内皮型一氧化氮合酶基因转染犬骨髓源内皮祖细胞的实验研究  

Experimental study on heNOS gene transfecting canine bone marrow-derived endothelial progenitor cells

作　　者：郭连瑞[1] 王国栋[1] 宋礼坡[1] 谷涌泉[1] 张淑文[1] 李建新[1] 汪忠镐[1] 张建[1] 

机构地区：[1]首都医科大学宣武医院血管外科,首都医科大学血管外科研究所,北京100053

出　　处：《南京医科大学学报（自然科学版）》2009年第12期1648-1652,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助(30471708)

摘　　要：目的:探讨人内皮型一氧化氮合酶(heNOS)基因转染犬内皮祖细胞(EPCs)的有效方法。方法:分别用5型腺病毒和pEGFP-N1质粒作为载体携带heNOS,对体外定向培养扩增的骨髓来源的犬EPCs进行体外转染,然后对转染heNOS基因后的EPCs进行鉴定及功能检测,观察heNOS基因的功能表达情况。结果:heNOS转染犬EPCs48h后,酶联免疫吸附试验(ELISA)检测到5型腺病毒转染组EPCs的eNOS表达量(2091.67±172.49pg/ml)较pEGFP-N1质粒转染组(173.67±36.76pg/ml)和未转染组(158.00±30.91pg/ml)均显著增多(P<0.01);硝酸还原酶法测定5型腺病毒转染组细胞培养上清中的NO含量(49.5±5.2μmol/L)较质粒转染组(38.5±7.1μmol/L)和未转染组(39.7±7.2μmol/L)均显著增多(P<0.01)。结论:5型腺病毒载体介导的heNOS基因能够成功地转染犬EPCs,并能在EPCs中有效表达。Objective：To establish an effective method to transfect canine bone marrow-derived EPCs with human eNOS gene（heNOS）. Methods：Canine endothelial progenitor cells（EPCs）were obtained,cultured and expanded with bone marrow-derived mononeuclear cells by our previously published ex vivo expansion method,then transfected by Ad5-heNOS recombinant adenovirus or pEGFP-N1-heNOS recombinant plasmid,EPCs without transfection as a control. The expression of heNOS protein in EPCs was measured by enzyme linked immunosorbent assay （ELISA） at 48 hours after gene transfection. The amount of the nitric oxide （NO） in the supernanant of culture fluid was detected by nitrate reductase assay. Results：The expression of heNOS protein in Ad5-heNOS transfected EPCs （2 091.67 ± 172.49 pg / ml） was significantly higher than that in pEGFP-N1-heNOS transfected EPCs （173.67 ± 36.76 pg / ml） and the control group （158.00 ± 30.91 pg / ml）（P 〈 0.01）. The amount of NO in the supernatant of the Ad5-heNOS group（49.5 ± 5.2 μmol / L） was significantly higher than that in the pEGFP- N1-heNOS group （38.5 ± 7.1 μmol / L） and the control group （39.7 ± 7.2 μmol / L）. Conclusions：The heNOS gene can be successfully transfected into and expressed in canine EPCs by Ad5-heNOS recombinant adenovirus.

关 键 词：内皮祖细胞 内皮一氧化氮合酶 腺病毒 转染 

分 类 号：R392.12[医药卫生—免疫学]