本文選題：膽道閉鎖 + Treg細(xì)胞�。� 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：研究目的： 研究調(diào)節(jié)性T細(xì)胞(Treg)和效應(yīng)性Th17細(xì)胞(Th17)在膽道閉鎖(BA)小鼠免疫炎癥發(fā)展中的作用；并過繼輸注Treg細(xì)胞或注射IL-17中和抗體,進(jìn)一步明確Treg/Th17細(xì)胞在膽道閉鎖免疫學(xué)發(fā)病機(jī)制中的作用。 研究方法： 利用RRV建立膽道閉鎖小鼠模型,新生Balb/c小鼠隨機(jī)分為以下四組：1,正常組：出生后24h內(nèi)腹腔注射生理鹽水；2,輪狀病毒組：出生后24h內(nèi)腹腔注射RRV；3, Treg細(xì)胞組：出生后12h內(nèi)腹腔過繼輸注Treg細(xì)胞,隨后12h內(nèi)注射RRV；4,IL-17抗體組：出生后12h內(nèi)注射RRV,隨后連續(xù)兩天注射不同劑量(15μg,30μg,50μg) IL-17中和抗體。觀察各組小鼠在不同時期的表型及組織病理學(xué)等變化；利用實(shí)時定量PCR (RT-qPCR)和Western blot分別檢測各組小鼠肝臟組織中Foxp3,IL-17A以及ROR-yt基因和蛋白表達(dá)；利用免疫組織化學(xué)檢測各組小鼠肝臟組織中CD4+細(xì)胞、Foxp3+Treg細(xì)胞及IL-17+Th17細(xì)胞表達(dá)部位及變化；流式細(xì)胞術(shù)檢測各組小鼠肝臟組織中Treg細(xì)胞及Th17細(xì)胞比例變化。利用ELISA檢測小鼠肝臟組織中炎性細(xì)胞因子變化,以及肝臟組織DC細(xì)胞的TLRs表達(dá)情況。分離DC細(xì)胞、CD4+T細(xì)胞并進(jìn)行培養(yǎng),利用ELISA檢測細(xì)胞培養(yǎng)上清中IL-6表達(dá)區(qū)別。體外利用細(xì)胞共培養(yǎng),觀察Treg細(xì)胞對Th17細(xì)胞分化的抑制作用,觀察膽道閉鎖小鼠體內(nèi)Treg細(xì)胞抑制功能的變化。 實(shí)驗(yàn)結(jié)果： 正常組23只,3天內(nèi)死亡1只,無1例出現(xiàn)發(fā)育遲緩和膽汁淤積癥狀,其余小鼠均健康存活到21天。24只輪狀病毒組小鼠中發(fā)生膽道閉鎖的有18只(75.0%),以發(fā)育遲緩(體重增長緩慢或停滯)和膽汁淤積(無毛區(qū)皮膚黃染,大便陶土樣)為主要癥狀；肝臟大體觀見淤膽明顯,表面顆粒狀,肝外膽管呈索條狀,膽囊顯示不清；肝臟及膽管組織切片HE染色可見肝外膽管閉鎖,匯管區(qū)大量炎性細(xì)胞浸潤。膽道閉鎖小鼠肝臟組織中Foxp3, IL-17和ROR-yt基因及蛋白表達(dá)均明顯高于正常組,在第7天達(dá)到峰值；免疫組織化學(xué)顯示膽道閉鎖小鼠肝臟組織尤其是匯管區(qū)中CD4+細(xì)胞、Foxp3+Treg細(xì)胞及IL-17+Ih17細(xì)胞均顯著增加；流式細(xì)胞術(shù)發(fā)現(xiàn)膽道閉鎖小鼠肝臟組織中Treg細(xì)胞比例降低,而Th17細(xì)胞比例升高；膽道閉鎖小鼠肝臟組織中IL-2、IL-4、IL-5、TGF-β1、IL-10, IL-17, IL-12、IL-23、IFN-γ和IL-6均高于正常對照組。膽道閉鎖小鼠肝臟DC細(xì)胞中TLR3、7、8基因和蛋白均高于正常對照組。膽道閉鎖小鼠肝臟DC細(xì)胞中IL-6明顯高于正常對照組,而CD4+T細(xì)胞中IL-6分泌無明顯差異。細(xì)胞共培養(yǎng)發(fā)現(xiàn)Treg細(xì)胞能抑制Th17細(xì)胞產(chǎn)生,而膽道閉鎖小鼠中Treg細(xì)胞抑制功能降低。過繼輸注Treg細(xì)胞后,小鼠膽道閉鎖發(fā)生率降低到23.1%,肝臟炎性表現(xiàn)明顯減輕,小鼠生存期延長,肝臟組織中Th17細(xì)胞相關(guān)基因和蛋白較膽道閉鎖小鼠明顯降低,免疫組織化學(xué)檢測發(fā)現(xiàn)IL-17+細(xì)胞表達(dá)減少,流式細(xì)胞術(shù)發(fā)現(xiàn)肝臟組織中Th17細(xì)胞比例降低(P0.05)；注射不同劑量IL-17中和抗體后,小鼠膽道閉鎖發(fā)生率均有不同程度降低,以注射50μg抗體組有統(tǒng)計學(xué)意義(P0.05),而且肝臟組織尤其是匯管區(qū)炎性表現(xiàn)減輕,伴隨有小鼠生存期延長,進(jìn)一步研究發(fā)現(xiàn)注射50μg IL-17抗體組的小鼠肝臟組織中IL-17+蛋白較病毒組明顯減少(P0.05),而免疫組化發(fā)現(xiàn)肝臟組織匯管區(qū)IL-17+細(xì)胞表達(dá)較病毒組明顯減少(P0.05)。 實(shí)驗(yàn)結(jié)論： 1.病毒感染后,引起肝臟微環(huán)境改變以及TLRs依賴的DC細(xì)胞等變化,進(jìn)而導(dǎo)致小鼠體內(nèi)Treg細(xì)胞減少及功能降低,而Th17細(xì)胞增多,二者失衡在膽道閉鎖膽管損傷的進(jìn)行性炎癥中發(fā)揮著重要作用。 2.過繼輸注Treg細(xì)胞后能明顯改善小鼠膽道閉鎖表型,延長生存期,減輕體內(nèi)炎癥表現(xiàn),表明Treg細(xì)胞對病毒感染后的炎性反應(yīng)起抑制作用,為膽道閉鎖的防治提出了可能。 3.通過注射IL-17中和抗體,可以達(dá)到使小鼠膽道閉鎖表型降低,生存期延長的目的,減輕體內(nèi)炎癥表現(xiàn),表明Th17細(xì)胞對膽道閉鎖的發(fā)病過程起促進(jìn)作用,為治療病毒感染后膽道閉鎖的進(jìn)行性免疫炎癥奠定了實(shí)驗(yàn)基礎(chǔ)。
[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 - 緯 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 .

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R726.5

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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