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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 kallikrein (HK) adeno-associated virus (AAV) vector and detect the expression of HK and endothelial-related genes after the recombinant virus is infected with human umbilical vein endothelial cell line (HUVEC). METHODS: Human adeno-associated virus vector pAAV-MCS was cloned by HK gene and 293 cells were co-transfected with adeno-associated virus packaging plasmid pAAV-RC and adenovirus helper plasmid pHe1per. The recombinant adeno-associated virus (rAAV-HK ). HUVECs were infected with rAAV-HK. The expression of HK, eNOS, caspase-3, ET-1, VEGF and ET-1 were detected by RT-PCR. Body and bradykinin B 1 receptor, bradykinin B 2 receptor mRNA transcription changes. ELISA assay HUVEC supernatant and intracellular HK content. Results rAAV-HK was successfully constructed. After transfected with rAAV-HK, the intracellular HKmRNA transcription (0.59 ± 0.12) in HUVEC group was significantly higher than that in blank group (0.26 ± 0.05) (P <0.01). The intracellular HK content (120.1 ± 40.9) was significantly higher than that in the blank group (30.8 ± 12.8) (P <0.01). The eNOS mRNA transcription in the experimental group was significantly higher than that in the blank group (1.19 ± 0.28) .66 ± 0.11) (P <0.05). The caspase-3 mRNA transcription in the experimental group (0.30 ± 0.25) was weaker than that in the blank group (0.67 ± 0.27) (P <0.05) ). There was no significant difference in the transcription of VEGF, endothelin-1, endothelin B 1 receptor, bradykinin B 1 receptor and bradykinin B 2 receptor mRNA in the two groups. Conclusion The rAAV-HK virus can effectively up-regulate the expression of HK after HUVEC infection and promote the intracellular transcription of eNOS mRNA and the transcription of caspase-3 mRNA in HUVEC. It suggests that the introduction of HK gene can improve endothelial function.