体外膜肺氧合支持对猪控制型心死亡模型供体小肠移植术后早期肠功能的影响

Influence of extracorporeal membranous oxygenation on early allograft function after intestinal transplantation

目的探讨体外膜肺氧合(ECMO)技术对控制型心死亡模型猪供体小肠移植后早期肠道功能的影响。方法选取体质量22~25 kg白色杂种猪48只,按数字表法随机分为正常对照组(LD组)、心死亡供体组(DCD组)、ECMO支持1 h组(E1组)和ECMO支持3 h组(E3组)共4组,每组12只;每组再随机分为供体组和受体组2个亚组,每个亚组各6只。LD组的供体亚组常规截取移植用小肠;DCD组的供体亚组先建立DCD模型,然后截取移植用小肠;E1、E3组的供体亚组,先建立DCD模型,再分别采用ECMO支持1、3 h后,截取移植用小肠。各受体亚组行小肠移植手术。分别于肠移植前、移植后再灌注1 h及移植后第1、3、5、7天经移植肠造口活检钳切取各受体亚组移植肠组织制备病理切片,采用Park/Chiu评分系统评估肠组织损伤程度,透射电镜下观察移植术后第7天小肠黏膜超微结构,免疫组织化学染色观察移植术后再灌注1 h时肠黏膜细胞凋亡因子caspase-3表达水平。分别于移植前和移植术后第1、3、5、7天采集各受体亚组猪颈外静脉血,检测血清D-乳酸水平评估肠黏膜受损情况及肠道通透性;移植术后第7天检测血浆D-木糖最大浓度评估移植肠吸收能力。结果(1)各受体亚组组织病理学评分:小肠移植术前,各组移植肠均有肠黏膜损伤,其中E3组肠黏膜损伤最重,肠黏膜损伤程度Park/Chiu评分E3组高于LD组、DCD组及E1组,差异均有统计学意义(P值均<0.05),而LD组、DCD组、E1组间Park/Chiu评分比较,差异均无统计学意义(P值均>0.05)。小肠移植术后再灌注1 h和术后第1天时,DCD组Park/Chiu评分最高,明显高于LD组、E1组、E3组,差异均有统计学差异(P值均<0.05);而LD组、DCD组、E1组Park/Chiu评分比较,差异均无统计学意义(P值均>0.05)。术后第3、5、7天,LD组、DCD组、E1组、E3组Park/Chiu评分差异均无统计学意义(P值均>0.05)。(2)各受体亚组肠黏膜超微结构:术后第7天各组移植肠超微结构示基本恢复正常,LD组与E1组、E3组紧密连接结构与微绒毛改变无明显差异,而DCD组紧密连接结构相对较短、间隙较大。(3)各受体亚组肠黏膜细胞凋亡因子caspase-3表达水平:小肠移植术后再灌注1 h时,DCD组、E3组caspase-3相对表达水平分别高于LD组和E1组,差异均有统计学意义(P值均<0.05);而DCD组与E3组以及LD组与E1组比较,差异均无统计学意义(P值均>0.05)。(4)各受体亚组血浆D-乳酸水平:术后第1天和第3天,DCD组、E3组血浆D-乳酸水平分别高于LD组和E1组,差异均有统计学意义(P值均<0.05);DCD组与E3组、LD组与E1组相比,差异无统计学意义(P值均>0.05)。术后第5、7天,DCD组血浆D-乳酸水平高于LD组、E1组及E3组,差异均有统计学意义(P值均<0.05),LD组、E1组、E3组血浆D-乳酸水平差异均无统计学意义(P值均>0.05)。(5)各受体亚组移植肠吸收功能:肠移植术后第7天各组血浆D-木糖最大浓度由高至低依次为LD组、E1组、E3组和DCD组,4组间的比较,差异均有统计学意义(P值均<0.05)。结论ECMO支持1 h可改善移植后早期移植肠吸收功能,减轻移植肠缺血再灌注损伤;降低再灌注时移植肠黏膜caspase-3蛋白表达、减少细胞凋亡可能是其保护机制之一。

Objective To evaluate the effect of extracorporeal membranous oxygenation(ECMO)on early allograft function after intestinal transplantation.Methods Forty-eight domestic crossbred pigs were assigned to living donation(LD),donation after cardiac death(DCD),E1,and E3 groups.Each group was randomly divided into two subgroups,with six pigs each.The small intestine donor of the LD subgroup was obtained for transplantation routinely.The DCD model of the DCD subgroup was first established,and then the small intestine donor was obtained for transplantation.The donor subgroups of the E1 and E3 groups were used to first establish the DCD model.Then,1 and 3 h ECMO was used to obtain a small bowel for transplantation,respectively.Small bowel transplantation was performed in each subgroup of recipients.The transplanted intestinal tissues of each recipient subgroup were obtained to evaluate pathological sections before bowel transplantation;1 h after reperfusion after transplantation;and 1,3,5,and 7 days after transplantation.The Park/Chiu scoring system was used to evaluate the degree of intestinal tissue damage.The ultrastructure of small intestinal mucosa was observed under a transmission electron microscope on day 7 after transplantation,and the expression of caspase-3 in the intestinal mucosa was observed by immunohistochemical staining 1 h after transplantation after reperfusion.The external jugular vein blood of each recipient subgroup was collected before transplantation and 1,3,5,and 7 days after transplantation,and the serum D-lactic acid level was measured to assess the damage of the intestinal mucosa and intestinal permeability.The maximum plasma D-xylose concentration was measured on the 7th day after surgery to evaluate the absorption capacity of the transplanted intestine.Results(1)Histopathologic evaluation of subgroup of recipients:Before transplantation,the Park/Chiu score in the E3 group was significantly higher than that in the LD,DCD,and E1 groups(all P values<0.05).The Park/Chiu score in all three groups showed no significant difference(all P values>0.05).One hour and 1 day after transplantation,the Park/Chiu score in the DCD group was significantly higher than that in the LD,E1,and E3 groups(all P values<0.05).The Park/Chiu score in all three groups showed no significant difference(all P values>0.05).On day 3,5,and 7 after surgery,no significant difference was observed in the Park/Chiu score among the LD,DCD,E1,and E3 groups.(2)Transmission electron microscopy of subgroup of recipients:The intestinal mucosal microvilli were arranged tightly and neatly,and the tight junctions appeared as typical membrane fusions with intact tight junction structure and desmosomes,which basically returned to normal in the E1,E3,and LD groups at POD 7.Conversely,the arrangement of the microvilli was slightly irregular,and the desmosomes were relatively shorter in the DCD group.(3)Intestinal mucosal apoptosis of subgroup of recipients:the expression level of caspase-3 in the DCD and E3 groups was significantly higher than that in the LD and E1 groups(all P values<0.05).No significant difference was observed between the DCD and E3 groups or LD and E1 groups(all P values>0.05).(4)Plasma D-lactic acid of subgroup of recipients:On the 1st and 3rd day after surgery,the plasma D-lactic acid level of the DCD group、E3 group were significantly higher than that of the LD and E1 groups,and the differences were statistically significant(all P values<0.05).There was no significant difference between DCD group and E3 group,LD group and E1 group(all P values>0.05).On the 5th and 7th postoperative day,the plasma D-lactic acid level in the DCD group was significantly higher than that in the LD,E1,and E3 groups(all P values<0.05).No statistical difference was observed in the plasma D-lactic acid level in the three groups(all P values>0.05).(5)Intestinal absorption function of subgroup of recipients:On the 7th day after intestinal transplantation,the maxmum plasma D-xylose concentration of each group was in order of LD group,E1 group,E3 group and DCD group.Pairwise comparison among the 4 groups showed statistically significant difference(all P values<0.05).Conclusion One hour ECMO intervention can improve the intestinal absorptive function in the early stage after transplantation and reduce the ischemia-reperfusion injury of the transplanted intestine.Reducing caspase-3 expression and cell apoptosis in the intestinal mucosa may be one of the protective mechanisms of ECMO intervention.