调节性和效应性T细胞在胆道闭锁小鼠免疫炎症发展中的作用及影响

发布时间：2018-05-04 18:50

本文选题：胆道闭锁 + Treg细胞　；参考：《华中科技大学》2014年博士论文

【摘要】：研究目的：研究调节性T细胞(Treg)和效应性Th17细胞(Th17)在胆道闭锁(BA)小鼠免疫炎症发展中的作用；并过继输注Treg细胞或注射IL-17中和抗体,进一步明确Treg/Th17细胞在胆道闭锁免疫学发病机制中的作用。研究方法：利用RRV建立胆道闭锁小鼠模型,新生Balb/c小鼠随机分为以下四组：1,正常组：出生后24h内腹腔注射生理盐水；2,轮状病毒组：出生后24h内腹腔注射RRV；3, Treg细胞组：出生后12h内腹腔过继输注Treg细胞,随后12h内注射RRV；4,IL-17抗体组：出生后12h内注射RRV,随后连续两天注射不同剂量(15μg,30μg,50μg) IL-17中和抗体。观察各组小鼠在不同时期的表型及组织病理学等变化；利用实时定量PCR (RT-qPCR)和Western blot分别检测各组小鼠肝脏组织中Foxp3,IL-17A以及ROR-yt基因和蛋白表达；利用免疫组织化学检测各组小鼠肝脏组织中CD4+细胞、Foxp3+Treg细胞及IL-17+Th17细胞表达部位及变化；流式细胞术检测各组小鼠肝脏组织中Treg细胞及Th17细胞比例变化。利用ELISA检测小鼠肝脏组织中炎性细胞因子变化,以及肝脏组织DC细胞的TLRs表达情况。分离DC细胞、CD4+T细胞并进行培养,利用ELISA检测细胞培养上清中IL-6表达区别。体外利用细胞共培养,观察Treg细胞对Th17细胞分化的抑制作用,观察胆道闭锁小鼠体内Treg细胞抑制功能的变化。实验结果：正常组23只,3天内死亡1只,无1例出现发育迟缓和胆汁淤积症状,其余小鼠均健康存活到21天。24只轮状病毒组小鼠中发生胆道闭锁的有18只(75.0%),以发育迟缓(体重增长缓慢或停滞)和胆汁淤积(无毛区皮肤黄染,大便陶土样)为主要症状；肝脏大体观见淤胆明显,表面颗粒状,肝外胆管呈索条状,胆囊显示不清；肝脏及胆管组织切片HE染色可见肝外胆管闭锁,汇管区大量炎性细胞浸润。胆道闭锁小鼠肝脏组织中Foxp3, IL-17和ROR-yt基因及蛋白表达均明显高于正常组,在第7天达到峰值；免疫组织化学显示胆道闭锁小鼠肝脏组织尤其是汇管区中CD4+细胞、Foxp3+Treg细胞及IL-17+Ih17细胞均显著增加；流式细胞术发现胆道闭锁小鼠肝脏组织中Treg细胞比例降低,而Th17细胞比例升高；胆道闭锁小鼠肝脏组织中IL-2、IL-4、IL-5、TGF-β1、IL-10, IL-17, IL-12、IL-23、IFN-γ和IL-6均高于正常对照组。胆道闭锁小鼠肝脏DC细胞中TLR3、7、8基因和蛋白均高于正常对照组。胆道闭锁小鼠肝脏DC细胞中IL-6明显高于正常对照组,而CD4+T细胞中IL-6分泌无明显差异。细胞共培养发现Treg细胞能抑制Th17细胞产生,而胆道闭锁小鼠中Treg细胞抑制功能降低。过继输注Treg细胞后,小鼠胆道闭锁发生率降低到23.1%,肝脏炎性表现明显减轻,小鼠生存期延长,肝脏组织中Th17细胞相关基因和蛋白较胆道闭锁小鼠明显降低,免疫组织化学检测发现IL-17+细胞表达减少,流式细胞术发现肝脏组织中Th17细胞比例降低(P0.05)；注射不同剂量IL-17中和抗体后,小鼠胆道闭锁发生率均有不同程度降低,以注射50μg抗体组有统计学意义(P0.05),而且肝脏组织尤其是汇管区炎性表现减轻,伴随有小鼠生存期延长,进一步研究发现注射50μg IL-17抗体组的小鼠肝脏组织中IL-17+蛋白较病毒组明显减少(P0.05),而免疫组化发现肝脏组织汇管区IL-17+细胞表达较病毒组明显减少(P0.05)。实验结论： 1.病毒感染后,引起肝脏微环境改变以及TLRs依赖的DC细胞等变化,进而导致小鼠体内Treg细胞减少及功能降低,而Th17细胞增多,二者失衡在胆道闭锁胆管损伤的进行性炎症中发挥着重要作用。 2.过继输注Treg细胞后能明显改善小鼠胆道闭锁表型,延长生存期,减轻体内炎症表现,表明Treg细胞对病毒感染后的炎性反应起抑制作用,为胆道闭锁的防治提出了可能。 3.通过注射IL-17中和抗体,可以达到使小鼠胆道闭锁表型降低,生存期延长的目的,减轻体内炎症表现,表明Th17细胞对胆道闭锁的发病过程起促进作用,为治疗病毒感染后胆道闭锁的进行性免疫炎症奠定了实验基础。
[Abstract]:Purpose of study :

To study the role of regulatory T cells ( Treg ) and effector Th17 cells ( Th17 ) in the development of immune inflammation in biliary atresia ( BA ) mice ;
The role of Treg / Th17 cells in the pathogenesis of biliary atresia immunological pathogenesis was further clarified by adoptive infusion of Treg cells or injection of IL - 17 and antibodies .

Study method :

The model of biliary atresia was established with RRV . Balb / c mice were randomly divided into four groups : 1 . Normal group : normal saline was injected intraperitoneally 24 hours after birth ;
2 . rotavirus group : intraperitoneal injection of RRV within 24 hours after birth ;
3 . Treg cell group : Treg cells were injected intraperitoneally 12 hours after birth , followed by injection of RRV in 12h .
4 . IL - 17 antibody group : RRV was injected in 12 hours after birth , followed by injection of different doses ( 15 渭g , 30 渭g , 50 渭g ) of IL - 17 and antibody in two consecutive days .
RT - qPCR and Western blot were used to detect the expression of Foxp3 , IL - 17A in liver tissues of each group , as well as mRNA and protein expression in liver tissues of each group .
The expression sites and changes of CD4 + cells , Foxp3 + Treg cells and IL - 17 + Th17 cells were detected by immunohistochemistry .
Flow cytometry was used to detect the changes of Treg cells and Th17 cells in liver tissues of each group . The expression of inflammatory cytokines in liver tissues of mice was detected by ELISA , and the expression of TLRs in liver tissue DC cells was detected . The expression of IL - 6 in culture supernatant was detected by ELISA . The inhibitory effect of Treg cells on Th17 cell differentiation was observed by means of ELISA .

Experimental results :

In the normal group 23 , 1 died within 3 days , none in 1 case developed developmental retardation and cholestatic symptoms , the rest of the mice survived to 21 days . Only 18 ( 75.0 % ) of 24 rotavirus group mice had biliary atresia , and the main symptoms were slow growth ( slow or stagnant body weight ) and cholestatic ( no hair area skin yellow stain , stool sample ) .
The general view of the liver is obvious , the surface is granular , the intrahepatic bile duct of the liver is in the form of a cord , and the gallbladder is not clear ;
The expression of Foxp3 , IL - 17 and ror - yt gene and protein in liver tissues of biliary atresia mice were significantly higher than those in the normal group , and peaked at 7th day .
Immunohistochemical analysis showed that CD4 + cells , Foxp3 + Treg cells and IL - 17 + Ih17 cells increased significantly in the liver tissues of biliary atresia mice .
Flow cytometry showed that the proportion of Treg cells decreased in the liver tissues of biliary atresia mice , and the proportion of Th17 cells increased .
IL - 2 , IL - 4 , IL - 5 , TGF - 尾1 , IL - 10 , IL - 17 , IL - 12 , IL - 23 , IL - 17 , IL - 12 , IL - 23 , IFN - 纬 and IL - 6 in liver tissues of biliary atresia mice were higher than those in control group .
After injection of different doses of IL - 17 and antibody , the incidence of biliary atresia in mice was reduced to some extent ( P0.05 ) .

Experimental Conclusion :

1 . After virus infection , the changes of microenvironment of liver and DC cells dependent on TLRs are caused , which leads to the decrease of Treg cells and decrease of function in mice , and the increase of Th17 cells , which play an important role in the progressive inflammation of biliary atresia bile duct injury .

2 . After the adoptive infusion of Treg cells , the biliary atresia phenotype of mice can be obviously improved , the survival time can be prolonged , the inflammatory response in vivo is alleviated , and it is shown that the Treg cell can inhibit the inflammatory response after virus infection and provides a possibility for the prevention and treatment of biliary atresia .

3 . By injection of IL - 17 and antibody , it is possible to achieve the aim of reducing the blocking phenotype of biliary tract in mice , prolonging the survival period , reducing the inflammatory expression in vivo , suggesting that Th17 cells play a role in the pathogenesis of biliary atresia , which lays a foundation for the treatment of the progressive immune inflammation of biliary atresia after virus infection .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R726.5

【共引文献】