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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 ,NOS活性和NO含量升
Objective To investigate the effect of recombinant human kallikrein (HK) gene adeno-associated virus vector (rAVV / HK) on cultured human umbilical vein endothelial cells (HUVECs). Methods HK gene was cloned into adeno-associated virus vector pAAV MCS to form recombinant adeno-associated virus (rAAV HK) plasmid. After PCR and DNA sequencing, rAAV HK, pAAV Helper and pAAV RC 3 The recombinant plasmids were co-transfected into 293 cells by liposomes and packaged into recombinant rAVV / HK harboring the HK gene. The expression of the reporter gene rAVV / LacZ in HT1080 was determined by in situ β-galactosidase staining, The virus titer was calculated and the expression of transfected genes in the passaged cells was observed. Under the same conditions, HUVECs (control group) and HUVEC transfected with rAVV / HK (transfected group) 48 h were cultured in vitro, and the expression of HKmRAN was detected by RT-PCR Nitric oxide (NO) content and nitric oxide synthase (NOS) activity of HUVECs in both groups were detected by nitrate reductase and spectrophotometry respectively. Results The titer of rAVV / HK virus was 6.2 × 10 7 cells / mL. LacZ gene could be stably and consistently expressed in the first passage and the sixth generation HT10 80 cells. Compared with the control group, the expression of HK mRNA in HUVECs transfected with HK group was increased (P <0.01 01). Moreover, the content of NO and the activity of NOS in HUVECs cultured with HK group were significantly increased (P <0.01 and <0 0 5. CONCLUSION: HK gene can be successfully transfected into HU VECs cultured in vitro by recombinant adeno-associated virus vector, which increases the expression of HKmRNA, the activity of NOS and the content of NO