血红素加氧酶-1修饰的BMMSCs改善大鼠减体积移植肝脏微循环的研究  

Role of HO-1/BMMSCs in improving hepatic microcirculation after reduced-size transplantation in rats

作　　者：杨柳[1] 曹欢 孙东 侯宾 沈中阳[2] 宋红丽[2] Yang Liu;Cao Huan;Sun Dong;Hou Bin;Shen Zhongyang;Song Hongli(First Central Clinical College,Tianjin Medical University,Tianjin 300192,China;Department of Organ Transplantation,First Central Municipal Hospital,Tianjin Organ Transplantation Clinical Medicine Research Center,Key Laboratory of Transplantation Medicine,Chinese Academy of Medical Sciences,National Health Commission Key Laboratory of Critical Care Medicine,Tianjin 300192,China)

机构地区：[1]天津医科大学一中心临床学院,300192 [2]天津市第一中心医院器官移植中心天津市器官移植临床医学研究中心中国医学科学院移植医学重点实验室国家卫生健康委员会危重病急救医学重点实验室,300192

出　　处：《中华器官移植杂志》2020年第1期42-48,共7页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金(81670574、81270528、81441022)。

摘　　要：目的探讨血红素加氧酶-1(HO-1)修饰的骨髓间充质干细胞(BMMSCs)在改善大鼠减体积移植肝脏的微循环中的作用。方法贴壁法分离培养BMMSCs,转染HO-1/腺病毒(Adv)构建HO-1/BMMSCs。"二袖套"法建立大鼠原位50%减体积肝移植急性排斥反应模型,术后即刻分别注射生理盐水(NS)、BMMSCs或HO-1/BMMSCs单细胞悬液1 ml,观察存活的入组大鼠术后即刻、3、7和14 d的指标变化,每组每个时间点5只大鼠。通过生化检测线粒体型天门冬氨酸氨基转移酶同工酶(mAST)的水平;化学比色法检测肝组织中Na^+-K^+-ATP酶活力;透射电镜技术检测术后7 d时的肝细胞线粒体超微结构;Power Lab检测肝移植术后7d时的门静脉压力;Western blot检测移植肝内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)和血管性假血友病因子(vWF)的表达水平;HE染色观察肝病理学改变;免疫组化观察肝组织vWF的表达情况;ELISA检测血清中透明质酸(HA)的水平。结果HO-1/BMMSCs能显著减轻50%减体积肝移植急性排斥反应模型中移植肝的病理损伤及排斥反应,改善线粒体损伤及能量代谢,同时能显著促进eNOS的表达,抑制iNOS的表达,降低门静脉压力,显著促进肝窦vWF的表达及HA的降解,保护肝窦内皮细胞,进而改善肝脏微循环,与NS组和BMMSCs组比较差异具有统计学意义(P<0.05)。结论HO-1/BMMSCs可以通过改善肝脏微循环,发挥保护大鼠减体积移植肝的作用。Objective To explore the role of heme oxygenase-1(HO-1)modified bone marrow mesenchymal stem cells(BMMSCs)in improving hepatic microcirculation after reduced-size liver transplantation(RLT)in rats.Methods BMMSCs were isolated and cultured in vitro by adherence method.Then HO-1/adenovirus(Adv)was transfected for constructing HO-1/BMMSCs.The"dual-sleeve"method was employed for establishing an acute rejection model after 50%RLT.Immediately post-operation,1 ml normal saline(NS)and BMMSCs or HO-1/BMMSCs single cell suspension were injected.The changes of surviving rats were observed by parameters at Day 3/7/14 post-operation.Five rats were observed at each timepoint.The serum level of mitochondrial aspartate aminotransferase(mAST)was detected;Na^+-K^+-ATPase in transplanted liver was measured by chemical colorimetry;mitochondrial ultrastructural changes were observed under a transmission electron microscope.Portal vein pressure was detected by Power Lab at Day 7 post-operation;the expressions of endothelial nitric oxide synthase(eNOS),inducible nitric oxide synthase(iNOS)and von Willebrand factor(vWF)in liver tissues were detected by Western blot.Liver histopathological changes were observed by hematoxylin&eosin stain.The expression of vWF was detected by immunohistochemistry and serum level of hyaluronic acid(HA)detected by enzyme-linked immunosorbent assay(ELISA).Results HO-1/BMMSCs could significantly lessen the pathological injury and rejection of 50%reduced-size transplanted liver,improve mitochondrial damage and energy metabolism,promote the expression of eNOS,suppress the expression of iNOS,reduce portal pressure,up-regulate the expression of hepatic sinus vWF and HA degradation,protect hepatic sinusoidal endothelial cells(SECs)and ultimately improve hepatic microcirculation.And the differences were statistically significant as compared with NS/BMMSCs group(P<0.05).Conclusions HO-1/BMMSCs may play an important role in protecting rat liver by improving hepatic microcirculation during RLT.

关 键 词：门静脉压力 急性排斥反应 肝细胞线粒体 肝窦内皮细胞 线粒体损伤 移植肝 减体积 大鼠 

分 类 号：R657[医药卫生—外科学]