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二氯甲烷对肝脏缺血再灌注损伤的影响

Effect of methylene chloride upon hepatic ischemic reperfusion injury

摘要:

目的 探讨二氯甲烷对减轻肝脏缺血再灌注损伤的机制.方法 将80只雄性SD大鼠随机分为4组(20只/组):对照组,无任何处理;CoPP(钴-原卟啉)组,供肝收获前24 h供体腹腔注射CoPP 5 mg/kg;ZnPP(锌-原卟啉)组,供肝收获前24 h供体腹腔注射ZnPP 20mg/kg;MC(二氯甲烷)组,供肝收获前7 d,供体口服MC 500 ms/(kg·d).受体不作处理,采用双袖套法施行大鼠同基因原位肝移植(每组供体和受体各10只),移植后3 d每组各处死5只受体鼠,取出供肝并检测:移植后受体肝功能;Western印迹法和免疫组化法检测供肝血红素氧合酶-1(HO-1)的表达;TUNEL法检测供肝细胞的凋亡;Suzuki标准评分评估供肝的缺血再灌注损伤程度.每组留下5只受体鼠观察移植后的生存时间.结果 MC组、CoPP组血清的丙氨酸转氨酶(ALT)[(75±16)、(65±28)U/L]、天冬氨酸转氨酶(AST)[(185±42)、(187±43)U/L]明显较对照组[ALT(346±45)U/L、AST(474±90)U/L]和ZnPP组[ALT(578±75)U/L、AST(1084±128)U/L]低(P<0.01).CoPP组肝脏移植物的HO-1表达量中位数为3.05,明显较对照组高(P<0.05),MC组与对照组间差异无统计学意义(P>0.05).MC组和CoPP组的移植物细胞凋亡指数分别为4.1%±0.6%、3.2%±0.8%,明显较对照组(12.5%±2.4%)和ZnPP组(25.8%±3.1%)低(P<0.05).MC组、CoPP组比对照组、ZnPP组的肝细胞损伤轻、炎症细胞浸润少,MC组、CoPP组的Suzuki评分分别为0.76±0.52、0.76±0.59,明显较对照组(1.32±0.74)和ZnPP组(1.80±0.70)低(P<0.05).MC组、CoPP组的中位生存时间分别为100、93 d,较对照组(85 d)、ZnPP组(12 d)延长(P<0.05).结论 HO-1上调和二氯甲烷对肝脏移植物缺血再灌注损伤均有保护作用.

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abstracts:

Objective To investigate the effects and mechanisms of methylene chloride (MC) in hepatic ischemic reperfusion injury.Methods Eighty SD-rats were divided equally into 4 groups:control group(n=20),donors without any treatment;CoPP group (n=20),donors injected with CoPP (5 mg/kg, ip) at 24 h;ZnPP group (n=20),donors injected with ZnPP (20 mg/kg,ip) at 24 h;MC group (n=20),donors fed with MC (500 mg/kg) per day for 7 days before graft procurement.Syngeneic orthotopie liver transplantation was performed in rats with modified Kamada's two-cuff technique.And SD rats were used as donors(n=10) and recipients respectively.5 recipients in each group were sacrificed and the grafts were procured at day 3 after transplantation,the pest-operative survival time was observed in the remnant.The tests were determined as following:the level of serum ALT,AST in recipients;heme oxygenase-1(HO-1)expression of graft was tested by immunohistochemistry and Western blot;the index of graft apoptosis examined by TUNEL method:the pathology of graft assessed by Suzuki's criteria.Results The level of serum ALT [(65±28)U/L],AST[(187±43)U/L] in CoPP and ALT [(75±16)U/L],AST[(185±42)U/L] in MC group was significantly lower than that ALT [(346±45)U/L],AST [(474±90)U/L]in control group and ALT [(578±75)U/L],AST [(1084±128)U/L] in ZnPP group (P<0.01).The median expression of HO-1 in MC group wag no significantly different with that in control group (P>0.05).While the median expression of HO-1 in CoPP group wag higher than that in control group (P<0.05).The index of graft apoptosis in MC group and CoPP group,4.1%±0.6%and 3.2%±0.8% respectively,was significantly lower than that (12.5% ± 2.4%) in control group and (25.8%±3.1%) in ZnPP group (P<0.05).Compared with the other two groups.MC and CoPP groups had lesser neutrophil infilibation and a lower grade of hepatocytie injury in grafts.Suzuki's scores in grafts of MC and CoPP groups were lower than that in control and ZnPP groups (P<0.05).The median post-transplantation survival time of the recipients in MC and CoPP groups was 1 00 and 93 days respectively while that in control and ZnPP groups was 85 and 12 days(P<0.05).Conclusion Over-expression of HO-1 and MC both have protective effects in hepatic ischemic reperfusion injury.

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