Th17、Treg及其平衡与大鼠肝移植术后免疫耐受的研究

Role of T-helper 17(Th17) and regulatory T(Treg) cells and Th17/Treg imbalance in immune tolerance in rats after liver transplantation

目的探讨肝移植术后肝组织T淋巴细胞Th17/Treg表达变化和肝移植免疫耐受之间的关系.方法以采用改良Kamada二袖套法建立LEWIS→BN原位肝移植大鼠急性排斥反应模型:LEWIS大鼠为供体,BN大鼠为受体,行原位肝移植(orthotopic liver transplantation,OLT).对照组为:BN→BN原位肝移植大鼠耐受模型:BN大鼠为供体,BN大鼠为受体,行OLT.术后观察大鼠一般情况,并于第1、3、5及7天分别处死大鼠取肝组织,HE染色观察肝组织结构.检测血清谷丙转氨酶(alanine transaminase,ALT)、谷草转氨酶(aspartate transaminase,AST)、白介素(interleukin,IL)-17、IL-23、IL-10和转化生长因子-β1(transforming growth factor beta 1,TGF-β1)的表达水平.结果与对照组比较,LEWIS→BN组各时间点肝功能指标ALT、AS T明显上调(P<0.05).对照组第7天ALT:819.29 IU/L±79.33 IU/L;OLT组第7天ALT:1305.62 IU/L±94.82 IU/L;对照组第7天AST:337.82 IU/L±32.17 IU/L;OLT组第7天AST:867.75 IU/L±73.97 IU/L;大鼠第7天外周血Th17相关因子IL-17(对照组:28.67 pg/m L±2.55 pg/m L,OLT组:92.36 pg/m L±9.00 pg/m L)、IL-23(对照组:26.82 pg/m L±8.17 pg/m L,OLT组:62.98 pg/m L±12.96 pg/m L)明显上升,而Treg相关因子IL-10(对照组:76.92 pg/m L±12.87 pg/m L,OLT组:47.92 pg/m L±7.00 pg/m L)、TGF-β1(对照组:129.47 pg/m L±18.37 pg/m L,OLT组:82.48 pg/m L±11.83 pg/m L)明显下降,Th17/Treg表达失去平衡.同时,TGF-β下游Smad2/3蛋白具有跟TGF-β1相同变化趋势,LEWIS→BN急性排斥组与BN→BN免疫耐受组相比,差异具有统计意义(P<0.05).结论IL-17、IL-23、IL-10、TGF-β1、Smad2/3参与了同种异体大鼠肝移植急性排斥反应,引起Treg向Th17的免疫偏移,可作为治疗大鼠肝移植免疫耐受的靶向指标.

AIM To investigate the relationship between Th17/Treg expression in liver tissue and immune tolerance after liver transplantation.METHODS A rat model of orthotopic liver transplantation from Lewis rats to Brown Norway（BN） rats（LEWIS→BN） was developed using Kamada＇s two-cuff technique. Control rats underwent orthotopic BN→BN liver transplantation. After operation, the general condition of rats was observed. Rats were sacrificed on days 1, 3, 5, and 7 after modeling to collect liver tissues for HE staining. The levels of serum alanine transaminase（ALT）, aspartate transaminase（AST）, interleukin（IL）-17, IL-23, IL-10, and transforming growth factor（TGF）-β1 were measured by ELISA.RESULTS Compared with the control group, serum levels of ALT and AST were increased obviously in the LEWIS→BN group at all the three time points（P 〈0.05, on the 7 th day, ALT in the control group： 819.29 IU/L ± 79.33 IU/L; in the LEWIS→BN group： 1305.62 IU/L ± 94.82 IU/L, AST in the control group： 337.82 IU/L ± 32.17 IU/L; AST in the LEWIS→BN group： 867.75 IU/L ± 73.97 IU/L）; the levels of Th17 related factors IL-17（the control group： 28.67 pg/m L ± 2.55 pg/m L, the LEWIS→BN group： 92.36 pg/m L ± 9.00 pg/m L） and IL-23（the control group： 26.82 pg/m L ± 8.17 pg/m L, the LEWIS→BN group： 62.98 pg/m L ± 12.96 pg/m L） in the peripheral blood were increased dramatically, while the levels of Treg related factor IL-10（the control group： 76.92 pg/m L ± 12.87 pg/m L, the LEWIS→BN group： 47.92 pg/m L ± 7.00 pg/m L） and TGF-β1（the control group： 129.47 pg/m L ± 18.37 pg/m L, the LEWIS→BN group： 82.48 pg/m L ± 11.83 pg/m L） were decreased dramatically in the LEWIS→BN group, suggesting the presence of Th17/Treg imbalance in acute rejection. The downregulation of Smad2/3 protein levels had the same trend as TGF-β1, and the difference between the Lewis→BN acute rejection group and the BN→BN immune tolerance group was statistically significant（P 〈0.05）.CONCLUSION IL-17, IL-23, IL-10, and TGF-β1 take part in acute rejection after orthotopic liver transplantation, causing Treg to Th17 immune deviation, which may be used as a target index in immune tolerance of liver transplantation.