型的由GP73抗体修饰的能够特异性结合肝癌细胞的靶向治疗载体;4.制备不同剂量的GP73抗体修饰脂质体,转染Hep G2肝癌细胞,流式细胞术筛选转染率最高的GP73抗体修饰组,确定脂质体中GP73抗体的最佳负载量;5.比较GP73抗体修饰的脂质体和普通阳离子脂质体对人肝癌细胞株Hep G2、Bel-7402、SMMC-7721和人正常肝细胞HL-7702以及人非肝癌细胞SW480和正常人胚肾细胞HEK293T的转染效率;6.研究由GP73抗体修饰的阳离子脂质体介导的HSVtk自杀基因联合更昔洛韦(GCV)对肝癌细胞的体外杀伤作用;7.研究由GP73抗体修饰的阳离子脂质体介导的HSVtk自杀基因联合GCV对肝癌荷瘤裸鼠体内肿瘤的杀伤作用。方法:1.用GP73的一抗(Anti-GOLPH2)连接二抗FITC分别孵育细胞Hep G2、SMMC-7721、Bel-7402、HL-7702、Hela、SW480和HEK293T,通过流式细胞仪检测细胞表面GP73的阳性表达率。2.乙醇注入法和挤压法联合制备普通阳离子脂质体(实验室前期制备方法)。3.将GP73抗体与阳离子脂质体偶联,制备GP73抗体修饰的脂质体。4.将普通阳离子脂质体和GP73抗体修饰脂质体分别与p Genesil-1质粒混匀、孵育,确定最佳的包裹比例。5.制备含量不同的GP73抗体修饰的阳离子脂质体,转染Hep G2肝癌细胞,流式细胞术筛选转染率最高的GP73抗体修饰脂质体组,确定脂质体中GP73的最佳负载量。6.分别使用GP73抗体修饰的阳离子脂质体转染p Genesil-1质粒,流式细胞仪检测Hep G2、Bel-7402、SMMC-7721、HL-7702、Hela、SW480、HEK293T细胞的报告基因绿色荧光蛋白(EGFP)的表达。7.用GP73抗体修饰的阳离子脂质体携带p Survivin-TK-PBI-CMV2质粒分别转染细胞Hep G2、Bel-7402和HL-7702,48小时后提取细胞的总RNA和总蛋白,并通过RT-PCR、Westernblot技术方法检测细胞中HSVtk基因的m RNA和蛋白表达水平。8.用GP73抗体修饰的阳离子脂质体携带p Survivin-TK-PBI-CMV2质粒分别转染肝癌细胞Hep G2、Bel-7402和肝正常细胞HL-7702,加入浓度为150μg/m L的前体药物GCV处理24 h,经流式细胞仪检测细胞凋亡情况。9.梯度剂量的GCV处理转染后的肝癌细胞48小时,MTT法检测HSVtk自杀基因系统对肝(癌)细胞生存率的影响情况。10.用生长状态良好的Hep G2肝癌细胞接种裸鼠成瘤,利用公式V=ab2/2,(a代表瘤体最长径,b代表瘤体最短径)计算瘤体积。11.HE染色检测干预后三组肿瘤组织坏死情况。12.TUNEL凋亡试剂盒检测干预后三组肿瘤组织细胞凋亡情况。13.裸鼠开始治疗后,观察裸鼠生长状态,并持续40天记录裸鼠存活状态,用Graphpad Prism v 6.0软件分析数据,绘制生长曲线图谱,比较三组荷瘤裸鼠生存期。结果:1.通过对细胞表面GP73阳性表达率的筛选,肝癌细胞Hep G2、Bel-7402、SMMC-7721细胞表面GP73阳性率高于HL-7702、Hela、SW480、HEK293T,差异有统计学意义(P0.05),表明了GP73在肝癌细胞表面表达有特异性;2.制备了一种普通阳离子脂质体;3.制备了一种GP73抗体修饰的能够特异性结合肝癌细胞的靶向治疗载体(Anti GP73-lipoplexes);4.GP73抗体修饰的脂质体对人肝癌细胞株Hep G2、Bel-7402和SMMC-7721的转染效率明显高于肝正常细胞和非肝癌细胞(P0.05);5.基因和蛋白水平检测表明,HSVtk基因在Hep G2和Bel-7402肝癌细胞中有表达,而在肝正常细胞HL-7702中未见表达;6.细胞增殖实验(MTT)结果可见,Hep G2和Bel-7402肝癌细胞转染组在GCV浓度不断增加的情况下,细胞存活率不断下降,与未转染加药组和正常肝细胞HL-7702相比有统计学差异(P0.05);7.流式细胞仪(FC 500)检测凋亡结果表明Hep G2、Bel-7402肝癌细胞死亡率明显高于肝正常细胞组(P0.05);8.肝肿瘤裸鼠造模成功,肿瘤体积达160~200 mm3;9.免疫组化和TUNEL检测肿瘤组织细胞凋亡结果表明,Anti GP73-lipoplexes运载HSVtk基因联合GCV治疗组肿瘤组织细胞凋亡明显,而lipoplexes+HSVtk+GCV组有少量细胞凋亡,空载组(lipoplexes+Vector+GCV)几乎未见凋亡细胞;10.生存曲线分析表明Anti GP73-lipoplexes+HSVtk+GCV治疗组裸鼠生存期明显长于对照组(P0.05)。结论:1.肝癌细胞Hep G2、Bel-7402和SMMC-7721表面GP73阳性表达率高于肝正常细胞和非肝癌细胞。2.GP73修饰的阳离子脂质体(GP73-lipoplexes)载体运输系统成功构建。3.Anti GP73-lipoplexes对肝癌细胞具有较高的转染效率。4.Anti GP73-lipoplexes介导自杀基因真核表达质粒p Survivin-TK-PBI-CMV2在GP73阳性高表达的Hep G2、Bel-7402肝癌细胞内成功表达。5.在细胞水平:Anti GP73-lipoplexes介导SUR启动子调控的HSVtk/GCV自杀基因系统对Hep G2、Bel-7402肝癌细胞具有选择性杀伤作用,对正常肝细胞HL-7702没有杀伤作用。6.Hep G2肝癌细胞异种移植瘤裸鼠接种成功。7.在动物水平:Anti GP73-lipoplexes介导SUR启动子调控的HSVtk/GCV自杀基因系统对Hep G2肝癌细胞异种移植瘤裸鼠肿瘤的生长与增殖有抑制作用,而且能够延长治疗组荷瘤裸鼠的生存期。
[Abstract]:Objective: 1. screening of hepatoma cells and other non HCC cell surface Golgi membrane protein 73 (GP73) positive expression rate, determine the specificity of GP73 expression in hepatocellular carcinoma cell surface; 2. the preparation of cationic liposome; 3. common cationic liposome (Lipoplexes) coupled with GP73 antibody, construct a novel by GP73 the modified antibody could specifically bind to hepatoma cells targeting vector; GP73 antibody modified liposome preparation of 4. different doses of Hep transfected with G2 hepatoma cells, the transfection efficiency of screening GP73 antibody modified group the highest flow cytometry, determine the optimal loading of GP73 antibody in liposome; Bel-7402 liposomes and cations 5. comparison of GP73 liposome modified antibody on human hepatocellular carcinoma cell line Hep, G2, SMMC-7721 and human normal liver cell HL-7702 and human hepatoma cells SW480 and non normal human embryonic kidney cells HEK293T transfection efficiency; 6. study by G Cationic liposome P73 antibody modified by HSVtk gene combined with ganciclovir (GCV) Dutch act killing effect on hepatocarcinoma cells in vitro; cytotoxicity 7. study by cationic lipid GP73 antibody modified body mediated HSVtk gene combined with GCV Dutch act on hepatocellular carcinoma tumor. Methods: 1. GP73 - (Anti-GOLPH2) connecting the two anti FITC were incubated with Hep, G2, SMMC-7721, Bel-7402, HL-7702, Hela, SW480 and HEK293T, the positive expression of GP73 cell surface was detected by flow cytometry.2. ethanol injection method and extrusion method combined with the preparation of cationic liposome (Lab preparation method).3. antibody and GP73 cationic liposome conjugated.4. liposome preparation of GP73 antibody modified ordinary cationic liposome and GP73 antibody modified liposome and plasmid Genesil-1 P were mixed and incubated to determine the best preparation for the proportion of.5. package Cationic lipid GP73 antibody modified with different content of the body, transfection of Hep G2 cells, the transfection efficiency of screening GP73 antibody modified liposome group by flow cytometry. The determination of GP73 liposome in the best load amount of.6. respectively using cationic lipid GP73 antibody modified P transfected Genesil-1 plasmid, Hep G2 detection, flow cytometry SMMC-7721, HL-7702, Bel-7402, Hela, SW480, reporter gene of HEK293T cells with green fluorescent protein (EGFP) expression of.7. using GP73 antibody modified cationic liposome with P Survivin-TK-PBI-CMV2 plasmid were transfected into Hep cells G2, Bel-7402 and HL-7702,48 hours after the extraction of total RNA cells and total protein, and the expression of m by RT-PCR. RNA and HSVtk protein gene Westernblot method of detecting the expression level of.8. with cationic lipid GP73 antibody modified body carrying P Survivin-TK-PBI-CMV2 plasmid respectively transfectedinto fine Hep cell G2, Bel-7402 and normal liver cells HL-7702, the concentration of 150 g/m L prodrugs of GCV treatment for 24 h, the cell apoptosis was detected by flow cytometry.9. doses of GCV treatment of hepatocellular carcinoma cells after transfection for 48 hours, MTT method for detection of HSVtk gene on liver Dutch Act (cancer) growth state good Hep G2 hepatocellular carcinoma cells in nude mice inoculated with.10. on cell survival rate, using the formula V=ab2/2 (a represents the longest diameter of the tumor, the tumor B represents the shortest path) to start treatment of the three groups of tumor cell apoptosis..13. nude mice tumor volume calculation.11.HE staining of tumor tissue necrosis after the intervention of three groups.12.TUNEL apoptosis detection kit after intervention, to observe the growth state of nude mice, and lasted for 40 days to record the survival status of nude mice, analysis of data using Graphpad Prism v 6 software, drawing the growth curves, compared with three groups of nude mice survival. Results: 1. through the screening of cell surface GP73 positive expression rate of Hep cells, G2, Bel-7402, SMMC-7721 positive rate of cell surface GP73 is higher than HL-7702, Hela, SW480, HEK293T, the difference was statistically significant (P0.05), showed that GP73 specific expression in HCC cell surface; a common cationic liposome preparation 2.; 3. were prepared by a modified GP73 antibody could specifically bind to hepatoma cells targeting vector (Anti GP73-lipoplexes); 4.GP73 antibody modified liposome on human hepatocellular carcinoma cell line Hep G2, Bel-7402 SMMC-7721 and the transfection efficiency was significantly higher than that of normal liver cells and non liver cancer cells (P0.05) show; detection of 5. gene and protein level, HSVtk gene expression in Hep G2 and Bel-7402 in HCC cells, whereas in normal liver cells HL-7702 no expression; 6. cell proliferation assay (MTT) showed that Hep, G2 and Bel-7402 hepatoma cells transfected group In the case of increasing the concentration of GCV, cell survival rate decreased, there was significant difference compared with untransfected dosing group and normal liver cells (P0.05 HL-7702 7.); flow cytometry (FC 500) apoptosis detection results showed that Hep G2, Bel-7402 cell mortality was significantly higher than normal liver cells group (P0.05); 8. liver cancer nude mice model, the tumor volume reached 160~200 mm3; 9. TUNEL and immunohistochemical detection of tumor cell apoptosis. The results showed that the Anti GP73-lipoplexes carrying HSVtk gene combined with GCV on the apoptosis of tumor cells was significantly, while the lipoplexes+HSVtk+GCV group has a small amount of apoptosis vectorgroup (lipoplexes+Vector+GCV) almost no apoptotic cells; 10. survival curves Anti analysis showed that GP73-lipoplexes+HSVtk+GCV treatment group was significantly longer than the control group of nude mice survival (P0.05). Conclusion: the liver cancer cell Hep 1. G2, Bel-7402 and GP73 on the surface of SMMC-7721 Cationic lipid positive expression rate is higher than the normal liver cells and non liver cells modified with.2.GP73 body (GP73-lipoplexes) carrier transport system was successfully constructed.4.Anti GP73-lipoplexes.3.Anti GP73-lipoplexes has high transfection efficiency on hepatoma cells by Dutch act gene eukaryotic expression plasmid P Survivin-TK-PBI-CMV2 in GP73 positive expression of Hep G2 and Bel-7402.5. cells in hepatoma cells the expression level of success: Dutch act system of Hep G2 Anti gene GP73-lipoplexes mediated by SUR promoter HSVtk/GCV, Bel-7402 cells with selective cytotoxicity on normal liver cells, HL-7702 had no killing effect of.6.Hep G2 cell xenografts in nude mice successfully.7. in animal level: Anti GP73-lipoplexes mediated by SUR promoter of HSVtk/GCV gene Dutch act the system of Hep G2 cell xenografts in nude mice and tumor growth Proliferation has inhibitory effect and can prolong the survival period of nude mice with tumor bearing.

【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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