雪腐镰刀菌烯醇能干扰培养软骨细胞表面黏附分子CD44的表达

背景：雪腐镰刀菌烯醇和硒缺乏与大骨节病的发生有一定的联系。透明质酸的代谢直接影响着蛋白聚糖的聚合和软骨的正常结构与功能。透明质酸代谢的关键环节是与软骨细胞表面透明质酸受体的结合。软骨细胞膜上的CD44作为透明质酸的主要受体，其表达直接影响透明质酸的代谢，继而影响软骨基质代谢，对维持软骨基质的结构与功能具有极其重要的意义。目的：探讨大骨节病有关病因因素对靶组织细胞的损伤和保护作用以及引起软骨细胞变性坏死的机制。设计：随机对照观察。单位：西安交通大学医学院遗传学与分子生物学系。材料：实验于2002-10／2004-07在西安交通大学环境与疾病相关基因教育部重点实验室完成。选择30d龄的新西兰纯种幼免1只，手术截取兔肱骨、股骨和胫骨。方法：采用细胞培养法于体外再建软骨组织模型，并加入不同浓度的大骨节病可疑致病因子雪腐镰刀菌烯醇和保护因子硒，检测软骨细胞膜上透明质酸受体CD44和细胞培养液中可溶性CD44。主要观察指标：①软骨细胞表面黏附分子CD44镜下观察。②软骨细胞培养液中可溶性CD44浓度。结果：①软骨细胞表面黏附分子CD44镜下观察。软骨细胞膜上CD44的表达随着雪腐镰刀菌烯醇浓度的增加而减少，加硒后有增加趋势。②软骨细胞培养液中可溶性CD44浓度：细胞培养液中可溶性CD44浓度随雪腐镰刀菌烯醇浓度升高逐渐降低，但高浓度组出现了增高，加硒后趋势不变；除空白对照组与加硒对照组外，组间差异显著（P〈0．05）。结论：雪腐镰刀菌烯醇能干扰软骨细胞表面黏附分子CD44表达，进而引起软骨细胞外基质代谢紊乱；补硒能够拮抗雪腐镰刀菌烯醇对软骨细胞的损伤，但作用有限。     

雪腐镰刀菌烯醇对培养软骨细胞蛋白聚糖合成的影响

背景:雪腐镰刀菌烯醇是真菌毒素之一,它可能与大骨节病的发生具有一定的相关性.目的:验证雪腐镰刀菌烯醇对软骨细胞蛋白聚糖合成的影响.方法:在体外单层培养的人胚软骨细胞中加入不同质量浓度(0.025,0.05,0.1,0.2,0.4,0.8,1.6 mg/L)的雪腐镰刀菌烯醇毒素,作用1～5 d后收集软骨细胞,用MTT法检测软骨细胞存活率;用紫外分光光度法检测细胞DNA含量,RT-PCR方法检测蛋白聚糖mRNA表达;用Western blot法检测人软骨细胞蛋白聚糖表达.结果与结论:0.025 mg/L雪腐镰刀菌烯醇毒素可刺激软骨细胞生长,其刺激作用呈先升高后降低的趋势.0.05～0.1 mg/L毒素作用早期的细胞存活率增加,随后细胞生长被毒素抑制.当毒素质量浓度升高至0.2 mg/L以上时,随着雪腐镰刀菌烯醇毒素质量浓度的增加及作用时间延长,细胞存活率明显下降.0.1～O.5 mg/L雪腐镰刀菌烯醇毒素可以抑制软骨细胞蛋白聚糖的合成及其mRNA表达.结果表明雪腐镰刀菌烯醇毒素对软骨细胞蛋白聚糖合成有明显的抑制作用,并以剂量和时间依赖性方式影响细胞的增殖.     

黏附分子CD44表达对白血病细胞黏附、迁移及浸润的影响

目的 探讨黏附分子CD44表达对白血病细胞黏附、迁移、浸润的影响.方法 选择对数生长期的白血病细胞株SHI-1、THP-1、NB4、K562细胞,采用逆转录-聚合酶链反应(RT-PCR)和Western blot法检测各种白血病细胞株CD44 mRNA和蛋白的相对表达水平,并将各株白血病细胞分为对照组(加入同种同型IgG)和实验组(加入CD44单抗),然后观察白血病细胞与人静脉内皮细胞系ECV304细胞的黏附率;用包被ECV304细胞的Transwell小室培养法观察细胞迁移率;用包被人工基质膜Matrigel的Transwell小室培养法观察白血病细胞穿过人工基质膜的浸润能力.结果 SHI-1、THP-1、NB4细胞均表达CD44 mRNA和蛋白,而K562细胞CD44 mRNA和蛋白表达量少甚至不表达;SHI-1、THP-1 、NB4细胞CD44 mRNA的相对表达水平分别为0.0731±0.0072、0.0827±0.0151、0.1473±0.0365,与K562细胞(0.0002±0.0000)相比,差异均有统计学意义(P值均<0.01).黏附实验结果显示:实验组SHI-1、THP-1、NB4细胞黏附率均较对照组下降(分别为72.78%、64.09%、57.42%),而实验组K562细胞黏附率为106.16%.迁移实验结果显示:对照组SHI-1、THP-1、NB4细胞迁移率分别为55%、29%、25%,实验组细胞迁移率下降(分别为32%、18%、12%),而两组中K562细胞无明显变化(均为2%).浸润实验显示:对照组SHI-1、THP-1、NB4细胞穿过Matrigel的细胞率分别为24%、15%、13%,实验组均有所下降(分别为12%、8%、4%),而两组中K562细胞不能穿过.结论 CD44抗原可能通过改变细胞的黏附、迁移及浸润能力,参与白血病细胞的髓外浸润过程.     

透明质酸对人骨关节炎软骨细胞骨桥蛋白和CD44表达的影响

背景：软骨进行性破坏为晚期骨关节炎的特征性病变,骨关节炎相关因子透明质酸、骨桥蛋白、CD44在骨关节炎软骨中表达增加。目的：通过透明质酸干预体外培养的人膝骨关节炎软骨细胞,探讨透明质酸对人膝骨关节炎软骨细胞CD44与骨桥蛋白表达的影响。方法：将软骨标本进行体外培养获取纯化的软骨细胞,分为3组：空白对照组、透明质酸干预组（100 mg/L）和透明质酸酶干预组（200 mg/L）。培养48 h后,采用Real-time Q PCR检测软骨细胞骨桥蛋白mRNA,CD44mRNA表达水平。用SPSS 17.0统计软件包分析骨桥蛋白mRNA和CD44 mRNA经透明质酸干预前后表达的差异。结果与结论：透明质酸组的骨桥蛋白mRNA表达水平较空白组高,透明质酸酶组的骨桥蛋白mRNA表达水平较空白组低;透明质酸组及透明质酸酶组的CD44 mRNA表达水平均较空白组低。结果提示透明质酸可以上调骨关节炎软骨细胞骨桥蛋白的表达;透明质酸在骨关节炎软骨细胞内对CD44表达的影响具有双相性,其影响结果可能与透明质酸的相对分子质量有关。     

面神经损伤后面神经核中黏附分子CD44表达与穴位电针刺激的影响

目的:观察穴位电针在面神经损伤后面神经再生中的作用及对黏附分子CD44表达的影响。方法:实验于2005-09/2006-02在泸州医学院神经生物学实验室完成。①实验材料:普通级6～8个月龄新西兰家兔36只,体质量2～2.75kg,雌雄不拘。②实验方法:采用随机数字法取兔32只,压榨损伤右侧面神经上颊支制备面神经损伤模型。③实验分组:随机分为手术对照组16只,不作穴位电针刺激,自然恢复;针刺组16只术后立即行翳风、颧髎、颊车、地仓、阳白、四白及合谷穴位电针治疗,1次/d,30min/次;另取4只家兔做正常对照组。④实验评估:于术后1,4,7,14d每组分别取4只兔麻醉后,经心灌注处死动物,苏木精-伊红染色观察面神经核的改变,免疫组织化学观察CD44的表达。结果:36只兔全部进入结果分析。①手术对照组和针刺组兔面神经核运动神经元的形态学改变:术后1d,两组细胞形态正常;术后4d,两组细胞肿胀、胞浆空泡化、染色质溶解、核仁偏移、尼氏小体消失;术后7d,手术对照组有明显神经元的死亡,针刺组仅有神经元变性表现,并出现核仁回归、尼氏小体重现;术后14d,手术对照组神经元的坏死更为明显,针刺组神经元肿胀及变性程度有明显改善。②各组CD44标记阳性面神经核运动神经元数目:正常组,面神经核中有CD44表达。术后1d,针刺组和手术对照组CD44有明显增加;术后4d,针刺组CD44阳性细胞数较手术对照组显著性增加(P<0.01);术后7d,手术对照组的CD44阳性细胞数目较针刺组增加(P<0.05)。术后14d,两组比较无差别,阳性细胞免疫染色着色不一。结论:穴位电刺激能促进黏附分子CD44在损伤面神经核的表达。     

一氧化氮对骨肉瘤细胞表面CD44表达的影响

目的研究一氧化氮（NO）对体外培养的骨肉瘤细胞株表面CD44表达的影响及意义。方法在体外培养骨肉瘤细胞株培养液中加入不同浓度的硝普钠（SNP）以产生外源性NO，用免疫组织化学的方法研究细胞表面CD44抗原成分的变化。结果肿瘤细胞表面CD44抗原在SNP存在的情况下表达明显下降。结论NO可以一定程度上抑制骨肉瘤细胞株CD44的表达，可能引起肿瘤细胞转移能力的改变。     

大肠癌患者外周血CD44和CD54的表达及临床意义

目的：探讨大肠癌患者外周血CD44、CD54的表达及临床意义。方法：采用流式细胞术对40例大肠癌患者及20例良性病变组、20例正常对照组外周血CD44、CD54的表达水平进行检测。结果：大肠癌患者外周血CD44、CD54的表达水平显著高于良性病变组及正常对照组（P〈0．05）；大肠癌患者外周血CD44和CD54的表达水平与淋巴结转移和临床分期相关；良性病变组与正常对照组比较差异无显著性。结论：外周血CD44及CD54的表达与肿瘤转移密切相关，为肿瘤的发生、发展及预后判断提供依据。     

恶性肿瘤患者血小板、红细胞黏附分子CD35、CD44、CD62P的表达与肿瘤转移的关系

摘 要 研究血小板以及红细胞CD35、CD44和CD62P分子的表达变化与肿瘤转移的生物学意义。通过流式细胞仪测定正常人和恶性肿瘤患者血小板和红细胞CD35、CD44和CD62P的表达情况。结果显示，恶性肿瘤患者血小板CD35和CD62P平均荧光强度明显高于正常人，转移组明显高于未转移组（P＜0.05）。转移组血小板CD44平均荧光强度明显高于正常人（P＜0.05）。恶性肿瘤患者红细胞CD35（P＜0.05）和CD44（P＜0.01）平均荧光强度均明显低于正常人，转移组红细胞CD44明显低于未转移组（P＜0.05）。提示恶性肿瘤患者血小板CD35、CD44和CD62P分子处于高表达状态，而红细胞CD35、CD44分子表达减少，和红细胞免疫功能下降有关，这些变化可能对肿瘤的生长和转移具有重要意义。     

膝关节腔注射透明质酸钠对成人大骨节病血清透明质酸、CD44、硫酸角质素含量的影响

目的探讨透明质酸钠治疗对大骨节病(KBD)患者血清中透明质酸(HA)、CD44、硫酸角质素(KS)含量的影响。方法分别检测关节腔注射透明质酸钠的成人KBD患者治疗前、治疗4周后,以及健康成人血清中的HA、CD44、KS含量。结果 KBD患者血清HA、CD44、KS含量明显高于健康成人,且不同性别HA、CD44、KS含量无差异。在经过关节腔注射透明质酸钠治疗后,血清中三指标含量均明显下降,其中HA含量男性高于女性。结论膝关节腔注射透明质酸钠可明显降低KBD患者血清中的HA、CD44、KS含量。血清HA、CD44、KS含量可作为KBD疗效评价指标之一。     

CD44v6在星形细胞瘤中的表达及临床病理意义

【目的】研究CD44v6在星形细胞瘤中的表达情况及相关性,探讨其与星形细胞瘤分级及术后复发的关系。【方法】应用免疫组化法检测58例星形细胞瘤中CD44v6的表达情况,以正常脑组织和反应性胶质细胞增生为阴性对照。【结果】CD44v6在正常脑组织和反应性胶质细胞增生中不表达,在星形细胞瘤中高表达,且在高级别的星形细胞瘤和术后复发星形细胞瘤中表达显著。【结论】CD44v6表达与星形细胞瘤的分级、术后复发密切相关,可作为星形细胞瘤的诊断和术后复发判断的重要依据。     

Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition

A cell adhesion model was previously used to select a series of monoclonal antibodies (mAbs), which were subsequently found to recognize CD44/Pgp-1. Interest in these reagents increased with the finding that they totally inhibited production of lymphoid or myeloid cells in long-term bone marrow cultures. Further investigation has now revealed that hyaluronate is a potential ligand for CD44 and that hyaluronate recognition accounts for the adhesion between B lineage hybridoma and stromal cells. The hybridoma cells adhered to hyaluronate-coated plastic wells as well as to monolayers of stromal cells. The adhesion in both cases was inhibited by treatment with hyaluronidases, and did not require divalent cations. Addition of exogenous hyaluronate also diminished binding of lymphoid cells to stromal cells. One of several mAbs to Pgp-1/CD44 was particularly effective at blocking these interactions. Since hyaluronate and Pgp-1/CD44 were present on both cell types, experiments were done to determine the cellular location of interacting molecules required for the adhesion process. Treatment of lymphoid cells with an anti-Pgp-1/CD44 antibody was more inhibitory than antibody treatment of the stromal cells. Conversely, hyaluronidase treatment of stromal cells reduced subsequent binding more than treatment of the lymphoid cells. Adhesive interactions that involve hyaluronate and CD44 could contribute to a number of cell recognition processes, including ones required for normal lympho-hemopoiesis.     

Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion.

The localization of circulating leukocytes within inflamed tissues occurs as the result of interactions with and migration across vascular endothelium, and is governed, in part, by the expression of adhesion molecules on both cell types. Recently, we have described a novel primary adhesion interaction between the structurally activated form of the adhesion molecule CD44 on lymphocytes and its major ligand hyaluronan on endothelial cells under physiologic laminar flow conditions, and have proposed that this interaction functions in an extravasation pathway for lymphocytes in vascular beds at sites of inflammation. While the regulation of activated CD44 on leukocytes has been characterized in depth, regulation of hyaluronate (HA) on endothelial cells has not been extensively studied. Here we demonstrate that the expression of HA on cultured endothelial cell lines and primary endothelial cultures is inducible by the proinflammatory cytokines TNFalpha and IL-1beta, as well as bacterial lipopolysaccharide. In addition, this inducibility appears strikingly restricted to endothelial cells derived from microvascular, but not large vessel, sources. The elevated HA levels thus induced result in increased CD44-dependent adhesive interactions in both nonstatic shear and laminar flow adhesion assays. Changes in mRNA levels for the described HA synthetic and degradative enzymes were not found, suggesting other more complex mechanisms of regulation. Together, these data add to the selectin and immunoglobulin gene families a new inducible endothelial adhesive molecule, hyaluronan, and help to further our understanding of the potential physiologic roles of the CD44/HA interaction; i.e., local cytokine production within inflamed vascular beds may enhance surface hyaluronan expression on endothelial cells, thereby creating local sites receptive to the CD44/HA interaction and thus extravasation of inflammatory cells.     

Effects of statins on adhesion molecule expression in endothelial cells

Summary.  Background : Inhibitors of HMG-CoA reductase are widely used to prevent atherosclerosis progression. The expression of adhesion molecules on activated endothelial cells (EC) is an important step in the initiation and progression of atherosclerosis. Objectives : We investigated whether adhesion molecule expression on activated EC is influenced by simvastatin, fluvastatin and pravastatin and, if so, by which mechanisms. Methods : Human EC from umbilical veins or saphenous veins were pretreated overnight with statins with or without mevalonate, and also for simvastatin or fluvastatin with the isoprenoid intermediates, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP). After 4–6 h activation with tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS), surface adhesion molecule expression was evaluated by ELISA and by flow cytometry. The same experiments were performed with selective inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277). Results : Pretreatment with simvastatin, fluvastatin or pravastatin potentiated the TNF-α and LPS-induced expression of E-selectin and VCAM-1, and mevalonate reversed the potentiating effect of these statins. GGPP also reversed the potentiating effect of simvastatin or fluvastatin on adhesion molecule expression, while FPP only partially reversed this effect. Furthermore, GGTI-286, but not FTI-277, mimicked the effect of simvastatin by increasing the TNF-α-mediated overexpression of E-selectin. Conclusions : Statins increase E-selectin- and VCAM-1-induced expression on vascular endothelial cells stimulated with TNF-α or LPS. The inhibition of geranylgeranylated proteins could contribute to this effect.     

CD44及CD44v6在胚胎各器官中的表达及意义

目的通过免疫组化染色观察CD44和CD44v6在胚胎各脏器中的表达,了解这两种表面标志物在不同器官中的表达情况。方法用免疫组化PV-9000二步法（非生物素）检测CD44和CD44v6在6例胚胎各器官的表达情况。CD44用已知阳性的淋巴结作阳性对照;CD44v6用已知阳性的扁桃体作阳性对照。两者均用PBS代替一抗作阴性对照。结果 CD44主要表达于间叶组织中,如食管、胃及肠管的深肌层,心脏中的部分小血管、神经束以及各器官中的纤维组织,肝和脾内的造血细胞也有表达,但不表达于骨、肾、睾丸、心肌和肝细胞。另外,在少数上皮组织中也见有表达,如食管黏膜和气管黏膜上皮的基底层细胞及气管腺上皮。CD44v6的表达仅见于食管上皮基底层细胞中,其它各器官均未见表达。结论通过免疫组化法观察CD44和CD44v6在胚胎发育过程中的表达情况,为成体干细胞特性的研究提供了良好直观的依据和有用的线索。