【作者】 李晓燕；

【导师】 朱平；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 类风湿关节炎(Rheumatoid arthritis,RA)是一种系统性的自身免疫性疾病,虽然其确切的发病机制还不清楚,但普遍认为可能是由外源性或内源性的抗原对具有某些遗传易感背景的人触发一系列在滑膜中的自身免疫反应。其临床特点为累及周身关节的增殖性和侵蚀性滑膜炎,是类似于“局限性恶性肿瘤(locally invasive tumor)”的增生性和破坏性病变,呈进行性发展,自行缓解罕见。以往的研究证实了RA的滑膜细胞及巨噬细胞通过分泌基质金属蛋白酶(matrix metalloproteinase,MMPs)对关节软骨的破坏及加重疾病的病情,而其中CD147分子则是已知的一个MMPs重要调节因子。CD147又称为细胞外基质金属蛋白酶诱导剂(extracellular matrix metalloproteinase inducer, EMMPRIN),广泛表达于人血管内皮细胞、外周血细胞、造血系统起源的细胞表面,属于免疫球蛋白超家族,细胞环亲素A(yclophilin A,CypA)的受体之一。在RA关节腔内大量存在的CypA同样作为一个重要的促炎因子,在其他许多炎性及肿瘤等疾病中的作用都见报道,本实验室以往的研究显示CypA对RA滑液中的单个核细胞的趋化作用显著高于正常人外周血单个核细胞,且这一作用可被抗CD147抗体所阻断,提示了单个核细胞膜CD147分子可能参与了CypA对单个核细胞的趋化调节作用。但是到目前为止,对于CypA与CD147相互作用于淋巴细胞,尤其是B淋巴细胞的报道未曾见到。RA的滑膜下层可有大量的T细胞(CD4+细胞为主)、B细胞,巨噬细胞及浆细胞浸润,侵入的单个核细胞集聚在一起可形成淋巴样滤泡,少数发展成生发中心;生发中心的浆细胞有合成免疫球蛋白和RF的能力。然而,RA滑膜局部B细胞活化增生以及浆细胞抗体类别转化的机制尚不明确,通过对CypA与CD147相互作用、CypA在RA滑液内大量存在及CD147在RA中单核/巨噬细胞高表达等文献回顾以及本实验在单个核细胞研究实验基础上,本课题拟观察这一相互作用在B淋巴细胞是否也存在,初步研究CypA对滑膜及滑液中B细胞上CD147的表达,以及对B细胞活化分泌自身抗体作用的研究。流式细胞仪(flow cytometry,FCM)分析正常个体的外周血提示:B细胞的一小部分,T细胞和单核细胞表达EMMPRIN,然而在ATLL患者中,表达EMMPRIN的T细胞数目是多的,蛋白的表达水平是异常高的。体外,EMMPRIN表达阳性的人类T细胞亲淋巴病毒Ⅰ型(human T-cell lymphoma virus,HTLV-1)转化的淋巴细胞(transformed lymphocyte,MT-2)和EMMPRIN表达阴性人成纤维细胞共培养比单独培养提高了前-MMP-2(明胶酶A)和活化MMP-2的产生。这些刺激可被拮抗EMMPRIN的活化阻断蛋白抑制。近年来大量的资料显示慢性炎症在一些肿瘤从癌前阶段演化为完全癌症中发挥重要角色。肿瘤和炎症之间的链接很长时间都成为人们的疑点。本实验室以往的研究显示CypA通过单个核细胞膜CD147分子参与对该细胞的趋化调节作用,CD147分子在健康人成熟单个核细胞的表达与增生的炎症细胞(癌前细胞)及肿瘤细胞的表达呈依次增高的趋势。但关于CD147在炎症和肿瘤中活化B细胞上的表达水平研究尚未见报道,以及在B细胞不同活化阶段cyclophilin-CD147作用发生疾病机制的研究尚未探讨。因此,本课题研究将CypA通过CD147分子相互作用、CD147分子介导信号通路的研究思路用于CD147在B细胞不同发育转化阶段表达中的研究,该结果对进一步认识CypA、CD147分子及两者相互作用复合物在炎症、癌前病变以及肿瘤转化过程中发病机制的作用有着重要的意义,并为探索炎症和肿瘤转化中的生物标记及靶向治疗提供了良好的理论基础。方法:1. Cyclophilin A-CD147活化B细胞在RA发病中的作用收集RA患者严重骨侵袭者取膝关节滑膜组织,首先制备组织切片,采用免疫组化染色(分别用CD19、CD35、CD38标记B淋巴细胞、滤泡样树突状细胞、浆细胞)观察RA滑膜组织中B淋巴细胞生发中心的形成,进而免疫组化染色观察CD147在其中的表达分布特点;分离RA滑液单个核细胞,加入CypA,B细胞活化因子(BAFF),脂多糖(LPS)作用细胞,于12、24、48h,分别收集培养悬浮单个核细胞,采用三重标记法(CD147-FITC、CD3-PrecP、CD19-APC),流式细胞术法测定相关分子。检测不同刺激后CD19+ B细胞表面CD147表达MFI的变化;收集CypA,BAFF,LPS作用后滑液细胞的培养上清,检测RF亚型,抗CCP抗体,及免疫球蛋白的分泌水平,并用滑液中总免疫球蛋白校正。观察不同刺激作用后自身抗体分泌水平。2.急性B淋巴细胞白血病为代表的血液系统肿瘤中B细胞CD147的表达为了研究CD147在急性白血病患者骨髓白血病细胞上的表达,尤其是幼稚B细胞上的表达,本实验采用流式细胞术三色免疫荧光直接标记技术检测,比较健康供者和急性白血病患者及其治疗缓解期患者骨髓中不同细胞亚群尤其是白血病细胞上CD147表达的差异,并追踪观察骨髓移植或者化疗后CD147表达的变化。3. CypA通过CD147促进B淋巴瘤细胞侵袭和转移的体内研究为了进一步深入研究是否CypA通过CD147途径参与B细胞的免疫学机制,并排除其它因素的干扰,本研究选取表型为CD19+CD10+CD147high的B细胞淋巴瘤细胞系Raji细胞,体外经CypA作用后经尾静脉注入Scid小鼠体内,观察Raji细胞在Scid小鼠体内侵袭和转移情况以及分泌MMP等方面的功能机制。结果:1. CD147在RA滑膜淋巴样组织中的表达与分布,CypA对滑液B细胞表达CD147的调节15例RA患者,均具有膝关节严重骨侵袭以及典型RA临床表现,取滑膜标本收集滑液分离单个核细胞。组织学和免疫组化结果显示,15例RA患者滑膜标本中13例有生发中心样的组织结构,并且13例RA滑膜组织滤泡样生发中心中均存在不同程度的CD147的表达。其中阳性6例,强阳性7例。镜下计数在13例RA滑膜淋巴滤泡中CD147表达阳性的细胞,淋巴细胞30±8%,滤泡样树突装细胞细胞平均为32±7%,血管内皮细胞平均为38±11%,浆细胞细胞几乎不表达;进一步本实验通过CypA,BAFF,LPS作用以上13例滑膜存在淋巴滤泡样组织结构RA患者滑液单个核细胞,收集悬浮细胞及上清,检测不同刺激后CD19+ B细胞表面CD147表达MFI的变化及自身抗体分泌水平,提示:CypA(200ng/ml),BAFF(25ng/ml), LPS(20ng/ml)作用后48h CD19+ B细胞表面CD147表达MFI、RF和anti-CCP水平最高,显著高于刺激前水平(p<0.01),且CypA与BAFF、LPS作用间无显著差异(p>0.05)。2.急性B淋巴细胞白血病为代表的血液系统肿瘤中B细胞CD147的表达流式细胞术分析显示,急性白血病初发患者的骨髓中,白血病细胞膜表面CD147表达的平均荧光强度(mean fluorescence intensity,MFI)(97.96±14.07,p< 0.01)显著高于健康供者(66.61±15.59)和治疗缓解期患者(73.72±8.54),其他细胞群(淋巴细胞,中性粒细胞,有核红细胞)CD147表达的MFI亦显著高于健康供者及治疗缓解期患者(p< 0.01);进一步比较急性B淋巴细胞白血病初发患者白血病细胞CD147的表达(105.96±5,p< 0.01)显著高于健康供者白血病细胞群中幼稚B系细胞(CD10/CD19) (35.76±8.49),而急性B淋巴细胞白血病骨髓移植治疗后CD147的表达亦降低(58.46±1.26,比较健康供者p< 0.05)。同时,分析比较不同急性白血病亚型的CD147表达时发现,急性B淋巴细胞白血病(ALL-B)骨髓中白血病细胞亚群CD147的表达(105.96±5)显著高于患者本人骨髓中成熟B淋巴细胞(43.46±12.34,p< 0.01)。并在ALL-B不同亚型的患者骨髓中白血病细胞亚群CD147的表达时发现,越幼稚亚型的ALL-B患者骨髓中白血病细胞亚群CD147的表达越高,随着幼稚细胞百分率的增高CD147的表达也增高,并且其表达在治疗缓解期患者的表达中有回归健康供者表达特点的趋势,这一免疫分型的分布特点有一定的规律性。然而分析其它急性白血病的幼稚细胞并没有发现明显的规律性。3. CypA通过CD147促进B淋巴瘤细胞侵袭和转移的体内研究流式细胞术显示Raji细胞大多为CD19+CD10+细胞(约占85%),证明其为较幼稚的B淋巴细胞,并且表面均表达CD147,MFI达到337.56,即为CD19+CD10+CD147high表达,符合进一步功能实验细胞株的要求。体外用CypA(200ng/ml)作用Raji细胞培养48h后,经尾静脉免疫注入Scid小鼠,3周后观察其PBMC,骨髓细胞,肝细胞,脾脏细胞中CD147+细胞转移情况,发现:用未经处理的Raji细胞免疫小鼠,其骨髓细胞中(15.07±5.43%)、肝细胞中分别为(25.75±6.58%)、脾脏细胞中分别为(19.13±4.33%),CD19+CD10+CD147+细胞百分率显著低于经CypA作用的Raji细胞免疫小鼠,分别为(21.11±6.02%)、(47.77±10.23%)、(39.65±7.23%,p <0.01)。而对小鼠的肝脏和脾脏的组织学和免疫组化研究显示,输注未经或CypA作用过的Raji细胞,两组小鼠CD19+CD10+CD147+细胞荧光表达没有显著差异。并且MMP-9组成以溶解形式存在于免疫小鼠血清中,体外CypA(200ng/ml)作用Raji细胞48h后,Scid小鼠血清MMP-9水平显著高于未经处理的Raji细免疫组。结论:本研究提示CD147可能作为生物标志用于急性白血病特别是急B淋的早期诊断和预测治疗后复发;CypA、CD147有可能是RA和急性白血病特别是急B淋的药靶。深入研究将有助于阐明CypA与CD147相互作用参与炎症、癌前病变以及肿瘤转化过程的发病机制,探索抗炎和抗肿瘤靶向治疗的新策略。而本研究的结果和研究体系为下一步的研究提供了理论和实验基础。更多还原

【Abstract】 Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammation at synovium. The clinical feature of RA is manifested by proliferative and erosive synovitis similar to locally invasive tumor. Until now, the pathogenesis of RA has not been understood well and the therapy of RA just controls the symptom and development of RA, but doesn’t cure it thoroughly.Previous studies showed that membrane CD147 expression on monocytes/ macrophages in RA synovium is much higher than that on monocytes/macrophages derived from RA patient’s peripheral blood and that CD147 overexpression on synoviocytes in RA synovium enhances the production of MMP-2, -9 and the invasiveness of synoviocytes. CD147 also named extracellular matrix metalloproteinase inducer (EMMPRIN), belongs to immunoglobulin superfamily and is one of Cyclophilin A (CypA)’s receptors. It is an important role in the embryonic development, organ formation, bony remodeling, wounds repairing in physiology, and tumor metastasis, arthritis and fibrosis in pathophysiology. CypA, involved in numerous inflammatory events, is abundant in RA synovial fluid and is the major target of immunosuppressive drug cyclosporin A (CsA). It has multiple functions, such as peptidyle-prolyl-isomerase activity (PPIase) for protein folding and transducting, taking part in the immunologic derangement and contributes to inflammation. The potential role of interaction between CypA and CD147 has been shown in some diseases, such as AIDS, SARS, and prostatic carcinoma.FACS analysis of peripheral blood from normal individuals revealed that small fractions of B-cells, T-cells, and monocytes expressed emmprin, whereas emmprin-expressing T-cells were much increased in number, and expressed this protein to a higher level, in ATLL patients. In vitro co-cultures of emmprin-positive HTLV-1-transformed lymphocytes (MT-2) and emmprinnegative human fibroblasts enhanced the production of pro-MMP-2 (gelatinase A) and active MMP-2, compared with cultures of either cell type alone. This stimulation was inhibited by an activity-blocking peptide against emmprin.In recent years a body of evidence has accumulated to show that chronic infl ammation can play an important role in the progression of some types of tumors from a premalignant state to full-blown disease. A link between cancer and infl ammation has long been suspected. Previous studies in our lab showed that CypA enhanced MMP-9 expression and adhesion of monocytes/macrophages by its direct binding to CD147. The expression of CD147 has been shown elevation on the surface of normal mononuclearcell, proliferative inflammatory cells and tumour cells successively. But CD147 expression on the actviated and malignant human B-cell subsets, and the pathogenesis of CypA-CD147 pathway in different activated B-cell stage has not been reported yet.So in this study, it is the first time that the interaction between CypA and CD147 will be studied for CD147 expression on B-cell development stage. The results of the study we believe may help us to gain a better understanding of the potential role of CypA-CD147 pathway in immflammation, precancerosis and neoplasia pathogenesis, and may give good ideas for the future biological tag and targeted therapy of autoimmune diseases and neoplasia therapy.Methods:1. Cyclophilin-mediated CD147 expression on B-cell from autoimmune diseases (RA)Samples of synovium and synovial fluid (SF) were obtained from 15 patients with active RA in Xijing Hospital. Streptavidin/peroxidase (SP) immunostainings were performed to detect the existence of B-cell germinal center (GC)-like reacrion and CD147 expression at the synovium derived from 15 patients with RA; FACS analyses were performed to quantify the levels of CD147 in synovial fluid B-cells (CD147-FITC、CD3-PrecP、CD19-APC) stimulated CypA, BAFF and LPS. At the same time, SF levels of RF and anti-CCP were quantified by rate nephelometry and ELISA, and corrected to synovial total IgM and IgG, respectively.2. CD147 expression on the malignant B-cell subsets in acute lymphoblastic leukemiaTo analyze CD147 expression by leukemic cell in boon marrow of acute lymphoblastic leukemia patients, especially by immature B-cell, were stained with a mixture of CD34/CD33 , CD10/CD19 , CD10/CD11b, and incubated with anti-CD147 monoclonal antibody were analyzed on properly compensated FACSCalibur. To study the relationship between CD147 and leukemia, we evaluated the expression of CD147 using this method on acute leukemia primary patient, post-treatment patients and contro and examined the availability of this procedure for evaluation of the quality of AL-remission.3. CypA stimulation to B lymphoma cell line invasion and malignant ability via CD147 pathway in vivoTo clarify the mechanism of in vivo invasion and metastases of Cyclophilin-CD147 interactions on B-cell, we examined the organ distribution of CD19+CD10+CD147high Raji cell (B lymphoma cell line) stimulated by CypA in severe combined immunodeficiency (SCID) mice using FACS and SP immunostainings.Results:1. Cyclophilin-mediated CD147 expression on B-cell from autoimmune diseases (RA)All the RA patients met the 1987 revised diagnostic criteria of the AmericanCollege of Rheumatology and have severe bone degradation. Expression of GC-like reacrion was detected at synovium from 13 of the 15 patients with RA, and the expression of CD147 of GC-like reacrion was detected in the 13 patients with RA. There was an apparently increased expression of CD147 in follicular synovitis the 7 patients with RA. Among the in folliculus lymphaticus of RA synovium expressing CD147, the lymphocytes share 30±8%, the folliculardendritic cells (FDCs) share 32±7%, the endothelial cells share 38±11%, and the plasma cells was negative, respectively; Mononuclearcells were isolated from synovial fluids of RA. The the mean expressional levels (MFI) of CD147 of CD19+ B-cell and corrected synovial fluid levels of RF and anti-CCP of RA patients were significantly higher after CypA, BAFFor LPS stimulation than those of control group(p<0.01), which was similar to result when stimulated by CypA, BAFFor LPS (p>0.05).2. CD147 expression on the malignant B-cell subsets in acute lymphoblastic leukemiaThe expression of CD147 on leukemic cells was evaluated by flow cytometry. The MFI of CD147 expression on leukemic cells (97.96±14.07,p< 0.01) was higher in the primary patient than in post-treatment patients (73.72±8.54) and normal control groups (66.61±15.59), with no significant difference between post-treatment patients and normal control (p >0.05). CD147 expression on lymphocyte, neutrophil, and erythroblast were also higher in the primary patient than in post-treatment patients and normal control groups (p< 0.01). The MFI of CD147 positive staining cells in leukemic B-cells (CD10/CD19) in primary B-ALL patient (105.96±5,p< 0.01) was higher than post-treatment patients (58.46±1.26) and normal control(35.76±8.49). At the same time, we found CD147 expression on leukemic cells (105.96±5) was higher than on mature B-cell (43.46±12.34,p< 0.01) in boon marrow of B-ALL. Relative to several differentiated B-ALL, the immaturer-B subtypes of ALL leukemia cells in the bone marrow of patients with the expression of CD147 higher, and with the increased percentage of immature cell, CD147 expression also increased. Its expression in the treatment of patients degraded to the normal expression, and there is certain degree of regularity of immune distribution. However, analysis of other acute leukemia leukemic cells and found no clear pattern.3. CypA stimulation to B lymphoma cell line invasion and migration ability via CD147 pathway in vivoOur next question was to address the correlation between CD147 expression and cyclophilin-mediated responses in malignant B-cell. Thus, we detected the capacity of CypA to induce malignant in Raji cell (B lymphoma cell line) by injected into SCID mice. For these studies, Raji cell expressed CD19+CD10+(about 85%), belong to immature B lymphocytes, and expressed CD147, MFI reached 337.56, which is CD19+CD10+CD147high expression, in line with further functional requirements of the experimental cells. The percentage of CD19+CD10+CD147+ cells in Raji cells stimulated by CypA in mice was significantly higher than in Raji cells alone immunized mice In mice immunized with the liver and spleen tissues observed (p <0.01), CD19+CD10+CD147+ cells expressed no significant difference in fluorescence in Raji cells stimulated by CypA and Raji cells alone after two groups of mice immunized. And CypA significantly increased MMP-9 expression, not MMP-2.Conclusions:Studies here showed that CD147 may be used for early diagnosis of ALL as biomarkers and for targeted therapy provide experimental basis. These findings may give some good ideas for the future biological tag and targeted therapy of autoimmune diseases and neoplasia therapy.更多还原