人白细胞介素10离体供肺基因转染对大鼠移植肺缺血再灌注损伤的保护作用

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5×109 PFU/ml hIL-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hIL-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-γ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.

Isografts on subsequent ischemia-reperfusion injury: experiment with rats

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 x 10(9) 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 degrees C 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-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). 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-alpha and IFN-gamma 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.