PUMA在大鼠胰腺移植缺血-再灌注损伤中的意义

The effect of PUMA protein on ischemia-reperfusion injury in rat model of pancreatic transplantation

目的:探讨PUMA在移植胰腺缺血再灌注损伤(I/RI)中的早期促凋亡作用。方法:对封闭群大鼠建立大鼠腔静脉内分泌引流、肠道外分泌引流的动物模型.再灌注后第0、1、2、3、4、6、9、12 h等时点,每时点处死5只大鼠,切取移植胰腺,石蜡包埋切片,原位DNA缺口末端标记(TUNEL)法测定胰腺组织细胞凋亡,Western blot方法测定移植胰腺组织PUMA,bcl-2、bax、caspase-3蛋白表达。结果:再灌注术后第0、1、2、3、4、6、9、12 h,胰腺细胞凋亡数分别为(29.42±4.93)、(47.65±6.43)、(74.80±9.73)、(106.35±16.80)、(148.71±19.50)、(123.96±15.54)、(97.32±10.60)和(57.42±9.56)个/高倍视野。各时点均显著高于正常胰腺(23.48±4.26)。第4小时细胞凋亡数明显增高,12 h降低到最低值,不同组细胞凋亡数的差异有统计学意义(P<0.01);PUMA以及其下游的bax和caspase-8蛋白表达在第4小时达到最高峰,12 h降低到最高值,bcl-2在第4小时达到最低值,蛋白表达差异均有统计学意义(P<0.05),以后逐渐升高,12 h降低到最低值,PUMA表达与各时点的细胞凋亡数有明显的正相关(r=1.00,P<0.05)。结论:I/RI后细胞早期凋亡与PUMA活化有关,PUMA是通过调节下游bax、caspase-8、bcl-2基因发挥凋亡促进作用。

Objective： To study the pro-apoptosis effect of P53 up-regulated modulator of apoptosis （PU- MA） protein on ischemia-reperfusion injury of rats stimulated during pancreatic transplantation. Methods： Rat models of pancreatic transplantation with venacava drainage and enteric drainage were established firstly. Then 5 animals in each group were sacrificed respectively at different time points including 0, 1, 2, 3, 4, 6, 9 andl2 h after reperfusion, with pancreatic grafts being removed for apoptosic analysis by using DNA nick end labeling tech- nique and protein expression analysis of PUMA, bcl-2, bax and caspase-3 by using western blot method. Results： Under high power field, the numbers of apoptosic cells in pancreatic grafts at each time point was 29.42 ± 4.93, 47.65 ±6.43, 74.8 ±9.73, 106.35 ±16.8, 148.71±19.50, 123.96 ±15.54, 97.32 ±10. 6 and 57. 42 ± 9.56, respectively which are all significantly higher than the number in normal pancreatic tissues （23.48 ± 4.26）. It indicated the numbers were obviously increased at 4h after profusion while decreased to the minimum at 12h with significant difference among each group （ P 〈 0.01 ）. The expression of PUMA, bax and caspase-3 all started to up-regulated at lh after profusion and increased to maximum at 4h, then gradually decreased to minimum at 12h. While for bcl-2, the expression started to down-regulated at lh after profusion and decreased to the minimum at 4h, then gradually increased to maximum at 12h. There are significant differences among the protein expression （ P 〈 0.05 ）. Besides, the PUMA expression is positively correlated to the number of apoptosic cells. （ r = t. 00, P 〈 0.05）. Conclusion： Cell apoptosis in ischemia-reperfusion injury is highly correlated to the activation of PUMA. The pro-apoptosis effect of PUMA is generated by regulating bax, caspase-8 and bcl-2.