原发性胆汁性肝硬化患者Th17细胞及IL-17表达的初步研究
|
摘要
背景
原发性胆汁性肝硬化(primary biliary cirrhosis, PBC)是一种肝内进行性、非化脓性小胆管破坏伴门静脉区炎症、纤维化的自身免疫性肝病,最终可进展为肝硬化和肝衰竭而死亡。PBC的病因和确切发病机制至今尚不完全清楚,肝胆管上皮细胞异常表达线粒体抗原、T细胞介导的异常免疫反应和宿主自身抗原发生变化等机制都可能参与PBC的发生。PBC在全球范围内都有发生,尤其在北美国家40岁以上妇女发病率逐年增多的趋势；在我国,近年来随着诊断技术的进步,从文献报道和来院就医的PBC患者数量看来,PBc发现率有增多的趋势。目前国内外对PBC尚无特效的治疗方法,对早中期患者通常认为熊去氧胆酸(ursodeoxycholic acid, UDCA)仍为其最有效的治疗方法。然而,UDCA不能阻断疾病进程,PBC发展到后期只能通过肝移植来解决,且肝移植后复发率很高。
Th17细胞是新近发现的一种CD4+T细胞亚群,通过分泌效应细胞因子IL-17在自身免疫性疾病中起着重要的作用。在实验性自身免疫性脑脊髓炎(EAE)动物模型中,发现过继输注Th17细胞比输注Thl细胞更能有效诱发EAE,注射IL-17也能加重EAE动物的炎性症状和严重程度,而采用IL-17基因缺陷小鼠则可以减弱疾病的进程。动物实验还发现,正常小鼠肝CD4+T细胞分泌IL-17高于其脾CD4+T细胞分泌的IL-17；PBC小鼠自身免疫模型中肝组织和外周血Th17细胞及IL-17过度表达,实验结果提示Th17细胞在肝细胞微环境中对肝脏自身免疫疾病过程可能起重要作用。
目的
基于以上国内外研究现状和存在问题,本研究拟对原发性胆汁性肝硬化患者(PBC)、乙肝肝硬化患者(HBV-PHC)及正常人(HC)分别行外周血Thl7细胞／CD4+T细胞比例和IL-17表达水平的检测,同时分析其血清IL-17表达水平与肝脏损伤标志物血清谷氨酰转肽酶(GGT)、碱性磷酸酶(ALP)浓度的关系,初步探讨PBC患者外周血Th17细胞比例和功能的变化及其临床意义。为我们今后进一步研究Th17细胞在自身免疫性肝病中作用,提供一定的实验依据,进而可能为PBC的免疫学治疗靶点提供一个新的思路,不仅具有重要的学术价值,而且具有巨大的应用前景和社会效益。
方法
1流式细胞术(FCM)检测研究对象外周血Th17细胞占CD4+T细胞比例。
2采用酶联免疫吸附法(ELISA)法检测研究对象血清IL-17水平,同时用酶动力速率检测血清GGT和ALP的浓度,比较IL-17水平与GGT、ALP浓度的相关性。
3实时荧光定量PCR方法检测研究对象外周血单个核细胞IL-17mRNA基因表达水平。
结果
1.经PMA/Ionomycin刺激三组研究对象外周血单个核细胞4h后流式检测,最终以散点图中IL-17染色阳性表示Th17细胞,用CellQuest软件分析数据。PBC患者组和乙肝肝硬化患者组外周血Thl7/CD4~+T细胞水平分别为[(0.83±0.58)%]、[(0.81±0.45)%],显著高于健康组[(0.58±0.20)%],差异有统计学意义(P<0.05),但PBC患者组和乙肝肝硬化患者组相比,差异无统计学意义(P>0.05)。
2.采用ELISA法对三组研究对象血清IL-17水平进行检测,PBC患者组和乙肝肝硬化患者组血清IL-17水平分别为[(24.6±9.7)pg/ml]、[(21.6±6.1)pg/ml],显著高于健康组[(18.0±2.1)pg/ml],差异有统计学意义(P<0.05),但PBC患者组和乙肝肝硬化患者组相比,差异无统计学意义(P>0.05)。同时,我们用酶动力速率法分别检测三组研究对象中的GGT和ALP这两个反映胆汁淤积的血清学指标。检测结果发现血清中GGT和ALP水平分别为：PBC患者组[(320±267)U/L]、[(204±126)U/L]；乙肝肝硬化患者组[(246±156)U/L]、[(132±113)U/L]；健康对照组[(23±12)U/L]、[(60±13)U/L],任意两组之间比较,差异均有有统计学意义(P<0.05)。
3.实时荧光定量PCR方法检测各组外周血单个核细胞IL-17mRNA表达水平。与乙肝肝硬化患者组、健康组相比,PBC患者组的IL-17mRNA的表达量均显著性升高,差异有统计学意义(P<0.05),尤其PBC患者组的表达量是健康组的2.21倍(P<0.05)：乙肝肝硬化患者组和健康组相比,差异有统计学意义(P<0.05)。
4.基于各组血清学IL-17水平和GGT、ALP浓度,分别对三组研究对象上述指标间进行相关性统计学分析发现：PBC患者组血清IL-17水平与GGT、ALP浓度呈显著正相关性,差异有统计学意义(P<0.05)而乙肝肝硬化患者组和健康组,未见明显的相关性,差异无统计学意义(P>0.05)。
结论
在本实验中我们发现PBC患者组和乙肝肝硬化患者组外周血Th17细胞及血清IL-17水平明显高于健康对照组,差异有统计学意义；PBC患者组外周血单个核细胞IL-17mRNA水平明显高于乙肝肝硬化患者组和健康组,差异有统计学意义。Th17细胞及其分泌的促炎因子IL-17,可能在PBC免疫发病和炎症性疾病预后中起着重要作用。目前PBC尚无特异性的治疗方法,因此进一步研究Th17细胞在PBC发病机理中的作用有望成为帮助患者重建免疫系统的又一新的靶点,为人类治疗PBC提供新的思路。
Background
Primary biliary cirrhosis (PBC) is an autoimmune chronic cholestatic liver disease characterized by destruction of intrahepatic bile ducts and deveo-pment of cirrhosis and liver failure. Serologically, PBC is characterized by the presence of antimitochondrial antibodies (AMA).T cell-mediated abnormal immune response、the abnormal changes in the host itself antigen and other mechanisms may be involved in the occurrence of PBC. The morbidity of PBC is increasing in recent years in middle-aged women in North American. In China, there is not systematically epidemic data with PBC, however, with the developing of diagnostic technique, the morbility of PBC is increasing. At present,there is no effectively curative method for PBC. Ursodeoxycholic acid(UDCA)is known as the most effective drug for PBC. It can only provide symptomatic relief of cholestasis but can not block disease course. At late stage of PBC, liver transplantation could be the last and only choice.
CD4-positive T helper type 17 (Th17) cells, characterized by the secretion of interleukin(IL)-17, have been implicated in the pathogenesisof autoimmune diseases. The researchers found that Th17 cells is more efficience to induce EAE than Thl cells in experimental autoimmune encephalomyelitis(EAE) in mice model. They also found that the EAE mice injected with IL-17 can enhance the symptoms of inflammatory and severity. Conversely, IL-17-/- mice can delay the progression of EAE. Animal experiments discovered that CD4+T cells can secrete higher IL-17 in mice. They also found the over-expression of Th17 cells and IL-17 in the autoimmune liver tissue and peripheral Blood in PBC mouse models.Those results suggest that Th17 cells play a important role in the pathogenesis of PBC.
Objective
This study is about three groups of patients with primary biliary cirrhosis (PBC) and HBV-related posthepatitic cirrhosis(HBV-PHC) and health controls (HC) which were performed inperipheral blood Thl7 cells/CD4+Tcells ratio and the expression levels of IL-17, Simultaneously analyzed the correlation between the serum levels of IL-17 and serum markers of liver injuryed glutamyl transpeptidase peptidase(GGT)、alkaline phosphatase (ALP), for the purpose of studying the function and its clinical significance of peripheral blood Th17 cells and IL-17 in PBC patients. Those results may provide some experimental evidenc with Th17 cells in autoimmune liver diseases and provide the immune target of a new idea for therapeutic.
Methods
1. The ratio of Thl7 cells/CD4+T cell in the peripheral blood were detected By Flow cytometry (FCM).
2. The levels serum of IL-17 were measured by enzyme-linked immunosorbent assay (ELISA) and the serum levels of GGT and ALP wre measured by enzyme kinetic rate. At the same time, compared the correlation between the Levels of IL-17 and GGT、ALP.
3. The IL-17 mRNA gene expression in the peripheral blood mononuclear cells were measured by real time quantitative RT-PCR.
Results
1. PBMC of three groups were stimulated by PMA/Ionomycin and detected by flow cytometry after cultured 4h, eventually IL-17+ in scatter staining indicated Th17 cells and using CellQuest software to analyze data. Compared to HC group [(0.58±0.20)%], Th17 cells in peripheral blood in PBC patient group[(0.83±0.57)%] and HBV-PHC[(0.81±0.45)%)] (P<0.05). But between PBC and HBV-PHC (P>0.05).
2. The serum leves of IL-17 in the three groups were measured by ELISA. The serum levels of IL-17 in PBC[(24.6±9.7)pg/mL] and HBV-PHC [(21.6±6.1)pg/mL] was significantly higher than HC[(18.0±2.1)pg/mL] (P<0.05). Between PBC and HBV-PHC(P<0.05).At the same time, the concentrations of serum GGT, ALT which are liver injuryed markers were measured by enzyme kinetic rate. The Concentration of GGT[(320±267)U/L] and [ALP(204±126)U/L] in PBC group, the concentration of GGT [(246±156) U/L] and ALP[(132±113)U/L] in HBV-PHC, the concentrationin of GGT[(23±12) U/L] and ALP [(60±3)U/L] in HC, any comp-Arison between the two groups (P<0.05).
3. IL-17mRNA gene expression in the peripheral blood mononuclear cells were measured by real time quantitative RT-PCR. Compared with HBV-PHC and HC the expression of IL-17 were significantly higher in PBC patient group (P<0.05) Excepally the expression of PBC groups is 2.21 times than HC group (P<0.05). Between HBV-PHC and healthy groups(P<0.05).
4. Based on the serum levels of IL-17 and GGT, ALP, we studied that correlation between the index statistics analysis of three groups. The results showed PBC patients with the serum levels of IL-17 and GGT, ALP were positively correlated(P<0.05). HBV-PHC and HC,no significant correlation(P>0.05).
Conclusion
In this study, the results showed that the levels of Th17 cells in periph-eral blood and the serum levels of IL-17 were significantly elevated in PBC group and HBV-PHC group. Compared to HC group, which the difference was significantly. The levels IL-17 mRNA of peripheral blood mononuclear cells were significantly elevated in PBC group compared to HBV-PHC group and HC group, which the difference were statistically significant. Th17 cells and their secretion of proinflammatory cytokines IL-17 may play a important role in the prognosis of PBC and the inflammatory immune disease. There is no specific cure for PBC, so further studies are required to clarify the effect of Th17 cells in the pathogenesis of PBC.It can help us rebuild the immune system, but also provide new ideas for the strateies of PBC.
原发性胆汁性肝硬化(primary biliary cirrhosis, PBC)是一种肝内进行性、非化脓性小胆管破坏伴门静脉区炎症、纤维化的自身免疫性肝病,最终可进展为肝硬化和肝衰竭而死亡。PBC的病因和确切发病机制至今尚不完全清楚,肝胆管上皮细胞异常表达线粒体抗原、T细胞介导的异常免疫反应和宿主自身抗原发生变化等机制都可能参与PBC的发生。PBC在全球范围内都有发生,尤其在北美国家40岁以上妇女发病率逐年增多的趋势；在我国,近年来随着诊断技术的进步,从文献报道和来院就医的PBC患者数量看来,PBc发现率有增多的趋势。目前国内外对PBC尚无特效的治疗方法,对早中期患者通常认为熊去氧胆酸(ursodeoxycholic acid, UDCA)仍为其最有效的治疗方法。然而,UDCA不能阻断疾病进程,PBC发展到后期只能通过肝移植来解决,且肝移植后复发率很高。
Th17细胞是新近发现的一种CD4+T细胞亚群,通过分泌效应细胞因子IL-17在自身免疫性疾病中起着重要的作用。在实验性自身免疫性脑脊髓炎(EAE)动物模型中,发现过继输注Th17细胞比输注Thl细胞更能有效诱发EAE,注射IL-17也能加重EAE动物的炎性症状和严重程度,而采用IL-17基因缺陷小鼠则可以减弱疾病的进程。动物实验还发现,正常小鼠肝CD4+T细胞分泌IL-17高于其脾CD4+T细胞分泌的IL-17；PBC小鼠自身免疫模型中肝组织和外周血Th17细胞及IL-17过度表达,实验结果提示Th17细胞在肝细胞微环境中对肝脏自身免疫疾病过程可能起重要作用。
目的
基于以上国内外研究现状和存在问题,本研究拟对原发性胆汁性肝硬化患者(PBC)、乙肝肝硬化患者(HBV-PHC)及正常人(HC)分别行外周血Thl7细胞／CD4+T细胞比例和IL-17表达水平的检测,同时分析其血清IL-17表达水平与肝脏损伤标志物血清谷氨酰转肽酶(GGT)、碱性磷酸酶(ALP)浓度的关系,初步探讨PBC患者外周血Th17细胞比例和功能的变化及其临床意义。为我们今后进一步研究Th17细胞在自身免疫性肝病中作用,提供一定的实验依据,进而可能为PBC的免疫学治疗靶点提供一个新的思路,不仅具有重要的学术价值,而且具有巨大的应用前景和社会效益。
方法
1流式细胞术(FCM)检测研究对象外周血Th17细胞占CD4+T细胞比例。
2采用酶联免疫吸附法(ELISA)法检测研究对象血清IL-17水平,同时用酶动力速率检测血清GGT和ALP的浓度,比较IL-17水平与GGT、ALP浓度的相关性。
3实时荧光定量PCR方法检测研究对象外周血单个核细胞IL-17mRNA基因表达水平。
结果
1.经PMA/Ionomycin刺激三组研究对象外周血单个核细胞4h后流式检测,最终以散点图中IL-17染色阳性表示Th17细胞,用CellQuest软件分析数据。PBC患者组和乙肝肝硬化患者组外周血Thl7/CD4~+T细胞水平分别为[(0.83±0.58)%]、[(0.81±0.45)%],显著高于健康组[(0.58±0.20)%],差异有统计学意义(P<0.05),但PBC患者组和乙肝肝硬化患者组相比,差异无统计学意义(P>0.05)。
2.采用ELISA法对三组研究对象血清IL-17水平进行检测,PBC患者组和乙肝肝硬化患者组血清IL-17水平分别为[(24.6±9.7)pg/ml]、[(21.6±6.1)pg/ml],显著高于健康组[(18.0±2.1)pg/ml],差异有统计学意义(P<0.05),但PBC患者组和乙肝肝硬化患者组相比,差异无统计学意义(P>0.05)。同时,我们用酶动力速率法分别检测三组研究对象中的GGT和ALP这两个反映胆汁淤积的血清学指标。检测结果发现血清中GGT和ALP水平分别为：PBC患者组[(320±267)U/L]、[(204±126)U/L]；乙肝肝硬化患者组[(246±156)U/L]、[(132±113)U/L]；健康对照组[(23±12)U/L]、[(60±13)U/L],任意两组之间比较,差异均有有统计学意义(P<0.05)。
3.实时荧光定量PCR方法检测各组外周血单个核细胞IL-17mRNA表达水平。与乙肝肝硬化患者组、健康组相比,PBC患者组的IL-17mRNA的表达量均显著性升高,差异有统计学意义(P<0.05),尤其PBC患者组的表达量是健康组的2.21倍(P<0.05)：乙肝肝硬化患者组和健康组相比,差异有统计学意义(P<0.05)。
4.基于各组血清学IL-17水平和GGT、ALP浓度,分别对三组研究对象上述指标间进行相关性统计学分析发现：PBC患者组血清IL-17水平与GGT、ALP浓度呈显著正相关性,差异有统计学意义(P<0.05)而乙肝肝硬化患者组和健康组,未见明显的相关性,差异无统计学意义(P>0.05)。
结论
在本实验中我们发现PBC患者组和乙肝肝硬化患者组外周血Th17细胞及血清IL-17水平明显高于健康对照组,差异有统计学意义；PBC患者组外周血单个核细胞IL-17mRNA水平明显高于乙肝肝硬化患者组和健康组,差异有统计学意义。Th17细胞及其分泌的促炎因子IL-17,可能在PBC免疫发病和炎症性疾病预后中起着重要作用。目前PBC尚无特异性的治疗方法,因此进一步研究Th17细胞在PBC发病机理中的作用有望成为帮助患者重建免疫系统的又一新的靶点,为人类治疗PBC提供新的思路。
Background
Primary biliary cirrhosis (PBC) is an autoimmune chronic cholestatic liver disease characterized by destruction of intrahepatic bile ducts and deveo-pment of cirrhosis and liver failure. Serologically, PBC is characterized by the presence of antimitochondrial antibodies (AMA).T cell-mediated abnormal immune response、the abnormal changes in the host itself antigen and other mechanisms may be involved in the occurrence of PBC. The morbidity of PBC is increasing in recent years in middle-aged women in North American. In China, there is not systematically epidemic data with PBC, however, with the developing of diagnostic technique, the morbility of PBC is increasing. At present,there is no effectively curative method for PBC. Ursodeoxycholic acid(UDCA)is known as the most effective drug for PBC. It can only provide symptomatic relief of cholestasis but can not block disease course. At late stage of PBC, liver transplantation could be the last and only choice.
CD4-positive T helper type 17 (Th17) cells, characterized by the secretion of interleukin(IL)-17, have been implicated in the pathogenesisof autoimmune diseases. The researchers found that Th17 cells is more efficience to induce EAE than Thl cells in experimental autoimmune encephalomyelitis(EAE) in mice model. They also found that the EAE mice injected with IL-17 can enhance the symptoms of inflammatory and severity. Conversely, IL-17-/- mice can delay the progression of EAE. Animal experiments discovered that CD4+T cells can secrete higher IL-17 in mice. They also found the over-expression of Th17 cells and IL-17 in the autoimmune liver tissue and peripheral Blood in PBC mouse models.Those results suggest that Th17 cells play a important role in the pathogenesis of PBC.
Objective
This study is about three groups of patients with primary biliary cirrhosis (PBC) and HBV-related posthepatitic cirrhosis(HBV-PHC) and health controls (HC) which were performed inperipheral blood Thl7 cells/CD4+Tcells ratio and the expression levels of IL-17, Simultaneously analyzed the correlation between the serum levels of IL-17 and serum markers of liver injuryed glutamyl transpeptidase peptidase(GGT)、alkaline phosphatase (ALP), for the purpose of studying the function and its clinical significance of peripheral blood Th17 cells and IL-17 in PBC patients. Those results may provide some experimental evidenc with Th17 cells in autoimmune liver diseases and provide the immune target of a new idea for therapeutic.
Methods
1. The ratio of Thl7 cells/CD4+T cell in the peripheral blood were detected By Flow cytometry (FCM).
2. The levels serum of IL-17 were measured by enzyme-linked immunosorbent assay (ELISA) and the serum levels of GGT and ALP wre measured by enzyme kinetic rate. At the same time, compared the correlation between the Levels of IL-17 and GGT、ALP.
3. The IL-17 mRNA gene expression in the peripheral blood mononuclear cells were measured by real time quantitative RT-PCR.
Results
1. PBMC of three groups were stimulated by PMA/Ionomycin and detected by flow cytometry after cultured 4h, eventually IL-17+ in scatter staining indicated Th17 cells and using CellQuest software to analyze data. Compared to HC group [(0.58±0.20)%], Th17 cells in peripheral blood in PBC patient group[(0.83±0.57)%] and HBV-PHC[(0.81±0.45)%)] (P<0.05). But between PBC and HBV-PHC (P>0.05).
2. The serum leves of IL-17 in the three groups were measured by ELISA. The serum levels of IL-17 in PBC[(24.6±9.7)pg/mL] and HBV-PHC [(21.6±6.1)pg/mL] was significantly higher than HC[(18.0±2.1)pg/mL] (P<0.05). Between PBC and HBV-PHC(P<0.05).At the same time, the concentrations of serum GGT, ALT which are liver injuryed markers were measured by enzyme kinetic rate. The Concentration of GGT[(320±267)U/L] and [ALP(204±126)U/L] in PBC group, the concentration of GGT [(246±156) U/L] and ALP[(132±113)U/L] in HBV-PHC, the concentrationin of GGT[(23±12) U/L] and ALP [(60±3)U/L] in HC, any comp-Arison between the two groups (P<0.05).
3. IL-17mRNA gene expression in the peripheral blood mononuclear cells were measured by real time quantitative RT-PCR. Compared with HBV-PHC and HC the expression of IL-17 were significantly higher in PBC patient group (P<0.05) Excepally the expression of PBC groups is 2.21 times than HC group (P<0.05). Between HBV-PHC and healthy groups(P<0.05).
4. Based on the serum levels of IL-17 and GGT, ALP, we studied that correlation between the index statistics analysis of three groups. The results showed PBC patients with the serum levels of IL-17 and GGT, ALP were positively correlated(P<0.05). HBV-PHC and HC,no significant correlation(P>0.05).
Conclusion
In this study, the results showed that the levels of Th17 cells in periph-eral blood and the serum levels of IL-17 were significantly elevated in PBC group and HBV-PHC group. Compared to HC group, which the difference was significantly. The levels IL-17 mRNA of peripheral blood mononuclear cells were significantly elevated in PBC group compared to HBV-PHC group and HC group, which the difference were statistically significant. Th17 cells and their secretion of proinflammatory cytokines IL-17 may play a important role in the prognosis of PBC and the inflammatory immune disease. There is no specific cure for PBC, so further studies are required to clarify the effect of Th17 cells in the pathogenesis of PBC.It can help us rebuild the immune system, but also provide new ideas for the strateies of PBC.
引文
[1]Kaplan M M, Gershwin M E. Primary biliary cirrhosis[J].N Engl J. Med,2005 ,353(12):1261-1273.
[2]Kumagi T, Onji M. Presentation and diagnosis of primary biliary cirrh osis in the 21st century. Clin Liver Dis,2008,12(5):243-259.
[3]Prince M, Amanda C, Newman W,et al. Survival and symptom progression in geographically based cohort of patients with primary biliary cirrhosis:a follow-up for up to 28 years[J]. Gastroenterology,2002,123 (9):1044-1051.
[4]David E,Jones J. Pathogenesis of primary biliary cirrhosis[J]. Journal of Hepatology,2003,39(9):639-648.
[5]Gershwin ME, Mackay IR. The causes of primary biliary cirrhosis:Convenient and inconvenient truths[J].Hepatology,2008,47(2):737-745.
[6]Selmi C, Mayo MJ, Bach N, et al. Primary biliary cirrhosis in monozygotic and dizygotic twins:genetics, epigenetics, and environment[J]. Gastroenter-ology,2004,127 (17):485-492.
[7]Kita H, Imawari M, Gershwin ME. Cellular immune response in primary biliary cirrhosis[J].HepatolRes,2004,28 (10):12-17.
[8]Rolland E, Dickson MD, Patricia M, et al.Grambsch Prognosis in primary biliary cirrhosis:Model for decision making[J]. Hepatology,2005,10(1):1-7.
[9]Ian Gan S, de Jongh M, Kaplan MM. Modafinil in the treatment of debilitating fatigue in Primary biliary cirrhosis:a clinincal experience[J]. Dig Dis Sci, 2009,54 (10):2242-2246.
[10]Wang C, Kang SG, Lee J, et al.The role of CCR6 in migration of Th17 cells and regulation of effector T cell balance in the gut[J]. Nature,2009,10 (1) 173-183.
[11]Caspar 0, Andrea P, Susan B, et al. Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells and results in spontaneous fatal autoimmunity[J]. Exp Med,2009,206:549-559.
[12]Golovanova EV,Il'chenko LIu, Tsaregorodtseva TM, et al. Cytokines in primary biliary cirrhosis (diagnostic and prognostic value)[J]. Ter Arkh, 2004,76(2):8-11
[13]Nagano T, Yamamoto K, Matsumoto S, et al.Cytokine profile in the liver of primary biliary cirrhosis[J].J Clin Immunol,1999,19(6):422-427.
[14]Park H, Li ZX, Yang X0, et al. A distinct lineage of CD4+T cell regulate tissue inflammation by producing interleukin 17[J]. Nat Zmmunol,2005,11 (6):1133-1141.
[15]Tesmer L, Lundy S, Sarkar S, et al. Th17 cells in human disease [J]. Immunolo-gical reviews,2008,223 (8):87-113.
[16]AshinoS, Wakita D, Shiohama Y, et al. A Th17-polarized cell population that has infiltrated the lung requires cells that convert to IFN-γ production in order to induce airway hyperresponsiveness [J]. Internatio-nal Immunology, 2010,22 (6):503-513.
[17]邓日辉,陈曲波。自身免疫性疾病中Thl7细胞效应因子的生物学功能研究进展[J].广东医学,2009,30(6)：993-995.
[18]Veldhoen M, Hocking R J, Atkins C J, et al.TGF beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17 produc-ing T cells [J]. Immunity,2006,24(2):179-189.
[19]Bettelli E, Vi jay K, Kuchroo H, et al. Thl, Th17, andTh9 effector cells induce Experimental autoimmune encephalomyelitis with different pathological Phen-otypes[J]. Immunol,2009,183(11):7169-7177.
[20]Zheng XF, Cintia S, Paiva D, et al. Desiccating Stress Promotion of Th17 differentiation by ocular surface tissues through a dendritic cell-mediated pathway[J]. Journal of vision,2010,51(2):3083-3091.
[21]Bettelli E, Korn T, Oukka M, et al.Inductin and effector functions of Th17 cells[J]. Nature,2008,19(5):1051-1057.
[22]Boniface K, Bak-Jensen KS, Li Y, et al. Prostaglandin E2 regulates Th17 cell differentiation and function through cyclic AMP and EP2/EP4 recept or signaling[J]. J Exp Med 2009,206(3):535-548.
[23]Su X, Ye J, Hsueh EC, et al. Tumor microenvironments direct the recruitment and expansion of human Th17 cells[J]. Immunol,2010,184(3):1630-1641.
[24]Komiyama Y, Nakae S, Matsuki T, et al. IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis[J]. Immunol,2006, 177(2):566-573.
[25]Ruth Y, Salunga TL, Tsuneyama K, et al. Hepatic IL-17 responses in human And murine primary biliary cirrhosis[J]. Journal of Autoimmunity,2009,32(1) :43-51.
[26]Joshi S, Cauch-Dudek K, Wanless IR, et al. Primary biliary cirrhosis with additional features of autoimmune hepatitis:response to therapy with ursode-oxycholic acid[J]. Hepatology,2002,35(6):409-413.
[27]Fukushima K, Ueno Y, Shimosegawa T. Treatment of Primary biliary cirrhosis: A new challenge?[J].HepatolRes,2010,40(1):61-68.
[28]Lindor KD, Gershwin ME, Poupon R, et al. Primary biliary cirrhosis[J]. Hepatology,2009,50(1):291-308.
[29]European Association for the study of the Liver, EAST Clinical pratice Guidelines:management of cholestatic liver diseases. J Hepatol,2009,51:237-267.
[30]Heathcote EJ, Management of primary biliary cirrhosis.The American Association for the Study of Liver Diseases practice guidelines. Hepatol-ogy,2000,4:1005-1013.
[31]中华传染病寄生虫学分会,肝脏病学分会,病毒性肝炎防治方案[J].中华肝脏病学杂志,2000,8(6)：324-329.
[32]Horie I, Abiru N, Nagayama Y, et al.T Helper type 17 immune response plays an indispensable role for development of Iodine-Induced autoimmunethyroid-itis in Nonobese Diabetic-H2h4Mice[J]. Endocrinology,2009,150(11):5135-5142.
[33]Benghiat FS, Charbonnier L M, Vokaer B, et al. Interleukin 17-producing T helper cells in alloimmunity[J]. Permissions& Reprints,2009,23(1):11-18.
[34]Chen Z, Tato CM, Muul L, et al. Distinct regulation of IL-17 in human helper T lymphocytes[J].Arthritis Rheum,2007,56(12):2936-2946.
[35]Hsu H C, Yang P, Wang J, et al. Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice[J].Nat Immunol,2008,9(2):166-175.
[36]Kawai T, Hosoya N, Moritoki Y, et al. Autoantibody IgG subclasses torecombi-nant antigens and the role of bacterial stimuli in primary biliary cirrhosis [J]. Hepatology Research,2009,39(9):874-881.
[37]Livak KJ, Schmittgen TD. Analysis of relative gene expression data Using real-time quantitative PCR and the 2-Δ ΔCt method[J]. Methods,2001,25(4): 402-408.
[38]Giulietti A, Overbergh L, Valckx D, et al. An overview of real-time quanti-tative PCR:applications to quantify cytokine gene expression [J]. Methods,2001 ,25(4):386-401.
[39]Seiderer J, Elben I, Diegelmann J, et al.Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease(IBD):upregulated colonicIL-17Fexpres-sion in active Crohn'sdisease and analysis of theIL-17F polymorphism in IBD[J].Inflamm Bowel Dis 2008,14 (4) 437-445.
[40]Stockinger B, Veldhoen M. Differentiation and function of Th17 T cells [J]. Current Opinion in Immunology,2007,19 (5):281-286.
[41]Nizri E, Irony-Tur-Sinai M, Lory 0, et al. Activation of the Cholinergic Anti-Inflammatory System by Nicotine Attenuates Neuroinflammation via Suppr-ession of Thl and Th17 Responses[J]. Immunology,2009,18 (10):6681-6688.
[42]Furuzawa-CarballedaJ, Vargas-Rojas M I, Cabral A R. Autoimmune inflam-mation from the Th17 perspective[J]. Autoimmunity Reviews,2007,6 (1):169-175.
[43]沉宏辉,赵景民,辛绍杰等.慢性乙型肝炎患者血清生化指标对肝组织炎症程度的评估价值.中华肝脏病杂志,2009,17：12-15.
[44]Ruth Y, Cheng CM, Lian ZX, et al. Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis[J]. Liver Failure and Liver Disease,2006,43(4):729-737.
[1]Park H, Li Z, Yang XO, et al a distinct lineage of CD4+T cell regulate tissue Inflammation by producing interleuki-17[J]. Nat zmmunol,2005,6(11): 1133-1141.
[2]Pene J, Chevalier S, Preisser L, et al.Chronically inflamed human tissue-sare infiltrated by high differentiated Th17 lymphocytes [J]. Immunol,2008,18 (11):7423-7430.
[3]Wang C, Kang SG,LeeJ, et al.The role of CCR6 in migration of Th17 cells and regulation of Effector T cell balance in the gut [J]. Nature,2009,10 (2):173-183.
[4]Nair RP, Ruether A, Stuart PE, et al. Polymorphisms of the IL-12 β and IL-23 R Genes Are Associated with Psoriasis[J].Invest Dermatol,2008,128(7):165 3-1661.
[5]Xin L, Li Y, Soong L. Role of interleukin-1 in activating the CDllchigh CD45RB-dendriticcell subset and priming Leishmania amazonensis-specific CD4+Tcell in vitro and in vivo[J].Infect Immun,2007,75(10):5018-5026.
[6]Bettelli E, Korn T, Oukka M et al.Inductin and effector functions of Th17 cells[J]. Nature2008,453(19):1051-1057.
[7]Korn T, Bettelli E, Gao W, et al. IL-21 initiates an alternative pathway to induce proinflammatory Th17 cells. Nature,2007,448(26):484-487.
[8]Yang X0, Pappu BP, Nurieva R,et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity,2008, 28 (10):29-39.
[9]Hayashia K, Altman A. Protein kinase C theta (PKC-θ):A key player in T cell life and death[J]. Pharmacol Res,2007,55 (6):537-544.
[10]Elias KM, Laurence A, Davidson TS, et al. Retinoic acid inhibits Th17 polari-zation andenhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway [J].Blood,2008,111 (3):1013-1020
[11]CHabaud M, Garnero P, Dayer JM, et al. Contribution of interleukin 17 to synovium matrix destruction inrheumatoid arthritis[J]. Cytokin,2000,12(7): 1092-1099.
[12]Shahrara S, Huang QQ, Mandelin AM, et al.Thl7 cells in rheumatoid arthri-tis [J]. Arthritis Research& Therapy,2008,10(4):1299-1305.
[13]Semik-Orzech A, Barczyk A, Pienchala W, et al.The role of in-terleukin 17A in inducing neutrophilic inflammation in the pulmonary tract[J]. PolMerkur Lekarski,2007,22(129):163-168.
[14]Atrey R, Neurath MF. New therapeutic strategies for treatment of inflamm-atory bowel disease[J]. Nature,2008,1 (3):175-182.
[15]李家文、陈旭娥、刘志香等.寻常型银屑病皮损中Th17细胞相关因子的表达[J].中国麻风病杂志,2008,24(3)：176-178.
[2]Kumagi T, Onji M. Presentation and diagnosis of primary biliary cirrh osis in the 21st century. Clin Liver Dis,2008,12(5):243-259.
[3]Prince M, Amanda C, Newman W,et al. Survival and symptom progression in geographically based cohort of patients with primary biliary cirrhosis:a follow-up for up to 28 years[J]. Gastroenterology,2002,123 (9):1044-1051.
[4]David E,Jones J. Pathogenesis of primary biliary cirrhosis[J]. Journal of Hepatology,2003,39(9):639-648.
[5]Gershwin ME, Mackay IR. The causes of primary biliary cirrhosis:Convenient and inconvenient truths[J].Hepatology,2008,47(2):737-745.
[6]Selmi C, Mayo MJ, Bach N, et al. Primary biliary cirrhosis in monozygotic and dizygotic twins:genetics, epigenetics, and environment[J]. Gastroenter-ology,2004,127 (17):485-492.
[7]Kita H, Imawari M, Gershwin ME. Cellular immune response in primary biliary cirrhosis[J].HepatolRes,2004,28 (10):12-17.
[8]Rolland E, Dickson MD, Patricia M, et al.Grambsch Prognosis in primary biliary cirrhosis:Model for decision making[J]. Hepatology,2005,10(1):1-7.
[9]Ian Gan S, de Jongh M, Kaplan MM. Modafinil in the treatment of debilitating fatigue in Primary biliary cirrhosis:a clinincal experience[J]. Dig Dis Sci, 2009,54 (10):2242-2246.
[10]Wang C, Kang SG, Lee J, et al.The role of CCR6 in migration of Th17 cells and regulation of effector T cell balance in the gut[J]. Nature,2009,10 (1) 173-183.
[11]Caspar 0, Andrea P, Susan B, et al. Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells and results in spontaneous fatal autoimmunity[J]. Exp Med,2009,206:549-559.
[12]Golovanova EV,Il'chenko LIu, Tsaregorodtseva TM, et al. Cytokines in primary biliary cirrhosis (diagnostic and prognostic value)[J]. Ter Arkh, 2004,76(2):8-11
[13]Nagano T, Yamamoto K, Matsumoto S, et al.Cytokine profile in the liver of primary biliary cirrhosis[J].J Clin Immunol,1999,19(6):422-427.
[14]Park H, Li ZX, Yang X0, et al. A distinct lineage of CD4+T cell regulate tissue inflammation by producing interleukin 17[J]. Nat Zmmunol,2005,11 (6):1133-1141.
[15]Tesmer L, Lundy S, Sarkar S, et al. Th17 cells in human disease [J]. Immunolo-gical reviews,2008,223 (8):87-113.
[16]AshinoS, Wakita D, Shiohama Y, et al. A Th17-polarized cell population that has infiltrated the lung requires cells that convert to IFN-γ production in order to induce airway hyperresponsiveness [J]. Internatio-nal Immunology, 2010,22 (6):503-513.
[17]邓日辉,陈曲波。自身免疫性疾病中Thl7细胞效应因子的生物学功能研究进展[J].广东医学,2009,30(6)：993-995.
[18]Veldhoen M, Hocking R J, Atkins C J, et al.TGF beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17 produc-ing T cells [J]. Immunity,2006,24(2):179-189.
[19]Bettelli E, Vi jay K, Kuchroo H, et al. Thl, Th17, andTh9 effector cells induce Experimental autoimmune encephalomyelitis with different pathological Phen-otypes[J]. Immunol,2009,183(11):7169-7177.
[20]Zheng XF, Cintia S, Paiva D, et al. Desiccating Stress Promotion of Th17 differentiation by ocular surface tissues through a dendritic cell-mediated pathway[J]. Journal of vision,2010,51(2):3083-3091.
[21]Bettelli E, Korn T, Oukka M, et al.Inductin and effector functions of Th17 cells[J]. Nature,2008,19(5):1051-1057.
[22]Boniface K, Bak-Jensen KS, Li Y, et al. Prostaglandin E2 regulates Th17 cell differentiation and function through cyclic AMP and EP2/EP4 recept or signaling[J]. J Exp Med 2009,206(3):535-548.
[23]Su X, Ye J, Hsueh EC, et al. Tumor microenvironments direct the recruitment and expansion of human Th17 cells[J]. Immunol,2010,184(3):1630-1641.
[24]Komiyama Y, Nakae S, Matsuki T, et al. IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis[J]. Immunol,2006, 177(2):566-573.
[25]Ruth Y, Salunga TL, Tsuneyama K, et al. Hepatic IL-17 responses in human And murine primary biliary cirrhosis[J]. Journal of Autoimmunity,2009,32(1) :43-51.
[26]Joshi S, Cauch-Dudek K, Wanless IR, et al. Primary biliary cirrhosis with additional features of autoimmune hepatitis:response to therapy with ursode-oxycholic acid[J]. Hepatology,2002,35(6):409-413.
[27]Fukushima K, Ueno Y, Shimosegawa T. Treatment of Primary biliary cirrhosis: A new challenge?[J].HepatolRes,2010,40(1):61-68.
[28]Lindor KD, Gershwin ME, Poupon R, et al. Primary biliary cirrhosis[J]. Hepatology,2009,50(1):291-308.
[29]European Association for the study of the Liver, EAST Clinical pratice Guidelines:management of cholestatic liver diseases. J Hepatol,2009,51:237-267.
[30]Heathcote EJ, Management of primary biliary cirrhosis.The American Association for the Study of Liver Diseases practice guidelines. Hepatol-ogy,2000,4:1005-1013.
[31]中华传染病寄生虫学分会,肝脏病学分会,病毒性肝炎防治方案[J].中华肝脏病学杂志,2000,8(6)：324-329.
[32]Horie I, Abiru N, Nagayama Y, et al.T Helper type 17 immune response plays an indispensable role for development of Iodine-Induced autoimmunethyroid-itis in Nonobese Diabetic-H2h4Mice[J]. Endocrinology,2009,150(11):5135-5142.
[33]Benghiat FS, Charbonnier L M, Vokaer B, et al. Interleukin 17-producing T helper cells in alloimmunity[J]. Permissions& Reprints,2009,23(1):11-18.
[34]Chen Z, Tato CM, Muul L, et al. Distinct regulation of IL-17 in human helper T lymphocytes[J].Arthritis Rheum,2007,56(12):2936-2946.
[35]Hsu H C, Yang P, Wang J, et al. Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice[J].Nat Immunol,2008,9(2):166-175.
[36]Kawai T, Hosoya N, Moritoki Y, et al. Autoantibody IgG subclasses torecombi-nant antigens and the role of bacterial stimuli in primary biliary cirrhosis [J]. Hepatology Research,2009,39(9):874-881.
[37]Livak KJ, Schmittgen TD. Analysis of relative gene expression data Using real-time quantitative PCR and the 2-Δ ΔCt method[J]. Methods,2001,25(4): 402-408.
[38]Giulietti A, Overbergh L, Valckx D, et al. An overview of real-time quanti-tative PCR:applications to quantify cytokine gene expression [J]. Methods,2001 ,25(4):386-401.
[39]Seiderer J, Elben I, Diegelmann J, et al.Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease(IBD):upregulated colonicIL-17Fexpres-sion in active Crohn'sdisease and analysis of theIL-17F polymorphism in IBD[J].Inflamm Bowel Dis 2008,14 (4) 437-445.
[40]Stockinger B, Veldhoen M. Differentiation and function of Th17 T cells [J]. Current Opinion in Immunology,2007,19 (5):281-286.
[41]Nizri E, Irony-Tur-Sinai M, Lory 0, et al. Activation of the Cholinergic Anti-Inflammatory System by Nicotine Attenuates Neuroinflammation via Suppr-ession of Thl and Th17 Responses[J]. Immunology,2009,18 (10):6681-6688.
[42]Furuzawa-CarballedaJ, Vargas-Rojas M I, Cabral A R. Autoimmune inflam-mation from the Th17 perspective[J]. Autoimmunity Reviews,2007,6 (1):169-175.
[43]沉宏辉,赵景民,辛绍杰等.慢性乙型肝炎患者血清生化指标对肝组织炎症程度的评估价值.中华肝脏病杂志,2009,17：12-15.
[44]Ruth Y, Cheng CM, Lian ZX, et al. Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis[J]. Liver Failure and Liver Disease,2006,43(4):729-737.
[1]Park H, Li Z, Yang XO, et al a distinct lineage of CD4+T cell regulate tissue Inflammation by producing interleuki-17[J]. Nat zmmunol,2005,6(11): 1133-1141.
[2]Pene J, Chevalier S, Preisser L, et al.Chronically inflamed human tissue-sare infiltrated by high differentiated Th17 lymphocytes [J]. Immunol,2008,18 (11):7423-7430.
[3]Wang C, Kang SG,LeeJ, et al.The role of CCR6 in migration of Th17 cells and regulation of Effector T cell balance in the gut [J]. Nature,2009,10 (2):173-183.
[4]Nair RP, Ruether A, Stuart PE, et al. Polymorphisms of the IL-12 β and IL-23 R Genes Are Associated with Psoriasis[J].Invest Dermatol,2008,128(7):165 3-1661.
[5]Xin L, Li Y, Soong L. Role of interleukin-1 in activating the CDllchigh CD45RB-dendriticcell subset and priming Leishmania amazonensis-specific CD4+Tcell in vitro and in vivo[J].Infect Immun,2007,75(10):5018-5026.
[6]Bettelli E, Korn T, Oukka M et al.Inductin and effector functions of Th17 cells[J]. Nature2008,453(19):1051-1057.
[7]Korn T, Bettelli E, Gao W, et al. IL-21 initiates an alternative pathway to induce proinflammatory Th17 cells. Nature,2007,448(26):484-487.
[8]Yang X0, Pappu BP, Nurieva R,et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity,2008, 28 (10):29-39.
[9]Hayashia K, Altman A. Protein kinase C theta (PKC-θ):A key player in T cell life and death[J]. Pharmacol Res,2007,55 (6):537-544.
[10]Elias KM, Laurence A, Davidson TS, et al. Retinoic acid inhibits Th17 polari-zation andenhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway [J].Blood,2008,111 (3):1013-1020
[11]CHabaud M, Garnero P, Dayer JM, et al. Contribution of interleukin 17 to synovium matrix destruction inrheumatoid arthritis[J]. Cytokin,2000,12(7): 1092-1099.
[12]Shahrara S, Huang QQ, Mandelin AM, et al.Thl7 cells in rheumatoid arthri-tis [J]. Arthritis Research& Therapy,2008,10(4):1299-1305.
[13]Semik-Orzech A, Barczyk A, Pienchala W, et al.The role of in-terleukin 17A in inducing neutrophilic inflammation in the pulmonary tract[J]. PolMerkur Lekarski,2007,22(129):163-168.
[14]Atrey R, Neurath MF. New therapeutic strategies for treatment of inflamm-atory bowel disease[J]. Nature,2008,1 (3):175-182.
[15]李家文、陈旭娥、刘志香等.寻常型银屑病皮损中Th17细胞相关因子的表达[J].中国麻风病杂志,2008,24(3)：176-178.