类风湿性关节炎细胞水平建模四种方法比较

目的 比较四种方法 在培养类风湿性关节炎(RA)滑膜细胞(FLS)中的异同,为研究RA提供较好的体外实验模型建模方法 .方法 20例滑膜组织标本均用组织块法、单胶原酶消化法、单胰酶温消化法和双酶消化法四种方法 分离、培养RA FLS,均取培养第7日细胞计数,取第4代细胞鉴定,免疫组化鉴定细胞,测定分离细胞的数量和纯度.结果 四种培养方法 的培养时间比较,单胶原酶法>双酶消化法>组织块法>单胰酶法(P＜0.05).组织块法及双酶消化法所得细胞数较单胶原酶法及双酶消化法少(P＜0.05).第4代细胞均以FLS为主(>95%).结论 四种方法 体外培养可获得生物学特性稳定的RA FLS细胞系,其中组织块法是细胞水平建立RA体外实验模型成功率较高的方法 ,单纯胰酶法是较快速的方法 .     

三种原代小胶质细胞纯化培养方法的对比及分析

目的寻找简捷、高效的原代小胶质细胞纯化培养方法。方法以小胶质细胞原代培养的经典培养方法为基础，通过提高初次接种密度、减少细胞离心过滤、进行营养丧失培养等改进，培养后期分别采用经典机械分离法、低浓度胰酶消化法和利多卡因分离纯化法行细胞分离与纯化，记录形态变化并采用同倍数视野动态计数，借助CD68及OX42抗体间接免疫荧光标记进行鉴定、计算纯度。结果三种分离方法均获得了较高纯度和产量的小胶质细胞，其中利多卡因分离纯化法获得细胞数及纯度明显高于其他分离方法，低浓度胰酶消化法次之。结论小胶质原代细胞培养过程中，利多卡因分离纯化法及低浓度胰酶消化法均可有效提高培养产量及纯度，以利多卡因分离纯化法最佳。     

低浓度混合酶改良消化法体外培养SD大鼠面颌肌细胞

目的观察低浓度混合酶改良消化法体外培养SD大鼠面颌肌细胞的结果。方法分离SD大鼠颌面部咬肌区肌肉组织,先用0.1%的Ⅰ型胶原酶消化后再用0.1%的Ⅰ型胶原酶和0.125%的胰蛋白酶(1∶1)低浓度混合酶消化法分离细胞,差速贴壁法纯化,所得细胞进行结蛋白(Desmin)免疫组化染色。结果体外培养所得细胞Desmin胞质阳性率达95%。结论采用低浓度混合酶消化和差速贴壁法体外培养SD大鼠面颌肌细胞可获得纯度高、产量高、活性强、稳定性好的面颌肌细胞。     

一种新生大鼠肝细胞分离改良方法的建立

目的尝试建立分离并获得稳定培养的新生大鼠原代肝细胞(HC)的简易方法。方法采用多步酶消化法和组织块培养贴壁法分离大鼠原代HC,经低速离心、选择性贴壁培养并纯化肝细胞。结果最初获得单细胞数量约1～2×10~6个/只,经0.4%台盼蓝染色细胞瞬时活率72%,单细胞悬液和组织块两种培养方法均在3～5 d发现细胞可长满瓶底的70%～80%,经传代纯化以后细胞经糖原染色和抗CK-18免疫组化染色鉴定,发现HC纯度95%,细胞生长状态良好,可供一般实验室使用。结论成功建立了获取原代培养HC的简易方法,细胞数量、纯度、活率较好,可供一般实验室实验研究。     

三种培养原代支气管成纤维细胞方法的比较

目的 探讨建立适用于SD大鼠支气管成纤维细胞原代培养的方法.方法 取重量为150~180 g的SD雄性大鼠的支气管组织,采用Ⅰ型胶原酶、木瓜蛋白酶联合消化+组织块黏附法、胰酶+胶原酶联合消化(双酶消化法)+组织块黏附法、组织块黏附法进行原代支气管成纤维细胞的培养.利用镜下观察细胞的生长特点;免疫荧光染色法鉴定细胞的类型及纯度;细胞的增殖活性用CCK-8检测并绘制生长曲线.结果 酶消化+组织块黏附法培养使细胞游出速度更快、细胞数量产生更多.组织块黏附法培养的细胞爬出相对缓慢、培养周期更长、获得的细胞数量相对较少.CCK8增殖曲线呈S形.培养48 h,经酶消化法分离得到的成纤维细胞已经游出,约有60%以上细胞已经贴壁,培养6~7 d可传代.组织块贴壁法培养5 d时,细胞开始从组织块边缘缓慢爬出,随后从组织块周围延伸长,14 d左右可传代.使用酶消化法培养的支气管成纤维细胞中可混有杂细胞团,为提高成纤维细胞的纯度可通过差速贴壁法纯化.结论 双酶消化法+组织块黏附法可以更高效、快速的分离和获取支气管成纤维细胞,为研究慢性阻塞性肺疾病(COPD)气道重塑提供了充足的种子细胞.     

体外原代培养脐带基质间充质细胞和人皮肤成纤维细胞

目的探讨体外原代培养脐带基质间充质细胞(UMC)和人皮肤成纤维细胞(HSF)的方法。方法用Ⅳ型胶原蛋白酶、Ⅰ型脱氧核糖核酸酶和0.25%胰蛋白酶消化法原代分离培养UMC细胞,用组织块贴壁法原代分离培养HSF细胞,镜下观察细胞的培养过程和生长状态。结果脐带组织分离培养第3天,有少量UMC贴壁生长,细胞膜周围有折光性,形态类似于成纤维细胞;皮肤组织块贴壁培养第7天,有HSF爬出,呈长梭形、不规则三角形。随着细胞培养时间延长,UMC和HSF数量逐渐增多,细胞生长状态良好。结论应用酶消化法和组织块贴壁法可成功分离培养出UMC和HSF。     

一种新生大鼠肝细胞分离改良方法的建立*

目的 尝试建立分离并获得稳定培养的新生大鼠原代肝细胞（HC）的简易方法。方法 采用多步酶消化法和组织块培养贴壁法分离大鼠原代HC,经低速离心、选择性贴壁培养并纯化肝细胞。结果 最初获得单细胞数量约1~2×10⁶个/只,经0.4%台盼蓝染色细胞瞬时活率>72%,单细胞悬液和组织块两种培养方法均在3~5 d发现细胞可长满瓶底的70%~80%,经传代纯化以后细胞经糖原染色和抗CK-18免疫组化染色鉴定,发现HC纯度>95%,细胞生长状态良好,可供一般实验室使用。结论 成功建立了获取原代培养HC的简易方法,细胞数量、纯度、活率较好,可供一般实验室实验研究。     

人心包外脂肪干细胞原代培养

目的：建立人心包外脂肪干细胞体外培养研究模型。方法采用胶原酶消化和差速贴壁法纯化细胞,倒置相差显微镜观察细胞形态,HE染色,免疫荧光检测细胞表面相关抗原表达。结果形态学特征以及脂肪干细胞特异性相关抗原检测证实,得到高纯度人心包外脂肪干细胞。结论利用胶原酶消化法能成功分离并培养人心包外脂肪干细胞,可用于组织工程学进一步研究。     

大鼠乳鼠心房肌细胞的分离和培养

目的探讨Wistar大鼠乳鼠心房肌细胞的原代分离及培养方法,为细胞电生理或房性心律失常的研究提供心房肌细胞。方法取出生1~3 d的Wistar大鼠的乳鼠,分离心室和心房,胰蛋白酶和Ⅱ型胶原酶消化分离的心房肌组织,差速贴壁法分离心房肌细胞与成纤维细胞,并加入0.5ml Brd U纯化心房肌细胞。高糖培养基培养心房肌细胞,显微镜下观察心房肌细胞形态,通过免疫荧光法测定心房肌细胞纯度。结果成功分离培养心房肌细胞并观察其形态,纯度为94.3%±1.4%,3~4 d出现同步搏动。结论应用差速贴壁法并加用Brd U,可以进一步纯化胰蛋白酶和Ⅱ型胶原酶分离的心房肌细胞。     

金雀异黄素对胶原诱导性大鼠成纤维样滑膜细胞分泌的血管新生相关因子的影响

目的 观察金雀异黄素(genistein)对Ⅱ型胶原诱导性关节炎(CIA)大鼠关节成纤维样滑膜细胞(FLS)分泌的血管内皮生长因子(VEGF)及基质金属蛋白酶(MMP)-1、2、3、9的影响.方法 采用Ⅱ型胶原和完全弗氏佐剂(CFA)建寺CIA大鼠模型;每周进行一次关节炎指数(AI)评分;于造模第6周摄取关节X线片,选取大鼠关节进行关节滑膜组织病理检测:取出关节滑膜组织,用胶原酶消化法分离培养原代成纤维样滑膜细胞;流式法检测滑膜细胞血管细胞黏附分子(VCAM)-1的表达;在FLS培养中加入用不同浓度的金雀异黄素(100,200.400 μmol/L),间接酶联免疫吸附试验(ELISA)法检测FLS培养上清中VEGF及MMP-1、2、3、9的表达.结果 Ⅱ型胶原和CFA共同致敏后3 d,大鼠开始出现关节肿胀,AI逐渐增高,于第3周时最明显,观察AI评分、关节X线以及关节滑膜组织病理发现,造模组与空白对照组比较差异有统计学意义.大鼠滑膜细胞培养至第4代,检测其VCAM-1的表达高达85.5%,提示所培养的细胞即为FLS.不同浓度的金雀异黄素(100,200,400μmol/L)和甲氨蝶呤(MTX,1 mg/L)作用于FLS后,FLS培养上清中VEGF和MMP-1、2、3、9的分泌受到抑制,且抑制作用强度与药物的浓度有关.结论 本实验使用Ⅱ型胶原和完全弗氏佐剂成功构建了CIA大鼠模型,用胶原酶消化法成功地分离并培养了原代大鼠成纤维样滑膜细胞.一定浓度的金雀异黄素能抑制CIA大鼠FLS分泌的VEGF和MMP-1、2、3、9,且抑制作用呈浓度依赖性.     

PCRRFLP单酶切法鉴别赫坎按蚊复合体的研究

目的建立一种新的赫坎按蚊复合体近缘种按蚊基因鉴别技术,应用于现场按蚊样本的鉴别。方法用分子生物学软件Vector NTI 9.0,对赫坎按蚊复合体的嗜人按蚊、雷氏按蚊、中华按蚊、八代按蚊4个近缘种按蚊rDNA基因的ITS2区段序列进行限制性内切酶酶切位点预测分析,选择特异性内切酶,建立一种能同时鉴别4种近缘种按蚊的聚合酶链反应限制性片段长度多态(PCR RFLP)单酶切法鉴别技术,并对现场捕获的452只按蚊样本进行基因鉴别。结果软件预测赫坎按蚊复合体近缘种4种按蚊的rDNA基因的ITS2区段可被限制性内切酶Dde I切成大小易于区分的不同片段,PCR RFLP单酶切实验结果和软件预测结果完全吻合。4个疟疾流行区捕获的452只按蚊样本经PCR RFLP Dde I单酶切法鉴定有20只嗜人按蚊、6只雷氏按蚊、391只中华按蚊、35只八代按蚊,与形态学鉴别结果符合率为93.4%,与PCR RFLP双酶切法结果符合率为100%。结论新建立的PCR RFLP Dde I单酶切法基因鉴别技术可准确鉴别赫坎按蚊复合体,比传统的形态学鉴别更为简便、可靠,适合用于复合媒介地区的媒介监测。     

Effects of carrageenin on human synovial cells in vitro: morphology, hyaluronic acid production, growth, and the lysosomal system.

Some in-vitro effects of the arthritogenic polysaccharide carrageenin were studied on cells from human synovium. Synovial cells were isolated from intact human knee joints, and cell lines were developed by passaging with trypsin. Carrageenin was ingested by the cells but did not significantly affect cell growth, numbers of lysosomes, intracellular lysosomal enzyme activity (N-acetyl-beta-D-glucosaminidase), or release of lysosomal enzyme from cells. Carrageenin produced a reduction in net hyaluronic acid synthesis. It also induced a striking morphological change in a high proportion of synovial cells, characterised by increased spreading over the culture surface and apparent condensation of the cytoplasm into a pattern of ridges. Nonrheumatoid and rheumatoid synovial cells behaved similarly to one another.     

Distribution of macrophages in rheumatoid synovial membrane and its association with basic activity

Summary Rheumatoid arthritis (RA) is an inflammatory disease of the synovial membrane, which results in the destruction of joints by inflammatory pannus. The synovial membrane shows proliferation and cellular infiltrates on microscopy with signs of chronic and acute inflammation. Macrophages are thought to play a central role in the pathogenesis of RA. We examined synovial membrane specimens of 21 RA patients using morphological, immunohistological and enzyme histochemical methods for number and distribution of macrophages. We were able to identify 41.5±8.8% of lining cells as macrophages, depending on the method used. In abundant diffuse lymphocellular infiltrates, 23.4±11.1% of mononuclear cells were macrophages. In addition, most cells in the region of tumorlike proliferation and a stromal population of fibroblastlike cells were detected by macrophage markers. Although cell number in synovial membrane increases drastically, we did not find correlations between the relative amount of macrophages in these regions and basic activity. Basic activity includes proliferative reaction as well as lymphoplasmacellular and mononuclear infiltration-both signs of an immunopathological process. In contrast, using enzymes or activation markers, there was a clear correlation. We consider that a constant high percentage of macrophages in RA synovial membrane is present regardless of any actual in flammatory process.     

Collagenase production by rheumatoid synovial cells: morphological and immunohistochemical studies of the dendritic cell.

The dendritic cells of dissociated, adherent rheumatoid synovial cell cultures are recognised by their distinctive morphological features--compact cytoplasm around the nucleus and long, branched cytoplasmic extensions. Such cells usually composed approximately 10% of the total adherent cell population but could vary from as few as 2% to as many as 40% with different synovial specimens. Histological studies have shown the cells to contain many mitochondria and large, spherical cytoplasmic inclusions which often distort the dendritic extensions. Although lysosomes were observed, no evidence for phagocytic activity was obtained. Immunolocalisation studies by means of a monospecific antibody to human collagenase have shown that the dendritic cell attached to a collagenous substratum produces and releases this enzyme in vitro. In contrast collagenase was detected in only a few of the fibroblast- and macrophage-like cells, and it was always intracellular. It is proposed that the dendritic cell may have an important role in the pathophysiology of the rheumatoid joint, particularly with regard to collagenase-mediated cartilage destruction.     

Interleukin-6 localisation in the synovial membrane in rheumatoid arthritis

Summary Polyclonal antibodies were raised in rabbits against Interleukin-6 (IL-6) by immunisation with a synthetic peptide of identical sequence to the amino terminal 12 amino acids of human IL-6. These antibodies reacted with recombinant IL-6 by ELISA and stained the cytoplasm of the IL-6 secreting bladder tumour cell line T24. Staining was abolished by prior incubation of the antibody with the IL-6 peptide. F(ab)₂ fragments made by pepsin digestion of the IgG were immunopurified, labelled with biotin and retained activity in the biochemical and histological assays. Sections of synovial membrane from patients with rheumatoid arthritis (RA) were stained with these antibodies, using an immunoperoxidase technique, and cells containing IL-6 were domonstrated in the thickened synovial lining layer and also in a perivascular distribution in the deeper synovium. In osteoarthritis there were fewer cells in the lining layer and hence localisation appeared similar in both the interstitial area and lining layer. Double-staining techniques with mouse monoclonal antibodies against cell subset markers in five RA synovial membranes showed that up to 13% of T-cells and 19% of antibody-producing cells stained or IL-6. However, up to 70% of the macrophages contained IL-6 and these were found in close proximity to lg-producing plasma cells. This study showed that Anacrophages were the major cells of the immune system in which IL-6 could be localised in RA, and suggests a cole for locally produced IL-6 in the stimulation of theumatoid factor production.     

Antigenic bacterial polysaccharide in rheumatoid synovial effusions.

Phenol-water extracted rheumatoid synovial fluids and synovial fluid leukocytes contain an antigen immunologically identical to the Proprionibacterium group bacteria. The antigen was identified by counter-immunoelectrophoresis in 70% of rheumatoid synovial fluid leukocyte pellets and in 60% of rheumatoid synovial fluids. It was also present in 6% of nonrheumatoid fluids and in 22% of nonrheumatoid inflammatory fluid leukocytes. Antigen was not detectable in synovial samples before extraction. Synovial and bacterial antigens were further purified by proteolytic digestion and Sepharose 4B column chromatography. Biochemical and enzymatic studies of bacterial and synovial antigens were similar and consistent with a high molecular weight polysaccharide. Serum antibody to bacterial and synovial antigens was significantly less frequent in rheumatoid sera than in normal controls. The significance of demonstrating a bacterial polysaccharide primarily in rheumatoid synovial effusions is discussed.     

Antigenic bacterial polysaccharide in rheumatoid synovial effusions

Phenol-water extracted rheumatoid synovial fluids and synovial fluid leukocytes contain an antigen immunologically identical to the Proprionibacterium group bacteria. The antigen was identified by counter-immunoelectrophoresis in 70% of rheumatoid synovial fluid leukocyte pellets and in 60% of rheumatoid synovial fluids. It was also present in 6% of nonrheumatoid fluids and in 22% of nonrheumatoid inflammatory fluid leukocytes. Antigen was not detectable in synovial samples before extraction. Synovial and bacterial antigens were further purified by proteolytic digestion and Sepharose 4B column chromatography. Biochemical and enzymatic studies of bacterial and synovial antigens were similar and consistent with a high molecular weight polysaccharide. Serum antibody to bacterial and synovial antigens was significantly less frequent in rheumatoid sera than in normal controls. The significance of demonstrating a bacterial polysaccharide primarily in rheumatoid synovial effusions is discussed.     

Ultrastructural localisation of muramidase in the human synovial membrane.

The synovial intimal cell layer comprises two morphological types of cell, A and B, with an intermediate type also postulated. Type A cells show features in common with other cells of the mononuclear phagocyte system, while type B cells appear similar to fibroblasts and are assumed to have synthetic activity. Muramidase is a marker of mononuclear phagocytic cells, and we have investigated the synovial membrane for the presence of this enzyme in cells by an immunogold labelling technique. Muramidase was localised within intracytoplasmic vacuoles in subintimal macrophages and type A synoviocytes. This finding provides further evidence that type A cells are closely related to macrophages.