大鼠附睾上皮细胞的原代培养及纯化鉴定

目的建立大鼠附睾上皮细胞原代培养及纯化方法。方法利用酶消化法和组织块法对大鼠附睾上皮细胞进行原代培养,然后用胰酶两步消化法进一步纯化附睾上皮细胞,最后分别利用免疫荧光和免疫组织化学染色对原代培养的细胞及相关蛋白表达情况进行鉴定。结果酶消化法较组织块法得到的附睾上皮细胞纯度高,免疫荧光染色结果证明所得附睾上皮细胞主要是主细胞,免疫组织化学结果证明培养的附睾上皮细胞中有雄激素受体和雌激素受体α的表达。结论利用酶消化法对大鼠附睾上皮细胞进行体外培养,方法简单易行,成功率高。     

成年SD大鼠心肌原代成纤维细胞分离及培养的优化方案

目的:摸索及优选成年SD大鼠心肌原代成纤维细胞的体外分离、培养及鉴定的实验方法。方法:将成年SD大鼠心脏剪成小组织块,采用以下四种方案(A:0.08%胰酶+0.1%胶原酶II消化15 min,B:0.2%胶原酶II消化15 min,C:0.2%胶原酶II消化60min,D:0.2%胶原酶II消化90 min)提取成年大鼠心脏原代成纤维细胞,再通过差速贴壁分离方法培养原代成纤维细胞。采用倒置显微镜观察成纤维细胞的基本形态特征,并进行Vimentiin免疫荧光染色对培养的原代细胞进行荧光鉴定;采用台盼兰染色对培养的原代成纤维细胞存活率进行鉴定;采用细胞计数对培养的成纤维细胞生长趋势进行鉴定。结果:四种方法均能培养成纤维细胞,但单酶消化60 min可一次性提取较多细胞,并且细胞状态佳,3 d即可传代。72 h成纤维细胞Vimentin免疫荧光染色阳性率高达97%。台盼兰染色可见其细胞死亡率明显降低,并且细胞计数可见细胞生长状态极佳。结论:单酶消化60 min是提取成年SD大鼠心肌原代成纤维细胞的高效、快速、稳定的实验方法,为心脏疾病的基础及临床研究提供了较为理想的细胞学实验模型。     

豚鼠肾小管上皮原代细胞的培养

目的建立一种简便易行的豚鼠原代肾小管上皮细胞培养方法。方法运用筛网分离法和多种酶消化法获取高纯度的肾小管上皮细胞。利用免疫组化法和形态学观察法鉴定培养的肾小管上皮细胞性质及纯度。结果通过肾小管节段贴壁,胶原酶消化组织节段和细胞等方法,有效地促进肾小管原代细胞增殖;胰酶节段消化法的细胞贴壁效果稍差,细胞传代状态不理想;胰酶消化法则细胞贴壁较少,细胞生长状态较差。结论培养豚鼠原代。肾小管上皮细胞是可行的。     

RA滑膜成纤维细胞的原代培养及鉴定

目的:改良体外分离、培养类风湿性关节炎滑膜成纤维细胞的方法,并进行鉴定。方法:取关节镜手术中获得RA患者滑膜组织进行机械分离、胶原酶消化后直接将所有消化产物置于细胞培养皿两次贴壁培养,差速消化法纯化成纤维细胞,倒置显微镜观察细胞形态、流式细胞术及免疫细胞化学的方法鉴定细胞纯度。结果:胶原酶消化后直接贴壁结合差速消化纯化法分离获得的原代滑膜细胞中呈梭形的成纤维样细胞占98%以上,细胞核呈椭圆形位于细胞中央,偶见少量圆形的滑膜巨噬细胞。流式细胞术显示98%以上的滑膜细胞具有vimeintin+CD68的成纤维细胞特征。免疫细胞化学提示滑膜细胞vimentin表达阳性、CD68不表达。结论:成功分离获得了纯度和活性很高的人滑膜成纤维样细胞,方法更简便,效率更高,为后续类风湿性关节炎滑膜侵袭机制的研究奠定了基础。     

成年小鼠原代皮肤成纤维细胞的分离培养及其生物学特性

目的:建立一种周期短、成本低的成年小鼠原代皮肤成纤维细胞分离培养方法,并探索其生物学特性。方法:取8~12周龄BALB/c小鼠背部、尾尖、耳部皮肤,配制2种血清的细胞培养液,采用组织块贴壁法、酶消化法、酶消化组织块贴壁法进行原代皮肤成纤维细胞的培养,通过显微镜观察比较原代细胞的数量、形态、培养周期及纯度;通过免疫荧光、CCK-8、UVB辐照、流式细胞术进行生物学特性鉴别。结果:背部皮肤组织块贴壁使用Gibco胎牛血清培养7 d无细胞游出,CLARK特级胎牛血清细胞游出较多。背部皮肤经酶消化法得到细胞贴壁少;经组织块贴壁法细胞生长慢,培养周期长;酶消化组织块贴壁法细胞游出速度快、数量多、呈长梭形。尾尖取材量少,得到细胞少;耳部皮肤取材方便,但细胞纯度低。CCK8增殖曲线呈S型;相较于对照组,UVB辐照后细胞凋亡率增高17%。结论:CLARK特级胎牛血清、背部皮肤取材、酶消化组织块贴壁法是培养成年小鼠原代皮肤成纤维细胞最优的方案,可增加细胞得量、缩短培养周期,降低成本。     

前列腺基质细胞的原代培养方法

目的:建立一种操作简单、成功率高、重复性好的前列腺增生组织原代基质细胞(PSC)培养方法。方法:采用胶原酶消化法、组织块贴壁法和胰酶消化组织块贴壁法,从70岁及以上男性的良性前列腺增生组织中分离培养PSC,通过显微镜观察比较PSC的数量、形态、培养周期,用免疫荧光染色法鉴定PSC的纯度。结果:胶原酶消化法得到的贴壁细胞少,细胞体积较小且形态无法铺展,增殖能力较弱;组织块贴壁法培养72h后细胞会从组织边缘缓慢爬出,生长周期长;胰酶消化组织块贴壁法,细胞培养7d后基本融合,折光性强,细胞多呈长梭形,通过免疫荧光染色鉴定,基质细胞纯度在95%以上。结论:利用胰酶消化组织块贴壁法建立了一种易行、高效且重复性好的前列腺增生组织基质细胞培养方法。     

组织块法和酶消化法体外培养原代鼠阴道上皮细胞的研究

为探讨更佳的阴道上皮细胞体外培养技术,为组织工程化阴道动物模型提供种子细胞,分别应用组织块法和酶消化法原代培养大鼠阴道上皮细胞,观察两种方法细胞生长所需时间、细胞形态和生长特性,免疫组化进行鉴定。结果表明,两种细胞培养方法均能获得不规则圆形或多边形的阴道上皮细胞,其传代后增殖特性和生长曲线基本一致,角蛋白染色阳性,但酶消化法较组织块法细胞贴壁快、生长时间短、产量大、细胞纯度高、能较迅速获得较多细胞用于组织工程阴道的构建。     

大鼠细小肺动脉平滑肌细胞原代培养和鉴定方法的研究

目的：建立一种重复性好、培养周期短及传代次数多的大鼠细小肺动脉平滑肌细胞（PASMCs）培养方法。方法：在无菌条件下,分离雄性SD大鼠肺细小动脉,剥离外膜和剔除内皮细胞,经胶原酶I消化,培养PASMCs。0.4%台盼蓝染色测定细胞活力;倒置相差显微镜观察;免疫细胞化学法和免疫荧光染色法,进行平滑肌α-肌动蛋白（α-SMactin）鉴定。结果：形态学观察、免疫细胞化学法及免疫荧光染色法鉴定表明培养细胞为PASMCs;细胞存活率在96.5%以上;原代培养后4～7d即可传代,并且生长特点、细胞形态不易发生改变。结论：采用胶原酶I消化法培养PASMCs,方法简单、酶消化时间易控制、培养周期短、重复性好,培养的原代PASMCs具有数量多和生长迅速的特点。     

成年大鼠阴茎海绵体cajal间质细胞的分离、培养与鉴定

目的:探索成年大鼠阴茎海绵体内cajal间质细胞(ICCs)的分离、培养和鉴定方法,为进一步研究其在阴茎海绵体中的作用提供条件.方法:取大鼠阴茎海绵体组织,采用酶消化法分离细胞,差速贴壁法相对纯化ICCs,将纯化后的细胞悬液接种于DMEM培养基中进行培养.通过倒置显微镜下观察细胞贴壁和形态,并用c-Kit特异性抗体标记细胞,免疫荧光法鉴定ICCs.结果:培养24小时后ICCs贴壁良好,细胞形态学观察显示ICCs呈纺锤状,有两个或多的突起,免疫荧光检验可见ICCs呈c-Kit抗体染色阳性.结果:用酶消化法可成功分离和培养大鼠阴茎海绵体ICCs,大鼠海绵体组织内ICCs的生理学功能有待进一步研究.     

SD大鼠胸腹主动脉平滑肌细胞的原代培养及鉴定

目的建立原代大鼠胸腹主动脉平滑肌细胞培养方法,为研究心脑血管动脉粥样硬化疾病提供重要的载体和工具细胞。方法选取6～8周龄SD大鼠2只,剪开胸腹腔,剥离主动脉,刮除血管内、外膜,经0.2%Ⅱ型胶原酶/弹性蛋白酶消化后,剪碎成块,种瓶进行原代培养。通过细胞形态学观察、α平滑肌肌动蛋白免疫细胞化学染色法鉴定所培养的目的细胞。结果接种于培养瓶中的血管组织块培养48h后开始贴壁;72h后细胞以组织块为中心,向外迁移,"岛屿状"细胞团簇初步形成;96h后原代细胞集落逐渐融合,铺满瓶底,呈现典型的"峰-谷"样生长;第一代传代细胞形态基本保持不变,高倍镜下细胞呈三角形或星形。免疫细胞化学染色显示,细胞α平滑肌肌动蛋白阳性率达99%以上。结论复合酶消化法结合组织块法能够成功高效地分离培养出原代大鼠胸腹主动脉平滑肌细胞。     

Activation of latent rheumatoid synovial collagenase by human mast cell tryptase

The functional role of mast cells in rheumatoid synovium was investigated by assessing the ability of mast cell tryptase to activate latent collagenase derived from rheumatoid synoviocytes. Tryptase, a mast cell neutral protease, was demonstrated in situ to reside in rheumatoid synovial mast cells, by an immunoperoxidase technique using a mouse mAb against tryptase, and in vitro to be released by dispersed synovial mast cells after both immunologic and nonimmunologic challenge. Each rheumatoid synovial mast cell contains an average of 6.2 pg of immunoreactive tryptase and the percent release values of this protease correlated with those of histamine (r = 0.58, p less than 0.01). The ability of purified tryptase to promote collagenolysis was demonstrated in a dose-dependent fashion using latent collagenase derived from rheumatoid synovium, synovial fluid, IL-1-stimulated cultured synoviocytes, and partially purified latent collagenase derived from conditioned media, with between 10 and 92% of the collagen substrate degraded. [3H] Collagen, treated with tryptase-activated latent collagenase, was subjected to electrophoresis on SDS polyacrylamide gels and autoradiography showed the collagen degradation pattern (A, B) characteristically produced by collagenase. Mast cell lysates also activated synovial latent collagenase yielding 24% digestion of collagen substrate. This activator in mast cell lysates could be inhibited by diisopropylflurophosphate or by immunoadsorption of tryptase. Thus, mast cells may activate metalloproteinases and play a role in the catabolism of collagen that occurs in rheumatoid synovium.     

不同二步酶灌注法分离大鼠肝星状细胞的比较研究

目的:在现有二步酶灌注法分离大鼠肝星状细胞(hepatic stellate cells,HSC)的基础上,探索更加高效的分离HSC方法。方法:分别采用链酶蛋白酶+胶原酶循环灌注、链酶蛋白酶非循环灌注+胶原酶循环灌注以及胶原酶单独循环灌注法分离大鼠HSC,比较三种方法的细胞获得率、活性和纯度差异。应用0.4%台盼蓝染色判断活性,结蛋白(desmin)、波形蛋白(vimentin)细胞免疫荧光方法鉴定纯度。结果:链酶蛋白酶非循环灌注+胶原酶循环灌注法细胞获得率高于另两种方法,细胞活力高于链酶蛋白酶循环灌注+胶原酶循环灌注法,三组得到的细胞纯度均高于90%且无显著差异。结论:在三种二步酶灌注方法中,链酶蛋白酶非循环灌注+胶原酶循环灌注法能显著提高HSC获得率,且对细胞活力影响小,不降低细胞纯度,是一种高效的分离方法,有利于HSC相关肝脏疾病的生物学研究。     

Profile of Exoglycosidases in Synovial Cell Cultures Derived from Human Synovial Membrane

OBJECTIVE: Determining the activity of lysosomal exoglycosidases in tissue cultures of synoviocytes derived from the knee joints of patients with injured anterior cruciate ligaments (ACL), juvenile idiopathic arthritis (JIA), and rheumatoid arthritis (RA). METHODS: The following exoglycosidases in cultured synoviocytes were analyzed with p-nitrophenyl derivatives of appropriate sugars as substrates: hexosaminidase (HEX) and its isoenzyme A (HEX-A), beta-glucuronidase (GluA), beta-galactosidase (GAL), alpha-mannosidase (MAN), and alpha-fucosidase (FUC). RESULTS: In our cell cultures, fibroblast-like synovial cells (FLS) dominated. In the group of patients with ACL-injuries, and in the groups of patients with JIA and RA, the activity of the investigated exoglycosidases was significantly higher in the intra- rather than in the extracellular compartment. Hexosaminidase was the predominant exoglycosidase. Stimulation of synoviocytes by IL-1beta in cell cultures significantly increased the activity of HEX, HEX-A, and GluA in both compartments, as well as of GAL, MAN, and FUC in the intracellular compartment. Stimulation by IL-1beta rheumatoidal synoviocytes increased by 128-201% the activity of HEX and HEX A in intracellular compartments and 33-72% in extracellular compartment. CONCLUSIONS: The profile of lysosomal exoglycosidases in a cell culture of human synoviocytes is similar, but not identical, to those in the knee joint. Hexosaminidase is the dominant glycosidase in cultured unstimulated and IL-1beta-stimulated human synoviocytes. The HEX inhibitors may be new drugs for the treatment of inflamed knee joints.     

The active form of leflunomide, HMR1726, facilitates TNF-alpha and IL-17 induced MMP-1 and MMP-3 expression.

BACKGROUND/AIMS: To elucidate the influence and mode of action of HMR1726 (the active metabolite of leflunomide) on TNF-alpha and IL-17 activated metalloproteinases expression in synoviocytes. METHODS: Synovial fibroblasts from RA and OA patients were stimulated with both cytokines and altered gene expression in the presence or absence of leflunomide was detected by microarray analyses and quantitative RT-PCR. Protein expression was detected by western blotting and commercial ELISAs. RESULTS: Microarray analyses revealed that the addition of HMR1726 (50 microM) to TNF-alpha and IL-17- stimulated synoviocytes induced gene expression of metallo-proteinases, especially MMP-1 and -3 in comparison to activated synoviocytes in the absence of leflunomide. To confirm these data, we examined the influence of different concentrations of HMR1726 in synoviocytes from further 5 OA and 7 RA patients by quantitative PCR. HMR1726 gradually induced MMP-1 and MMP-3 gene expression in a dose-dosedependent manner. Similar results were observed on protein levels. Examination of signal transduction pathways participating in the regulation of leflunomideinduced MMPs expression showed that the mechanism underlying activation of MMP-1 is in part p38- and activation of MMP-3 was MEK1/2- dependent. CONCLUSION: Leflunomide was not able to abolish expression of metallo-proteinases in synoviocytes activated with TNF-a and IL-17.     

Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity (TIMP-1/EPA).

This study examines the role of interleukin-6 (IL-6) in connective tissue metabolism. Effects of different preparations of IL-6 on production of collagenase and tissue inhibitor of metalloproteinases-1/erythroid potentiating activity production are studied in human fibroblasts, synoviocytes, and articular chondrocytes. In contrast to interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha), IL-6 does not stimulate the production of collagenase, nor does it modulate the stimulatory effects of IL-1 beta and TNF alpha on the production of this proteinase. Furthermore, IL-6 has no detectable effect on prostaglandin E2 production, an additional proinflammatory response induced by IL-1 beta and TNF alpha. IL-6, however, is identified as a potent inducer of de novo synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity in all types of connective tissue cells examined. These results define new biological activities of IL-6 and provide further insight into the regulation of connective tissues by cytokines.     

NF-kappa B regulation by I kappa B kinase-2 in rheumatoid arthritis synoviocytes

IkappaB kinase-1 and IkappaB kinase-2 (IKK1 and IKK2; also called IKKalpha and IKKbeta, respectively) are part of the signal complex that regulates NF-kappaB activity in many cell types, including fibroblast-like synoviocytes (FLS). We determined which of these two kinases is responsible for cytokine-induced NF-kappaB activation in synoviocytes and assessed the functional consequences of IKK1 or IKK2 overexpression and inhibition. FLS were infected with adenovirus constructs encoding either wild-type (wt) IKK1 or IKK2, the dominant negative (dn) mutant of both kinases, or a control construct encoding green fluorescence protein. Analysis of the NF-kappaB pathway revealed that cytokine-induced IKK activation, IkappaB degradation, and NF-kappaB activation was prevented in cells expressing the IKK2 dn mutant, whereas baseline NF-kappaB activity was increased by IKK2 wt. In addition, synthesis of IL-6 and IL-8, as well as expression of ICAM-1 and collagenase, was only increased by IKK2 wt, and their cytokine-induced production was abrogated by IKK2 dn mutant. However, the IKK1 dn mutant did not inhibit cytokine-mediated activation of NF-kappaB or any of the functional assays. These data indicate that IKK2 is the key convergence pathway for cytokine-induced NF-kappaB activation. Furthermore, IKK2 regulates adhesion molecule, matrix metalloproteinase, and cytokine production in FLS.     

Hsp72对类风湿关节炎滑膜细胞IL-6、IL-8表达及NFκ-B活化的影响

目的:探讨热休克蛋白(Hsp)72对类风湿关节炎患者滑膜细胞IL-6、IL-8表达的影响,从NFκ-B信号通路活化的角度阐明其作用机制。方法:原代培养类风湿关节炎患者的滑膜细胞;采用酶联免疫吸附试验(ELISA)法检测细胞培养上清中IL-6和IL-8的含量;采用Western blot检测滑膜细胞NFκ-B和ΙκBα蛋白的表达变化;采用免疫荧光技术检测NFκ-B核移位的变化。结果:Hsp72抑制TNFα-所诱导的IL-6和IL-8的生成;Hsp72抑制TNFα-所诱导NFκ-B在核内的表达和移位;Hsp72抑制TNFα-所诱导ΙκBα蛋白降解。结论:Hsp72可能通过抑制滑膜细胞IL-6、IL-8表达及抑制NF-κB信号通路活化而对类风湿关节炎发挥抗炎作用。