滑膜间充质干细胞分离培养、免疫表型鉴定及在混合淋巴反应体系中的抑制效应*

背景：滑膜组织中可分离出具有多向分化潜能干细胞特性的细胞。  目的：探索滑膜间充质干细胞体外分离、培养的可行性、免疫表型的鉴定及对混合淋巴细胞反应体系中淋巴细胞增殖的影响,评价其免疫学特性。  方法：关节镜下获取10例半月板损伤患者的膝关节滑膜组织,胶原酶消化获得有核细胞。挑选单细胞克隆,筛选获得滑膜间充质干细胞。检测其增殖能力、细胞活力,流式检测其细胞免疫表型,采用人双向混合淋巴细胞反应体系及植物血凝素刺激的淋巴细胞增殖反应体系,根据滑膜间充质干细胞与外周血单个核细胞的不同比例加入丝裂霉素处理滑膜间充质干细胞。MTT法检测并计算其抑制率。 结果与结论：10组滑膜间充质干细胞系增殖能力和细胞活力差异无显著性意义(P > 0.05)。10组细胞系免疫表型：CD44、CD90、CD105呈阳性,CD14、CD34、CD45和HLA-DR呈阴性。滑膜间充质干细胞抑制混合淋巴细胞反应体系中及植物血凝素刺激的淋巴细胞增殖反应体系中淋巴细胞的增殖、抑制率与加入的滑膜间充质干细胞数量成正比。提示关节滑膜组织可以分离、培养获得滑膜间充质干细胞,其具有间充质干细胞特异性表型,且滑膜间充质干细胞具有免疫调节作用。      

兔膝骨性关节炎滑膜细胞体外培养和鉴定

目的 探讨复制膝骨性关节炎的实验动物模型;探讨兔膝关节滑膜细胞体外分离、培养的方法及生长特性.方法 改良Hulth法复制膝骨性关节炎,于造模后4周机械分离滑膜组织;用改良酶消化法分离滑膜细胞进行培养,观察滑膜细胞的生长情况.结果 改良Hulth法复制膝骨性关节炎模型,滑膜增生明显;酶消化法分离培养获得大量滑膜细胞且细胞...     

塞来昔布对膝关节炎大鼠肿瘤坏死因子α信号通路及关节滑膜细胞的影响*

目的：探究塞来昔布对膝关节炎大鼠肿瘤坏死因子α（ TNF-α）信号通路及关节滑膜细胞的影响。方法： SD大鼠随机分为模型组、假手术组和治疗组,治疗组大鼠制作膝关节炎模型后给予塞来昔布进行治疗。通过 TUNEL 法、qRT-PCR 法、CCK-8 法、免疫印迹等方法检测3 组大鼠滑膜细胞凋亡情况、TNF-α mRNA表达含量、 TNF-α 蛋白含量、关节炎指数及细胞增殖情况。结果：模型组、治疗组、假手术组关节滑膜细胞凋亡率分别为 89.38%、46.84%、13.68%。模型组与假手术组、治疗组相比较,细胞凋亡率显著升高,治疗组细胞凋亡数量多 于假手术组。假手术组、治疗组细胞增殖OD值比模型组高,差异具有统计学意义,假手术组关节滑膜细胞的增 殖数量最多,模型组关节滑膜细胞的增殖数量最少,治疗组关节滑膜细胞的增殖情况比假手术组相比明显减少。 与假手术组、治疗组比较,模型组关节滑膜细胞中TNF-α 蛋白表达水平显著上升,治疗组TNF-α 蛋白表达水平较 假手术组明显升高。与假手术组、治疗组比较,模型组TNF-α mRNA含量显著升高,治疗组TNF-α mRNA表达 量较假手术组明显升高。治疗组和模型组大鼠关节炎指数相近,随着给药时间的增加,治疗组大鼠膝关节炎情况 逐渐好转,而未服用药物的模型组大鼠病情逐渐恶化。结论：塞来昔布胶囊治疗膝关节炎大鼠效果显著,能够显 著降低TNF-α 信号通路的表达水平,改善关节滑膜细胞的凋亡情况。     

类风湿性关节炎患者关节滑液和滑膜中γδT 细胞的体外建系及功能研究

目的　探究在类风湿性关节炎（ Ｒ Ａ） 患者的关节滑液和滑膜中选择性存在,且有数量升高的γδ Ｔ 细胞的免疫学功能,以阐述类风湿性关节炎的发病机理。方法　应用固相抗体扩增法,建立关节滑液、滑膜和外周血γδ Ｔ 细胞系,并在此基础上,探讨了γδ Ｔ 细胞的细胞表型、细胞毒活性、增殖活性和细胞因子分泌情况。结果　所获得的滑液、滑膜γδ Ｔ 细胞系,多数以 Ｖδ１ 亚型细胞占优势;呈现出 Ｉ Ｌ２ 和 Ｉ Ｌ１５ 依赖的细胞增殖应答和显著增强的细胞毒活性;γδ Ｔ 细胞对自体和异体滑膜细胞的细胞毒作用无显著差异; Ｍ．ｔｂ（ 结核分枝杆菌） 、 Ｍ．ｔｂ 蛋白等抗原及有丝分裂原的刺激不能有效的提高建系的γδ Ｔ 细胞的细胞毒作用或增殖活性; 与异体滑膜细胞共同培养可以促进滑液γδ Ｔ 细胞的增殖; 细胞因子 Ｉ Ｌ２ 、 Ｔ Ｎ Ｆα的分泌维持在较低的水平。结论　存在于局部关节中的γδ Ｔ 细胞可能通过对滑膜细胞的细胞毒作用参与类风湿性关节炎的发病机制。     

小鼠骨髓间充质干细胞的分离培养及形态学观察

目的建立一种简单实用的小鼠骨髓间充质干细胞(MSCs)的分离培养方法,通过了解其细胞生物学特性,为MSCs的应用提供实验依据。方法采用差速贴壁法体外分离、扩增MSCs,倒置显微镜观察原代及传代细胞的形态及生长过程。结果在小鼠骨髓间充质干细胞的培养过程中,血液系细胞在换液过程被去除,成纤维细胞污染经差速贴壁法也可去除。获得的骨髓间充质细胞形态较均一,生长状态良好。结论采用差速贴壁培养法可获得一定纯度的MSCs,此法简单、实用,并且获得的细胞生长状态良好,增殖能力强.     

成体小鼠骨髓间充质干细胞的培养和纯化

目的建立一种简单易行的小鼠骨髓间充质干细胞(MSC)的分离培养方法,并对其表面标志进行鉴定,为其应用提供实验依据.方法采用差速贴壁法体外分离、扩增MSC,倒置显微镜观察其生长过程,HE染色观察原代培养细胞的生长特性,免疫细胞化学检测MSC表面抗体的表达及细胞的大概纯度.结果用此法进行小鼠骨髓间充质干细胞的培养过程中,血液系细胞在换液过程中被去除,成纤维细胞污染可经差速贴壁法去除.经鉴定获得的骨髓间充质细胞能达到理想的纯度,生长状态良好.结论采用差速贴壁培养法可获得一定纯度的MSC,这种方法简单、便捷、实用,并且用这种方法所分离获得的细胞生长状态好,在体外条件下能大量增殖,原代及传代生长都可形成均一的细胞集落.     

成体小鼠骨髓间充质干细胞的培养和纯化

目的 建立一种简单易行的小鼠骨髓间充质干细胞（MSC）的分离培养方法，并对其表面标志进行鉴定，为其应用提供实验依据。方法 采用差速贴壁法体外分离、扩增MSC，倒置显微镜观察其生长过程，HE染色观察原代培养细胞的生长特性，免疫细胞化学检测MSC表面抗体的表达及细胞的大概纯度。结果 用此法进行小鼠骨髓间充质干细胞的培养过程中，血液系细胞在换液过程中被去除，成纤维细胞污染可经差速贴壁法去除，经鉴定获得的骨髓间充质细胞能达到理想的纯度，生长状态良好。结论 采用差速贴壁培养法可获得一定纯度的MSC。这种方法简单、便捷、实用，并且用这种方法所分离获得的细胞生长状态好，在体外条件下能大量增殖，原代及传代生长都可形成均一的细胞集落。     

构建糖氧剥夺模型大鼠少突胶质细胞体外培养与分离纯化

目的:对原有少突胶质细胞祖细胞培养与分离纯化方法进行改进,以简单、高效获得少突胶质细胞并建立少突胶质细胞糖氧剥夺(oxygen glucose deprivation,OGD)模型。方法:取SD乳鼠脑皮质,采用两种不同的细胞增殖法和两种不同的分离纯化法分四组培养,分别为细胞因子增殖联合摇床震荡分离纯化法组,B104CM增殖联合摇床震荡分离纯化法组,细胞因子增殖联合EDTA消化机械吹打分离纯化法组,B104CM增殖联合EDTA消化机械吹打分离纯化法组。倒置显微镜下观察神经细胞形态学变化并进行细胞计数。A2B5和髓鞘碱性蛋白鉴定少突胶质细胞并分析其纯度。纯化后的少突胶质细胞祖细胞分化培养3 d后分四组培养,正常组正常培养,低氧组分三组,换无糖DMEM培养基,OGD 1、2、3 h。然后采用MTT比色法检测细胞活力;流式细胞术分析细胞的凋亡率。结果:(1)B104CM增殖联合EDTA消化机械吹打分离纯化法较其他3种培养方法收获的少突胶质细胞祖细胞数量多(P0.05)。(2)A2B5和MBP特异性标记,细胞纯度可达到95%。(3)MTT结果显示:OGD 1 h少突胶质细胞的细胞活力即降低(P0.05),随着缺氧时间延长细胞活力呈降低的趋势更加明显(P0.05)。(4)流式细胞测细胞凋亡的结果显示:OGD 1 h、2 h、3 h,细胞的凋亡率分别为14.43%±0.20%,21.99%±0.42%和44.71%±0.20%,均高于正常对照组(6.86%±0.05%,P0.05)。结论:(1)B104CM增殖联合EDTA消化机械吹打分离纯化培养法与其他三种方法比较实验操作简单,收获的少突胶质细胞祖细胞数量最多,可用于少突胶质细胞的培养。(2)OGD可致少突胶质细胞损伤,且与OGD持续时间密切相关,实验成功的建立OGD损伤模型。     

动物组织细胞分离纯化方法学

要了解某种特定细胞的结构、功能或表面标志等特征,就必须将其在体外培养增殖获得足够的数量以供研究,这首先就必须获取种子细胞。从血液、淋巴液等液体中分离细胞时,可直接进行纯化,从心脏、肝脏等器官分离细胞时,一般的方法是先获得细胞悬液,再对其进行分离     

犬关节软骨细胞的体外分离与培养

背景：自1967年Manning等提出的通过胰蛋白酶和细菌胶原酶联合消化法分离软骨细胞以来,软骨细胞的体外分离培养研究较多,但目前尚无统一标准。 目的：研究犬关节软骨细胞在体外分离以及培养的条件,寻求体外扩增软骨细胞较简便、可行、高效的实验方法。 方法：取3周龄幼犬关节软骨组织,应用胰酶、胶原酶制定8种消化方法获取软骨细胞,对比不同方法所获取细胞数量及细胞成活率,分离细胞进行原代及传代培养,观察各代软骨细胞形态。 结果与结论：在体外分离犬关节软骨细胞的各种方法中,单纯应用Ⅱ型胶原酶消化法获得的软骨细胞数最多,细胞成活率最高。软骨细胞可以通过体外培养获得扩增,并且维持良好的细胞形态及表型,但仅限于5代以内。     

Regulation of TNF-alpha-mediated hyperplasia through TNF receptors,TRAFs, and NF-kappaB in synoviocytes obtained from patients with rheumatoid arthritis

Although the etiology of rheumatoid arthritis (RA) has not been clearly understood to date, the hyperplasia of the synovial membrane imposed by pro-inflammatory cytokines has been suggested to play a crucial role in the progression of this disease. TNF-alpha, a potent pro-inflammatory cytokine, was detected at highly enhanced concentrations in the blood and synovial fluids of patients with RA relative to those of patients with osteoarthritis and normal subjects. To evaluate the role of TNF-alpha in the synovial hyperplasia during the pathogenic state, we investigated cellular outcomes and molecular mechanisms of synoviocytes in response to TNF-alpha. Following TNF-alpha treatment, fibroblast-like synoviocytes (FLS) obtained from patients with RA proliferated, unlike the cells from a normal subject that were unaffected. This TNF-alpha induced proliferation of synoviocytes obtained from RA patients coincided with down-regulation of TNFR1 and up-regulation of TNFR2 and TRAF1-6, as well as NF-kappaB activation. TNF-alpha-induced proliferation of synoviocytes was inhibited by transfection with a dominant negative mutant form of I-kappaBalpha cDNA (I-kappaBalphadN). Moreover, following TNF-alpha treatment, transfectants with I-kappaBalphadN underwent apoptosis, whereas mock-transfectants did not. Taken together, these results suggest that high levels of TNF-alpha present in RA synovium play an important role in the synovial hyperplasia of RA by suppressing apoptosis and promoting proliferation of synoviocytes through NF-kappaB-dependent signaling pathways mediated by up-regulated TNFR2 and TRAF1-6 molecules.     

类风湿关节炎大鼠滑膜细胞的类肿瘤样增生与相关基因表达的关系

目的:探讨佐剂性关节炎(AA)大鼠滑膜细胞的类肿瘤样增生和相关基因表达的机制。方法:将Wistar大鼠随机分两组,每组10只,并应用弗氏完全佐剂诱发大鼠AA实验性模型,定期观测踝关节肿胀度至第40天。分别取AA模型组和正常对照组大鼠踝关节进行组织切片,用HE染色后观察病理学变化。取大鼠膝关节滑膜组织体外进行原代细胞培养,用MTT比色法检测大鼠滑膜细胞的增生。同时采用RTPCR法,检测模型组及正常对照组大鼠滑膜组织中相关基因Cmyc和ODCmRNA的转录水平。结果:①体外培养的AA模型组大鼠关节滑膜细胞的增殖显著高于正常组大鼠(P<0.01),并呈类肿瘤样增生。②AA模型组大鼠关节滑膜组织中,Cmyc和ODCmRNA的转录水平明显高于正常对照大鼠(P<0.01)。结论:AA模型组大鼠关节滑膜细胞的类肿瘤样增生,可能与其相关基因Cmyc和ODCmRNA的转录水平增高有关。     

Three-dimensional ultrastructure of synoviocytes in the knee joint of rabbits and morphological changes in osteoarthritis model

The synovial intima is composed of two types of synoviocytes: absorptive macrophages and secretory, fibroblast-like F cells. Many studies have tried to observe synoviocytes by scanning electron microscopy (SEM) but failed to reveal the entire shape of synoviocytes because they are deeply embedded in the interstitial matrix. The present study, primarily employing SEM observation of NaOH macerated samples, reveals the distribution and three-dimensional ultrastructure of the synoviocytes in the normal knee joint of rabbits, and the morphological changes of synoviocytes in an osteoarthritis model of this animal. F cells were broadly distributed throughout the synovial intima, while macrophages showed a restricted distribution on fatty tissues around the patella. F cells were classified into a flat type, which covered the surface of synovial membrane like an epithelium, and a dendritic type, which extended long processes to form a characteristic meshwork on the surface. The flat type predominated in regions adhering to the femur, while the dendritic type predominated in ambilateral parts of both the patella and tendon of the musculus quadriceps femoris, and on the peripatellar fatty tissue. Intermediate forms of flat and dendritic types appeared in middle regions between the patella and periphery of the joint capsule. In the synovial membrane of the osteoarthritis model, both types of synoviocytes increased in number and changed their morphology, indicating their elevated activities in absorption and secretion. It is suggested that the ultrastructural changes in synoviocytes reflect pathological conditions of the synovial membrane, and synoviocytes play important roles in the pathogenesis of osteoarthritis.     

Expression and production of the long pentraxin PTX3 in rheumatoid arthritis (RA)

PTX3 is a secreted molecule which consists of a C-terminal domain similar to classical pentraxins (e.g. C-reactive protein (CRP)) and of an unrelated N-terminal domain. Unlike the classical pentraxins, the long pentraxin PTX3 is expressed in response to IL-1beta and tumour necrosis factor-alpha (TNF-alpha), but not to IL-6, in various cell types. The present study was designed to investigate the expression of PTX3 in RA. Dissociated RA and osteoarthritis (OA) type B synoviocytes were cultured in the presence and in the absence of inflammatory cytokines. PTX3 mRNA expression in synoviocytes was evaluated by Northern analysis. PTX3 protein levels in synovial cell cultures and synovial fluid were estimated by ELISA, and PTX3 distribution in synovial tissues by immunohistochemical techniques. OA synoviocytes were induced to express high levels of PTX3 mRNA by TNF-alpha, but not by other cytokines including IL-1beta and IL-6. RA synoviocytes, unlike OA synoviocytes, constitutively expressed high levels of PTX3 in the absence of deliberate stimulation. The constitutive expression of PTX3 in RA synoviocytes was not modified by anti-TNF-alpha antibodies, IL-1 receptor antagonist or a combination of the two agents. In contrast, interferon-gamma and transforming growth factor-beta inhibited PTX3 constitutive expression in RA synoviocytes. The joint fluid from RA patients contained higher levels of immunoreactive PTX3 than controls and the synovial tissue contained endothelial cells and synoviocytes positive for PTX3 by immunohistochemistry. In conclusion, PTX3 may play a role in inflammatory circuits of RA, and its relevance as a marker of disease activity deserves further study.     

Synovitis and bovine syncytial virus isolation in experimentally induced malignant catarrhal fever

Synovial membranes and fluid from joints of cattle with experimentally induced and naturally-occurring malignant catarrhal fever (MCF) were examined grossly and by histological, ultrastructural, immunological and virological means. Synovitis, characterized by focally roughened synovial membranes and cloudy synovial fluid with occasional fibrin strands, was frequently found, particularly in tibiotarsal joints. Histologically, there was variable mononuclear cell infiltration of synovial tissues with necrosis, and fibrin exudation in severe cases. Ultrastructurally, most synoviocytes were intermediate between Types A and B. Variably severe degenerative changes were observed in synoviocytes and lymphoid cells. Lymphoid vasculitis was common in severely involved joints. Synovial fluid contained markedly increased numbers of mononuclear cells and elevated total protein, but no significant elevation in serum titres of rheumatoid factor was associated with the development of MCF in the experimental disease. Bovine syncytial virus was isolated from cells of synovial fluid of 9 of 10 cattle with experimental MCF and from other tissues of 8 of 9 cattle with MCF. The significance of this finding is unclear.     

Pentraxin 3 is a marker of early joint inflammation in patients with juvenile idiopathic arthritis

Pentraxin 3 (PTX3) is an acute phase protein produced in different body tissues. The aims of this study were to characterize PTX3 secretion in synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients and to analyze the correlation of PTX3 levels in SF with clinical characteristics and the course of the disease. SF-PTX3 levels were measured in a cohort of 75 consecutive JIA patients followed in a single center. Patients’ clinical characteristics, disease course, and therapies were analyzed for their correlation with SF-PTX3 levels. A synovial cell line was used to study the kinetics of PTX3 secretion by synoviocytes. SF-PTX3 levels varied over a wide range. Elevated SF-PTX3 levels were detected in patients who subsequently required treatment with disease-modifying antirheumatic drugs during the follow-up period. SF-PTX3 levels were found to be inversely correlated with the length of time from onset of joint swelling. No correlation was found between synovial and serum PTX3 or C-reactive protein (CRP). Following in vitro stimulation of synovial cell line with TNFa or IL1, the secretion of PTX3 increases transiently in the first 48–72 h. A similar increase was obtained in patients’ synovial fluids but not with IL6. Higher SF-PTX3 levels were found when tested closer to arthritis exacerbation and 48–72 h after in vitro stimulation of cells from a synovial cell line, implying that PTX3 plays a role in early stages of inflammation. Higher SF-PTX3 levels were associated with several clinical features reflecting disease severity and prognostic data. Measuring SF-PTX3 levels may help in providing a more focused and patient-adjusted treatment.     

Regulation of GRB2 and FLICE2 expression by TNF-alpha in rheumatoid synovium

Chronic rheumatoid arthritis (RA) is characterized by the hyperplasia of synovial tissue, which results from dysregulation of proliferative and antiapoptotic signals transduced in the synovial cells by unknown mechanisms. To identify candidate factors involved in the regulation of synovial hyperplasia, the expression profile of 205 apoptosis-related genes was compared between tissues from patients with RA and osteoarthritis (OA) using a cDNA microarray. Upregulated genes in the RA synovium included TNFR2, FLICE2, and signaling molecules involved in a MAP kinase pathway (GRB2, MAPK p38). In contrast, genes encoding SARP1 and various cell cycle regulators were down-regulated in the RA synovium relative to OA. Importantly, the expression levels of GRB2 and FLICE2 genes were remarkably enhanced in RA synoviocytes but not in OA synoviocytes in response to tumor necrosis factor (TNF)-alpha treatment. Thus, these results suggest that over-expression of GRB2 and FLICE2 in RA synovium is caused by TNF-alpha inducibility differentially regulated in RA synoviocytes and provide potential pathogenic roles of these genes in the hyperplasia of the RA synovium.     

Three-dimensional ultrastructure of synoviocytes in the horse joint as revealed by the scanning electron microscope.

The synovial membrane displays a superficial cellular lining composed of two types of synoviocytes: "absorptive" macrophages (type A cells) and "secretory" fibroblast-like cells (type B cells). The types are intermingled and extend a variety of processes, rendering the cellular architecture of the synovial membrane difficult to visualize. Previous electron microscopic and histochemical studies failed to demonstrate the entire shape of synoviocytes, except our immunohistochemical study for protein gene product 9.5 in the horse joint. The present SEM study is the first to demonstrate the three-dimensional ultrastructure of synoviocytes as well as their distribution in the synovial membrane, using macerated samples from the horse carpal joints. The equine synovial membrane was largely covered by conspicuously developed synovial villi. Type A synoviocytes were closely similar to macrophages in regard to surface structure, and showed uneven distribution with the densest occurrence around the tips of the synovial villi. In the basal half of villi, type B synoviocytes, which were situated in close proximity to the synovial cavity, projected thick processes horizontally and intertwined to form a regular network of processes on the synovial surface. Those in the upper half of the villi were located in the abluminal layers and protruded an antenna-like process into the joint cavity with tips covered with long microvilli, in addition to forming the superficial plexus of processes. Type B cells were also provided with fine, membranous extensions that tended to cover the surface of synovial intima. The meshwork of horizontal processes, the antenna-like processes, and the membranous processes imply advantages in not only secretion but also sensation and regulation of the barrier function in the synovial membrane.