【摘要】 目的探讨离体经肺静脉灌注途径转染目的基因的可行性,观察移植肺局部转染人白细胞介素（hIL）-10基因对移植肺缺血再灌注损伤（IRI）的影响。方法采用改良的三袖套法建立大鼠左肺原位移植模型,移植前供肺离体经肺静脉逆行灌注5×10⁹PFU/ml hlL-10基因重组的腺病毒载体（转基因组）,保存3 h 后移入受鼠;另设空载体对照组、空白对照组和假手术对照组。再灌注后4 h,测定动脉血 PaO₂;检测移植肺湿-干重比率（W/D）、丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、髓过氧化物酶（MPO）活性;观察组织学改变;采用 RT-PCR 法和免疫组化染色法检测移植肺hlL-10基因的表达情况,酶联免疫吸附法（ELISA）测定移植肺肿瘤坏死因子（TNF）-α、干扰素（IFN）-γ的表达水平。结果空载体对照组和空白对照组出现明显的移植肺 IRI,转基因组移植肺IRI 明显减轻,PaO₂提高、W/D 比率降低、MDA 含量和 MPO 活性下降（P<0.01）、SOD 活性升高（P<0.05）;转基因组移植肺 TNF-α、IFN-β的表达下调（P<0.01）,肺泡间质水肿减轻,炎细胞浸润减少,并有外源性 hIL-10基因的表达。结论 （1）离体供肺经肺静脉灌注途径转染外源性基因有效可行;（2）腺病毒介导 hIL-10转基因能有效地减轻移植肺 IRI,改善早期移植肺功能。更多还原

【Abstract】 Objective To investigate the feasibility of ex vivo adenovirus-mediated gene transfer of human interleukin10（hIL10）via the pulmonary vein into lung isografts,and to investigate the effect of hiL- 10 gene transfer on subsequent ischemia-reperfusion injury（IRI）.Methods Fifty-six male SD rats were randomly divided into 4 equal groups:Group D,undergoing left lung isotransplantation with the improved cuff anastomosis technique(the Isografts were transvascularly transfected 5 ml of 5×10⁹ plaque-forming units/ml adenovec-hIL-10 complex,Group C,with the Isografts transvascularly transfected with blank adenovirus vector Adenovec,Group B,with the Isografts transvascularly transfected with diluent,and Group A,undergoing sham operation.All allografts were preserved for 3 hours at 10℃ before transplantation.Four hours after reperfusion blood samples were collected from hr abdominal aorta to undergo blood air analysis. Lung function was evaluated by partial pressure of oxygen（PaO₂）.Then the rats were killed with their left lung taken out to undergo pathological examination.The graft lung wet-to-dry（W/D）weight ratio was measured.SABC immunohistochemistry was used to detect the expression of hIL-10 in the cytoplasm.ELISA was used to detect the expression of tumor necrosis factor-α（TNF-α）and interferon-γ（IFN-γ）.The levels of malonyldialdehyde（MDA）,superoxide dismutase（SOD）,and myeloperoxidase（MPO）were measured by.Pathological morphologic change was also analyzed.Results The PaO₂ level of Group D was significantly higher than those of Groups B and C（both P<0.01）.The W/D ratio,and levels of MDA and MPO of Group D were significantly lower than those of Groups B and C（both P<0.01）,but the SOD level of Group D was significantly higher than those of Groups B and C（both P<0.05）.The TNF-α and IFN-γ levels of Group D were significantly lower than those of Groups B and C（both P<0.01）.Fewer tissue edema and interstitial inflammation were found in lungs.Of Group D RT-PCR showed hIL-10 expression in the lungs of the rats of Group D,but not in other groups.Conclusion Ex vivo adenovirus-mediated gene transfer of hIL-10 via the pulmonary vein into the lung isografts is feasible and effective,hIL-10 gene transfer into lung isografts ameliorates subsequent IRI and improves early posttransplant graft function.更多还原