摘要
目的研究血红素加氧酶-1(H0-1)修饰的骨髓间充质干细胞(BMMSCs)联合常温机械灌注(NMP)保存大鼠心脏死亡后捐献(DCD)供肝移植后对胆管上皮细胞(BEC)的作用。方法体外培养鉴定和扩增SD大鼠的第三代BMMSCs,以及通过Ad/H0-1修饰的BMMSCs(Ad/H0-1/BMMSCs),建立体外稳定的NMP系统装置;采用220只6~8周龄体重200.0~220.0g雄性SD受体大鼠构建心脏死亡后热缺血30分钟DCD移植肝模型;将220只SD受体大鼠按照随机数表法随机分成假手术组(S组)、冷保存组(SCS组)、单纯NMP组(NMP组)、BMMSCs联合NMP组(BP组)和Ad HO-1修饰的BMMSCs联合NMP组(HBP组),每组44只,其中NMP组、BP组和HBP组均进行4h体外灌注。各组分别于移植术后0d、1d、7d和14d取材并检测相关指标,观察受体生存时间、肝功能以及胆管病理学改变。通过蛋白质免疫印迹法以及免疫组化染色检测BEC中细胞角蛋白19(CK19)表达,TUNEL染色测定凋亡的BEC,免疫组化法检测凋亡相关蛋白caspase-3的表达。结果HBP组大鼠术后生存时间较其他各组明显延长[SCS组(5.6±0.8)d比NMP组(18.4±2.0)d、比BP组(20.5±1.5)d、比HBP组(82.5±3.2)d],差异均具有统计学意义(均P<0.05);与其他各组相比,BP组和HBP组在肝功能、胆管病理学等方面得到显著改善,BEC中CK19蛋白表达显著增加[术后14d:S组(0.81±0.02)比SCS组(0.35±0.03)、比NMP组(0.47±0.02)、比BP组(0.63±0.02).比HBP组(0.77±0.01)],差异均具有统计学意义(均P<0.05);BEC的凋亡数量以及凋亡相关蛋白caspase-3的表达明显降低[术后14d:S组(10.0±1.2)%比SCS组(57.3±5.5)%、比NMP组(40.1±4.6)%、比BP组(32.0±2.2)%、比HBP组(13.7±3.1)],差异均具有统计学意义(均P<0.05);而HBP组较BP组相比保护作用更明显,差异具有统计学意义(P<0.05)。结论Ad/HO-1修饰BMMSCs较单用BMMSCs联合NMP体外保存大鼠DCD供肝可以更好地保护移植肝中BEC功能,提高移植术后生存率。
Objective To study the effect of bone marrow mesenchymal stem cells(BMMSCs)combined with normothermic mechanical perfusion(NMP)on biliary epithelial cells(BEC)after DCD donor liver transplantation in rats.Methods The third generation of BMMSCs and the BMMSCs modified by Ad/H 0-1(Ad/H O-l/B M M SCs)were cultured,identified and expanded in vitro.To establish a stable NMP system device in vitro.The DCD liver transplantation models were constructed in rats after cardiac ischemia for 30 minutes,220 SD recipient rats were randomly divided into sham operation group(S group,n=44)static cold storage(SCS group,n=44)group,and simple NMP group(P group,n=44),BMMSCs combined with NMP group(B P group,n=44)and BMMSCs modified by Ad/H 0-1 combine with NMP group(HBP group,n=44),NMP group,BP group and HBP group were subjected to vitro perfusion for 4h.The group were taken at0,1,7 and 14 days after transplantation and the relevant indicators were detected,n=6 in each group.The survival rate of the recipient rats,liver function and pathological changes of the bile duct were observed.The expression of cytokeratin 19(CK19)protein in BEC was detected by immunohistochemistry and Western blot.Apoptotic biliary epithelial cells were detected by TUNEL staining and the expression of apoptosis-related protein caspase-3 was detected by immunohistochemistry.Results The survival time of HBP group was significantly prolonged for(5.6±0.8)d in SCS group vs.(18.4±2.0)d in NMP group,(20.5±1.5)d in BP group,(82.5±3.2)d in HBP group,the differences were statistically significant(all P<0.05).Compared with other groups,the HBP group and the BP group were significantly improved in liver function and biliary pathology,and the expression of CK19 protein in BEC was significantly increased[(0.81±0.02)in S group vs.(0.35±0.03)in SCS group,(0.47±0.02)in NMP group,(0.63±0.02)in BP group,(0.77±0.01)in HBP group on postoperative day(POD)14],the differences were statistically significant(all P<0.05).The number of apoptosis and the expression of apoptosis-related protein caspase-3 in HBP group were significantly decreased[(10.0±1.2)in S group vs.(57.3±5.5)in SCS group,(40.1±4.6)in NMP group,(32.0±2.2)in BP group,(13.7±3.1)in HBP group on POD 14],the difference was statistically significant(all P<0.05).Compared with the BP group,the protective effect of the HBP group was more obvious,and the difference was statistically signifi-cant(P<0.05).Conclusion By the method of the BMMSCs modified by Ad/HO-1 combined with NMP in vitro preservation of rat,DCD donor liver can significantly improve the effect of BEC on rats and the survival rate after liver transplantation.
作者
侯宾
曹欢
杨柳
孙东
史源
沈中阳
宋红丽
Hou Bin;Cao Huan;Yang Liu;Sun Dong;Shi Yuan;Shen Zhongyang;Song Hongli(The First Central Clinical College,Tianjin Medical University,Tianjin 300192,China;Department of of Org rgan Transplantation,Tianjin First Central Hospital,Tianjin Clinical Ressearch Center for Organ Transplantation,Key Laboratory of Transplantation Medicine,Chinese Academy of Medical Sciences,Tianjin 300192,China)
出处
《中华肝胆外科杂志》
CAS
CSCD
北大核心
2020年第2期139-144,共6页
Chinese Journal of Hepatobiliary Surgery
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
间充质基质细胞
胆管上皮细胞
凋亡
Liver transplantation
Normothermic mechanical perfusion
Mesenchymal stromal cells
Biliary epithelial cells
Apoptosis
