EPO对同种异体心脏移植中供心保护作用的实验研究

目的 通过建立同种异体心脏移植模型来探讨促红细胞生成素(EPO)预处理供受体对供心的保护作用及其可能机制。 方法 健康雄性SD大鼠60只随机分成3组,每组20只。对照组:摘取供心前30min经尾静脉注射生理盐水0.5ml;EPO供体预处理组:供体摘取供心前30min经尾静脉注射EPO 5000U/kg,受体不处理;EPO受体预处理组:受体于移植前30min经尾静脉注射EPO 5000U/kg,供体不处理。利用4℃ HTK液保存供心16h后建立大鼠同种腹腔心脏移植模型。观察手术成功情况并评价供心功能;移植后8h和24h采血化验:CK-MB和TnT变化;移植24h后处死大鼠取供心留取组织标本检测:SOD和LPO的活性;SABC法检测组织中caspase-3蛋白表达;Tunel法评价心肌细胞凋亡。 结果 供心全部成功复跳,并存活到实验结束;EPO预处理组心功能较对照组明显改善(P<0.05);移植8h,供、受体预处理组 CK-MB、TnT均显著低于对照组(P<0.05),移植24h供体预处理组CK-MB、TnT及受体预处理组CK-MB、TnT低于对照组(P<0.05);供、受体预处理组间各指标比较差异均无统计学意义(P>0.05)。移植后8、24h组内比较,移植后24h两组各指标均显著低于8h(P<0.05)。心肌组织中SOD和LPO活性:供受体预处理组SOD活性明显高于对照组,LPO活性显著低于对照组(P<0.05);供、受体预处理组间比较无统计学差异(P>0.05)。EPO预处理组心肌组织中细胞凋亡指数分别为:3.36±0.36、3.48±0.22明显低于对照组3.21±0.50,差异有统计学意义(P<0.05)。EPO供体预处理组细胞凋亡指数与受体预处理组(3.36±0.36 vs 3.48±0.22)相比,差异无统计学意义(P>0.05)。供、受体预处理组caspase-3蛋白表达量均低于对照组(P<0.05),但受体预处理组caspase-3蛋白表达量高于供体预处理组(P<0.05)。 结论 EPO预处理供受体可通过减少氧自由基产生,提高氧自由基清除能力,抑制心肌细胞凋亡来发挥保护供心作用,提高移植心脏功能。

Experimental Study of Effect of EPO on Donor Heart Preservation in Allogeneic Heart Transplantation

Objective To establish the model of cardiac allograft to explore erythropoietin (EPO) pretreatment of donor and receptor and study the protective effect on donor heart and its possible mechanism. Methods Sixty healthy male SD rats were randomly divided into 3 groups, with 20 rats in each group.In normal group,0.5ml normal saline was infused via caudal vein at 30min before donor harvesting. In donor preconditioning group, EPO (5000U/kg) was infused via caudal vein at 30min before donor harvesting, but no treatment in recipients. And in recipient preconditioning group, EPO (5000U/kg) was infused via caudal vein at 30min before recipient transplantation, but no treatment in donors. The donor was preserved in HTK solution after16h.The model of allogeneic heart transplantation was established in abdominal cavity in rat. Blood was drawn at 8 and 24h after reperfusion for analysis of CK-MB,TnT as markers of graft injury. The rats were killed after 24h transplantation. The heart specimens collected for detecting myocardial tissue was harvested to determine the superoxide dismutase (SOD) and lipid hydroperoxide (LPO) activity at 24 hours after reperfusion. Graft active caspase-3 protein expression was measured by immunohistochemistry staining, and apoptosis index (AI) was calculated by TUNEL. Results The hearts can successfully jump, and survive to the end of the experiment. heart function EPO pretreatment group of was better than that of control group (P<0.05).The serum levels of CK-MB and TnT in donor and recipient preconditioning groups were significantly lower than those in control group at 8 hours after reperfusion (P<0.05). The levels of CK-MB and Tnt in donor preconditioning group and the levels of CK-Mb and TnT in recipient preconditioning group were significantly lower than those in control group at 24 hours (P<0.05),and no significant difference was found between donor and recipient perconditioning groups (P>0.05). The levels of CK-MB and TnT at 24 hours were significantly lower than those at 8 hours in each group (P<0.05). SOD activity in donor/receptor preconditioning group was significantly higher than that in the control group. The activity of LPO was significantly lower than the control group (P<0.05). There is no significant difference between groups for pretreatment, receptor (P>0.05).AI in EPO pretreatment groups were (3.36±0.36) and (3.48±0.22).They were significantly lower than those of the control group (3.21±0.50) (P<0.05). Pretreatment with EPO donor group,apoptsis index (3.36±0.36) and receptor preconditioning group(3.48±0.22) showed no significant differentce(P>0.05).Caspase-3 in recipient pretreatment group was significantly higher than that in donor preconditioning group and control group (P<0.05). Caspase-3 protein expression in receptor preconditioning group was higher than that of donor pretreatment group (P<0.05). Conclusion EPO can protect the donor heart by reducing oxidative stress,and improving the ability of scavenging oxygen free radicals and inhibiting apoptosis and improving the function of donor heart.