氯喹抑制体外培养星形胶质细胞的激活

目的研究氯喹对体外培养大鼠海马星形胶质细胞激活的抑制作用,为癫痫的治疗提供实验依据。方法分离新生SD大鼠海马,体外培养星形胶质细胞,经纯化鉴定后分为:对照组、戊四氮(PTZ)组、氯喹干预组(25、50和75 mg/L),经相应处理后,分别用MTT法、免疫荧光、Western blot测定星形胶质细胞数量及活性、星形胶质纤维酸性蛋白(GFAP)及CyclinD1的表达量。结果与对照组比较,PTZ可激活星形胶质细胞的增殖,使GFAP、CyclinD1的表达量增加(P<0.05);与PTZ组比较,氯喹阻滞了PTZ激活的星形胶质细胞的增殖(P<0.05);氯喹可抑制PTZ激活星形胶质细胞异常增加的GFAP的表达;氯喹可抑制PTZ激活星形胶质细胞的CyclinD1的表达量(P<0.05);与对照组相比,3种结果均显示75 mg/L氯喹对体外培养星形胶质细胞激活的抑制作用较强,并可维持其在正常范围。结论氯喹具有抑制PTZ激活体外培养星形胶质细胞的作用,其可能通过抑制星形胶质细胞的增殖来发挥抗癫痫作用。     

布洛芬对戊四氮点燃癫痫大鼠星形胶质细胞的影响

目的:探讨不同剂量布洛芬对戊四氮(PTZ)点燃癫痫大鼠的影响及其作用机制。方法:雄性SD大鼠60只,随机分为对照组、PTZ组和PTZ+布洛芬组(按布洛芬剂量分为4组),分别对其进行干预,观察记录各组大鼠行为学及脑电图变化,同时观测布洛芬的不良反应,采用免疫荧光染色及Western Blot检测GFAP的表达情况。结果:PTZ组与对照组相比,痫样发作和星形胶质细胞增生明显(P 0. 05); PTZ+布洛芬各组痫样发作和星形胶质细胞增生情况较PTZ组降低,且剂量越高抑制作用越明显(P 0. 05),但不良反应的发生也越多。结论:布洛芬可通过抑制星形胶质细胞增生影响癫痫发作,且剂量越高抑制作用越强,但其不良反应也随剂量的增加而增加。     

活化小胶质细胞致星形胶质细胞激活

目的探讨活化小胶质细胞培养液对星形胶质细胞的影响。方法 LPS激活原代培养小胶质细胞,采用活化的小胶质细胞条件培养液刺激星形胶质细胞,观察星形胶质细胞GFAP及IL-1β和TNFα的表达。结果 LPS刺激后,小胶质细胞OX42表达量上升,IL-1β和TNFα的表达量增高;小胶质细胞条件培养液可致星形胶质细胞激活,GFAP表达量上升,IL-1β和TNFα的表达量增加。结论活化小胶质细胞的条件培养液可致星形胶质细胞激活,激活的小胶质细胞和星形胶质细胞表达前炎症介质IL-1β和TNFα。     

柴胡皂苷a抑制PTZ诱导的小鼠海马星形胶质细胞活化

目的:探讨柴胡皂苷a(SSa)对戊四氮(PTZ)诱导的小鼠海马星形胶质细胞活化的抑制作用。方法:分离培养小鼠海马星形胶质细胞,将细胞随机分为对照组、PTZ组、PTZ+0.625 mg/L SSa组和PTZ+1.25 mg/L SSa组。通过免疫荧光染色检测胶质细胞原纤维酸性蛋白(GFAP)的表达来鉴定细胞;用MTT检测评估细胞活力;用流式细胞术检测各组细胞的周期变化;ELISA法检测各组细胞中GFAP和间隙连接蛋白43(Cx43)的表达水平;流式细胞术和Hoechst 33258染色检测各组细胞的凋亡情况。结果:体外原代培养的星形胶质细胞贴壁生长,细胞突起明显。免疫荧光显示星形胶质细胞呈GFAP阳性表达。与对照组比较,PTZ组细胞活力和G_2/M期细胞百分比显著增加(P0.05),GFAP和Cx43的表达水平也显著上调(P0.05);与PTZ组比较,PTZ+0.625 mg/L SSa组和PTZ+1.25 mg/L SSa组细胞活力和G_2/M期细胞百分比均明显下降,GFAP和Cx43的表达水平也降低,但细胞凋亡水平显著增加(P0.05)。结论:SSa能够显著抑制PTZ诱导的海马星形胶质细胞活化,抑制细胞增殖并诱导凋亡。     

体外炎性诱导星形胶质细胞激活及功能分析

目的 观察体外培养的星形胶质细胞在炎性刺激下的激活及功能变化。方法 体外培养C57BL/6小鼠皮层的星形胶质细胞,应用免疫细胞化学和Western blot等方法,测定炎性刺激下各组细胞形态学及促炎因子（iNOS、IL-6、TNF-α）和抗炎因子（ARG1、IL-10、TGF-β1）表达水平的变化情况。结果炎性刺激后,免疫荧光细胞染色结果显示,M1(LPS+IFN-γ)组细胞被异常激活,胞体出现肿胀变大、扭曲等形态改变,且GFAP阳性细胞数量明显增多（P<0.001 vs. control））。CCK-8细胞活力检测结果显示,LPS组、IFN-γ组及M1组的吸光度均升高,其中M1组显著升高（P<0.001 vs. control）。Western blot结果表明,M1组促炎因子（iNOS、IL-6和TNF-α）表达均明显升高（P<0.001 vs. control）;各组抗炎因子（ARG1、IL-10和TGF-β1）的表达也显著升高（P<0.01 or 0.001 vs. control）。结论 体外炎性刺激异常激活星形胶质细胞,形态和功能显著变化,且A1样星...     

雷公藤甲素对LPS诱导的神经炎症中星形胶质细胞和小胶质细胞激活的抑制作用

选用健康成年雄性SD大鼠,用不同剂量的雷公藤甲素[10、25、50μg/(kg·d)]预处理5d后,海马注射内毒素(LPS,4μg)24h。采用免疫组织化学和荧光组织化学方法观察海马内星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)和小胶质细胞标记物蓖麻凝集素-1(RCA-1)表达的变化。结果显示:与生理盐水对照组比较,LPS注射引起注射部位GFAP表达增加,RCA-1阳性细胞增多、变大。而雷公藤甲素[50μg/(kg·d)]可明显下调LPS诱导的GFAP和RCA-1的表达,其抑制程度与药物剂量呈正相关。本研究结果提示,雷公藤甲素对海马内LPS诱导的星形胶质细胞和小胶质细胞的激活有明显的抑制作用。     

低氧诱导体外培养大鼠星形胶质细胞VEGF的表达

目的　血管内皮细胞生长因子 (VEGF)是一种高度特异性的促血管内皮细胞生长的因子 ,探讨其在低氧情况下缺血组织血管形成的影响。方法　用免疫组织化学法和RT RCR法检测了正常培养和低氧复氧诱导情况下体外培养大鼠星形胶质细胞中VEGF蛋白及VEGFmRNA表达。结果　正常培养的大鼠星形胶质细胞中有VEGF表达 ,但表达量较低 ,低氧复氧诱导后星形胶质细胞中VEGF表达量会增加 ,且随着复氧时间延长表达量增加 ,6h到达高峰 ,以后又逐渐降低。结论　低氧复氧可能诱导大鼠星形胶质细胞代偿性增生 ,使VEGFmRNA表达上调而促进血管再生 ,这在一定程度上可减少缺血对神经元的影响。     

氨对星形胶质细胞超微结构的影响

目的：在体外实验条件下观察氨对星状胶质细胞超微结构的影响。材料与方法：用ＮＨ４Ｃｌ造成高高ＮＨ４环境，对体外培养之星形胶质细胞的形态变化进行了超微结构观察。结果：高ＮＨ４环境下，星菜胶质细胞首先表现出损伤性变化，细胞电子密度降低，线粒体、内质网等细胞器肿胀，在胞质内形成许多单位膜包绕的电子密度降低的空泡区域，以后随着氨浓度加大或／和氨作用的时间延长，细胞内成份开始出现增生修复现象，次级溶酶体数量增     

芍药甙对体外培养大鼠星形胶质细胞活性的作用

目的　观察中药芍药有效成分芍药甙 (paeoniflorin ,PF)对培养的大脑皮层星形胶质细胞活性的影响。方法　采用体外星形胶质细胞培养的方法 ,对新生SD大鼠 (P2 )大脑星形胶质细胞进行培养 ,7d后纯化 ,再分别进行有血清和无血清培养。以MTT法观察PF对星形胶质细胞活性的影响 ,运用免疫组织化学的方法进行形态学观察。　结果　PF对星形胶质细胞活性有明显的抑制作用 ,不同浓度PF组星形胶质细胞活性均低于对照组 (P <0 0 1) ,并呈现出一定的量效关系 ,这种对星形胶质细胞活性的抑制作用与血清的存在与否无关。　结论　本实验表明芍药甙能抑制星形胶质细胞的活性 ,从而提示PF促进神经细胞活性的作用是一种直接的作用 ,而不是通过胶质细胞间接影响神经细胞     

Effect of Verapamil on T-Lymphocyte Activation in Vitro

Verapamil, a calcium-inhibitory drug, was found to inhibit T-lymphocyte proliferation in mixed lymphocyte culture and the proliferative response to phytohaemagglutinin and mumps antigen. It also inhibited production of interleukin 2 (IL-2). To exert inhibition, verapamil had to be added early in the culture period. Verapamil also had a relatively small inhibitory effect on IL-2-dependent growth. The effects were clearly seen only at concentrations exceeding the therapeutic serum level of verapamil.     

抗凝剂、温度和性别对体外中性粒细胞自发性激活的影响

从细胞形态学和细胞酶组织化学的角度出发，用伪足法和ＮＢＴ法检测体外中性粒细胞自发性激活，研究了抗凝剂、孵育温度和性别对检测结果的影响。发现：（１）ＥＤＴＡ可显著抑制中性粒细胞自发性激活，血标本以小剂量肝素抗凝为宜：（２）孵育温度对中性粒细胞自发性激活有影响，３７℃比２５℃的激活百分率高，同激活百分率出现较２５℃早１ｈ；（３）女性中性粒细胞自发性激活百分率较男性高，同激活百分率出现较男性早１ｈ。研究     

Inhibitory Effects of Rutin on the Endothelial Protein C Receptor Shedding In Vitro and In Vivo

Endothelial cell protein C receptor (EPCR) has important functions in regulation of coagulation and inflammation. EPCR shedding from the cell surface is mediated by tumor necrosis factor-α converting enzyme (TACE). Rutin is one of the major flavonoids from the buckwheat plant Fagopyrum tataricum. In this study, we investigated the effects of rutin on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding. We used a CLP model because this model more closely resembles human sepsis. Data showed rutin was a potent inhibitor of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding by suppression of TACE expression. Treatment with rutin resulted in a decrease of PMA-stimulated phosphorylation of p38, extracellular regulated kinases 1/2, and c-Jun N-terminal kinase. These results suggest the potential application of rutin for treatment of PMA and CLP-mediated EPCR shedding.     

Regulation of In Vitro Immunocyte Activation

Supernatants from incubated normal mouse spleen cells suppressed the DNA synthetic response induced by polyclonal B- and T-cell activators and the primary immune response to sheep erythrocytes in normal spleen cells in vitro. The inhibitory effects of the supernatants were found to be dependent on the culture system used. The main cell population producing or releasing the inhibitory factors was nonadherent spleen cells, but macrophages and bone marrow cells could also give supernatants with inhibitory effects. Thymocytes did not release any inhibitors under the same conditions. Separation of the supernatants showed that there were at least two factors with inhibitory effects on proliferation in lymphocytes. One of these factors had a molecular weight of less than 10,000 (not degradable by protease) and suppressed activation by all tested polyclonal B- and T-cell activators. Another factor (a protein with a molecular weight of more than 30,000) inhibited activation by some polyclonal B-cell activators (lipopolysaccharide, type III pneumococcal polysaccharide) whereas the proliferation induced by dextran sulfate was less or not at all affected under the same culture conditions. Trivial mechanisms for inhibition by supernatant factors, such as medium depletion, general toxicity, and accumulation of free thymidine in the medium, were excluded.     

Activation of Macrophages by in Vitro Allostimulated T Cells

Murine peritoneal macrophages, parabiotically co-cultured with combinations of in vitro H-2 sensitized thymus-derived lymphocytes obtained from drug-pretreated mice, possessed an increased cytotoxicity against alloantibody-coated target cells. This heightened activity appeared to be accentuated by and dependent on T-cell synergy. After 5 days of in vitro allosensitization at 37 degrees C, cortisone-resistant thymocytes allosensitized in combination with cyclophosphamide-pretreated splenic T cells released molecules that produced strong antibody-dependent macrophage-mediated cytotoxicity (ADCC). This enhanced ADCC correlated with increased macrophage rosetting with IgG-sensitized erythrocytes. These heightened activities resulted from soluble mediators released by the activated T cells which diffused across a 0.22-microns Millipore filter and were not dependent on lymphocyte-macrophage contact. Evidence that these molecules originated from the highly enriched T-cell populations and were not synthesized de novo by macrophages was supported by results of pretreatment with protein and RNA synthesis inhibitors. Evidence that soluble Fc receptors released from the alloactivated T cells were responsible for the increased macrophage EA binding and ADCC was obtained in affinity chromatography experiments in which activity could be depleted by passage over a Sepharose-Fc-coupled column and recovered in the column eluate.     

Characterization of an Inhibitory Allogeneic Effect on Humoral Responsiveness In Vitro

Evidence is presented that the induction of a humoral response is inhibitable by a thymus-derived cell (TI) that acts on the antigen-sensitive precursors of both the thymus-derived cooperating and the bone marrow-derived antibody-secreting cell-that is, the tC and B cell respectively. The inhibition of induction of the tC and B cell by the TI cell is shown to be reversed by increasing the effective level of cooperation. This competitive interaction between the inhibitory (TI) and cooperating (TC) systems is postulated to be part of the mechanism for regulating the class of the response, cell-mediated or humoral. The following properties of the inhibitory system were demonstrated: [1] The tI cell--the antigen-sensitive precursor of the TI cell--is both paralyzable and inducible. [2] The TI cell appears during the induction of a cell-mediated response and, if not identical to the effector cytotoxic ('killer') TK cell, the TI cell is induced in parallel with it. [3] The effector function of the TI cell, like that of the TK cell, is H-2-restricted.     

T细胞体外活化模型的建立

目的：比较并建立T细胞体外活化的条件,为后续功能及机制相关实验奠定基础。方法以Jur-kat细胞为模型,选择T细胞早期活化的指标CD69分子作为评价指标,通过比较不同刺激条件下CD69分子的表达量的差别,来判定合适的体外刺激条件,并通过增殖实验、抑制性信号表达量以及PBMC细胞进行验证。结果多克隆刺激剂PMA和ionomycin在剂量分别为20和500ng/ml时,Jurkat细胞活化效率较高;单克隆刺激剂anti-CD3包被和anti-CD28游离,且剂量在5μg/ml和1．5μg/ml时Jurkat细胞活化效率较高;作用48h检测多克隆刺激剂活化效率略高于单克隆刺激剂,细胞发生了增殖、抑制性分子CTLA-4的表达增加。结论建立了Jurkat及PBMC细胞的稳定的体外刺激条件,为后续研究T细胞的功能等奠定了实验基础。