重组人组织激肽释放酶基因转染人脐静脉内皮细胞  

Human umbilical vein Endothelial Cells by Recombinant Human Tissue Kall ikrein Gene Transfer

作　　者：陈慧[1] 朱鹏立[2] 李体远 余福玲[1] 阮景明[2] 李世峰[1] 吴小盈[1] 杜珙 戴勇 

机构地区：[1]福建省立医院省心血管病研究所内科,福建福州350001 [2]福建省立医院高干病房,福建福州350001 [3]深圳市人民医院中心实验室,广东深圳518020

出　　处：《高血压杂志》2004年第5期456-460,共5页Chinese Journal of Hypertension

基　　金：福建省青年科技人才创新项目 ( 2 0 0 1 J 0 6)

摘　　要：目的　探讨重组人组织型激肽释放酶 (HK)基因腺相关病毒载体 (rAVV/HK)对体外培养的人脐静脉内皮细胞 (HUVECs)作用。方法　将HK基因定向克隆入腺相关病毒质粒 pAAV MCS中 ,形成重组腺相关病毒 (rAAV HK)质粒 ,经聚合酶链反应 (PCR)检测和DNA测序后 ,将rAAV HK、pAAV Helper和 pAAV RC 3种质粒通过脂质体共转染 2 93细胞 ,包装成带有HK目的基因的重组rAVV/HK ,采用 β半乳糖苷酶原位染色法测定报告基因rAVV/LacZ在HT10 80中的表达 ,间接计算病毒滴度 ,并观察转染基因在传代细胞中的表达。在相同的条件下体外分别培养HUVECs(对照组 )和rAVV/HK转染的HUVEC(HK转染组 ) 4 8h ,采用逆转录聚合酶链反应 (RT PCR)技术检测两组HUVECsHKmRAN的表达 ,并利用硝酸还原酶法和分光光度法分别检测两组HUVECs一氧化氮(NO)含量和一氧化氮合酶 (NOS)活性。结果　rAVV/HK病毒滴度为 6 .2× 10 7个 /mL ,LacZ基因能够在第一传代和第六代HT10 80细胞内稳定持续表达。与对照组比 ,HK转染组HUVECsHKmRNA表达增加 (P <0 0 0 1) ;而且HK转染组HUVECs细胞培养液中NO的含量和NOS活性明显增高 ,P值分别 <0 0 1和 <0 0 5。结论　HK基因通过重组腺相关病毒载体可成功转染体外培养的HU VECs ,使其HKmRNA表达增加 。Objective To investigate the effect of recombinant aden o-associated virus vector(AVV) expressing the human tissue kallikrein(HK)ge ne (rAVV/HK) on human umbilical vein endothelial cells(HUVECs) in vitro. Methods The HK gene was inserted into the AAV cassettes(pAAV-MC ), then rAVV/HK were generated by cotransfection AAV-293 cells with three plas mids (rAAV-HK, pAAV-RC and pAAV-Helper) by means of lipidosome after rAAV-HK plasmids was formed and was detected by PCR(polymerase chain raction)meanwhil e being sequenced. The viral titer of rAVV/HK and its expression were measured b y β-galactosidase staining method,which could determine rAVV/LacZ report gene in HT1080 The cultured HUVECs in vtro was divided into HK transfection group s and control groups for 48 hours. The expression of HK mRAN was detected by RT -PCR(reverse transcription-PCR), NO contents and NOS activity were determine d by the nitrate reductase method and spectrophotometry respectively in the two groups. Results The viral titer of the rAVV/HK was 6.2×10 7 particles/mL. LacZ gene could be stably expressed to go down to the first a nd sixth generation in HT1080 Compared with control group, the expression of HK mRAN in HUVECs transfected by HK was significantly increased(P<0001). NO contents and NOS activity in HUVECs transfected by HK were significantly increased(P<001 and P< 005, respectively). Conclusions HK gene by recombinant adeno-associated virus vector can successfully transfect HUVECs in vitro. The expression of HK mRAN, NOS activit y and NO contents were incresed after the HUVECs by the HK gene transfection. T his result would provid the objective evidence for gene therapy of human vascula r endothelial cell abnormality.

关 键 词：重组人组织激肽释放酶基因 转染 脐静脉 内皮细胞 腺相关病毒载体 

分 类 号：R346[医药卫生—基础医学]