【作者】 余福玲；

【导师】 陈慧； 朱鹏立；

【作者基本信息】 福建医科大学 ， 内科学， 2004， 硕士

【摘要】 目的:1、构建携带人组织激肽释放酶基因(human kallikrein,HK)的腺相关病毒(Adeno-associated virus,AAV)载体,检测带有目的基因的重组AAV载体(rAAV/HK)滴度。2、体外培养人脐静脉内皮细胞HUVEC,将构建的rAAV/HK导入内皮细胞中,观察感染后内皮细胞一氧化氮(NO)、一氧化氮合酶(cNOS)的分泌情况。方法:1、试验一:在深圳市人民医院李体远博士合成的HK质粒基础上,扩增HK基因,用Xba I, EcoR I分别酶切HK基因和AAV-MCS质粒,然后连接HK和AAV-MCS的线性片断,形成重组腺相关病毒rAAV-HK质粒,经PCR检测和测序后,将rAAV-HK质粒与辅助质粒pHelper、pAAV-RC共转染到HEK293(人胚肾细胞)内包装成rAAV/HK载体,通过β-半乳糖苷酶原位染色法检测报告基因pAAV-LacZ在人纤维肉瘤细胞(HT1080)中的表达,计算rAAV/HK载体病毒滴度。2、试验二:在相同条件下体外培养2组血管内皮细胞,一组用不同浓度携带目的基因的rAAV/HK(6.2×103mmol/l、6.2×104mmol/l、6.2×105mmol/l)的病毒液感染HUVEC,以硝酸还原酶法和一氧化氮合酶检测试剂盒分别检测48小时后HUVEC细胞培养液中NO的浓度值和cNOS活力,选取其中NO和cNOS表达最高的为HK干预组。另一组为单纯HUVEC细胞(对照组),同时分别检测对照组培养48小时后HUVEC细胞培养液中NO的浓度值和cNOS活力。结果:1、经PCR检测和测序证实rAAV/HK载体存在HK目的基因片断,其长度为800bp,说明rAAV/HK载体构建成功,rAAV/HK的滴度为6.2×107/ml。RT-PCR表明HK的表达有增加。2、病毒液浓度为6.2×103mmol/l的HUVEC细胞培养液中NO表达高于其他浓度值病毒液。与对照组相比,HK干预组细胞液中的NO、cNOS均增加。体外培养48小时后HK干预组细胞液NO浓度值和cNOS活力分别为40.16±7.61μmol/l和7.71±1.25U/ml;而对照组分别为30.82±3.07μmol/l和5.72±1.23U/ml。经SPSS10.0统计处理,48小时后2组差别具有显著性意义(P<0.05=。<WP=4>结论:本试验构建的rAAV/HK载体能将HK导入人血管内皮细胞,并使内皮细胞的NO、cNOS的分泌增多。本研究结果为今后激肽释放酶-激肽系统在人类高血压的基因治疗提供了理论依据。更多还原

【Abstract】 Objectives: 1、To clone the human kallikrein(HK) gene and Adeno-associated virus plasmid (AAV-HK) to become the recombined AAV vector containing HK gene (rAAV/HK) and detect its concentration. 2、To study the changes of endothelial function through nitric oxides(NO) and nitric oxide synthase(cNOS) in cultured endothelial cell(HUVEC) with rAAV/HK vector.Methods:1、To amplify the HK coding region. The PCR product and AAV-MCS were cut by Xba I and EcoR I respectively. The HK was cloned into Adeno-associated virus(AAV) plasmid to become the recombined AAV plasmid (rAAV-HK).The rAAV-HK was confirmed by PCR and sequence, then co-transfecting the helper plasmid pHelper、plasmid RC and the rAAV-HK containing the human kallikrein cDNA into the packaging cell line 293 cell. The titer of rAVV/HK was measured indirectly using the pAAV-LacZ control plasmid through β-Galactosidase Staining Kit. 2、The cultured human endothelial cell HUVEC was divided into two groups. One group were infected by different concentrations of rAAV/HK vector (6.2×103mmol/l、6.2×104mmol/l、6.2×105mmol/l), then the contents of NO and vitality of cNOS were detected by nitrate reductase respectively after 48 hours and choose the highest expression of NO and cNOS as HK group. The other group was measured at the same time as control group.Results: 1、The successful construction of rAAV/HK vector was confirmed by PCR and sequence. The titers of its virus were determined to be about 6.2×107 per ml. The expression of human tissue kallikrein gene increase by RT-PCR. 2、Compared with control group, No and cNOS of endothelial cell in HK group were increased. The contents of NO and vitality of cNOS in HK group were 40.16±7.61μmol/l and 7.71±1.25U/ml respectively, while the contents of NO and vitality of cNOS in control group were 30.82±3.07μmol/l and 5.72±1.23U/ml after the HUVEC were cultured in 48 hours; There was a significant difference in 48 hours(P<0.05).Conclusions: The rAAV/HK vector can increase the NO and NOS <WP=6>expression of endothelial cell and improve the function of the endothelial cell. Our work provides a theoretical warranty for the use of KKS in gene therapy of human hypertension.更多还原