摘要
目的构建激肽释放酶(HK)腺相关病毒(AAV)载体,检测重组病毒感染脐静脉内皮细胞系(HUVEC)后HK基因和内皮功能相关基因表达的变化。方法HK基因克隆人腺相关病毒载体质粒pAAV-MCS,和腺相关病毒包装质粒pAAV-RC、腺病毒辅助质粒pHe1per共转染293细胞,包装成带有HK基因的重组腺相关病毒(rAAV-HK)。以rAAV-HK感染HUVEC。RT-PCR法检测HK基因、内皮型一氧化氮合酶(eNOS)、凋亡蛋白酶(caspase-3)、内皮素-1(ET-1)、血管内皮生长因子(VEGF)、内皮素B_1受体以及缓激肽B_1受体、缓激肽B_2受体的mRNA转录变化。ELISA法测定HUVEC上清液和胞内HK的含量。结果成功构建了rAAV-HK。rAAV-HK感染HUVEC后,实验组胞内HKmRNA转录(0．59±0．12)比空白组(0．26±0．05)明显增加(P＜0．01)；实验组胞内HK含量(120．1±40．9)比空白组(30．8±12．8)显著升高(P＜0．01)；实验组eNOSmRNA转录(1．19±0．28)较空白组(0．66±0．11)增加(P＜0．05),实验组caspase-3mRNA转录(0．30±0．25)较空白组(0．67±0．27)减弱(P＜0．05)。VEGF、内皮素-1、内皮素B_1受体、缓激肽B_1受体、缓激肽B_2受体mRNA转录两组差异无统计学意义。结论rAAV-HK病毒感染HUVEC后能高效表达HK,并促进HUVEC胞内eNOSmRNA转录,抑制caspase-3mRNA转录,提示导入HK基因能够改善内皮功能。
Objective To construct the recombinant adeno-associated virus vector （rAAV） expressing the human tissue kallikrein （HK） gene and to detect the expression of interested gene in human umbilical vein endothelial cells （HUVEC） which were infected with different titers of rAAV. Methods The HK gene was cloned into the pAAV- MCS and co-transfected AAV-293 cells with other two plasmids （the pAAV-RC, and pHelper） by lipofectamine. The recombinant AAV（rAAV- HK）particles was harvested and its viral titer was measured. HUVEC were infected with different titers of rAAV-HK particles. Seventy-two hours later, the expression of the interested gene was detected by RT-PCR and ELISA. Results The AAV expression system of human tissue kallikrein gene was successfully constructed. The viral titer of recombinant AAV reached 6.2×10^7 particles/ml. When compared with the control group, the transcription of HK gene in the group which was infected with rAAV-HK increased significantly [（0.59 ± 0.12） vs （0.26± 0. 05 ） （P 〈 0.05 ）], and the expression of HK in the HUVEC was three times more than that in the control[（120.1±40.9）vs （30. 8±12.8）] （P〈0. 001）. The transcription of eNOSmRNA in the infected HUVEC was higher than that of the control [（1.19±0. 28）vs（0.66±0.11）]（P〈0.05）. The transcription of caspase-3 mRNA was lower than that of the control[（0. 30±0. 25）vs（0. 67±0. 27）] （P〈0.05）. And there was no obvious variation happened in the transcription of VEGF, ET-1, TR-B, bradykinin B1 receptor and Bradykinin B2 receptor. Conclusions When co-transfected AAV-293 cells with three plasmids （pAAV-HK, pAAV-RC, pHelper）, the HK gene expression is significantly and stably increased. Introducing HK gene into HUVEC can improve endothelial transfection efficiency.
出处
《中华老年医学杂志》
CAS
CSCD
北大核心
2006年第12期925-929,共5页
Chinese Journal of Geriatrics
基金
福建省青年科技人员创新基金(2001J-06)
