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目的探讨人内皮型一氧化氮合酶基因(heNOS)转染对犬骨髓源内皮祖细胞(EPC)功能的影响。方法用携带heNOS基因的5型腺病毒作为载体(Ad5-heNOS),对体外定向培养扩增的骨髓源犬EPC进行转染,以未转染的犬EPC作为对照。用酶联免疫吸附试验(ELISA)和硝酸还原酶法检测转染后的EPC中heNOS蛋白的表达和NO的产量,并检测转染后EPC的增殖、黏附、迁移、抗衰老和成血管能力等功能。结果 Ad5-heNOS转染48 h后,转染组EPC的eNOS蛋白表达量和NO产量均明显高于未转染组[(2091.67±172.489)pg/mL vs.(158.00±30.914)pg/mL;(49.5±5.16)μmol/Lvs.(39.7±7.24)μmol/L](均P<0.01);转染组EPC的细胞增殖数、黏附数、迁移数均明显高于未转染组(0.52±0.03 vs.0.31±0.02;28.00±1.41 vs.11.83±1.45;109.67±6.95 vs.72.67±6.29)(均P<0.01),而衰老细胞百分数明显低于未转染组(0.22±0.02 vs.0.32±0.01)(P<0.01);转染heNOS基因的EPC在体外基质胶中培养可形成血管样结构。结论 heNOS基因转染能促进犬EPC的增殖并增强其功能。
Objective To investigate the effect of endothelial nitric oxide synthase (henos) gene transfection on the function of endothelial progenitor cells (EPCs) of canine bone marrow. Methods The adenovirus type 5 adenovirus carrying heNOS gene was used as the vector (Ad5-heNOS) to transfect the EPCs from bone marrow-derived dogs in vitro. The untransfected canine EPCs were used as controls. The expression of heNOS protein and the production of NO in transfected EPCs were detected by enzyme-linked immunosorbent assay (ELISA) and nitrate reductase assay, and the proliferation, adhesion, migration, anti-aging and angiogenesis ability of EPCs were detected after transfection Features. Results After transfection with Ad5-heNOS for 48 h, the expression of eNOS protein and NO production in transfected EPCs were significantly higher than those in untransfected cells [(2091.67 ± 172.489) pg / mL vs. (158.00 ± 30.914) pg / mL) (49.5 ± 5.16 μmol / L vs 39.7 ± 7.24 μmol / L, respectively) (all P <0.01). The numbers of proliferation, adhesion and migration of EPC in transfection group were significantly higher than that in non-transfected group 0.03 vs.0.31 ± 0.02; 28.00 ± 1.41 vs.11.83 ± 1.45; 109.67 ± 6.95 vs.72.67 ± 6.29, respectively), while the percentage of senescent cells was significantly lower than that of the untransfected cells (0.22 ± 0.02 vs.0.32 ± 0.01) (P <0.01). EPCs transfected with heNOS gene could form vascular-like structures when cultured in vitro. Conclusion HeNOS gene transfection can promote EPC proliferation and enhance its function.