骨髓间充质干细胞联合常温机械灌注对肝移植急性排斥反应大鼠肠道菌群的影响  

Effect of bone marrow mesenchymal stem cells combined with normothemic machine perfusion on the intestinal flora in rats with acute rejection of liver transplantation

作　　者：林玲[1] 袁梦淑 吴龙龙 田轩 郑卫萍[3] 曹欢 宋红丽[3] Lin Ling;Yuan Mengshu;Wu Longlong;Tian Xuan;Zheng Weiping;Cao Huan;Song Hongli(First Center Clinical College,Tianjin Medical University,Tianjin 300192,China;School of Medicine,Nankai University,Tianjin 300074,China;Department of Organ Transplantation,Tianjin First Central Hospital,Tianjin Key Laboratory of Organ Transplantation,Tianjin Organ Transplantation Clinical Medical Research Center,Tianjin 300192,China)

机构地区：[1]天津医科大学一中心临床学院,300192 [2]南开大学医学院,天津300074 [3]天津市第一中心医院器官移植科天津市器官移植重点实验室天津市器官移植临床医学研究中心,300192

出　　处：《中华实验外科杂志》2022年第3期447-450,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82070639、81670574)。

摘　　要：目的探讨骨髓间充质干细胞(BMSCs)联合常温机械灌注(NMP)对肝移植急性排斥反应大鼠肠道菌群的影响。方法2020年4月至10月,选取24只雄性Lewis大鼠为供体,24只雄性Brown Norway大鼠为受体,"二袖套"法建立大鼠肝移植急性排斥反应模型,根据以下处理肝脏方法将大鼠分为四组:假手术组(Sham组)、NMP组(NMP组)、NMP/BMSCs组(BMP组)和他克莫司组(FK506组),在术后第7天处死各组大鼠,苏木精-伊红(HE)染色观察肝脏病理学;生化分析仪检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和碱性磷酸酶(ALP)的含量;酶联免疫吸附测定(ELISA)和实时荧光定量聚合酶链反应(qRT-PCR)分别检测血清和肠道组织中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的含量;对各组大鼠粪便标本进行高通量测序,获得16SrDNA数据。两组间数据比较采用独立样本t检验,多组数据比较采用单因素方差分析。结果BMP组肝窦内充血、炎性细胞浸润程度减轻;排斥活动指数(RAI)(4.17±0.75)低于NMP组(7.67±0.82,t=7.720,P<0.05),差异有统计学意义;血清ALT、AST和ALP[(254.02±35.55)U/L、(457.42±131.22)U/L、(169.60±65.61)U/L]水平低于NMP组[(362.22±57.73)U/L、(741.67±87.19)U/L、(269.93±43.51)U/L,t=3.909、4.419、3.196P<0.05],差异有统计学意义;肠道菌群丰度增加,变形菌门占比下降,阿克曼菌科占比上升,花生四烯酸代谢、上皮细胞的细菌侵袭通路丰度下降;血液中炎性因子TNF-α和IL-6[(66.01±12.46)pg/ml、(130.3±9.67)pg/ml]浓度低于NMP组[(127.1±17.85)pg/ml、(169.9±8.25)pg/ml,t=6.276、6.975,P<0.05],差异有统计学意义;肠组织中炎性因子TNF-α和IL-6[(1.85±0.17)、(3.47±0.22)]的mRNA水平低于NMP组[(2.71±0.34)、(9.45±1.34),t=3.955、7.604,P<0.05],差异有统计学意义。结论BMSCs联合NMP可以减轻大鼠肝移植术后急性排斥反应,同时对大鼠肠道菌群紊乱具有调节作用。Objective To study the effect of bone marrow mesenchymal stem cells(BMSCs)combined with normothemic machine perfusion(NMP)on the intestinal flora of rats with acute rejection of liver transplantation.Methods From April to October 2020,24 male Lewis rats were selected as donors and 24 male brown Norweg rats as recipients using the"two-cuff"method to establish the acute rejection model of liver transplantation in rats,and the rats were divided into four groups according to different liver treatment methods:Sham group,NMP group,BMP group and FK506 group.Rats in each group were sacrificed on the 7 d after operation.Hematoxylin and eosin(HE)staining was used to detect liver histopathological changes.Biochemical analyzer was used to detect the contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and alkaline phosphatase(ALP).Enzyme linked immunosorbent assay(ELISA)and quantitative real-time polymerase chain reaction(qRT-PCR)were used to detect the contents of tumor necrosis factorα(TNF-α)and interleukin 6(IL-6)in serum and intestinal tissue.Rat stool specimens were subjected to high-throughput sequencing to obtain 16S rDNA data.Data comparison between the two groups was performed by independent sample t-test,and multi-group analysis was performed by one-way analysis of variance(ANOVA).Results The degree of hepatic sinusoidal obstruction and inflammatory cell infiltration in the BMP group was reduced,and the rejection activity index(RAI)(4.17±0.75)was lower than the NMP group(7.67±0.82),with the difference being statistically significant(t=7.720P<0.05).The rat serum contents of ALT,AST and ALP in BMP group[(254.02±35.55),(457.42±131.22),(169.60±65.61)U/L]were lower than those in the NMP group[(362.22±57.73),(741.67±87.19),(269.93±43.51)U/L],with the difference being statistically significant(t=3.909,4.419,3.196,P<0.05).The abundance of intestinal flora increased,the proportion of Proteobacteria decreased while Akkermanaceae increased,arachidonic acid metabolism and bacterial invasion of ep

关 键 词：肝移植 骨髓间充质干细胞 急性排斥反应 肠道菌群 

分 类 号：R657.3[医药卫生—外科学]