摘要
目的探讨骨髓间充质干细胞(BMMSC)联合常温机械灌注(NMP)保存大鼠心脏死亡后捐献(DCD)供肝肝移植后对胆管上皮细胞(BEC)的保护作用。方法取2~3周龄、体重40~60 g雄性SD大鼠,体外提取、培养、鉴定和扩增BMMSC至第3代。取6~8周龄、体重200~220 g雄性SD大鼠,按随机数字表法分为假手术组(Sham组)、冷保存组(SCS组)、单纯NMP组(NMP组)和BMMSC联合NMP组(BMMSC+NMP组),每组44只。建立心脏死亡后热缺血30 min DCD供肝模型;Sham组仅游离肝脏韧带,不阻断肝脏血供,30 min后缝合。SCS组供肝采用器官保存液(UW)冷保存4 h;NMP组和BMMSC+NMP组建立体外稳定的NMP系统装置,给予灌注液或联合BMMSC灌注4 h;建立原位肝移植术模型,并分别于肝移植术后0、1、7、14 d观察受体存活率。各组于术后各时间点采用生化法检测血清肝功能指标;通过苏木素-伊红(HE)染色观察胆管组织形态学改变;采用免疫组化法和蛋白质免疫印迹试验(Western Blot)定性、定量检测BEC中细胞角蛋白19(CK19)的表达。结果经BMMSC的形态、分化功能和表型鉴定,证实本实验中采用的干细胞为符合标准的BMMSC。NMP组和BMMSC+NMP组大鼠术后0、1、7、14 d存活率均较SCS组明显升高,以BMMSC+NMP组升高更为显著,术后0 d存活率达100%,14 d存活率仍明显高于SCS组和NMP组〔80.0%(16/20)比20.0%(4/20)、70.0%(14/20),均P<0.05〕。随肝移植术后时间延长,SCS组大鼠肝功能指标异常逐渐明显,术后1~7 d达峰值;光镜下显示,胆管组织损伤逐渐加重,术后7 d损伤程度最严重,表现为肝细胞出现大量"气球样"改变,胆管扩张明显并伴有大面积炎性细胞浸润;免疫组化和Western Blot均显示,BEC胞质内CK19表达逐渐减少,7 d达谷值,随后逐渐回升。BMMSC+NMP组和NMP组在肝功能、胆管病理形态学观察以及BEC中CK19表达等方面均明显优于SCS组,以BMMSC+NMP组改善更为显著,表明BMMSC联合NMP对BEC的保护作用明显优于SCS和NMP。结论通过BMMSC联合NMP体外保存大鼠DCD供肝,可以显著减轻肝移植术后BEC损伤,从而改善大鼠肝移植预后,提高移植术后存活率。
Objective To investigate the protective effect of bone marrow mesenchymal stem cells(BMMSC)combined with normothermic mechanical perfusion(NMP)on biliary epithelial cells(BEC)in rats receiving donation after cardiac death(DCD)donor liver transplantation.Methods The BMMSC were isolated from male Sprague-Dawley(SD)rats aged 2-3 weeks and weighing 40-60 g,and then cultured,identified and expanded to the third generation in vitro.Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group(Sham group),static cold storage(SCS group),simple NMP group(NMP group)and BMMSC combined with NMP group(BMMSC+NMP group)by random number table method with 44 rats in each group.The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time.While the rats in Sham group merely received perihepatic ligaments-separation,which did not affect their liver blood supply,and then their incisions were sutured after 30 minutes.The DCD donor grafts in SCS group were preserved in the University of Wisconsin(UW)cold storage solution for 4 hours.While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system.The orthotopic liver transplantation model was reproduced,and the survival rate of the recipients was observed at 0,1,7 and 14 days after liver transplantation.The biochemical liver function of rats in different groups was determined at each time point after operation.The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin(HE)staining,and the expression of cytokeratin 19(CK19)was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples.Results The morphology,differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC.The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0,1,7 and 14 days after operation.The increase was more significant in the BMMSC+NMP group,with 100%on postoperative day(POD)0,and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group[80.0%(16/20)vs.20.0%(4/20),70.0%(14/20),both P<0.05].As the time after liver transplantation prolonged,the liver function parameters of rats in the SCS group were deteriorated gradually,which reached the peak at 1-7 days after operation.The damage of biliary tissue increased gradually under the microscope,and the injury was most serious on POD 7 in the SCS group,showing a lot of balloon-like changes in hepatocytes,with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration.Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group,reached the lowest on POD 7,and then gradually increased.The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function,pathological injury of biliary tract and CK19 expression in BEC,and the improvement was more significant in the BMMSC+NMP group.These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP.Conclusion Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly,thus improving both the prognosis and the survival rate after transplantation.
作者
侯宾
宋红丽
曹欢
杨柳
孙东
沈中阳
Hou Bin;Song Hongli;Cao Huan;Yang Liu;Sun Dong;Shen Zhongyang(The First Central Clinical College,Tianjin Medical University,Tianjin 300192,China;Department of Organ Transplantation,Tianjin First Center Hospital,Tianjin Key Laboratory of Organ Transplantation,Tianjin Clinical Research Center for Organ Transplantation,NHC Key Laboratory of Critical Care Medicine,Key Laboratory of Transplantation Medicine,Chinese Academy of Medical Sciences,Tianjin 300192,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2019年第9期1137-1142,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金(81670574,81270528,81441022)。
关键词
肝移植
常温机械灌注
缺血/再灌注损伤
骨髓间充质干细胞
胆管上皮细胞
Liver transplantation
Normothermic mechanical perfusion
Ischemia/reperfusion injury
Bone marrow mesenchymal stem cell
Biliary epithelial cell
