MiR-155在大鼠肝移植术后排斥反应中作用机制的研究

Study of miR-155 in the mechanism of rejection after liver transplantation in rats

目的探讨微小核糖核酸(miRNA,miR)-155在大鼠肝移植术后排斥反应中的作用机制。方法将大鼠分为异系移植模型组(排斥组,n=10),供体为雄性Lewis大鼠,受体为雄性BN大鼠;同系移植模型组(对照组,n=10),供、受体均为雄性Lewis大鼠;两组均按照"二袖套法"建立大鼠原位肝移植模型。术后第7日处死大鼠取血标本和肝组织。检测肝功能指标丙氨酸转氨酶(ALT)、总胆红素(TB)以及细胞因子白细胞介素(IL)-2、IL-4、干扰素(IFN)-γ的水平;光学显微镜下观察肝组织的病理变化情况。对两组大鼠肝组织样本每组选取3例进行高通量测序,检测排斥反应相关miRNA,进行生物信息学分析,预测并分析其相关的信号通路以及基因。结果肝功能检测显示排斥组大鼠血清中ALT、TB水平均高于对照组,差异均有统计学意义(均为P<0.01)。外周血细胞因子检测显示,与对照组比较,排斥组的IL-2和IFN-γ水平升高(均为P<0.01),IL-4水平下降(P<0.01)。病理检查结果提示,与对照组比较,排斥组发生了明显的排斥反应。高通量检测结果示排斥组miR-155表达上调明显,为对照组5.89倍。生物信息学分析提示,miR-155的上调表达与哺乳动物雷帕霉素靶蛋白(m TOR)、丝裂原活化蛋白激酶(MAPK)及T细胞受体信号通路有关,可能参与调节的基因有酵母自噬基因(ATG1)及其同源基因ULK2、胰岛素样生长因子-1(Igf-1)以及G蛋白偶联受体的调节基因(Arrb1)等。结论 miR-155可能促进了大鼠肝移植术后排斥反应的发生发展,涉及此过程的信号通路可能有m TOR、MAPK信号通路及T细胞受体信号途径,ATG1、ULK2、Igf-1以及Arrb1等基因可能参与了此过程。

Objective To explore the mechanism of micro RNA（mi RNA, mi R）-155 in the rejection after liver transplantation in rats. Methods The rats were divided into two groups. In the xenograft model group（rejection group, n=10）, the donors were male Lewis rats and the recipients were male BN rats. In the allograft model group（control group, n=10）, both the donors and recipients were male Lewis rats. The rat models with orthotopic liver transplantation were established by two-cuff technique in two groups. At postoperative 7 d, the animals were sacrificed for the collection of blood and liver tissue samples. The serum levels of alanine aminotransferase（ALT）, total bilirubin（TB）, and cytokines of interleukin（IL）-2, IL-4, interferon（IFN）-γ were quantitatively measured. The pathological changes of liver tissues were observed under light microscope. In each group, three liver tissue samples were prepared and subject to highthroughput sequencing. The mi RNAs related to rejection were identified for bioinformatics analysis to predict and analyze relevant signaling pathways and genes. Results In the rejection group, the serum levels of ALT and TB were significantly higher than those in the control group（both P〈0.01）. Compared with the control group, the levels of IL-2 and IFN-γwere considerably up-regulated（both P〈0.01）, whereas the level of IL-4 was dramatically down-regulated（P〈0.01）. Pathological examination demonstrated that more evident rejections were observed in the rejection group than the control group. High-throughput sequencing revealed that the expression level of mi R-155 was significantly up-regulated in the rejection group, which was 5.89 times of that in the control group. Bioinformatics analysis demonstrated that up-regulation of mi R-155 was associated with the mammalian target of rapamycin（m TOR）, mitogen-activated protein kinase（MAPK） and T cell receptor signaling pathways. The genes which were probably responsible for regulation included the yeast autophagy related gene 1（ATG1） and its homologous gene ULK2, insulin-like growth factor-1（Igf-1） and G proteincoupled receptor regulatory gene（Arrb1）, etc. Conclusions mi R-155 might promote the incidence and progression of rejection after liver transplantation in rats. The involved signaling pathways probably include the m TOR, MAPK signaling pathway and T cell receptor signaling pathway. ATG1, ULK2, Igf-1, and Arrb1 genes may participate in this process.