【作者】 史旭华；

【导师】 张奉春； 张烜； 唐福林；

【作者基本信息】 中国协和医科大学 ， 风湿病学， 2006， 博士

【摘要】 第一部分药物治疗原发性胆汁性肝硬化的临床疗效观察目的前瞻性研究熊去氧胆酸(UDCA)、UDC联合泼尼松龙、UDCA联合硫唑嘌呤(Azp)三种治疗方案对原发性胆汁性肝硬化(PBC)的疗效并评价影响疗效的危险因素。材料与方法82例初诊PBC患者随机分为UDCA(U组，28人)、UDCA联合泼尼松龙(UP组，27人)、UDCA联合Azp(UA组，27人)三个治疗组，在治疗第0、3、6、12月采集详细的临床及实验室资料。记录肝硬化失代偿、死亡、肝移植等事件以及药物副作用。入选标准符合2000年美国肝脏病协会制定的PBC诊疗指南；排除标准为合并其他肝病，年龄＞70岁，肝功失代偿(Child分级B／C)，预计存活期不超过1年，合并其他自身免疫病。药物剂量为UDCA 13-15mg·d-1·kg-1；泼尼松龙起始剂量0.5mg·d-1·kg-1，4周开始减量，减至7.5mg·d-1时维持；Azp 1mg·d-1·kg-1。疾病进展规定为：观察期内肝硬化失代偿、肝移植或肝脏相关的死亡、胆红素升高到原水平两倍以上。生化缓解定义为：治疗后总胆红素(TBIL)正常同时碱性磷酸酶(ALP)和谷氨酰转肽酶(GGT)水平降至正常值高限1.5倍以下。结果UP组患者乏力和瘙痒程度改善(分别为P=0.015和P=0.037)，U组、UA组症状无改善。三组患者谷氨酸氨基转移酶(ALT)、天门冬酸氨基转移酶(AST)、ALP、GGT、总胆红素(TBIL)、直接胆红素(DBIL)、IgM均下降，组内效应分别为P=0.000、0.000、0.000、0.000、0.009、0.001、0.000，在3个月内下降最明显；三种治疗方案相比无差异。疾病进展分别为U组中2人，UP组3人(其中1人因食道静脉曲张破裂大出血死亡)，UA组1人。发生疾病进展的患者Mayo危险性评分(MRS)(4.4±0.15 vs 5.7±0.55，P=0.018)高、凝血酶原时间(PT)(11.6±0.12 vs 12.4±0.32，P=0.042)延长。U组未出现药物副作用；UP组血糖升高2例、满月脸5例、多毛1例；UA组白细胞下降2例，胆绞痛1例(UDCA相关)。基线ALP、GGT、总胆固醇(CHO)水平是生化缓解的危险因素(β=-2.095，P=0.015，Exp(β)=0.123，95％CI 0.023-0.663)，DBIL、TBIL、总胆汁酸(TBA)、PT也不利于生化缓解(β=-1.162，P=0.075，Exp(B)=0.313，95％CI 0.083-1.124)；联合泼尼松龙(β=-0.747，P=0.350，Exp(B)=0.474，95％CI 0.099-2.266)或Azp(β=-1.479，P=0.223，Exp(B)=0.228，95％CI 0.021-2.453)不能使生化缓解率提高。结论三种方案对PBC患者的肝脏生化指标、IgM的改善作用相同，UP方案可减轻乏力瘙痒症状：生化缓解多发生在治疗3月内，高水平的基线ALP、GGT、CHO是生化缓解的危险因素，高水平的TBIL、DBIL、TBA、PT不利于生化缓解，两种联合治疗方案不增加生化缓解率。MRS高、PT延长的患者易疾病进展。U方案副作用发生率低。第二部分原发性胆汁性肝硬化外周血淋巴亚群、细胞因子特点及药物治疗后的变化目的原发性胆汁性肝硬化(PBC)患者外周血淋巴细胞亚群及细胞因子的特点及其对治疗的反应。材料与方法全部病例来自论文第一部分熊去氧胆酸(UDCA)(U组)、UDCA联合泼尼松龙(UP组)、UDCA联合硫哗嘌呤(Azp)(UA组)治疗PBC的前瞻性、随机、对照研究中的82例PBC患者。参加者在治疗前和治疗3、6月时检测外周血淋巴细胞亚群(溶血法)和细胞因子(酶联免疫吸附分析，ELISA)水平。设未治疗的慢性乙型肝炎患者(CHB)29例和干燥综合征(PSS)21例，健康对照组(HS)20例与药物治疗前PBC患者相应的指标对比。结果与PSS患者相比，PBC患者B细胞比例低(P=0.010)、CD4+T细胞比例升高(P=0.003)、记忆细胞(CD4+CD45RO+)增多(P=0.051)、IFN-γ、TNF-α、IL-2均升高(分别为P=0.004、P=0.003、P=0.000)。与健康对照者比，PBC患者B细胞比例升高(P=0.366)但未达到统计学意义上的差异，CD4+T细胞比例升高(P=0.044)而CD8+T细胞比例降低(P=0.277)、CD4／CD8+比例升高(P=0.042)，记忆T细胞比例升高(P=0.049)，IFN-γ(P=0.001)、TNF-α(P=0.000)、IL-2(P=0.000)、IL-4(P=0.010)、IL-6(P=0.029)升高，IL-10低但未达到统计学意义上的差异(P=0.453)。外周血CD8+T细胞数与MRS(P=0.001)、胆红素水平(TBIL为P=0.010，DBIL为P=0.013)呈负相关而与白蛋白水平(P=0.008)正相关。外周血TNF-α水平与血ALT(p=0.005)、AST(p=0.002)、TBIL(p=0.001)、DBIL(p=0.002)、MRS(p=0.020)正相关。治疗后PBC患者B细胞比例下降(U组治疗6个月时P＜0.05)。三组CD8+T细胞升高(UP组6月时P=0.002)，U组和UP组CD4+T细胞比例(U组3、6月时为P=0.047，P=0.025)和CD4／CD8下降(UP组6个月时P=0.020)。治疗后三组患者记忆细胞比例减少、纯真细胞增多但尚未达统计学意义上的差异。三组治疗后CD4+CD28+T细胞比例的变化无统计学上差异；治疗后U组CD8+CD28+T细胞比例继续降低(6个月时P=0.032)，UP、UA组变化无统计学差异。治疗3月时U组IFN-Y、IL-2、IL-4、IL-6水平均降低，其中IFN-Y、IL-4、IL-6在6个月时再次升高，而UP组和UA组患者在治疗后均低于基线水平。三组TNF-α水平均较治疗前低(UP、UA组6月时分别为P=0.042和P=0.043)。结论CD4+T细胞比例的增加伴随CD8+T细胞比例的下降及CD8+T细胞与病情的相关性提示其在PBC发病机制中的重要作用。外周血同时存在Thl型和Th2型细胞因子的异常且以Th1细胞占主导地位，提示PBC以细胞免疫为主。TNF-α是参与PBC免疫损伤的重要因子并与病情相关。三种治疗方案对免疫系统的影响不完全相同。第三部分原发性胆汁性肝硬化自身抗体特征及其对药物治疗的反应目的研究PBC患者抗线粒体抗体(AMA)、抗线粒体抗体M2亚型(AMA-M2)、抗GP210抗体和抗SP100抗体的临床相关性及对药物治疗的反应。材料与方法82例初诊未治疗的PBC患者随机分为熊去氧胆酸(U组，28人)、熊去氧胆酸联合泼尼松龙(UP组，27人)、熊去氧胆酸联合硫唑嘌呤组(UA组，27人)三组，治疗前检测AMA(间接免疫荧光法，IIF)、AMA-M2(酶联免疫吸附分析法，ELISA)、抗GP210抗体和抗SP100抗体(免疫印迹法，IBT)，治疗第3、6月复查AMA、AMA-M2。结果PBC患者的AMA滴度、AMA-M2浓度与临床表现(乏力、瘙痒、黄疸)、肝生化指标(ALT、AST、ALP、GGT、TBIL、DBIL、ALB、TBA、CHO)、肝组织学病变程度(纤维化、汇管区炎症、汇周碎屑坏死、肝细胞坏死性损害、胆管增生或减少)间无相关性，AMA与IgM正相关(P=0.046)；AMA与外周血IL-6、IL-1β正相关(P=0.002，P=0.024)，AMA-M2与IL-6正相关(0.024)。三种治疗方案对AMA、AMA-M2水平无影响(分别为组内效应P=0.270，组间效应P=0.648和组内效应P=0.613，组间效应P=0.764)。疾病进展和疾病未进展的患者间、生化缓解和生化未缓解的患者间基线AMA、AMA-M2水平无差异。抗GP210抗体阳性患者比阴性患者黄疸发生率高(P=0.003)且程度重(P=0.005)、食道静脉曲张发生率高(P=0.005)、Mayo危险性评分高(P=0.005)、DBIL(P=0.035)和TBA水平(P=0.002)高而ALB水平(P=0.002)低、肝组织学分期晚(P=0.013)、纤维化重(P=0.002)；抗GP210抗体阳性者治疗6月时的生化缓解率低(16.7％vs 52.0％，P=0.012)，按胆红素水平将抗GP210抗体阴性与阳性患者1∶1匹配后两组生化缓解率无统计学意义上的差异(16.7 vs 33.3％，P=0.266)。抗SP100抗体阳性与阴性患者的症状、肝脏生化指标、IgM、肝组织学病变程度无差异。结论AMA、AMA-M2对PBC患者有诊断意义，但其水平与病情无关，既不受药物治疗影响，也不影响药物疗效。抗GP210抗体阳性的PBC患者病情重，生化缓解率低，但尚无证据表明抗GP210抗体本身对药物治疗有抵抗作用。抗SP100抗体可作为一项辅助诊断的手段，但与PBC患者的病情无关。第四部分原发性胆汁性肝硬化的肝脏病理与淋巴细胞浸润特征及其临床相关性目的研究PBC患者肝脏病理、淋巴细胞浸润特征及其临床相关性。材料与方法24例初诊未经治疗的PBC患者的肝脏穿刺标本常规进行HE染色，记录病理分期(LudwigⅠ-Ⅳ期，Ⅰ-Ⅱ为早期、Ⅲ-Ⅳ为晚期)、纤维化(0-3级，0.1级为轻度、2-3级为重度)、汇管区炎症(0-3级，0.1级为轻度、2-3级为重度)、汇管区周围碎屑坏死(0-2级，0级为轻度，1-2级为重度)、肝细胞坏死性损害(0-2级，0级为轻度，1-2级为重度)、胆管增生及胆管减少(0-2级，0级为轻度，1-2级为重度)；其中10例标本连续切片，进行免疫组化染色(CD20、CD45RO、CD8)，计数每个汇管区每种淋巴细胞亚型的平均数，分析前述指标的临床相关性。结果病理分期、纤维化程度与黄疸程度、总胆红素(TBIL)、直接胆红素(DBIL)、总胆汁酸(TBA)、总胆固醇(CHO)、IgG均呈正相关，与白蛋白(ALB)、血嗜酸细胞(EOS)比例负相关，纤维化程度还与Mayo危险性评分(MRS)正相关。汇周碎屑坏死与黄疸和瘙痒程度、MRS、碱性磷酸酶(ALP)、TBIL、DBIL、TBA正相关，还与血TNF-α水平正相关(相关系数0.617，P=0.006)。胆管减少程度与黄疸、MRS、天门冬酸氨基转移酶(AST)、TBIL、DBIL、TBA、IgG正相关而与ALB呈负相关。不同病理分期及不同严重程度的各种肝组织病变形态的PBC患者临床指标间的差异与前述相关分析中的结果一致。汇管区及汇管区周围CD20+淋巴细胞数与肝脏生化指标间无相关性，CD45RO+、CD8+淋巴细胞浸润程度与ALT、AST、GGT、TBA正相关，CD8+淋巴细胞还与ALP正相关(相关系数=0.775，P=0.041)，也与血IFN-γ水平正相关(相关系数0.901，P=0.006)。结论肝脏病理分期、纤维化程度、汇管区周围碎屑坏死、胆管减少程度是与PBC患者病情轻重及病程有关的指标。TNF-α是反映汇管区周围碎屑坏死程度的有用指标，二者反映了疾病处在进展期。PBC的组织学损害以细胞免疫为主，CD8+T细胞起重要作用，升高的IFN-γ水平增强CD8+T细胞对胆管的破坏。更多还原

【Abstract】 Part one Prospective, randomized, controlled study ofursodeoxycholic acid, ursodeoxycholic acid combined withprednisonlone or azatharaprine therapy for primary biliary cirrhosisObject The aim of this study was to evaluate the effects of thecombination therapy with prednisonlone/azatharaprine and ursodeoxycholic acid(UDCA) for primary biliary cirrhosis (PBC), compared to UDCA monotherapy.Materials and methods Eighty two patients with untreated PBC were dividedrandomly into three groups. Group U (28 patients) received UDCA alone for 12months, group UP (27 patients) received UDCA and prednisonlone, while group UA(27 patients) received UDCA and Azp. The clinical and laboratory data was followedafter 3, 6 and 12 months. Inclusion criteria was the guideline for management of PBCwhich AASLD presented in 2000. The dose of these dugs were defined as UDCA13-15mg·d-1·kg-1, prednisonlone started with 0.5mg·d-1·kg-1, and tapered to7.5mg·d-1 from the fifth week, Azp 1mgod-1·kg-1. Disease progression wasdescriptived as liver decompensation, liver transplantation, two times elevation ofTBIL. Biochemical remission was defined as the normalization of serum bilirubinwith gamma-glutamyl transferase (GGT) with alkaline phosphatase (ALP)＜1.5×ULN (upper limit of normalization). Result Fatigue and pruritus were improvedonly in group UP. ALT, aspartate aminotransferase (AST), ALP, GGT, total bilirubin(TBIL), direct bilirubin (DBIL), IgM all decreased below or reached the ULN values(within-subject effects, P=0.000、0.000、0.000、0.000、0.009、0.001、0.000,respectively), and there had no ststistic significance between three groups(between-subject effects, P＞0.05 for all). The number of patients with diseaseprogression were two in group U, three in group UP, and one in group UA. Thepatients with disease progression had higher Mayo risk score(P=0.018) and prolongedProthrombin time (PT) (P=0.042). No side-effects occurred in group U. In group UP, side-effect related with Glucocorticosteroids occurred in eight patients. In group UA,two patients had side-effects related with Azp and one had side-effect related toUDCA. The level of ALP、GGT、CHO in baseline were risk factor for biochemicalremission (β=-2.095, P=0.015, Exp(β)=0.123, 95%CI 0.023-0.663), high level ofDBIL、TBIL、total bile acid (TBA)、PT of baseline were not beneficial forbiochemical remission. (β=-1.162, P=0.075, Exp(B)=0.313, 95%CI0.083-1.124),wether combined with prednisonlone (β=-0.747, P=0.350, Exp(B)= 0.474, 95%CI0.099-2.266) orAzp (β=-1.479, P=0.223, Exp(B)=0.228, 95%CI0.021-2.453)with UDCA were not related with biochemical remission. CONCLUSIONS Thereare no difference between three groups for improve biochemical data of liver and IgM.High level of ALP, GGT, CHO in baseline are risk factor for biochemical remission.High level of TBIL、DBIL、TBA、PT are also not beneficial for biochemical remission.The combination with prednisonlone or Azp are not related with the ratio ofbiochemical remission. The disease of patients with higher Mayo risk score andprolonged PT tend to progress.Part two Character of peripheral lymphocytic subsets and cytokinesin patients with primary biliary cirrhosis and their change aftertherapyObject The aim of this study was to describe the Character ofperipheral lymphocytic subsets and cytokines in patients with primary biliary cirrhosisand their change after therapy. Materials and methods Eighty two patients withuntreated PBC were divided randomly into three groups. Group U (28 patients)received UDCA for 12 months, group UP (27 patients) received UDCA andprednisonlone, while group UA (27 patients) received UDCA and azatharaprine (Azp).Peripheral lymphocytic subsets and cytokines were detected at 0, 3, 6 months aftertherapy using tricolour flowcytometry analysis and ELISA respectively. Twenty ninepatients with chronic hepatitis B (CHB), twenty one patients with pimary Sj(?)gren’ssyndrome (PSS) and twenty healthy people were also included as controls. ResultCompared with PSS, the percent of B cells were decreased (P=0.010), the percent of CD4+T cells (P=0.003) and memory cells (CD4+CD45RO+) (P=0.051) wereincreased, and the level of IFN-γ, TNF-α, IL-2 were all increased (P=0.004, P=0.003P=0.000 respectively) in PBC. Compared with healthy control (HC), the percent ofCD8+T cells (P=0.277) decreased companied with the increase of percent of CD4+Tcell (P=0.044) and the ratio of CD4/CD8 (P=0.042) in PBC. And the patients withPBC also had higher percent of memory cells (P=0.049) than HC. The level of IFN-γ(P=0.001), TNF-α(P=0.000), IL-2 (P=0.000), IL-4 (P=0.010), IL-6 (P=0.029)were increased in PBC comparing with HC. The lower level of IL-10 was observed inPBC than in HC but didn’t reach the statistic significance. The number of CD8+Tcells in peripheral were correlated with lower Mayi risk score (MRS) (P=0.001), TBIL(P=0.010), DBIL (P=0.013), and higher albumin (P=0.008). The level of TNF-αinperipheral were correlated with higher ALT (p=0.005) , AST (P=0.002) , TBIL(P=0.001) , DBIL (P=0.002) , MRS (P=0.020) . The percent of B cells decreasedin patients with PBC after therapy and reached the statistic significance in group U atsixth month. The percent of CD8+T cells increased in the three groups(P=0.002 ingroup UP at the sixth month). The percent of CD4+T cell (P=0.047, P=0.025 in groupU at the third and sixth months) and the ratio of CD4/CD8 (P=0.020 in group UP atthe sixth month) were decreased in group U and UP. Though didn’t reach the statisticsignificance, the percent of memory cells decreased in the three groups. The percentof CD8+CD28+T cells in CD8+T cells was dereased in group U after UDCA therapy.The level of IFN-γ, IL-2, IL-4, IL-6 all decreased after therapy at the third month ingroup U, and rebounded thereafter. In group of UP and UA, all these cytokines weresurpressed below the level of baseline. TNF-αdecreased in all the three group aftertherapy (P=0.042 and P=0.043 in group UP and UA at the sixth month).CONCLUSIONS The reduction of CD8+T cells and the correlations with diseaseactivity revealve it’s important role in the pathogenesis of PBC. PBC is a disease withpredominant cellular immunity as described with the pattern of peripheral cytokines.TNF-αis an important cytokine in the pathogenesis of patients with PBC, andcorrelated with the severity of the disease. The effects on the peripheral lymphocyticsubsets and cytokines of these three regime for the therapy of PBC are not identical.But the increase of CD8+T and the suppression of cytokines after therapy wereresponsive for the clinical amelioration of the disease. Part three Character of self antibody in patients with primary biliarycirrhosis and their change after therapyObject The aim of this study was to describe the character ofAnti-mitochondrial antibodies (AMA), Anti-mitochondrial M2 antibodies (AMA-M2),anti GP210 antibody (anti-GP210 Ab) and anti SP100 antibody (anti-SP100 Ab) inpatients with primary biliary cirrhosis (PBC) and their correlation with therapy.Materials and methods Eighty two patients with untreated PBC were dividedrandomly into three groups. Group U (28 patients) received UDCA, group UP (27patients) received UDCA and prednisonlone, while group UA (27 patients) receivedUDCA and azatharaprine (Azp). AMA (IIF), AMA-M2 (ELISA), anti-GP210antibody and anti-SP 100 antibody (IBT) were detected in these patients before therapy.AMA and AMA-M2 were repeated after therapy at the third and sixth months. ResultThere had no correlations between the titer of AMA, concentration of AMA-M2 andsymptoms, biochemical data of liver, histopathologie character in liver. AMA wascorrelated with peripheral IL-6 and IL-1β. The three protocol of therapy had noimpact on the level of AMA or AMA-M2 (within-subjects effects, P=0.270,between-subjects effects, P=0.648 for AMA; within-subjects effects, P=0.613,between-subjects effects P=0.764 for AMA-M2) . Whether the patient had diseaseprogression or biochemical remission, the difference of AMA or AMA-M2 didn’treach the statistic significance. The patients with anti-GP210 antibody had moresevere icterus(P=0.005), more frequent incident of esophageal varices (P=0.005),higher Mayo risk score (P=0.005). DBIL (P=0.035 ), TBA (P=0.002) in patients withthe antibody were higher and ALB was lower than those without. The patients withanti-GP210 antibody had later pathologic stage (P=0.013) and severe fibrosis(P=0.002). The biochemical remission was lower in patient with anti-GP210 antibodythan those without (16.7% vs 52.0%, respectively, P=0.012) . Being matched withBIL and MRS, the ratio of biochemical remission in two groups didn’t reach thestatistic significance (16.7 vs 33.3%, P=0.266). There had no difference betweenpatients with anti-SP100 and those withought in symptoms, biochemical data of liver,histopathologic character in liver. CONCLUSIONS AMA, AMA-M2, anti-SP100 antibody were valuble in the diagnosis of PBC, but they are not related with theseverity of the disease. Patients with anti-GP210 antibody had more severe diseaseand this antibody correlate with lower ratio of biochemical remission. But it’s notanti-GP210 antibody itself resist the role of drugs for PBC but because there are morerelatively severe cases in patients with positive anti-GP210 antibody.Part four Clinicopathological features of primary biliary cirrhosisObject The aim of this study was to describe the clinicopathologicalfeatures and character of lymphocytic subsets in portal tract of primary biliarycirrhosis (PBC). Materials and methods The liver biopsy speciments were obtainedfrom twenty four patients with PBC who participate in a prospective, randomized,controlled trial (Part one) through percutaneously needle puncture. These sampleswere fixed in formaldehyde and embedded in paraffin for routine histologicalexamination. Pathologic stage according to Ludwig (stage 1-4), fibsosis (grade 0-3),portal and periportal inflammation (grad 0-3), lymphocytic periportal piecemealnecrosis (grade 0-2), ductular proliferation (grade 0-2), intralobular hepatocytenecrosis (grade 0-2) and the degree of ductopenia (0-2) were record. Serial sections,4um-thick, were prepared for immunohistochemistry (CD20, CD45RO, CD8). Theaverage numbers of every lymphocytic subsets per portal area were computed. ResultThe pathologic stages, degree of fibrosis were positively correlated with ictetus, totalbilirubin (TBIL), total bile acid (TBA)、Cholesterol (CHO)、IgG, and negtivelycorrelated with albumin (ALB)、percent of eosinophilic cell ( EOS ). Fibrosis was alsopositively correlated with Mayo risk score (MRS). Lymphocytic periportal piecemealnecrosis was positively correlated with the degree of icterus and pruritus, MRS,alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirunbin (DBIL), total bileacid (TBA), and also TNF-α(correlations 0.617, P=0.006). the degree ofductopeniawas positively correlated with the extent of icterus, MRS, aspartate aminotransferase(AST), TBIL, DBIL, TBA, IgG, and negtively correlated with ALB. No correlationswere observed between the numbers of CD20+T cells in portal area and biochemicaldata of liver. But the numbers of CD45RO+, CD8+ T cell were positively with ALT、 AST、gamma-glutamyl transferase (GGT)、TBA, and CD8+T cells also positivelycorrelated with ALP (correlations 0.775, P=0.041 ) and IFN-Γ(correlations 0.901,P=0.006). CONCLUSIONS The pathologic stage and the degree of fibsosis,lymphocytic periportal piecemeal necrosis, ductopenia are correlated with the diseaseseverity and reflect the process of the disease. TNF-αis a usful cytokine which canreflect the degree of lymphocytic periportal piecemeal necrosis, and they all reflectthe progression of the disease. Cellular immunity is important in the process of PBC.Acompanied with elevated IFN-γ, CD8+T cells destroy the interlobular bile duct.更多还原