缺血预处理对大鼠移植胰腺缺血再灌注损伤的保护作用及其机制的研究

Effects of Ischemic Postconditioning on Ischemic Reperfusion Injury and Apoptosis of the Graft after Pancreas Transplantation in Rat

目的:探讨缺血预处理对大鼠移植胰腺冷缺血再灌注损伤和细胞凋亡的保护作用及其机制。方法:24只糖尿病SD大鼠随机分为缺血再灌注组(I/R组,n=6)和缺血预处理组(IPO组,n=18),IPO组又根据不同方法分为3个亚组:IPO1组(再灌注30s,缺血30s1次,n=6)、IPO2组(再灌注30s,缺血30s3次,n=6)和IPO3组(再灌注30s,缺血30s6次,n=6)。24只SD大鼠为供体,I/R组和IPO组均行同种胰腺移植。另取6只SD大鼠为对照组。检测各组再灌注前、后血糖及再灌注后2h移植胰腺组织中SOD和MDA含量;TUNEL法观察移植胰腺组织细胞凋亡情况,WesternBlot检测移植胰腺组织Bax和Bcl-2蛋白表达情况。结果:1)再灌注后IPO各组相对于I/R组血糖低(P<0.05,P<0.01,P<0.05);IPO2组较IPO1组和IPO3组血糖低(P均<0.05);2)再灌注后IPO组较I/R组移植胰腺组织中SOD含量高(P均<0.01),MDA含量低(P均<0.01),IPO2组相对于IPO1组和IPO3组SOD含量高(P均<0.05)、MDA含量低(P均<0.05)。3)再灌注后IPO组较I/R组移植胰组织中AI值低(P均<0.01),IPO2组相对于IPO1组和IPO3组AI值低(P均<0.05);4)I/R组胰组织Bax蛋白高表达,Bcl-2蛋白低表达,IPO各组再灌注后移植胰组织Bax蛋白低表达,Bcl-2蛋白高表达,而IPO2组Bcl-2蛋白表达最高,Bax蛋白表达最低。结论:缺血预处理对大鼠移植胰腺的缺血再灌注损伤具有保护作用,并可以减少移植胰腺缺血再灌注后的细胞凋亡,机制与减少氧自由基、上调Bcl-2蛋白和下调Bax蛋白有关。再灌注30s缺血30s重复3次是最佳的大鼠移植胰缺血预处理诱导办法。

Objective：To evaluate the effects of ischemic postconditioning on ischemic reperfusion injury and apoptosis of the graft after pancreas transplantation in the rat,and analyze the possible mechanism.Methods：Group Sham which consist 6 normol SD rats suffered with sham operation,24 steptozozin-induced diabetic SD rats were randomly assigned to 2 groups：group I/R consisted of 6 diabetic rats which received pancreas transplantation normally,without additional intervention,Group IPO consisted of 18 diabetic rats received pancreas transplantation exposed IPO with 30-second reperfusion followed by 30-second reocclusion for once（IPO1,n=6）,thrice（IPO2,n=6）,six times（IPO3,n=6）at the onset of reperfusion after cold ischemia.The blood glucose,MDA,SOD,apoptotic cells（TUNEL）,the expression of Bcl-2 and Bax protein（Western Blot）in graft were monitored at 2 hours after long-time reperfusion.Results：1）The mean blood glucose level in Group IPO was lower than Group I/R（P0.05,P0.01,P0.05）,and that in Group IPO2 was lower than IPO1 and IPO3 2 hours after reperfusion（P0.05）.2）The mean SOD level in Group IPO was higher than Group I/R（P0.01）,that in Group IPO2 was higher than IPO1 and IPO3 2 hours after reperfusion（P0.05）.The mean MDA level in Group IPO was lower than Group I/R（P0.01）,that in Group IPO2 was lower than IPO1 and IPO3 2 hours after reperfusion（P0.05）.3）The apoptotic index in Group IPO was lower than Group I/R（P0.01）,and that in Group IPO2 was lower than IPO1 and IPO3 at 2 hours after reperfusion（P0.05）.4）The expression of the Bax protein in Group I/R was higher than Group IPO and that in Group IPO2 was lowest.The expression of the Bcl-2 protein in Group IPO was higher than Group I/R,and that in Group IPO2 was highest.Conclusions：1）Ischemic postconditioning can protect graft from I/R injury during pancreas transplantation,which may achieve through reducing oxygen radical.2）Ischemic postconditioning can reduce apoptosis of the graft,which may achieve through regulating expressions of Bcl-2 and Bax protein.3）30s ischemic and 30s reperfusion twice is the best way to induce ischemic preconditioning in rat pancreas transplantation.