Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constr...Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.展开更多
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载体克隆法克隆PR39基因，将编码PR39、穿膜肽TA...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载体克隆法克隆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AAV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AAV—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6XHis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。展开更多
基金sponsored by the National Natural Science Foundation of China,No.30970992
文摘Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.
文摘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载体克隆法克隆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AAV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AAV—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6XHis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。
