【作者】 岳红红；

【导师】 朱平；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 目的:类风湿关节炎(rheumatoid arthritis, RA)是一种以关节滑膜增生、关节软骨侵蚀破坏为特征的全身性、炎性自身免疫性疾病,迄今尚无根治疗法。研究表明,RA患者比普通人群病死率增高70%,其中35～50%为心血管疾病所致,心血管疾病尤其是快速发展的动脉粥样硬化是RA患者的首要死亡原因。多种炎症介质及炎性细胞参与了RA患者长期慢性的炎症性反应,导致一系列致动脉粥样硬化的代谢功能改变。这些代谢紊乱之间相互作用,最终促使RA患者动脉粥样硬化加速进展。在这些炎症介质中,新近发现的细胞膜表面粘附分子CD147日益受到了人们广泛的关注。CD147分子,又称为细胞外基质金属蛋白酶诱导剂(extracellular matrix metalloproteinase inducer, EMMPRIN),在多种肿瘤细胞和组织中高表达,通过诱导成纤维细胞分泌基质金属蛋白酶(matrix metalloproteinase, MMP)促进了肿瘤的侵袭和转移。研究表明,CD147在RA患者滑膜以及外周血和滑液中的各种炎细胞表面均有较高表达,在RA的病理损伤中起到了重要的作用。此外,在人动脉粥样硬化斑块及纤维帽中,CD147、MMP亦有高表达,推测CD147在人动脉粥样硬化中可能通过诱导活化MMP促进组成纤维帽的细胞外基质降解,强度减小,导致斑块纤维帽变薄破裂。因此,CD147可能在动脉粥样硬化的过程中发挥了重要作用。新近研究发现,氧化型低密度脂蛋白(ox-LDL)可以促进细胞膜表面CD147 ( membrane CD147 , mCD147 )从心脏冠状动脉平滑肌细胞(HCA-SMCs)表面脱落,并以可溶性的形式(soluble CD147,sCD147)继续发挥其生物学效应,加速动脉粥样硬化的发生。本实验室先前工作发现,CD147可以从外周血单核细胞(PBMo)表面脱落或被直接分泌进入外周血或关节液中,提示sCD147可能参与RA关节损伤与发病。为了深入研究sCD147与RA患者动脉粥样硬化发生和进展的相关性,全面评价RA动脉粥样硬化的危险因素,探讨在免疫调节的基础上进行治疗性干预,从而预防RA患者心血管事件的发生。本课题旨在:①检测RA患者外周血中sCD147的含量并与其血脂水平进行相关性分析;②建立单核(Mo)/巨噬细胞(Mac)来源的泡沫细胞模型,研究:CD147在U937-Mo源性泡沫细胞膜表面的动态表达;CD147在分化中的Mo/Mac及泡沫细胞中表达的变化;泡沫细胞的形成对MMP-2, 9合成的影响;③RA、冠心病患者和健康对照的Mac源性泡沫细胞的mCD147/sCD147表达对ox-LDL浓度的依赖性;sCD147对心脏成纤维细胞(HCF)合成MMP-2, 9的影响及CD147拮抗剂AP-9的抑制作用。方法:1.采用ELISA检测RA、冠心病患者及健康对照血清中sCD147含量,分析sCD147水平与RA患者病情活动度(DAS28)的相关性;以全自动生化分析仪测定RA患者血脂水平,分析血清中sCD147与血脂水平的相关性。2.分离RA、冠心病患者及健康对照PBMo,以GM-CSF刺激分化为Mac后,分别加不同浓度ox-LDL作用48h,建立PBMo-Mac源性泡沫细胞模型;将PMA诱导分化前、后的U937细胞加入不同浓度ox-LDL作用48h,建立U937-Mo/Mac源性泡沫细胞模型,并通过高效液相色谱法及油红“O”特殊染色鉴定泡沫细胞。3.将U937-Mo细胞分别在不同浓度的ox-LDL条件下培养48h,收集细胞;将U937-Mo细胞与80mg/L ox-LDL共培养,于不同时间点收集细胞。采用流式细胞术,RT-PCR检测CD147及其mRNA在U937-Mo源性泡沫细胞中的表达与ox-LDL浓度及培养时间的依赖性。4. U937细胞在PMA诱导下分化7天后,与不同浓度ox-LDL作用48h,使之成为U937- Mac源性泡沫细胞。采用流式细胞术及RT-PCR检测CD147在单核细胞分化过程中和泡沫细胞膜表面的表达及其mRNA水平;采用ELISA、western blotting、明胶酶谱检测不同浓度ox-LDL对泡沫细胞产生sCD147及MMP-2, -9的影响。5.选取15例RA患者、17例冠心病患者及12例健康对照,分离其PBMo,经GM-CSF诱导分化为巨噬细胞后,与不同浓度ox-LDL作用48h建立泡沫细胞模型。采用流式细胞术,western blotting检测泡沫细胞mCD147的表达; ELISA,western blotting检测相应细胞培养上清中sCD147的表达; RT-PCR检测泡沫细胞中CD147 mRNA的表达;明胶酶谱、RT-PCR检测ox-LDL (100mg/L)与巨噬细胞作用48h后,细胞培养上清+心脏成纤维细胞(HCF)、细胞上清+HCF+AP9、HCF等三组细胞MMP-2,9的表达以及CD147拮抗剂AP-9对MMP-2, 9产生的抑制作用。结果:1.RA患者血清中sCD147水平显著高于冠心病患者及健康人(P<0.05),且病情高度活动RA组其sCD147水平显著高于病情低度活动组及中度活动组(P<0.05)。总胆固醇(TC)升高组及甘油三酯(TG)升高组RA患者的TC/TG水平与其血清中sCD147水平呈正相关(r=0.84,P<0.05;r=0.87,P<0.05),而TC轻度升高及正常组或TG正常组患者的TC/TG水平则与其血清中sCD147水平无相关性(r=0.41,P=0.21;r=0.14,P=0.57;r=0.49,P=0.87);低密度脂蛋白胆固醇(LDL-C)升高及轻度升高组RA患者的LDL-C水平与其血清中sCD147含量呈正相关(r=0.86,P<0.05;r=0.81,P<0.05),LDL-C正常组两者则无相关性(r=0.78,P=0.22);RA患者高密度脂蛋白胆固醇(HDL-C)水平与其血清中sCD147水平无相关性(r=0.04,P=0.96;r=0.13,P=0.87)。2.未吞噬ox-LDL的细胞内胆固醇酯约占总胆固醇的15.52%,吞噬后约占细胞内总胆固醇的60%以上,符合泡沫细胞生物学特征。吞噬组细胞经油红“O”特殊染色后,光镜下可见胞浆呈泡沫样改变,并有红染颗粒,表明脂质染色阳性。建立的泡沫细胞模型在生物和形态学上均符合泡沫细胞的定义。3.ox-LDL浓度和作用时间动态分析:CD147表达高峰的浓度为25 mg/L;固定ox-LDL浓度为80mg/L时,CD147表达高峰的作用时间为6h;而CD147mRNA表达高峰时ox-LDL作用浓度及时间分别为25 mg/L、6小时,此后其mRNA水平不变。4. U937细胞在PMA诱导后的第1,3 d, mCD147平均荧光强度(MFI)较诱导前显著升高(P<0.05);但在第5,7d,mCD147较诱导前显著降低(P<0.05);U937细胞经PMA诱导分化的7天中,CD147mRNA水平较分化前升高约4～6倍(P<0.05)。随着ox-LDL作用浓度增加,泡沫细胞mCD147 MFI较作用前呈逐渐减弱趋(P<0.05) ;同步采用western blotting及ELISA检测显示,细胞培养上清中sCD147水平较作用前呈逐渐上升趋势(P<0.05)。在此过程中,CD147mRNA表达不变。随着ox-LDL浓度增加,泡沫细胞MMP-2 , 9的分泌及活性逐渐增强。5.流式细胞术及western blotting结果显示,随着ox-LDL作用浓度的增加,泡沫细胞mCD147表达较作用前呈逐渐减弱趋势(P<0.05);同步ELISA和western blotting检测结果显示,细胞培养上清中sCD147含量较作用前呈逐渐上升趋势(P<0.05),且分子量与细胞膜表面相近;上述减弱和上升趋势由强到弱分别RA、冠心病患者,健康对照,但在此过程中泡沫细胞CD147 mRNA的水平无变化。明胶酶谱及RT-PCR结果表明,富含sCD147的细胞培养上清与HCF共培养后可促进后者MMP-2,9的合成, CD147拮抗剂AP-9可以部分抑制这种促进作用。结论:1.RA患者体内sCD147分子可能参与了RA的发病,且与病情活动度及血脂的异常相关。2.所建立的动脉粥样硬化的病理细胞模型在生物和形态学上均符合泡沫细胞的定义。3.CD147在U937-Mo源性的泡沫细胞膜表面表达升高;短时间、低浓度ox-LDL促进CD147表达;泡沫细胞中CD147 mRNA水平与ox-LDL的作用浓度及时间无关。4.CD147可能仅维持单核细胞向巨噬细胞分化时的表达水平,并可能足以使分化的巨噬细胞在其成熟期执行特殊功能;ox-LDL促进泡沫细胞合成MMP-2, 9。5.ox-LDL可能通过促进分化后的RA PBMo释放sCD147;sCD147促进HCF合成MMP-2, 9;CD147拮抗剂AP-9可以抑制这种促进作用。更多还原

【Abstract】 Objective:Rheumatoid arthritis (RA) is a chronic destructive autoimmune disease characterized by the inflammation and progressive destruction of distal joints. 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. Studies suggest that the mortality rate is approximately twice as high in patients with RA compared with the general population. Cardiovascular disease accounts for 70% of excess mortality in RA patients. Especially , accelerated atherosclerosis could be responsible for it. Systemic inflammatory response in RA is central to the accelerated atherogenesis. Among these inflammatory cells and cytokines , CD147 , a cell surface glycoprotein , arouses great interest.Extracellular matrix metalloproteinase inducer (EMMPRIN) / CD147 is enriched on the surface of most tumor cells and shown to stimulate underlying stromal cells to produce elevated levels of MMPs. These elevated levels of MMPs become concentrated and provide a mechanism for tumor cells to invade and metastasize into the extracellular matrix. Most of the inflammatory cells in RA express CD147,indicating that CD147 may play an important role in the RA pathology.In addition, CD147 and MMP have also been shown to be expressed in human coronary atherectomy tissue, which may provide a mechanism for atherosclerotic plaque growth and potentially for lesion destabilization.Recently study showed that oxidized low-density lipoproteins stimulate CD147 release by coronary smooth muscle cells , an effect which seems to result from enhanced CD147 shedding. In turn , soluble CD147 might accelerate extracellular matrix degradation in atherosclerotic plaques and thereby promote plaque growth and plaque destabilization. Similarly, our laboratory has demonstrated that CD147 can shed from the human peripheral blood monocytes of RA patients or it can be directly secreted into the synovia, which play a role in the RA pathology.Although sCD147 expression appeared to be associated with atherosclerosis development, there is no detailed mechanism between atherosclerotic risk factors and sCD147 in RA. Studying these problems facilitated us to evaluate the atherosclerotic risk factors , pave the way of finding new therapy and decrease the risk of cardiovascular events in RA. Therefore, we aimed to :1. study the relationship between sCD147 and lipid level in serum of RA; 2. establish the Mo/Mac-derived foam cells model; 3. investigate the expression kinetics of CD147 in U937 cell-derived foam cells, and their relationship with atherosclerosis; 4. demonstrate the involvement of CD147 in the development of atherosclerosis and investigate its expression profile in monocytes, macrophages, and foam cells as well as the effects of foam-cell formation on MMP-2, 9 expression; 5. study the influence of oxidized low-density lipoproteins (ox-LDLs) on cell-associated CD147 and sCD147 expression in GM-CSF–differentiated human peripheral blood monocytes; MMP-2, 9 production by HCF after co-culturing with sCD147. Meanwhile the role of antagonistic peptide(AP-9)in the interaction of sCD147 with HCF.Methods:1. The sCD147 level in patients with RA, CAHD and healthy volunteers were detected by ELISA ; The disease activity score (DAS28) in RA patients was evaluated and the correlation between sCD147 level and DAS28 score was analyzed; The serum lipid level in patients with RA was detected by an automatic biochemical analyzer and the correlation between sCD147 and lipid level in serum was analyzed.2. GM-CSF–differentiated human peripheral blood monocytes incubated with different concentration of ox-LDL for 48 h in RA, CAHD and healthy then Mac-derived foam cells were established; After and before PMA–differentiated U937 cells incubated with different concentration of ox-LDL for 48 h then U937 Mo/Mac- derived foam cells were established.The foam cells were identified by High performance liquid phase chromatography and oil red O dyeing.3. The U937 cells were incubated with different concentrations of ox-LDL for 48 h. Then under 80mg/L ox-LDL , the U937 cells were incubated for different time duration. CD147 was analyzed at protein and mRNA levels in U937 foam cells by flow cytometry, and RT-PCR.4. U937 cells were stimulated with PMA and were allowed to differentiate into macrophages during culture for 7 d in plastic dishes. On day 7, the macrophages were incubated with various concentrations of oxidized low-density lipoprotein in a serum-free medium for 48 h to permit their development into foam cells. The protein and mRNA expression levels of CD147 in the U937 cells, macrophages, and foam cells were determined by immunofluorescence flow cytometry and RT-PCR. To assess the effects of lipid loading in macrophages on the CD147 level and MMP-2 , 9 expression in supernatants , ELISA, western blotting, and gelatin zymography was conducted.5. Sample of human peripheral blood were obtain from patients with RA(15), CAHD(17) and healthy donor volunteers(12). Monocytes were isolated from peripheral blood. GM-CSF–differentiated human peripheral blood monocytes incubated with different concentration of ox-LDL for 48 h. CD147 expression in foam cells was analyzed by Flow cytometry and western blotting; sCD147 level in supernatants was analyzed by ELISA and western blotting; CD147 mRNA in foam cells was analyzed by RT-PCR; Zymography analysed MMP-2, 9 release by HCF after co-culturing with sCD147. In additional , the influence of the inhibitor AP-9 on MMP-2, 9 was also observed.Results:1.The level of sCD147 in serum with RA patients was significantly higher than that in patients with CAHD and healthy volunteers. sCD147 level in the RA group with high DAS28 score was significantly higher than that with low or media DAS28 score. In RA patients , elevated total cholesterol(TC)and triglyceride(TG)was positively correlated with serum sCD147 level(r=0.84,P<0.05; r=0.87,P<0.05; while lightly elevated , normal TC and normal TG had no correlation with serum sCD147 level (r=0.41,P=0.21;r=0.14,P=0.57;r=0.49,P=0.87). Elevated or lightly elevated LDL-C was positively correlated with serum sCD147 level(r=0.86,P<0.05;r=0.81,P<0.05), while there was no correlation in the group with normal LDL-C level (r=0.78,P=0.22). The high density lipoprotein-cholesterol (HDL-C)level in RA patients had no correlation with serum sCD147 level(r=0.04,P=0.96;r=0.13,P=0.87).2.The CE/TC in lipid-loaded foam cells is 60% , which was higher than that in nonlipid-loaded cells(15.52%). After lipid-loaded foam cells treated with Oil red O, many red pellets were found in the plasma of the cells. Thus the cell model accorded with foam cell in biology and morphology.3. The expression dose-kinetics demonstrated that the CD147 showed the peak expression induced by ox-LDL 25mg/L;Time-kinetic studies revealed that the CD147 levels showed the peak expression at 6 h ;CD147 mRNA levels showed the peak expression induced by ox-LDL 25 mg/L,and the maximum mRNA level of CD147 was shown at 6h. However, ox-LDL enrichment of foam cells had no further effect on CD147 expression.4.During the PMA-induced differentiation of U937 cells into macrophages in the 7-d culture, CD147 mRNA expression increased 4- to 6-fold in the differentiated monocytes when compared with the undifferentiated ones. The mean fluorescence intensity of CD147 in the differentiated U937 cells was higher than that in the undifferentiated U937 cells (P<0.001) on days 1 and 3. On days 5 and 7, the mean fluorescence intensity of CD147 in the differentiated U937 cells was lower than that in the undifferentiated ones (P<0.05).The mean fluorescence intensity of CD147 gradually reduced in the foam cells that were treated with various concentrations of ox-LDL when compared with the untreated macrophages. In contrast, the expression of CD147, MMP-2 and MMP-9 was steadily upregulated in the corresponding culture supernatant (P<0.05), however, the mRNA expression of CD147 remained unaltered throughout the experiment.5. CD147 expression gradually reduced in the foam cells that were treated with various concentrations of ox-LDL when compared with the untreated macrophages(P<0.05). The declining trend is RA, CAHD and healthy respectively. In contrast, sCD147 was steadily upregulated in the corresponding culture supernatant. The trend of escalation is RA, CAHD and healthy respectively. In addition, sCD147 could stimulate HFC to produce elevated levels of MMP-2 and MMP-9 , which could be suppressed by AP-9.Conclusions:1. sCD147 may be involved in the pathogenesis of RA and associated with the disease activity. Elevated sCD147 level may associated with dyslipidemia in RA.2. The cell model was established in vitro and accorded with foam cells in biology and morphology.3. CD147 expression was enhanced in the U937 foam cells; CD147 expression was mainly up-regulated by low dose of ox-LDL; Ox-LDL has no further effect on CD147 mRNA expression.4. CD147 mRNA and protein expression were markedly increased 4- 6 fold on GM-CSF–induced monocyte to macrophage differentiation. Such increases in a protein maybe provide the differentiated macrophage with a mechanistic framework to perform specific functional activities at this stage of cellular maturation. In addition, lipid loading in macrophages reduced cell-associated CD147 expression, while it up-regulated the CD147 levels and secretion and activation of MMP-2, 9 in the culture supernatant. Howerver, lipid loading in macrophages had no further effect on CD147 mRNA expression. we hypothesize that the sCD147 maybe shed from the foam cells surface.5. Ox-LDLs maybe stimulate the release of sCD147 in differentiated PBMo; sCD147 stimulated MMP-2, 9 synthesis in HCF; AP-9 treatment could inhibit this process.更多还原