诱导体内产生一氧化碳对小鼠心脏移植排斥反应的抑制作用

Inhibitory effect of methylene chloride-induced on cardiac allograft rejection in a routine heart transplantation model

目的探讨用二氯甲烷（MC）诱导同种心脏移植受鼠体内产生c0以减轻排斥反应及延长受鼠存活时间的作用，并探讨其机制。方法以C57BL／6小鼠为供者，Balb／c小鼠为受者，建立小鼠颈部异位心脏移植模型。实验分3组，MC1组和MC2组受鼠分别于术前1d至术后6d和至13d，用含MC的橄榄油100mg／kg灌胃，移植对照组受鼠术前1d至术后6d用等体积不含MC的橄榄油灌胃。术后监测移植心存活时间；检测受鼠肿瘤坏死因子α（TNF-α）、白细胞介素10（IL-10）及叉状头螺旋转录因子（Foxp3）mRNA及蛋白的表达；检测移植心脏细胞间黏附分子-1（ICAM-1）及胱天蛋白酶3的表达；观察移植心组织病理学变化。结果受鼠碳氧血红蛋白（COHb）浓度在MC灌胃前为（0．85±0．28）％，灌胃后3h达到峰值（5．24±0．45）％（P〈0．01）。移植对照组、MC1组、MC2组移植心脏的平均存活时间分别为6．3、12．1和19．4d，两MC组移植心存活时间较移植对照组明显延长（P〈0．01）。与移植对照组相比，两MC组TNF-α和TNF-α mRNA、ICAM-1及胱天蛋白酶3的表达均得到明显抑制（P〈0．01）；移植心肌淋巴细胞和单核细胞浸润的程度得到明显减轻，尤以MC2组最为明显。各组间血清IL-10和IL-10 mRNA及Foxp3蛋白和Foxp3 mRNA表达的差异均无统计学意义（P〉0．05）。结论诱导同种心脏移植受鼠体内产生CO能明显减轻排斥反应，延长移植心存活时间，其主要机制可能与CO的抗炎症和抗凋亡功能密切相关，而并不依赖于Foxp3表达的上调。

Objective To investigate the role of induction of carbon monoxide （CO） with methylene chloride （MC） in recipients in ameliorating allograft rejection and prolonging allograft survival and to explore the possible mechanisms in a routine heart transpiantation model.. Methods Inbred male C57BL/6 mice were used as donors and inbred male Balb/c mice as recipients respectively to establish cervical heterotopic heart transplantation model. The experiments were divided into 3 groups. Recipients were treated with MC （100 mg/kg, per os） day 1 prior to transplantation to day 6 posttransplantation （group MC1w） or to day 13 posttransplantation （group MC2w）, or treated with isovolumic olive oil day 1 prior to transplantation to cardiac arrest of allograft （group Tx）. The serum TNF-α and IL-10 proteins, TNF-α and IL-10 mRNA, and Foxp3 mRNA and protein in cardiac grafts were measured respectively. The intercellular adhesion molecule-1 （ICAM-1） and Caspase-3 protein in cardiac grafts, and the histopathologic changes of cardiac grafts were also observed. Results Serum COHb levels in untreated mice were （0. 85 ± 0. 28）%. After MC application, serum COHb peaked within 3 h in recipients （5.24 ± 0. 45）% （P〈0. 01）. The median survival time of cardiac grafts in group MC1w（12. 1 days） and group MC2w（19. 4 days） was longer than that in group Tx （6. 3 days） （P 〈0. 01 ）. As compared with group Tx, induction of CO in group MC1w and group MC2w down- regulated significantly the levels of serum TNF-α （P〈0. 0l） and TNF-α mRNA （P〈0. 01） of cardiac grafts and spleen in recipient mice, inhibited the protein expression of ICAM-1 （P〈0. 01 ） and Caspase-3 （P〈0. 01） of cardiac grafts, and inhibited, especially in group MC2w, the proliferation of lymphocytes and monocytes infiltration in cardiac grafts. There was no significant difference in serum IL-10 and Foxp3 mRNA and protein in cardiac grafts and spleen of recipients among the groups （P〉 0. 05）. Conclusion Induction of CM in recipients could relieve cardiac allograft rejection and prolong cardiac allograft survival via its anti-inflammation and anti-apoptotic effects, not via up-regulation of Foxp3 in recipient mice.