【作者】 孟繁立；

【导师】 王凯；

【作者基本信息】 山东大学 ， 内科学， 2011， 博士

【摘要】 第一部分：I型干扰素受体和IL-17及其受体在慢性乙型病毒性肝炎患者中的变化研究背景：干扰素-alpha (Interferon-alpha IFN-α)是治疗慢性乙型病毒性肝炎(chronic hepatitis B CHB)患者的一线抗病毒药物,I型干扰素受体(type I interferon-α/βreceptor, IFNAR)在干扰素α的起效作用过程中至关重要,我们已经研究发现辅助性T17细胞(T helper 17 cells Th17)可能参与CHB的发病机制。然而慢性乙型病毒性肝炎患者体内IFNAR的变化以及其在干扰素α2b治疗疗效评价中的作用尚不清楚,而且IFNAR与Th17分泌的白介素17(interleukin-17 IL-17)之间的关系有待于进一步研究。研究目的：本研究检测了慢性乙肝患者体内外周血单个核细胞(peripheral blood mononuclear cells PBMCs)以及肝组织内IFNAR以及IL-17及其受体的变化以揭示IFNAR其在应用干扰素α2b治疗疗效预测中的作用,同时探讨慢性乙型病毒性肝炎患者IFNAR与IL-17的关系。研究方法：共入选84例慢性乙型病毒性肝炎患者以及10例健康志愿者,其中25例接受了肝穿刺活检术。84例慢性乙肝患者中有25例符合抗病毒治疗的患者接受至少3个月的干扰素α2b治疗。分离PBMCs,淋巴细胞以及单核细胞内IFN-α/βR-1和IFN-α/βR-2的表达用流式细胞仪进行测定,提取PBMCs中的RNA, IFNAR1, IFNAR2, IL-6, IL-8,IL-17A, IL-17F, IL-17RA, IL-17RC, TGF-β1和TNF-α的mRNA水平用SYBR Green RT-PCR进行监测。肝组织中IFNAR-1和IFNAR-2用免疫荧光以及免疫组化染色进行观察。提取肝组织RNA,RT-PCR检测肝组织内IFNAR1,IFNAR2,IL-6,IL-8,IL-17A,IL-17F, IL-17RA, IL-17RC, TGF-β1和TNF-α的mRNA水平。在应用干扰素a2b治疗的患者中,每一个月监测外周血淋巴细胞以及单核细胞内IFN-α/βR-1和IFN-α/βR-2的表达以及外周血单个核细胞内IFNAR1, IFNAR2, IL-17A, IL-17F, IL-17RA和IL-17RC的mRNA变化。研究结果：慢性乙肝患者外周血单核细胞和淋巴细胞中IFN-α/βR-1和IFN-α/βR-2的表达升高,肝组织内IFNAR-1和IFNAR-2的水平也明显升高,并与与肝组织HBV-DNA正相关。在应用干扰素α2b治疗的慢性乙肝患者中,单核细胞和淋巴细胞内IFN-α/βR-1和IFN-α/βR-2的表达在第一个月明显升高,而在随后的两个月治疗中逐步降低。在治疗应答组(12/25)单核细胞和淋巴细胞中IFN-α/βR-1和IFN-α/βR-2的表达明显高于非应答组(13/25)。治疗前单核细胞内IFNα/βR-2表达较高的患者其干扰素应答率明显高于较低的患者。另外慢性乙型病毒性肝炎患者肝组织和PBMC中IFNAR,IL-17A, IL-17F,IL-17RA,IL-17RC,IL-6,IL-8,TGF-β1和TNF-α的表达明显升高,IL-17及其受体的表达与血清转氨酶的水平呈正相关,并且与炎症细胞因子IL-6,IL-8, TGF-β1和TNF-α的水平呈正相关。PBMCs中IFN-α/βR1和IFN-α/βR2的表达与IL-17及其受体呈负相关,并且随着干扰素的应用,IL-17及其受体的水平明显降低。结论：慢性乙型病毒性肝炎外周血单个核细胞以及肝组织内IFN-α/βR-1和IFN-α/βR-2的表达升高。在干扰素治疗有效组,其外周淋巴细胞和单核细胞IFNAR的表达均高于无效组,提示IFNAR在外周血单个核细胞的表达影响干扰素的疗效；干扰素治疗前,外周血单核细胞表面IFNAR-2的表达可以作为预测干扰素治疗慢性乙型病毒性肝炎疗效的预测指标。第二部分：IL-17及其信号系统参与肝纤维化的形成研究背景：研究发现产生IL-17的Thl7细胞与慢性炎症反应以及自身免疫性疾病广泛相关。而且我们发现慢性乙型病毒性肝炎患者外周血单个核细胞以及肝组织内IL-17及其受体的水平明显升高。提示Th17可能参与慢性肝病的发病机制。近来有研究发现IL-17可以导致肺间质纤维化,为我们进一步探讨IL-17在肝纤维化发病中的作用提供了依据。研究目标：确定IL-17在肝纤维化发病机制中的作用,包括IL-17对肝脏枯否氏细胞以及肝星状细胞的激活作用,以及其在肝脏原代细胞中的信号转导。研究方法：应用IL-17A和IL-17F刺激人LX-2和hTERT (human telomerase reverse transcriptase)星状细胞株,分析IL-17对星状细胞株的激活作用以及其在星状细胞株中的信号传导。星状细胞α-SMA, collagenα1(Ⅰ), TGF-β1和TIMP1mRNA的表达用RT-PCR方法检测,IL-17诱导的ERK1/2(extracellular regulated proteinkinases ERK), AKT (serine Threonine Kinase)和NF-κB (nuclear factorκB)的磷酸化用Western blot方法检测,NF-κB在星状细胞内的细胞核内移位用细胞免疫荧光染色观察。分离原代肝脏枯否氏细胞、星状细胞和肝细胞,分别应用IL-17A, IL-17F以及IL-17A+IL-17F进行刺激,IL-17对肝脏原代肝脏枯否氏细胞、星状细胞和肝细胞的作用用RT-PCR, Western blot以及细胞免疫荧光的方法进行检测,IL-17A依赖的胶原蛋白激活的机制进一步由体外实验野生型以及IL-17R敲除型collagen-GFP小鼠原代星状细胞中加以证实。我们利用纯合子STAT3 floxed等位基因(homozygous for STAT3 floxed allele STAT3f1/f1)小鼠与(胶质细胞原纤维酸性蛋白-cre Glial fibrillary acidic protein GFAP-cre)小鼠进行杂交,获得定靶于星状细胞敲除STAT3的小鼠,分离小鼠的星状细胞,应用IL-17A进行刺激,观察星状细胞的活化情况,应用STAT3f1/f1小鼠作为对照。从而确定IL-17A对星状细胞的激活是否依赖于STAT3的激活。野生型(WT)以及IL-17RA敲除型小鼠用胆总管结扎(bile duct ligationBDL)和四氯化碳(carbon tetrachloride CCl4)腹腔内注射诱导肝纤维化,通过天狼腥红(Sirius Red)染色观察肝组织病理,分离BDL模型组以及对照组肝脏内实质性细胞(non-parenchymal cells NPCs)。用流式细胞仪检测NPCs各细胞群中IL-17A的表达。α-SMA免疫组化染色评估肝纤维化的程度,提取肝组织RNA, RT-PCR评估肝组织内MMP3,TIMP, TNF-α, TGF-β1, IL-6, IL-1-β的表达。提取肝组织蛋白质Western-blot检测肝组织蛋白中α-SMA的表达。研究结果：IL-17A在体外实验中可以激活枯否氏细胞(表达IL-17A, IL-17F, TGF-β1, IL-6 and IL-1-β)以及肝脏星状细胞(表达α-SMA, collagenα1 (I), TGF-β1, and TIMP1),表明IL-17A可以独立激活枯否氏细胞以及产胶原细胞。IL-17A激活星状细胞的信号通路包括STAT3, ERK1/2, AKT, NF-κB的磷酸化,其中STAT3的磷酸化是IL-17A激活星状细胞的首要信号通路,定靶于星状细胞敲除STAT3后,IL-17A激活星状细胞的功能被明显阻断。进一步研究发现清除IL-17A信号通路(在IL-17RA敲除型小鼠)与野生型相比可以明显减轻BDL (65±7%)以及CCl4(45±8%)诱导产生的肝纤维化。BDL诱导的肝纤维化模型中IL-17A主要在炎症细胞中明显升高(CD4+, CD8+ T cells>Kupffer cells)。IL-17A的作用与星状细胞内I型胶原蛋白的表达增高相关,结论：IL-17在肝纤维化的发病机制中有重要的作用,IL-17A可以明显激活肝脏枯否氏细胞和星状细胞,IL-17A激活星状细胞主要依赖于STAT3的磷酸化。第三部分：枯否氏细胞IL-17A/IL-17RA信号通路在Th17导致肝纤维化的发病机制中至关重要研究背景：枯否氏细胞被认为是参与肝内炎症反应的首要靶细胞。在肝脏受损伤后,枯否氏细胞可以激活并释放一系列验证前细胞因子以及活性氧介质,可以激活星状细胞,从而导致肝纤维化。我们研究发现IL-17可以激活枯否氏细胞和星状细胞,敲除IL-17RA可以明显减轻由BDL和CCl4诱导的肝纤维化。同时发现IL-17在体外激活枯否氏细胞的作用明显强于星状细胞。为此我们应用体外枯否氏细胞与星状细胞联合培养,体内进行IL-17RA和WT小鼠之间的骨髓移植(bone marrow transplantation BMT),试图揭示IL-17导致肝纤维化的机制。研究目标：确定IL-17导致肝纤维化的发病机制,探讨枯否氏细胞和星状细胞内IL-17及其信号通路在肝纤维化发病机制中的地位。研究方法：体外实验中我们分离WT和IL-17RA小鼠原代枯否氏细胞,同时分离WT-collage-GFP和IL-17R-/--collagen-GFP小鼠原代星状细胞,联合培养后应用IL-17A进行刺激24小时,观察星状细胞表达胶原蛋白-GFP的阳性率。体内试验我们采用了骨髓移植的实验方法,受体小鼠经氯膦酸盐脂质体(Clodronate-Liposomes)静脉注射清除单核-巨噬细胞系统和放射性照射清除淋巴细胞之后完成IL-17A-/-,IL-17RA-/-与WT小鼠之间的骨髓移植,共获得5组嵌合体小鼠,分别为：WT移植入WT组(枯否氏细胞为野生型,HSCs为野生型)；IL-17A-/-移植入WT组(枯否氏细胞无IL-17A; HSCs为野生型)；WT移植入IL-17RA-/-组(枯否氏细胞为野生型,HSCs无IL-17RA);IL-17RA-/-移植入WT组(枯否氏细胞无IL-17RA, HSCs为野生型)以及IL-17RA-/-移植入IL-17RA-/-组(枯否氏细胞和HSCs均无IL-17RA)。2个月后嵌合体小鼠行胆管结扎构建肝纤维化模型,通过天狼腥红染色(Sirius Red),α-SMA免疫组化染色以及RT-PCR评估肝纤维化。研究结果：WT枯否氏细胞与IL-17RA-/--collagen-GFP星状细胞联合培养组经IL-17A刺激24小时后,其星状细胞表达collagen-GFP的阳性率明显高于IL-17RA-/-枯否氏细胞与WT-collagen-GFP星状细胞联合培养组(34±11%比25±7%)。而在骨髓移植实验中,清除骨髓来源的IL-17A/IL-17RA信号通路系统(IL-17RA-/-移植入WT或者IL-17A移植入WT)组,可以分别减轻肝纤维化55±9%和50±12%。但是清除肝脏内生性细胞(肝细胞和星状细胞)IL-17A/IL-17RA信号通路组(WT移植入IL-17RA-/-),其肝纤维化程度仍有所减轻,但不是很明显(25±7%)。结论：枯否氏细胞内IL-17A/IL-17RA信号通路对于诱导肝纤维化至关重要更多还原

【Abstract】 PartⅠ:Alteration of Interferon-α/βReceptors and Relationship with Interleukin-17 in Chronic Hepatitis B PatientsBackground:Interferon-alpha (IFN-α) is among the first line of anti-virus drugs applied in the treatment of chronic hepatitis B (CHB) patients. TypeⅠinterferon receptor (interferon-a/p receptor, IFNAR) plays a key role in the effective action of interferon-alpha. We have found out that T helper 17 cells (Th17) may be involved in the pathogenesis of CHB. The predicable role of IFNAR during IFN-a treatment and its relationship with interleukin-17 (IL-17) secreted by Th17 cells in patients with chronic hepatitis B have not been intensively studied. Aims:The present study determined type I interferon receptor (interferon-α/βreceptor, IFNAR) and its predicable role in interferonα2b treatment in chronic hepatitis B (CHB) patients. At the same time to explore the relationship between IFNAR and IL-17 in chronic hepatitis B patients.Methods:84 CHB patients were enrolled,25 accepted liver biopsy and 25 were treated with interferonα2b for at least three months. The expression of IFN-α/βR-1 and IFN-α/βR-2 in lymphocytes and monocytes was measured by flow cytometry. IFNAR1, IFNAR2, IL-17A, IL-17F, IL-17RA, IL-17RC, IL-6, IL-8, TGF-β1 and TNF-αmRNA from peripheral blood mononuclear cells (PBMCs) and liver tissue were quantified using SYBR Green RT-PCR. Immunofluorescence double staining with IFN-α/βR-1 and IFN-α/βR-2 antibodies was performed, and immunohistochemistry was measured using IFN-α/βR-2. During three-month application of IFN-α2b, monitored the alteration of IFN-α/βR-1 and IFN-α/βR-2 in PBMCs by flow cytometry every month. Changes of IL-17 and IL-17 receptors were measured in PBMCs in the same time.Results:Our results found that IFN-α/βR-1,2 in monocytes and lymphocytes increased in CHB patients. Expression of IFNAR-1 and IFNAR-2 in liver was also increased and had positive correlation with HBV-DNA in liver tissue. Expression of IFN-α/βR-1,2 in lymphocytes and monocytes increased in the first month, but then decreased during the subsequent interferon-α2b treatment, patients who had higher levels of IFN-a/(3R-2 in monocytes prior to therapy showed better viral response than those with lower levels. mRNA expression of IFNAR, IL-17, IL-17R, IL-6, IL-8, TGF-β1 and TNF-a significantly increased in PBMCs and liver tissues of all chronic HBV patients, while the expression of IFN-α/βR1, IFN-α/βR2 had a negative correlation with IL-17 and IL-17 receptors in PBMCs. mRNA levels of IL-17 cytokines and receptors simultaneously decreased after IFN-α2b administration.Conclusions:Expression of IFN-α/βR-2 in monocytes can be used as a predictable parameter to evaluate the effect of IFN-a treatment in CHB patients. IL-17 and IL-17 receptors were increased in CHB patients, and they had negative correlation with IFNAR. PartⅡ:Interleukin-17 and its cell signal pathway contribute to liver fibrosisBackgrounds:Interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) have been broadly linked to chronic inflammation and human autoimmunity. We have found out that IL-17 and IL-17 receptors are increased both in PBMCs and in liver tissue of CHB patients, and are down-regulated by application of IFN-α2b. TGF-β1 and IL-6 who were involved in the induction of Th17 differentiation are both up-regulated during development of liver fibrosis. And most recently, it is reported that IL-17 contributed to the formation of lung fibrosis. So we set up this project to explore the role of IL-17 in liver fibrosis.Aims:To determine the role of IL-17A in pathogenesis of liver fibrosis, including the activation of kupffer cells and hepatic stellate cells (HSCs).Methods:Effect of IL-17 on hLX-2 and hTERT hepatic stellate cell lines was determined by RT-PCR, Western blot, fluorescent microscopy. The effect of IL-17A on liver resident cells was studied in primary cultures of mouse KC, HSCs and hepatocytes by RT-PCR, Western blot, fluorescent microscopy, and flow cytometry. We generated IL-17RA-/--collagen-GFP mice, isolated primary quiescent HSCs stimulated with IL-17A to evaluated the GFP positive cells compared with WT-collagen-GFP HSCs. We took advantage of STAT3fl/fl and GFAP-cre mice, so we got mice without STAT3 focus on hepatic stellate cells. After isolation of quiescent HSCS, we stimulated the HSCs with IL-17A to compare the expression of collagenα1(Ⅰ) with STAT3fl/fl HSCs. The development of liver fibrosis in response to bile duct ligation (BDL) or carbon tetrachloride (CCl4) was compared in wild type mice and IL-17RA-/-mice, devoid of the IL-17A receptor. Liver fibrosis was assessed by Sirius Red staining, a-SMA staining, and RT-PCR.Results:IL-17A in vitro stimulated Kupffer cells (to express IL-17A, TGF-β1, IL-6 and IL-1) and HSCs (to expressα-SMA, collagenα1(1), TGF-β1, and TIMP1), suggesting that IL-17A independently contributes to the activation of inflammatory and collagen producing cells. In support of this hypothesis, abrogation of IL-17A signaling in IL-17RA-/- mice attenuated development of BDL-(by 65±7%) or CCl4-(by 45±8%) induced liver fibrosis in comparison with wild type mice. IL-17A and IL-17F was mainly increased in inflammatory cells (CD4+,CD8+ T cells> Kupffer cells) of BDL mice. The activation of IL-17A on hepatic stellate cells depends on the phosphorylation of STAT3, ERK1/2, AKT and NF-κB. This effect of IL-17A correlated with increased expression of the collagenα1(Ⅰ) gene. Phosphorylation of Stat3 in HSCs is the primary cell signal pathway during the activation of IL-17A.Conclusion:IL-17 plays as an important role in the pathogenesis of liver fibrosis. IL-17 can significantly activate kupffer cells and HSCs. IL-17A has a direct role on the activation of HSCs via the activation of STAT3’s phosphorylation. PARTⅢIL-17A/IL-17RA signal pathway in kupffer cells is critical in the pathogenesis of Th17 involved in liver fibrosisBackgrounds:Kupffer cells are thought to be the key target cells involved in the chronic inflammatory reaction in liver. After liver injury, kupffer cells can be activated to produce a series of pro-inflammatory cytokines and reactive oxygen species, so as to induce liver fibrosis. We have found out that IL-17 can induce the activation of kupffer cells and HSCs, and knockout IL-17RA can amerolate liver fibrosis induced by BDL and CCl4 injection in mice. Most surprisingly, the activation effect of kupffer cells by IL-17A was more significantly than HSCs. So we accomplished co-culture of kupffer cells and HSCs in vitro and applied bone marrow transplantation between IL-17A-/-, IL-17RA-/- and WT mice in vivo, to explore the mechanism of IL-17 in the pathogenesis of liver fibrosis.Aims:To determine the role of IL-17 and its cell signaling transduction in kupffer cells and HSCs in pathogenesis of liver fibrosis, and focus on the effect of kupffer cells in the IL-17’s fibrosis induction effect.Methods:After the isolation of WT and IL-17RA-/- kupffer cells, we co-cultured them with quiescent WT-collage-GFP and IL-17R-/--collagen-GFP HSCs. After 24-hour stimulation with IL-17A, percentage of GFP positive cells was calculated under fluorescent microscopy.After clodronate-liposome injection to deplete mononuclear phagocyte system and irradiation to deplete lymphocytes, we accomplished bone-marrow transplantation (BMT) using IL-17A-/-, IL-17RA-/- and WT mice. Total five groups of chimera mice were generated, WT into WT group (kupffer cells and HSCs both have IL-17RA); IL-17A-/- into WT group (with no IL-17A, kupffer cells and HSCs both have IL-17RA); IL-17RA-/- into WT group (kupffer cells with no IL-17RA, HSCs have IL-17RA); WT into IL-17RA-/- group (kupffer cells have IL-17RA, HSCs with no IL-17RA) and IL-17RA-/- into IL-17RA-/- group (both kupffer cells and HSCs have no IL-17RA). Two months after BMT, we applied BDL to induce liver firosis on all the chimera mice. Liver fibrosis was assessed by Sirius Red staining, a-SMA staining, and RT-PCR.Results:After stimulated with IL-17A for 24 hours, the percentage of GFP positive HSCs in WT kupffer cells co-cultured with IL-17RA-/--collagen-GFP HSCs is much higher than in IL-17RA-/- kupffer cells co-cultured with WT HSCs (34±11% vs. 25±7%). In all the chimera mice after bone marrow transplantation, Disruption of IL-17A/IL-17RA signaling in the BM-derived cells of chimera mice (IL-17RA-/- into WT or IL-17A-/- into WT) resulted in reduction of fibrosis by 55±9% and 50±12%, respectively. However, disruption of IL-17A/IL-17RA signaling in endogenous liver cells including HSCs reduced liver fibrosis by 25±7% in chimera mice (WT into IL-17RA-/- mice).Conclusion:IL-17A/IL-17RA signaling pathway in kupffer cells has a critical role in development of liver fibrosis in mice.更多还原