【作者】 叶红；

【导师】 王凯；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2014， 博士

【摘要】 中文摘要第一部分CD163在慢加急性乙型肝炎肝衰竭患者中的表达及临床意义乙型肝炎病毒感染是一个重要的世界卫生问题。它被认为是各种肝脏急慢性疾病的主要病因之一。由于某些病因的急剧发展,一些慢性乙型肝炎(CHB)患者有可能发展为肝衰竭。我们将这些由慢性乙型病毒性肝炎发展而来的肝衰竭称为慢加急性乙型肝炎肝衰竭(ACHBLF),其在所有肝衰竭病因中所占比例高达70%。ACHBLF的发病机制尚不完全清楚,其死亡率高,预后差,因而降低肝衰竭患者的发病率和死亡率成为研究热点。寻找能预测肝衰竭的生物标志物能加强对患者的监测和治疗,降低ACHBLF患者的发病率和死亡率。目前大量临床研究发现,ACHBLF的发病可能与自身强烈的免疫反应有关,因此,充分理解ACHBLF发病过程的免疫应答十分重要。而CD163及sCD163是近期发现的与急性炎症反应过程密切相关的免疫因子。CD163的研究尚在起步阶段,CD163在ACHBLF患者的免疫过程中可能起到重要作用,但其在ACHBLF患者中扮演的角色尚不完全明确,对诊断及预后评估意义仍未确定,而这正是我们研究的重点及方向。目的：检测ACHBLF患者中外周血单个核细胞表面CD163及外周血血浆中sCD163的表达,明确其在急性炎性反应过程中扮演的角色及临床意义；为临床治疗ACHBLF患者寻找新的临床监测因子。方法：ELISA方法检测ACHBLF患者、CHB患者及正常对照组(HCs)血浆中sCD163的表达水平;流式细胞术检钡ACHBLF患者、CHB患者及HCs外周血单个核细胞CD163的阳性率和CD163平均荧光强度(MFI),应用逆转录聚合酶链反应(RT-PCR)测量ACHBLF患者、CHB患者及HCs外周血单个核细胞中CD163mRNA的表达。结果：1、外周血中CD163阳性的单个核细胞比率在ACHBLF患者中要明显高于CHB患者及HCs,具有显著性差异。外周血中CD163MFI在ACHBLF患者中较其他两组升高,但三组无显著性差异。2、CD163mRNA在ACHBLF患者中的表达要明显高于CHB及HCs,具有显著性差异。3、血浆中sCD163表达在ACHBLF患者中明显升高,且与患者预后有明显相关,在生存组患者中的血浆sCD163浓度要明显低于死亡组患者sCD163浓度,具有显著性差异。4、血浆中sCD163表达在ACHBLF患者中与反映患者预后的MELD Score呈正相关,与HBV-DNA正相关,凝血酶原活动度负相关,与总胆红素水平无相关。5、外周血中CD163阳性的单个核细胞比率在CHB患者中要明显高于HCs,具有显著性差异。6、CD163mRNA在CHB患者中的表达要高于HCs,但并不具有显著性差异。7、血浆sCD163表达在CHB患者中明显升高,高于HCs,但是升高幅度远远小于其在其ACHBLF患者中升高幅度,具有显著性差异。结论：作为在巨噬细胞表面特异表达的CD163在ACHBLF患者、CHB患者发病过程中起重要作用,本研究提示CD163与sCD163都可以作为ACHBLF患者有用的标志物,其中sCD163检测更为简单直接,组间差异更为明显,与疾病的严重程度明显相关。CD163与sCD163都可以作为肝衰竭患者预后的评价指标,与患者预后呈负相关。中文摘要第二部分CD163在原发性肝癌患者中的表达及临床意义感染乙型肝炎病毒的患者易罹患肝癌,有学者统计发现,慢性乙型肝炎及慢性乙肝后肝硬化患者罹患肝癌的几率是正常人的9-10倍。80%以上的原发性肝癌患者有乙型肝炎病毒感染,原发性肝癌(HCC)是肝脏的恶性肿瘤,在所有的恶性肿瘤中死亡率高达第5位,其五年生存率不足5%,这些数据表明,了解HCC的发病过程,进行早期诊断与风险评估对HCC患者非常重要,但是目前尚缺乏非常有效的早期预测手段及监测方法。与肿瘤相关的增高的巨噬细胞被认为在肿瘤的病因及不良预后中扮演了重要角色,其中最近引起人们广泛兴趣的是,巨噬细胞的M2分型副群具有抗炎的作用,而抗炎因子CD163是M2副群的特异性标记,且为巨噬细胞活化的标记物,它的高表达可促进肿瘤的血管生成,纤维组织细胞重组,影响组织重建过程,在某些肿瘤的病理过程及在急慢性肝脏炎症疾病中起着重要作用。其在大量炎症性疾病及肿瘤性疾病中的广泛发现,使其有望成为近年来非常有效且易于监测的临床指标。而且在前一部分试验中,通过对ACHBLF患者中外周血及血浆中CD163与sCD163表达水平的检测,我们明确了在急性炎症反应过程中CD163及sCD163的表达与临床预后的关系。在本实验中,我们选取了HCC患者作为研究对象,明确CD163及sCD163在HCC患者中的表达,明确其在HCC发病过程中的重要作用,了解其与临床指标及预后的联系,寻找新的发病机理及治疗方向。目的：探讨HCC患者中外周血单个核细胞CD163及血浆中sCD163的表达,可明确其在HCC疾病过程中扮演的角色及临床意义,为临床治疗HCC寻找监测因子,探寻新的发病机理及治疗方向。方法：ELISA方法检测HCC患者、LC患者、CHB患者及HCs血浆中sCD163的表达水平；流式细胞术检测HCC患者、LC患者、CHB患者及HCs外周血单个核细胞CD163的阳性率和CD163平均荧光强度；应用逆转录聚合酶链反应(RT-PCR)测量HCC患者、LC患者、CHB患者及HCs组外周血单个核细胞中CD163mRNA的表达。结果：1、外周血CD163阳性的单个核细胞比率在HCC患者中明显升高,组间有着显著性差异；CD163免疫荧光强度组间有显著性差异。2、HCC患者中,CD163mRNA表达明显升高,组间具有显著性差异。3、血浆中sCD163浓度在HCC患者中明显升高,组间具有显著性差异。4、外周血中表达CD163的单个核细胞比率在LC患者中要明显高于HCs,具有显著性差异。5、CD163mRNA在LC患者中的表达要高于HCs,组间具有显著性差异。6、血浆sCD163表达在LC、CHB患者中明显升高,高于HCs,LC患者血浆sCD163升高幅度远远大于其在其CHB患者中升高幅度,具有显著性差异。7、血浆sCD163表达水平与HCC患者的AFP、HBV-DNA、HBeAg无明显相关。8、根据意大利CLIP评分分析,sCD163浓度与评分之间不存在明显相关性,TNM分期,Ⅲ期存在远处转移及Child C级的HCC患者的sCD163浓度要高于无远处转移Ⅰ、Ⅱ期患者,但是无显著性差异。结论：作为在巨噬细胞表面特异表达的CD163在HCC、LC患者发病过程中起重要作用,本研究提示CD163与sCD163都可以作为HCC及LC患者有用的标志物,其中sCD163检测更为简单直接,组间差异更为明显,与疾病的严重程度明显相关。但是sCD163与临床指标在HCC患者中均无明确相关,与患者预后的联系仍需进一步实验证实。中文摘要第三部分CD163综述CD163结构及表达形式单核细胞和巨噬细胞在人类免疫系统中发挥着重要作用。巨噬细胞可分为M1型亚群和M2型亚群,两种细胞分别有促炎和抗炎的作用。CD163是在已知的M2型巨噬细胞表面特异性表达的,属于清道夫受体半胱氨酸富集结构域家族B。它是一个有着130kD重量的Ⅰ型蛋白,共有5种同源异构体,结构中含有9个清道夫受体半胱氨酸富集结构域。CD13在体内共有两种存在形式：一种是以跨膜大分子的形式存在于单核巨噬细胞上；另一种是存在于血浆或其它组织液内,具有可溶性,即sCDl63。CD163的作用CD163可以特异地识别血红蛋白：结合珠蛋白复合体,是体内血红蛋白的特异性清道夫受体。通过清除游离的血红蛋白,CD163可起到调节人体免疫的作用。CD163还可识别肿瘤坏死因子样凋亡弱诱导因子(TWEAK),并与其特异性结合,发挥抗炎作用,担当着革兰氏阳性菌及革兰氏阴性菌监控器的角色；在炎症反应刺激下,CD163可以和自己的配体结合,转导信号,促进单核细胞与上皮细胞的黏附,诱导炎性介质IL-10等炎症因子的释放。CD163与各种疾病的关系CD163及sCD163与免疫系统关系密切。大量的临床及实验室研究证实,CD163及sCD163与各种炎症性、肿瘤性、心脑血管疾病密切相关。在严重烧伤、急性肝衰竭等严重炎性疾病时,CD163明显升高,与预后有明显相关;在肝癌、淋巴瘤等恶性肿瘤中,亦可见CD163的明显升高,在淋巴结转移及治疗反应不佳的病人中尤为明显,提示CD163在肿瘤患者预后监测中的重要作用。糖尿病、高血脂、冠状动脉粥样硬化性心脏病等疾病中,CD163及sCD163均已被明确证实与疾病预后明显相关。CD163及sCD163在抗炎、促肿瘤方面的广阔前景,使其成为人们在未来研究的热门生物靶点,并已取得了初步成效。更多还原

【Abstract】 INCREASED CD163AND SCD163IS ASSOCIATED’ ACUTE-ON-CHRONIC HEPATITIS B LIVER FAIL ABSTRACTHepatitis B virus (HBV) infection is an important health problem worldwide. It is thought to be one of the main causes of liver related chronic and acute diseases. With severe acute exacerbation of the disease, some chronic hepatitis B (CHB) patients may progress to liver failure. We define this progression as acute-on-chronic hepatitis B liver failure (ACHBLF). It constitutes about70%of all ACLF in areas with a high incidence of hepatitis B.ACHBLF has an extremely poor prognosis due to a lack of understanding of its pathogenesis. Thus reducing the morbidity and mortality of hepatic failure patients has become a research hotspot. The search for biomarkers which can predict hepatic failure can strengthen the monitoring and treatment of patients with liver failure and reduce the incidence and mortality of ACHBLF. Current clinical research found that there was very close relationship between the pathogenesis of ACHBLF and strong immune response. Therefore, it is very important to fully understand the immune response of ACHBLF.In recent years, the extensive research in CD163make it possible to be an effective indicator and therapeutic target, which can be detected easily. The research of CD163is still in its infancy. However, their diagnostic and prognostic potential in patients with ACHBLF patients has not been investigated yet. That is the focus and direction of our research.Objective:To study the expression and significance of CD163in ACHBLF patients. We detected the surface CD163in peripheral blood mononuclear cells (PBMCs) and sCD163in peripheral blood in ACHBLF patients. We want to know the relationship between the expression of CD163, sCD163and the prognosis of patients with ACHBLF patients, and their relationship with clinical indexes and clinical significance. Methods:We detected the frequency of CD163+PBMCs using flow cytometry(FCM). We also evaluated the average mean fluorescence intensity (MFI) of CD163+PBMCs using FCM. Real-time transcription-polymerase chain reaction (RT-PCR) was performed to assess relative CD163mRNA levels in PBMCs. We evaluated the plasma levels of sCD163by enzyme linked immunosorbent assay (ELISA).Results:1. CD163positive in PBMCs in patients with ACHBLF is significantly higher than CHB patients and healthy controls(HCs), there were significant differences within three groups. However, there were no significant differences in MFI in CD163+PBMCs within three groups.2. CD163mRNA expression in ACHBLF patients was significantly higher than CHB patients and HCs, there were significant differences within three groups.3. Plasma sCD163expression was significantly elevated in patients with ACHBLF, and was significantly correlated with the prognosis of patients. There were significant differences within three groups.4. Plasma levels of sCD163were positively correlated with MELD score, plasma HBV-DNA levels and negatively correlated with PTA, but had no correlation with TBIL.5. CD163positive in PBMCs in CHB patients was significantly higher than that in HCs.6. CD163mRNA expression in CHB patients was higher than that of HCs, there was no significant difference between two groups.7. Plasma sCD163expression was significantly elevated in patients with CHB, there was significant difference between two groups.Conclusion:As expressed in the macrophage surface specific, CD163plays an important role in patients with ACHBLF and CHB in the pathogenesis.The research suggested that CD163and sCD163can be used as useful biomarkers for ACHBLF. SCD163detection is more simple and direct, with more significant group differences. It is significantly correlated with the severity of the disease. CD163and sCD163can be used as index of prognosis in patients with liver failure, and negatively correlated with the prognosis of the patients. 英文摘要第二部分INCREASED CD163AND SCD163IS ASSOCIATED HEPATOCELLULAR CARCINOMA ABSTRACTPeople who have hepatitis B are prone to have a much higher risk of suffering from hepatocellular caicinoma(HCC). Some scholars think that people who have chronic hepatitis B and liver cirrhosis with hepatitis B may have the probability of suffering from HCC9to10times higher than that of normal people. More than80%of HCC patients have hepatitis B virus infection. In all of the malignant tumors, HCC have mortality rates as high as the fifth. Its five-year survival rate is less than5%. These data show that understanding of the pathogenesis, early diagnosis and risk assessment in patients with HCC are very important. However, we lack of effective early prediction and monitoring method of HCC.It is widely interested that TAMs have an anti-inflammatory function resembling M2macrophages. CD163is a M2macrophages specific marker and macrophage activation marker. Its high expression can promote tumor angiogenesis, restructuring, affecting fibrosis tissue cells, organizing reconstruction process. It plays an important role in the pathological process of tumors and acute or chronic inflammation of the liver diseases.Nevertheless in the former part of experiment, we have found the high levels of CD163expression in patients with ACHBLF. We also found the relationship between the expression of CD163and sCD163and clinical prognosis in acute inflammation process. In this experiment, we selected the patients with HCC as the research object. CD163and sCD163may play an important role in patients with HCC. We are eager to know its relationship between the prognosis and clinical indexes, and look for the new direction of treatment.Objective:To study the expression and significance of CD163in HCC. We evalues the surface CD163on peripheral blood mononuclear cells (PBMCs) and the expression of sCD163in peripheral blood. We would like to understand the relationship between the expression of CD163and sCD163and the prognosis of patients with HCC and their relationship with clinical indexes and clinical significance.Methods:We determined the frequency of CD163+PBMCs using flow cytometry (FCM). We also evaluated the average mean fluorescence intensity (MFI) of CD163+PBMCs using FCM. Real-time transcription-polymerase chain reaction (RT-PCR) was performed to assess relative CD163mRNA levels in PBMCs. We evaluated the plasma levels of sCD163by enzyme linked immunosorbent assay(ELISA).Results:1. CD163+mononuclear cells increased significantly in patients with hepatocellular carcinoma (HCC), compared with liver cirrhosis (LC), chronic hepatitis B (CHB) and healthy controls (HCs) with significant difference. There was significant difference in mean fluorescence intensity(MFI) of CD163+PBMCS within groups.2. In patients with HCC, CD163mRNA expression is much higher than the patients of CHB and HCs.There was significant difference within groups.3. sCD163in plasma increased significantly in patients with HCC, and has close relationship with the prognosis of patients with HCC. There were significant differences within groups.4. CD163+mononuclear cells increased significantly in patients with LC, compared with CHB and HCs with significant difference.5. In patients with LC, CD163mRNA expression is higher than the patients of CHB and HCs. There was significant difference within groups.6. sCD163in plasma increased higher in patients with LC, compared with the patients of CHB and HCs with significant differences. 7. sCD163in plasma increased significantly in patients with HCC had no associations with AFP、HBV-DNA and HBeAg.8. There is no correlation between sCD163concentration and Italian CLIP scores. According to a2002AJCC international staging, namely the TNM staging, sCD163concentration in patients with HCC in phase Ⅲ of distant metastasis and Child C level was higher than patients in the stage Ⅰ, stage Ⅱ, but with no significance.Conclusion:CD163plays an important role in patients with HCC、LC in the pathogenesis. The research suggested that CD163and sCD163can be used as useful biomarkers in the treatment of patients with HCC. SCD163detection is much simpler and direct. It is significantly correlated with the severity of the disease. 英文摘要第三部分CD163REVIEW ABSTRACTForm and structure of CD163Monocytes and macrophages play an important role in the human immune system. Macrophages can be divided into M1subsets and M2subsets, which have proinflammatory and anti-inflammatory effects respectively. CD163only express on the macrophage surface of M2type. It belongs to the scavenger receptor cysteine rich domain family B and is a I type protein with130kD weight. It has5homologous isoforms, with structure containing9scavenger receptor cysteine rich domains. CD163has two forms in vivo:one is a transmembrane molecule existing in the form of mononuclear macrophages; the other one present in the blood or other tissue fluid, soluble, namely sCD163.Role of CD163CD163can specifically recognize haptoglobin hemoglobin complex and is a hemoglobin specific scavenger receptor. Through the clearance of free hemoglobin, it can adjust the body’s immune function. CD163can also identify factor of apoptosis (TWEAK). With its specific binding, it plays an important role in monitoring gram positive bacteria and gram negative bacteria. With inflammation stimulation, it can also promote monocyte adhesion to epithelial cells and induction of inflammatory mediators, such as inflammatory factors IL-10.Relationship between CD163and various diseasesCD163and sCD163have a close relationship with the immune system. Several clinical and laboratory studies found that CD163and sCD163are very closely related with various diseases, such as inflammation, tumor, cardiovascular or cerebrovascular diseases. In severe burns, acute liver failure and other serious inflammatory diseases, CD163increased significantly and related with prognosis. CD163increased significantly in HCC and malignant lymphoma, especially in patients with lymph node metastasis and poor response to therapy. It suggests an important role of CD163in prognosis of tumor patients. CD163and sCD163have been clearly proved to be significantly associated with the prognosis of the diabetes、hyperlipidemia and coronary heart disease.CD163and sCD163play important roles in anti-inflammation and promoting cancers, which make them popular in biological targets for future research and have already achieved initial success.更多还原