【作者】 戚朝霞；

【导师】 王凯；

【作者基本信息】 山东大学 ， 内科学， 2012， 博士

【摘要】 慢性乙型肝炎发病机制十分复杂,宿主免疫调节紊乱是导致不能有效清除病毒、病情迁延不愈的重要原因。许多研究表明Th1、Th2细胞亚群的比例失衡、功能失调是导致慢性乙型肝炎发病的重要因素。Th17细胞是近年来发现的一种新的效应性CD4+T细胞亚群,以产生白介素17(IL-17)为特征。Th17细胞的分化需要IL-6存在条件下转化生长因子(TGF-β)的活化。维甲酸相关孤核受体γt(RORγt)作为维甲酸相关孤核受体亚家族的成员之一,属于类固醇受体超家族。大量研究表明RORγt和RORα是调节Th17细胞分化的重要因子,尤其RORγt发挥主要作用。大量证据表明Th17细胞亚群比例增加在慢性乙型肝炎的发生、发展中具有重要的作用,调节慢性乙型肝炎患者中Th17细胞的异常可能成为慢性乙型肝炎治疗的新策略。但是,对于RORγt和RORα在慢性乙型肝炎的发病中如何发挥作用尚很少有研究。目的本研究的目的是通过对慢性乙型肝炎患者外周血RORγt和RORα基因表达,Th17细胞亚群及血清细胞因子的检测,进一步探讨RORγt和RORα在慢性乙型肝炎发病机制中的作用及评价它们的临床相关性。方法应用三色流式细胞术检测慢性乙型肝炎患者和正常对照外周血中Th17细胞占CD3+T细胞的比例。应用逆转录聚合酶链反应(RT-PCR)方法检测]RORγt和RORα在慢性乙型肝炎患者和正常对照外周血单个核细胞(PBMC)中的表达。应用ELISA技术检测慢性乙型肝炎患者和正常对照血清中IL-6,TGF-β,IL-17,IL-23和IFN-γ的表达水平。结果1、慢性乙型肝炎患者Th17细胞亚群的比例(1.77±0.52%)较正常对照组(0.33±0.15%)显著升高(P<0.05)。2、慢性乙型肝炎患者外周血RORα的mRNA水平(6.442×10-2±3.17×10-2)明显高于正常对照组(1.666×10-3±8.77×104,P<0.05),慢性乙型肝炎患者外周血RORyt的mRNA水平(1.217×10-3±0.787x10-3)明显高于正常对照组(3.207×10-4±1.38×10-4,P<0.05)。3、慢性乙型肝炎患者血清IL-6水平(13.36±3.79pg/ml)较正常对照组明显升高(3.43±0.55pg/ml,P<0.01)；血清TGF-(3水平(35.22±2.4ng/ml)较正常对照组明显升高(18.28±1.8ng/ml,P<0.01);慢性乙型肝炎患者血清IFN-y水平(10.17±3.38pg/ml)较正常对照组明显降低(17.14±2.28pg/ml,P<0.05);慢性乙型肝炎患者血清IL-17和IL-23水平(65.38±11.65pg/ml,51.10±6.69pg/ml)较正常对照组(61.07±8.84pg/ml,45.87±4.47pg/ml)无明显差异(P≥0.05)。4、在慢性乙型肝炎患者中,RORγt.IL-6、IL-23与Th17细胞亚群比例存在明显的正相关性(r=0.563,P=0.01；r=0.403,P=0.027；r=0.447,P=0.013),但RORα、 TGF-β、IFN-γ与Th17细胞亚群比例无相关性。5、慢性乙型肝炎患者外周血中Th17细胞亚群比例与ALT呈明显正相关(r=0.587,P=0.005),RORγt与ALT呈明显正相关(r=0.384,P=0.036),但RORα与ALT无相关性(r=0.064,P=0.738)；RORγt、RORα和Thl7细胞亚群比例与外周血HBV-DNA均无相关性(r=0.031,P=0.871；r=0.315,P=0.090；r=0.222,P=0.239)。结论我们的研究结果进一步表明,在慢性乙型肝炎发生和发展中,Th17细胞亚群比例增加具有重要的作用；研究首次表明,作为Th17细胞分化的重要调节因子,RORγt在慢性乙型肝炎的发病中可能具有重要作用。HBV感染可引起人类急慢性肝炎,与肝硬化和肝细胞癌的发生和发展密切相关,其中每年约有1%的乙型肝炎患者发展为重型肝炎。重型肝炎可分为急性重型肝炎(acute severe type hepatitis、亚急性重型肝炎(subacute severe type hepatitis, SSH)、慢性重型肝炎(chronic severe type hepatitis,CSH)。其中慢性重型肝炎又可分为慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)和慢性肝衰竭(chronic liver failure,CLF)。慢加急性肝衰竭是在慢性肝病基础上出现的急性肝脏功能失代偿。在我国,慢性乙型病毒性肝炎是引起慢加急性肝衰竭的主要病因,由慢性乙型病毒性肝炎所致的慢加急性肝衰竭又称为慢加急性重型乙型病毒性肝炎(ACHBLF)。ACHBLF进展迅速,病情凶险,临床上缺乏特异、有效的治疗手段,绝大部分患者预后较差。ACHBLF的发病机制尚不完全明确。在我们的第一部分实验中,我们已经发现在慢性乙型肝炎异常免疫反应中,Th17细胞亚群比例增加具有重要的作用；研究首次表明,作为Th17细胞分化的重要调节因子,RORγt在慢性乙型肝炎的发病中可能具有重要作用。目的本研究的目的是通过对慢加急性重型乙型病毒性肝炎患者外周血RORγt和RORα基因表达,Th17细胞亚群及血清细胞因子的检测,进一步探讨RORγt和RORβ在慢加急性重型乙型病毒性肝炎患者发病机制中的作用。方法应用三色流式细胞术检测慢加急性重型乙型病毒性肝炎患者和正常对照外周血中Th17细胞占CD3+T细胞的比例。应用逆转录聚合酶链反应(RT-PCR)方法检测RORγt和RORα在慢加急性重型乙型病毒性肝炎患者和正常对照外周血单个核细胞(PBMC)中的表达。应用ELISA技术检测慢加急性重型乙型病毒性肝炎患者和正常对照血清中IL-6,TGF-β,IL-17,IL-23和IFN-γ的表达水平。结果1、慢加急性重型乙型病毒性肝炎患者外周血Th17细胞亚群比例(3.87±0.97%)明显高于慢性乙型肝炎患者(1.77±0.52%,P<0.01)和正常对照组(0.33±0.15%,P<0.01)。2、慢加急性重型乙型病毒性肝炎患者外周血RORα的mRNA水平(2.38×10-1±1.04×10-1)明显高于慢性乙型肝炎患者(6.442×10-2±3.17×10-2,P<0.05)和正常对照组(1.666×10-3±8.77×104,P<0.05),慢加急性重型乙型病毒性肝炎患者外周血RORγt的mRNA水平(2.904×10-2±1.01×10-2)明显高于慢性乙型肝炎患者(1.217×10-3±0.787×10-3,P<0.05)和正常对照组(3.207×10-4±1.38×10-4,P<0.01)。3、慢加急性重型乙型病毒性肝炎患者血清IL-6水平(44.56+16.55pg/ml)较慢性乙型肝炎患者(13.36±3.79pg/ml)和正常对照组明显升高(3.43±0.55pg/ml,P<0.01)；血清TGF-β水平(55.78±1.6ng/ml)较慢性乙型肝炎患者(35.22±2.4ng/ml,P<0.05)和正常对照组明显升高(18.28±1.8ng/ml,P<0.01);血清IFN-γ水平(13.61±5.6pg/ml)较慢性乙型肝炎患者(10.17±3.38pg/ml)明显升高(P<0.05),但较正常对照组明显降低(17.14±2.28pg/ml,P<0.05);血清IL-17和IL-23水平(77.24±9.69pg/ml,55.96±25.45pg/ml)较慢性乙型肝炎患者(65.38±11.65pg/ml,51.10±6.69pg/ml)和正常对照组(61.07±8.84pg/ml,45.87±4.47pg/ml)无明显差异(P≥0.05)。4、在慢加急性重型乙型病毒性肝炎患者中,RORγt、IL-6、IL-23与Th17细胞亚群比例存在明显的正相关性(r=0.321,P=0.044；r=0.360,P=0.027；r=0.383,P=0.015),但RORα、TGF-β、IFN-γ与Th17细胞亚群比例无相关性。5、慢加急性重型乙型病毒性肝炎患者RORγt与PTA, TBIL和MELD存在明显的正相关性(r=-0.393,P=0.012；r=0.391,P=0.013；r=0.359,P=0.023)；但是RORγt-与ALT和HBV-DNA无相关性(r=-0.122,P=0.458；r=-0.303,P=0.057)。RORα与上述各指标均无相关性。6、根据患者的结局,将慢加急性重型乙型病毒性肝炎患者分为生存组和死亡组,发现死亡组患者RORγt和RORα水平(4.48×10-2±1.3×10-2,4.78×10-1±9.19×10-2)高于生存组(3.34×10-2±1.5×10-,23.55×10-1±7.65×10-2),但无明显差异(P>0.05)。同样,外周血Thl7细胞亚群比例在死亡组(3.97±2.21%)高于生存组(2.25±±0.78%),但无明显差异(P>0.05)。RORγt、RORα水平和外周血Th17细胞亚群比例在HBeAg阳性和HBeAg阴性患者中无明显差异(P>0.05)。结论我们的研究结果进一步表明,在慢加急性重型乙型病毒性肝炎的异常免疫反应中,Th17细胞在发病中具有重要的作用；研究首次表明,RORγt和RORα在慢加急性重型乙型病毒性肝炎的发病过程中可能具有重要作用,RORγt-与疾病的严重程度密切相关,可能作为判断慢加急性重型乙型病毒性肝炎疾病预后情况的因子之一。原发性肝癌是我国常见恶性肿瘤之一。原发性肝癌约90%以上为肝细胞肝癌,5%左右为胆管细胞肝癌,两者混合的肝癌罕见。本病病因和发病机制尚未确定,我国肝癌患者中约90%有乙型肝炎病毒(HBV)感染背景。原发性肝癌死亡率高,我国每年死于肝癌的人数超过11万,在恶性肿瘤死亡顺位中仅次于胃癌、食道癌而居第三位。肝癌患者体内细胞免疫功能受损,抗肿瘤能力下降,可能是肝癌患者抗肿瘤免疫应答缺陷的机制之一。近年来,Th17在肿瘤发病机制的研究受到越来越多人的重视。有研究认为Th17促进肿瘤发展,也有研究认为Thl7可以清除肿瘤细胞。多项研究提供的证据表明RORs在癌症中的作用,RORs表达或活性的改变可能会导致Th17细胞及其产生的炎性细胞因子的变化,从而正向或负向地影响癌症的发展。在我们的前两部分实验中,我们已经发现在慢性乙型肝炎、ACHBLF的异常免疫反应中,Th17细胞亚群比例增加具有重要的作用；研究首次表明,作为Th17细胞分化的重要调节因子,RORγt在慢性乙型肝炎的疾病进展过程中可能具有重要作用。本研究有助于我们揭示Th17和RORγt在肝癌中所起到的作用以及与疾病进展的关系,从而为临床治疗提供理论依据和新的途径。目的本研究的目的是通过对原发性肝癌患者外周血RORγt和RORa基因表达,Th17细胞亚群及血清细胞因子的检测,进一步探讨RORyt和RORa在原发性肝癌患者发病机制中的作用。方法应用三色流式细胞术检测原发性肝癌患者和正常对照外周血中Th17细胞占CD3+T细胞的比例。应用逆转录聚合酶链反应(RT-PCR)方法检测RORγt和RORa在原发性肝癌患者和正常对照外周血单个核细胞(PBMC)中的表达。应用ELISA技术检测原发性肝癌患者和正常对照血清中IL-6,TGF-β,IL-17,IL-23和IFN-γ的表达水平。结果1、原发性肝癌患者外周血Thl7细胞亚群比例(3.58±1.07%)明显高于慢性乙型肝炎患者(1.77±0.52%,P<0.05)和正常对照组(0.33±0.15%,P<0.01)。2、原发性肝癌患者外周血RORβ的mRNA水平(9.96×10-2±3.74x10-2)明显高于慢性乙型肝炎患者(6.442×10-2±3.17×10-2,P<0.05)和正常对照组(1.666×10-3±8.77×104,P<0.05),原发性肝癌患者外周血RORγt的mRNA水平(1.32×10-2±2.08x10-3)明显高于慢性乙型肝炎患者(1.217×10-3±0.787×10-3,P<0.05)和正常对照组(3.207×104±1.38×10-4,P<0.01)。3、原发性肝癌患者血清IL-6水平(34.86±3.02pg/ml)较慢性乙型肝炎患者(13.36±3.79pg/ml)和正常对照组明显升高(3.43±0.55pg/ml, P<0.01);血清TGF-β水平(44.95±11.16ng/ml)较慢性乙型肝炎患者(35.22±2.4ng/ml,P<0.05)和正常对照组明显升高(18.28±1.8ng/ml,P<0.01);原发性肝癌患者血清IFN-γ水平(12.8±3.49pg/ml)较慢性乙型肝炎患者(10.17±3.38pg/ml)明显升高(P<0.05),但较正常对照组明显降低(17.14±2.28pg/ml,P<0.05);原发性肝癌患者血清IL-17和IL-23水平(71.24±6.43pg/ml,56.38±6.49pg/ml)较慢性乙型肝炎患者(65.38±11.65pg/ml,51.10±6.69pg/ml)和正常对照组(61.07±8.84pg/ml,45.87±4.47pg/ml)无明显差异(P≥0.05)。4、在原发性肝癌患者中,RORγt、IL-6与Th17细胞亚群比例存在明显的正相关性(r=0.396,P=0.049；r=0.402,P=0.047),RORα、TGF-β、IL-23、IFN-γ与Th17细胞亚群比例无相关性(r=0.126,P=0.548；r=0.143,P=0.496；r=0.247,P=0.234；r=0.310,P=0.132)。5、按照TNM分期标准,将原发性肝癌患者分为Ⅰ期5例,Ⅱ期6例,Ⅲ期12例,Ⅳ期7例,结果显示RORγt和RORα水平随着疾病的进展逐渐上升,但无明显差异(P>0.05),分别为Ⅰ期(7.75×10-3±0.94×10-3,7.60×10-2±2.0×10-2),Ⅱ期(1.28×10-2±0.50×10-2,8.43×10-2±3.78×10-2),Ⅲ期(1.47×10-2±0.75×10-2,1.02×10-1±5.95×10-2),Ⅳ期(1.86×10-2±0.83×10-2,1.28×10-1±8.06×10-2)。外周血Th17细胞亚群比例同样随着疾病的进展逐渐上升,分别为Ⅰ期(3.28±0.36%),Ⅱ期(4.00±1.51%),Ⅲ期(4.14±0.77%),Ⅳ期(4.35±1.37%),无明显差异(P>0.05)。根据患者的病情,将原发性肝癌患者分为肝功能代偿组和肝功能失代偿组,发现失代偿组患者RORγt和RORa水平(1.48×10-2±0.66×10-2,1.11×10-1±6.19×10-2)高于代偿组(1.38×10-2±0.6×10-2,8×10-2±3.16×10-2,),但无明显差异(P>0.05)。外周血Th17细胞亚群比例在失代偿组(4.58±1.21%)高于代偿组(3.48±0.59%),有明显差异(P<0.05)。RORγt、RORa水平在HBeAg阳性的HCC患者分别为(1.46×10-2±0.46×10-2,1.04×10-1±6.08×10-2),与HBeAg阴性患者相比(1.17×10-2±0.66×10-2,9.50×10-2±5.58×10-2),无明显差异(P>0.05)。外周血Th17细胞亚群比例在HBeAg阳性患者(4.06±1.01%)和HBeAg阴性患者(3.91±1.16%)无明显差异(P>0.05)。结论我们的研究结果进一步表明,在原发性肝癌患者的异常免疫反应中,Th17细胞在发病中具有重要的作用；研究首次表明,RORγt和RORa在原发性肝癌的发病过程中可能具有重要作用,与疾病的严重程度密切相关,可能作为判断疾病预后情况的指标。更多还原

【Abstract】 Background The pathogenesis of chronic hepatitis B is very complex, the immune disorders of host cause by the virus inducing chronic inflammation, is important cause of liver disease. Th17cells have been provided as a third CD4+T cell effector subset besides the well-described Th1and Th2CD4+T cells. Retinoid orphan nuclear receptor (ROR) γt and RORα, which belong to the steroid hormone receptor superfamily, were reported to be required for the differentiation of naive CD4+T helper cells into Th17cells.Th17cells and their effector cytokines are increasingly being recognized as key determinant in chronic hepatitis B (CHB), but there was still little study about RORγt and RORα in this field.Objective T helper cells17(Th17) have accurate but inconclusive roles in the pathogenesis of chronic hepatitis B. Retinoic acid-related orphan receptor γt(RORγt) and RORα are two lineage-specific nuclear receptors directly mediating Th17differentiation.This study was aimed to evaluate the gene expression of RORα and RORγt and their potential role in CHB patients.Methods Thirty CHB patients and twenty healthy controls were included in our present study. The frequency of peripheral Th17cells were determined using flow cytometry. The mRNA levels of RORα and RORγt in peripheral mononuclear cells (PBMCs) were determined by quantitative real-time polymerase chain reaction (RT-PCR).The serum levels of interleukin-6(IL-6), transforming growth factor-β (TGF-β), interleukin-17(IL-17), interleukin-23(IL-23), and interferon-γ (IFN-γ) were measured by enzyme-linked immunosorbent assay (ELISA).Results The frequency of peripheral Th17cells in CHB was significantly increased than controls. The peripheral mRNA levels of RORα and RORγt in CHB were significantly higher than controls. The serum levels of IL-6and TGF-β in CHB were significantly higher than controls; the serum level of IFN-γ in CHB was significantly lower than controls. RORγt, IL-6and IL-23were positively correlated with the frequency of Th17cells, while RORα, TGF-β and IFN-γ had no correlation with the latter. RORγt and the frequency of Th17cells had positive correlation with serum alanine aminotransferase in CHB patients.Conclusion Our study data strongly support that Th17is an important determinant in the evolution of CHB. More important, our study demonstrates for the first time that RORγt rather than RORα maybe act important role in the pathogenesis of CHB. Background Hepatitis B virus (HBV) infection still poses a major public health threat in a large part of the world.1%CHB patients may rapidly progress to severe type hepatitis, a condition referred to as acute-on-chronic liver failure (ACLF). In China, acute-on-chronic hepatitis B liver failure (ACHBLF) accounts for more than80%of ACLF cases due to a high incidence of HBV infection. ACHBLF may progress to multiple organ dysfunction and death with a high fatality. The precise mechanisms of ACHBLF remain unclear.There are a mount of data indicating that Th17cells act as important role in liver failure, but there was little study about RORyt and RORa.To further investigate the role of RORyt and RORa, as well as their relationship in the pathogenesis of ACHBLF, we examined the mRNA expression of RORyt and RORa in ACHBLF patients.Objective Acute-on-chronic hepatitis B virus liver failure (ACHBLF) has been shown to carry poor prognosis, however, the pathogenesis of ACHBLF is still not fully understood. RORγt and RORa are two lineage-specific nuclear receptors directly mediating Th17differentiation.This study was aimed to evaluate the gene expression of RORa and ROR(?)t and their potential role in ACHBLF.Methods40ACHBLF patients were included. The frequency of peripheral Th17cells were determined using flow cytometry. The mRNA levels of RORa and RORyt in peripheral mononuclear cells (PBMCs) were determined by quantitative real-time polymerase chain reaction (RT-PCR).The serum levels of interleukin-6(IL-6), transforming growth factor-β (TGF-β), interleukin-17(IL-17), interleukin-23(IL-23), and interferon-γ (IFN-γ) were measured by enzyme-linked immunosorbent assay (ELISA).Results The frequency of peripheral Th17cells in ACHBLF was significantly increased than CHB and controls. The peripheral mRNA levels of RORα and RORγt in ACHBLF were significantly higher than CHB and controls. The serum levels of IL-6and TGF-β in ACHBLF were significantly higher than CHB and controls; the serum level of IFN-γ in ACHBLF was significantly higher than CHB, but lower than controls. In ACHBLF patients, RORyt, IL-6and IL-23were positively correlated with the frequency of Th17cells, while RORa, TGF-β and IFN-γ had no correlation with the latter.The mRNA level of RORγt was positively correlated with model of end stage liver disease (MELD) score, but there was no correlation of RORα and MELD score.Conclusion Our study strongly support that Th17cells act as an important determinant in the evolution of ACHBLF.More important, our study demonstrates for the first time that RORγt play an important role in pathogenesis of ACHBLF and might be considered to be a candidate factor consistent with the severity of disease. Background Primary liver cancer is one of the common malignant tumors in China. More than90%of primary liver cancer is hepatocellular carcinoma (HCC), about5%is cholangio carcinoma, a mixture of both is rare. The etiology of this disease and the pathogenesis has not been clear. It may be related to the combined effects of a variety of factors.Chronic liver disease may play an important role in the development and progress of liver cancer. Approximately90%of patients with liver cancer in China have hepatitis B virus infection. Some studies show that Th17promote tumor development, studies suggest Th17remove the tumor cells. Numbers of studies have provided evidence that the role of RORs in cancer, the RORs expression may result in the change of Th17cells activity and influent the development of cancer positively or negatively.Objective Primary liver cancer has been shown to carry poor prognosis, however, the pathogenesis of HCC is still not fully understood. RORγt and RORa are two lineage-specific nuclear receptors directly mediating Th17differentiation.This study was aimed to evaluate the gene expression of RORα and RORγt and their potential role in HCC.Methods30HCC patients were included. The frequency of peripheral Th17cells were determined using flow cytometry. The mRNA levels of RORα and RORγt in peripheral mononuclear cells (PBMCs) were determined by quantitative real-time polymerase chain reaction (RT-PCR).The serum levels of interleukin-6(IL-6), transforming growth factor-β (TGF-β), interleukin-17(IL-17), interleukin-23(IL-23), and interferon-γ (IFN-γ) were measured by enzyme-linked immunosorbent assay (ELISA).Results The frequency of peripheral Th17cells in HCC was significantly increased than CHB and controls. The peripheral mRNA levels of RORa and RORyt in HCC were significantly higher than CHB and controls. The serum levels of IL-6and TGF-β in HCC were significantly higher than CHB and controls; the serum level of IFN-γ in HCC was significantly higher than CHB, but lower than controls. In HCC patients, RORyt was positively correlated with the frequency of Thl7cells, while RORa had no correlation with the latter.The mRNA level of RORyt and RORa were correlated with disease progress.Conclusion Our study strongly support that Thl7cells act as an important determinant in the evolution of HCC.More important, our study demonstrates for the first time that RORyt and RORa play an important role in pathogenesis of HCC and might be considered to be candidate factors consistent with the severity of disease.更多还原