摘要目的 探讨腺相关病毒(AAV)介导的融合基因NT4-TAT-His-PR39对缺氧人脐静脉内皮细胞株ECV304内缺氧诱导因子1α(HIF-1α)表达的影响及其对缺氧环境鸡胚血管生成的影响.方法 PCR技术和T载体克降法克隆PR39基因,将编码PR39、穿膜肽TAT、6×His及NT4信号肽四个基因片段相连.磷酸钙沉淀法获得重组携带NT4-TAT-His-PR39的从V载体,然后AAV-PR39感染ECV304,免疫细胞化学检测ECV304胞内6×His表达情况.在1%O2条件培养ECV304,免疫细胞化学测定AAV-PR39组和PBS组胞内HIF-1α蛋白表达.30只鸡胚随机分为从V-PR39组、EV组和PBS组(各组n=10),分别在鸡胚尿膜囊(CAM)上加AAV-PR39、EV、PBS,然后每组各取5只鸡胚分别在低氧(5%O2)和常氧(21%O2)条件培养.图像软件Image Pro Plus(IPP)分析CAM血管密度.结果 AAV-PR39组ECV304中检测到6×His表达.低氧环境下,AAV-PR39组ECV304中HIF-1α蛋白表达明显高于PBS组,AAV-PR39组CAM血管密度明显大于其他两组.结论重组携带NT4-TAT-His-PR39的AAV载体能在ECV304中分泌表达,并提高缺氧细胞HIF-1α蛋白表达水平.重组AAV载体显著促进缺氧CAM血管生成,而对常氧CAM无此作用.

abstractsObjectives To investigate the impact of AAV-encoding NT4-TAT-His-PR39 fusion gene expression on HIF-1α level in ECV304 cultured under hypoxic condition ( 1% O2) and on angiogenesis in hypoxic chick embryo. Methods PR39 cDNA was connected with NT4, TAT, 6 x His cDNA by molecular biology methods. The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells. Then ECV304 were respectively infected with AAV-NT4-TAT-His-PR39, 6 x His expression and HIF-la level in ECV304 were detected by immunocytochemistry. The chicken embryos were randomized into the AAV-PR39, EV and PBS groups ( n = 10 each) subject to hypoxia (5% O2, n = 15 ) or normoxia environments (n = 15) , the vessel density of the chicken chorioallantoic membrane (CAM) were measured by Image Pro Plus (IPP) software. Results The expression of 6 x His protein was detected in AAV-PR39 infected ECV304 cells. HIF-1α protein activity was significantly increased in AAV-PR39 infected ECV304 underwent hypoxia compared to PBS and non-infected ECV304 groups ( P < 0. 05). The vessel density of chicken CAM in hypoxia environment but not in normoxia environment was also significantly higher in AAV-PR39 group than in EV group and PBS group (all P <0. 05). Conclusion AAV-encoding NT4-TAT-His-PR39 fusion gene expression significantly increased HIF-la level in ECV304 exposed to hypoxia and promoted angiogenesis in hypoxic chicken embryo.