【作者】 余克强；

【导师】 罗仁；

【作者基本信息】 南方医科大学 ， 中西结合临床， 2009， 博士

【摘要】 研究背景类风湿关节炎（RA）是一种累及全身多个组织和脏器的慢性、炎症性疾病。主要病理过程是滑膜受累导致滑膜炎症,引起骨和软骨的损伤,最后发展为关节畸形和关节强直。RA也可以产生全身性弥漫性炎症,包括肺间质的改变、心包、胸膜、巩膜的受累,以及常见的皮下组织的皮肤结节。虽然RA的发病机制尚未完全阐明,但自身免疫在RA发病机制、发生和发展中发挥的关键作用已经得到广泛的认可。RA在全世界的发病率约为1%,女性与男性的比例为3:1。所有年龄均可发病,最常见的发病年龄40岁到50岁。RA是主要的致残性疾病之一,造成关节活动功能的丧失,给社会和家庭带来巨大的压力和负担。在RA的发病机制中,在免疫基因易感宿主的自身抗原诱导活化的T细胞发挥着重要的作用。T细胞活化后导致多种免疫效应,包括滑膜细胞的增殖,内皮细胞的活化。骨髓和外周血中前炎症细胞活化,巨噬细胞、成纤维样滑膜细胞、B细胞分泌大量的细胞因子和自身抗体。树突状细胞（DC）是体内功能最为强大的抗原提呈细胞,是唯一可以激活初始T细胞的抗原提呈细胞。DC起源于骨髓干细胞,平时寄居在组织和外周血中。外周组织中的DC识别抗原后在细胞因子的刺激下逐渐分化,高表达MHC分子和协同刺激分子,在向次级淋巴器官迁移的过程中逐渐成熟,而后在淋巴器官中与T细胞结合,刺激其活化或诱导其耐受。研究表明,RA患者滑膜和关节中浸润的T细胞周围可见成熟的DC。这些DC高表达MHCⅡ类分子和CD40、CD80、CD86以及DC-SIGN等粘附分子和趋化因子受体,对T细胞的活化和组织的损伤起着重要作用。RA属祖国传统医学痹证范畴。痹证的分类,按病因可分为风痹、寒痹、湿痹、热痹、风湿热痹等;从病理特点可分为行痹、痛痹、着痹。痹证的发生主要是由于正气不足,感受风、寒、湿、热之邪所致。内因是基础,素体虚弱、正气不足、腠理不密、卫外不固,是引起痹证的内在因素,如再感受外邪,使肌肉、关节、经络痹阻而形成痹证。痹证的治疗以祛风通络为基本大法,首先应辨明寒热,治以祛风、除湿、舒筋活络,随其寒热而兼行清热或祛寒。对于病程日久,气血损伤,脏腑亏虚的痹证患者,应配合运用补益之法。青藤碱是中药清风藤的单体提取物,用于治疗RA已经有2000年的历史。研究发现青藤碱有抗炎、抗风湿的药理作用。尽管青藤碱广泛的药理作用已经得到阐明,但其作用机制,尤其是免疫抑制的机理尚有待进一步研究。为了进一步阐明青藤碱的药理作用,探讨其免疫抑制和RA的机理,本课题观察了青藤碱对RA患者DC趋化因子及其受体表达的影响和机制。目的:1.观察RA患者DC表面趋化因子受体的表达,研究DC表面趋化因子受体与RA患者疾病活动的相关关系;2.建立DC趋化因子及其受体的荧光定量PCR检测体系;3.观察青藤碱对RA患者DC趋化因子及其受体表达的影响;4.观察青藤碱对RA患者DC泛素和E3连接酶表达的影响;5.揭示青藤碱免疫抑制的机理,为青藤碱治疗RA提供理论和实验依据。方法:1.人外周血DC体外培养体系的建立根据课题组已经建立的方法,分离人外周血单核细胞,RPMI-1640完全培养基培养,采用GM-CSF和IL-4联合刺激,于培养第8天收获。2.DC表面趋化因子受体与RA患者疾病活动性的关系选取28名RA患者和10名正常健康人分为四组:正常组、低活动组、中活动组和高活动组。采用流式细胞仪检测RA患者DC表面CCR5和CCR7的表达,收集患者类风湿因子（RF）、C反应蛋白（CRP）和抗CCP抗体。采用直线相关分析CCR5、CCR7和RA患者疾病活动指标的相关关系。3.青藤碱对RA患者外周血DC趋化因子及其受体表达的影响采用1mM,2mM,5mM青藤碱溶液和空白对照溶液干预DC 12h。收集干预后的细胞提取DC总RNA并逆转录为cDNA。PCR扩增CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH cDNA片段,回收并纯化PCR产物。将CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH的PCR产物分别按10⁸,10⁷,10⁶,10⁵,10⁴copies/μl梯度倍比稀释,荧光定量PCR仪检测并自动计算出样品的定量结果,描绘标准曲线。将各样本cDNA按照建立体系中的反应条件在荧光定量PCR仪上进行扩增,由电脑自动分析并计算出反应体系中待测样品cDNA达到设定阈值的循环数（Ct值）。以公式2^-△△t计算出相对于空白对照组的青藤碱干预各组的DC中CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH mRNA的表达水平。收集DC培养上清,检测经不同浓度青藤碱干预后DC培养上清中CXCL9、CXCL10、CXCL11的表达。4.青藤碱对RA患者外周血DC中泛素和E3表达的影响收集青藤碱干预后的RA患者外周血DC,提取DC总RNA和蛋白。Real timePCR检测DC中泛素和E3连接酶mRNA的表达,western-blotting检测青藤碱对RA患者DC中泛素和E3连接酶表达的影响。5.统计学处理采用SPSS13.0统计软件进行数据分析,结果以（?）±S表示,多组间均数比较采用One way-ANOVA,多个组分别与对照组比较采用Dunnett检验;连续变量间的直线相关采用Pearson相关分析,P<0.05为有统计学意义。结果:1.在GM-CSF和IL-4联合刺激的作用下,单核细胞成功的分化为DC。在光镜和电镜下观察可见其呈现典型的树突状形态,与对照组相比,RA低、中、高活动组患者CCR5（F=107.53,P=0.000）和CCR7（F=46.283,P=0.000）表达较高。2.在低疾病活动组（RF,r=0.775,P=0.014;CRP,r=0.802,P=0.009）、中疾病活动组（RF,r=0.0854,P=0.002;CRP,r=0.818,P=0.004）、高疾病活动组（RF,r=0.773,P=0.015;CRP,r=0.699,P=0.036）表达在RA患者DC表面CCR5与RF和CRP存在直线相关关系,相关性有统计学意义;与CCR5相似,在低疾病活动组（RF,1=0.754,P=0.019;CRP,r=0.968,P=0.000）、中疾病活动组（RF,r=0.0854,P=0.002;CRP,r=0.949,P=0.000）、高疾病活动组（RF,r=0.938,P=0.000;CRP,r=0.968,P=0.000）表达在RA患者DC表面CCR7与RF和CRP存在直线相关关系,相关性有统计学意义。3.流式细胞结果显示,青藤碱干预后DC表面CCR5（F=89.236,P=0.000）和CCR7（F=94.731,P=0.000）表达有显著差异,差异有统计学意义;与空白对照组相比,2mM、5mM sinomenine干预后DC表面CCR5和CCR7（P<0.05 or P<0.01）表达显著降低,差异有统计学意义;GAPDH、CCR5、CCR7、CXCL9、CXCL10、CXCL11阳性梯度定量模板数与Ct值关系对数拟合作图,得到不同梯度定量模板的对数值与循环数（Ct值）之间关系的标准曲线,分别有着非常好的相关关系（0.9943、0.9955、0.9926、0.9981、0.9938、0.9994）。荧光定量PCR检测结果显示,与空白对照相比,经2mM和5mM青藤碱干预后的DC中CCR5、CCR7、CXCL9、CXCL10、CXCL11 mRNA表达均有所有降低（P<0.05 or P<0.01）;ELISA结果显示,与空白对照相比,经青藤碱干预后的DC培养上清中CXCL9、CXCL10、CXCL11差异有统计学意义。4.荧光定量PCR结果显示,青藤碱4个剂量组之间RA患者外周血DC中泛素（F=41.222,P=0.000）、E3连接酶（F=59.657,P=0.000）mRNA表达有显著性差异;与空白对照组相比,经2mM和5mM青藤碱干预后DC中泛素和E3连接酶表达显著降低;Western-blot检测结果显示,经青藤碱干预后4组泛素和E3连接酶的表达均有所改变,差异有统计学意义。结论:1.建立了RA患者外周血树突状细胞趋化因子受体检测体系;2.不同疾病活动情况下RA患者DC表面趋化因子受体的表达与RA患者疾病活动指标存在直线相关关系,DC表面CCR5和CCR7可能是监测RA患者疾病活动和治疗效果的有效指标;3.青藤碱可有效抑制RA患者DC趋化因子及其受体的表达,青藤碱的免疫抑制机理可能与抑制DC的迁移有关,青藤碱可能通过抑制DC迁移到次级淋巴器官与T细胞结合,从而达到治疗RA的作用;4.青藤碱可能抑制泛素-蛋白酶体中重要蛋白的表达,抑制了泛素-蛋白酶体的功能,降低了NF-（?）B途径的磷酸化,从而影响了DC的免疫功能,达到免疫抑制的效果。更多还原

【Abstract】 BackgroundRheumatoid arthritis （RA） is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks the joints producing a inflammatory synovitis that often progresses to destruction of the articular cartilage and ankylosis of the joints. Rheumatoid arthritis can also produce diffuse inflammation in the lungs, pericardium, pleura, and sclera, and also nodular lesions, most common in subcutaneous tissue under the skin. Although the cause of rheumatoid arthritis is unknown, autoimmunity plays a pivotal role in its chronicity and progression. About 1% of the world’s population is afflicted by rheumatoid arthritis, women three times more often than men. Onset is most frequent in 40 to 50 years, but no age is immune. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility.The activation of T cells by as yet unknown antigens in the immunogenetically susceptible host is most probably the event that initiates the rheumatoid process. T cell activation subsequently leads to multiple effects including activation and proliferation of synovial lining and endothelial cells, recruitment and activation of additional proinflammatory cells from the bone marrow and circulation, secretion of cytokines and proteases by macrophages and fibroblast-like synovial cells, and autoantibody production. Dendritic cells are the most potent subset of antigen presenting cells. They are derived from bone marrow stem cells and reside in peripheral tissues or blood. Upon exposure to antigens and cytokines the peripheral DC s, express high amounts of peptide-MHC, and upregulate their costimulatory molecules, migrate to draining lymph nodes, and interact with T cells to stimulate or tolerize them. Dendritic cells have been found in synovium and joint fluid in rheumatoid arthritis, often at the center of a cluster of T cells. These DC s express MHCⅡ, the costimulatory molecules CD40, CD80, CD86, adhesion molecules such as DC-SIGN and chemokine receptors.According to Traditional Chinese Medicine, this condition is called Bi Zheng, which is typically divided into four types: Wind-Cold Bi, Cold-Bi, Dampness-Bi and Heat-Bi. Through a thorough examination and consultation, including an assessment of the pulse and tongue, a diagnosis is made. Therapeutic principles For incipient bi-syndrome, expelling pathogenic factors should be the main therapeutic principle, including dispelling wind, dispersing cold, clearing away heat, eliminating dampness and dredging meridians and collaterals. For a weak patient suffering from bi-syndrome or a chronic case with deficiency of healthy qi, in addition to the therapy for expelling pathogenic factors, tonifying the spleen, liver, kidneys and blood should also be employed. For cases complicated by phlegm and blood stasis, activating blood circulation, dissipating blood stasis and masses and eliminating phlegm should be applied. Sinomenine, an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, has been utilized to treat RA in China for over 2000 years. A wide range of pharmacological actions which includes anti-inflammatory and anti-rheumatic effects can be mediated by sinomenine. Although sinomenine exhibits a wide range of pharmacological activities, mechanistic study on sinomenine is very limited yet. In order to further illustrate the immunosuppressive action of sinomenine, we deeply investgated the effect and mechanism of sinomenine on chemokines and their receptors of DCs in patients with RA.Objectives 1.To explore the relationship between disease activity and expression of chemokine receptors on the surface of dendritic cells in patients with RA;2. To build up detection system of real time PCR for measuring chemokines and their receptors of dendritic cells;3. To investigate the effect of sinomenine on expression of chemokines and chemokines receptors of dendritic cells in patients with RA;4. To evaluate the effect of sinomenine on the expression of ubiquitin and ubiquitin-protein ligase of dendritic cells in patient with RA.Methods1. Culture of monocyte-derived DCs in patients with RA in vitroPeripheral blood was collected and blood mononuclear cells were isolated. DCs were stimulalted with GM-CSF and IL-4 by the methods built in our past research.2. Determining the relationship between expression of chemokine receptors of RA patients and disease activityTweenty eight patients and ten healthy control were chosen and disease activity score 28 were calculated. DCs from patients with RA were divided into four groups according to the disease activity of patients or healthy control. The expression of CCR5 and CCR7 were detected by flow cytometer. The level of serum rheumatoid factor, C reactive protein, anti-CCP antibody were observed. The correlation of expression of CCR5, CCR7 and disease activity of RA patients were analyzed.3. Detecting the effect of sinomenine on the expression of chemokines and their receptors of DCs in patients with RADCs harvested from patients with RA were exposed to sinomenine 1mM, 2mM, 5mM sinomenine and medium for 12h, respectively. After treated with sinomenine, total RNA of DCs in patients with RA was isolated. cDNA of CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH was amplified by PCR and products were purified. Templates were diluted in different concentration. Real time PCR was performed to build up standard curves for CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH mRNA. Expression of CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH mRNA for each sample was detected by real time PCR. The mRNA level of each sample for each gene was normalized to that of the GAPDH mRNA. The relative mRNA level was represented as 2^-△△t and expressed as the fold increase compared to the untreated cells. Then, expression of CXCL9, CXCL10, CXCL11 were detected by ELISA.4. Observing the effect of sinomeine on expression ubiquitin and E3 ligase of DCsAfter exposed to sinomenine, DCs were collected and total RNA and protein was extracted. Expression of ubiquitin and E3 were analyzed by real time PCR and western blotting, respectively.5. Statistical analysisAll data are expressed as means±SD. Differences among one-way designed groups were analyzed by one-way ANOVA followed by Dunnett test. Linear relationship between two random variables were tested by Pearson correlation. A value of p<0.05 was considered statistically significant.Result1. Monocytes were cultured in the presence of GM-CSF and IL-4. Cells became nonadherent and clustered, exhibiting protruding veils typical of DCs. In contrast to healthy control, higher expression of CCR5 and CCR7 were observed in low, middle and high activity group.2. There are linear correlation relationship between CCR5 expressed on DCs and disease activity RF, CRP, anti-CCP antibody of RA patients in low activity（RF, r=0.775, P=0.014; CRP, r=0.802, P=0.009）, middle activity（RF, r=0.0854, p=0.002; CRP, r=0.818, P=0.004） and high activity（RF, r=0.773, P=0.015; CRP, r=0.699, p=0.036） group, respectively. And the same relationship were also observed in low activity（RF, r=0.754, P=0.019; CRP, r=0.968, P=0.000）, middle activity（RF, r=0.0854, P=0.002; CRP, r=0.949, P=0.000） and high activity（RF, r=0.938, P=0.000; CRP, r=0.968, P=0.000） group for CCR7.3. Significant differences of expression of CCR5（F=89.236, P=0.000） and CCR7（F=94.731, P=0.000） were observed in DCs exposed to 1mM, 2mM, 5mM sinomenine or control. Compared with control, CCR5 and CCR7（P<0.05 or P<0.01） were down-regulated in DCs treated with sinomenine at dose of 2mM or 5mM. Standard curves were obtained by real time PCR. Perfect linear correlations between different multiproportion dilution template for quantitation and cycle number （0.9943 for GAPDH, 0.9955 for CCR5, 0.9926 for CCR7, 0.9981 for CXCL9, 0.9938 for CXCL10, 0.9994 for CXCL11） were found in each group. Sinomenine appeared to effectively inhibit the expression of CCR5, CCR7, CXCL9, CXCL10, CXCL11 mRNA in DCs. Compared with control, significant differences were found in DCs treated with 2mM and 5mM sinomenine （P0.05 or P<0.01）. Significant down-regulation of CXCL9, CXCL10, CXCL11 secretion were noticed in DCs exposed to sinomenine comparing to that treated with medium alone.4. There were significant differences at expression of ubiquitin（F=41.222, P=0.000） and E3 ligase（F=59.657, P=0.000） mRNA between control and sinomenine groups. In contrast to control, DCs treated with 2mM, 5mM sinomenine expressed lower ubiquitin and E3 ligase. The similar result was also observed in the expression of ubiquitin and E3 ligase in protein level detected by Western Blot assay.Conclusion1. Chemokine receptors are expressed on DCs in RA patients with different disease actitivy. Chemokine receptors can be used as a index to monitor the disease activiy of RA.2. Sinomenine can effectively suppress the expression of chemokines and their receptors of DCs in patients with RA at mRNA and protein level. Sinomenine exhibits the effect of relieving RA through inhibiting the expression of chemokines and their receptors, which can lead to DCs migration.3. DCs can effectively decrease the expression of ubiquitin and E3 ligase of DC. The influence of sinomenine on DCs may relate to inhibit the ubiquitin system, which account for the maturation and migration of DCs.更多还原