转染HO-1基因的巨噬细胞减轻急性心肌梗死区炎症和氧化应激损伤

Macrophages transfected with heme oxygenase-1 gene reduce the inflammation and oxidative stress in acute myocardial infarction area

目的探讨移植转染血红素氧合酶1（HO-1）基因的巨噬细胞在减轻大鼠急性心肌梗死（AMI）区炎症和氧化应激损伤中的作用与机制。方法体外培养大鼠肺泡巨噬细胞（Mφ）并分为对照组、腺病毒（Ad）空载体转染巨噬细胞组（Ad-Mφ组）、含HO-1基因的Ad转染巨噬细胞组（Ad-HO-1-Mφ组）。检测各组的细胞增殖活力、HO-1蛋白表达量及培养液中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、IL-10等细胞因子表达量。另以冠状动脉结扎法建立SD大鼠AMI模型并随机分为对照组、Ad-Mφ移植组和Ad-HO-1-Mφ移植组,分别于心肌梗死区多点注射磷酸缓冲液（PBS）、含Ad-Mφ（2×109/L）的PBS液、含Ad-HO-1-Mφ（2×109/L）的PBS液各100μL,5天后处死各组大鼠,取梗死区心肌组织检测HO-1蛋白及IL-12、TNF-α、IL-10、单核细胞趋化蛋白1（MCP-1）等表达量,同时检测总抗氧化能力（T-AOC）和丙二醛（MDA）含量。结果腺病毒转染本身不影响巨噬细胞活力,但却能将HO-1基因转入巨噬细胞并获得高效表达（P〈0.05）,转染HO-1基因的巨噬细胞可明显降低促炎因子TNF-α、IL-6表达并升高抗炎因子IL-10的表达（均为P〈0.05）。与对照组及Ad-Mφ移植组比较,Ad-HO-1-Mφ移植组心肌梗死区HO-1蛋白表达明显升高（P〈0.05）,促炎因子TNF-α、IL-12、MCP-1表达减少而抗炎因子IL-10表达增加,对应的T-AOC升高而MDA显著降低（均为P〈0.05）。结论 Ad-HO-1-Mφ能够高表达HO-1蛋白并向具有抗炎特性的M2型极化。将Ad-HO-1-Mφ移植到大鼠急性心肌梗死区仍能高表达HO-1蛋白并显著减轻梗死区的炎症和氧化应激水平。

Aim To explore the effects and mechanisms of transplantation of macrophages transfected with heme oxygenase-1（ HO-1） gene by adenovirus vectors on inflammation,oxidative stress in acute myocardial infarction（ AMI） area. Methods Rat alveolar macrophages were cultured in vitro and divided into three groups and treated differently：control group,the cells were not transfected with adenovirus; Ad-Mφ group,the cells were transfected with blank adenovirus vector; Ad-HO-1-Mφ group,the cells were transfected with adenovirus carrying the HO-1 gene. The proliferative activity of macrophages and the expression of HO-1 protein as well as the inflammatory cytokines such as tumor necrosis factor（ TNF-α）,interleukelin-6（ IL-6）,IL-10 in the supernatant were detected. In addition,The AMI model in rats were established by ligating coronary artery and randomly divided into three groups： the simple AMI group,the Ad-Mφ transplantation group and the Ad-HO-1-Mφ transplantation group. PBS,PBS containing 2×105 Ad-Mφ and PBS containing 2×105 Ad-HO-1-Mφ were injected into the myocardial infarction area of rats in each group respectively. 5 days later,the expression of HO-1 protein and the inflammatory cytokines such as IL-12,TNF-α,IL-10,monocyte chemotactic protein 1（ MCP-1） as well as the total antioxidant capacity（ T-AOC） and malondialdehyde（ MDA） content in infarcted myocardium were detected. Results Adenovirus transfection itself did not affect macrophage proliferation activity,but could transfer HO-1 gene into macrophage and obtain high expression. Ad-HO-1-Mφ could significantly reduce the expression of pro-inflammatory cytokine TNF-α,IL-6 and increase the expression of anti-inflammatory factor IL-10（ P〈0. 05）. Compared with the simple AMI group and the Ad-Mφ transplantation group,the infarcted area of Ad-HO-1-Mφ transplantation group showed much more expression of HO-1 protein and IL-10 and less expression of TNF-α,IL-12,MCP-1 while T-AOC increased and MDA content decreased significantly（ P〈0. 05 respectively）. Conclusion Ad-HO-1-Mφ could overexpress HO-1 protein and polarize into M2 phenotype which has powerful anti-inflammatory properties. After being transplanted into myocardial infarcted area,the Ad-HO-1-Mφ could still overexpress HO-1 protein and significantly reduce the inflammation and oxidative stress levels in myocardial infracted area.