脂多糖对新生小鼠肺血管内皮细胞的影响及机制探讨

该文旨在探讨脂多糖(lipopolysaccharide, LPS)对新生小鼠肺血管内皮细胞的影响及机制。将分离培养的肺血管内皮细胞随机分为空白对照组和LPS组;用10μg/mL的LPS处理细胞后分别于0 h、6 h、12 h、24 h、48 h时间点收集细胞标本进行检测;采用划痕实验观察LPS对肺血管内皮细胞迁移的影响;用荧光定量PCR(Real-time PCR, RT-PCR)检测白介素-1β(IL-1β)、巨噬细胞炎性蛋白-1α(MIP-1α)、单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-α(TNF-α) mRNA水平变化; Western blot检测血管内皮生长因子(VEGF)、血管内皮生长因子受体2(VEGFR2)及核因子κB(nuclear factor kappa-B, NF-κB)相关蛋白P65水平的变化。结果显示,体外分离培养的肺血管内皮细胞成鹅卵石样排列, VIII因子相关抗原和CD31表面抗原荧光染色阳性。划痕实验中, LPS组细胞在12 h的迁移高于对照组(P0.001);荧光定量PCR检测到LPS组分泌的炎症因子IL-1β、TNF-α和趋化因子MIP-1α、MCP-1的mRNA表达明显高于对照组(P0.001); Western blot显示, LPS组与对照组相比, VEGF蛋白表达在24 h、48 h处降低(P0.05), VEGFR2蛋白表达在各时间段都明显降低(P0.001),同时, NF-κB相关蛋白P65活性显著升高(P0.05)。研究表明,脂多糖诱发的炎症反应影响肺血管内皮细胞的发育,其机制可能与NF-κB通路激活,诱导炎症因子、趋化因子表达升高和VEGF/VEGFR2表达下降有关。     

幽门螺杆菌感染激活NF-κB信号通路促进胃癌SGC-7901细胞炎症因子释放

为了探讨幽门螺杆菌对胃癌SGC-7901细胞炎症因子释放的影响,本研究将幽门螺杆菌感染SGC-7901细胞后,采用细胞计数盒(CCK-8)检测SGC-7901细胞活力,酶联免疫吸附实验(ELISA)检测炎症因子TNF-α、IL-1β以及IL-8的水平,Real-time PCR检测细胞TNF-α、IL-1β以及IL-8 m RNA的表达,蛋白免疫印迹法(Western blotting)检测NF-κB信号通路相关蛋白NF-κB p65蛋白表达以及IκBα磷酸化水平。研究结果表明,幽门螺杆菌感染后,SGC-7901细胞活力显著增加;幽门螺杆菌感染明显上调SGC-7901细胞TNF-α、IL-1β以及IL-8 mRNA的表达;本研究还进一步发现幽门螺杆菌感染显著增加SGC-7901细胞TNF-α、IL-1β以及IL-8的水平;此外,幽门螺杆菌处理的SGC-7901细胞,其NF-κB p65的蛋白表达以及IκBα磷酸化水平均显著上调。本研究的结论初步表明,幽门螺杆菌感染促进胃癌SGC-7901细胞炎症因子的释放,其机制可能涉及激活NF-κB信号通路。     

不同诱导因子对人外周血单个核细胞P2X7受体表达的作用

ATP激活P2X7受体可产生一系列的白细胞功能反应,因此P2X7受体的表达调控引起我们的兴趣。然而P2X7受体在正常人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)、单核细胞中的表达调控机制尚未阐明。本文用半定量RT-PCR方法检测多种细胞因子、细菌抗原、丝裂原对P2X7受体表达的诱导作用,探索P2X7受体的诱导表达模式。结果表明,单个核细胞和单核细胞可检出P2X7受体的表达;白细胞介素2、4、6(interleukin-2、-4、-6,IL-2、IL-4、IL-6)、肿瘤坏死因子仪(tumour necrosis factor-α,TNF-α)等细胞因子和金黄色葡萄球菌CowanⅠ株(Staphylococcus aureus Cowan strainⅠ,SAC)、脂多糖(lipopolysaccharide,LPS)能上调PBMC的P2X7受体表达,而γ干扰素(interferon-γ,IFN-γ)、粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)、巨噬细胞集落刺激因子(macmphage colony-stimulating factor,M-CSF)和植物血凝素(phytohemagglutinin-M,PHA-M)等则没有作用;LPS和M-CSF可以提高单核细胞的P2X7受体表达,IFN-γ、TNF-α、GM-CSF作用较弱,但是这些因子的预处理并不能增强LPS对P2X7受体表达的诱导。炎症因子促进P2X7受体的表达,提示P2X7受体可能在对抗细菌感染的免疫反应中起一定作用,这有待于进一步研究。     

Aβ_(1-42)寡聚体通过PI3K-AKT信号通路诱导神经胶质细胞内TNF-α表达的机制研究

本文主要应用Western blot和Real-Time PCR等实验方法考察Aβ_(1-42)寡聚体通过AKT信号通路对人源胶质母细胞瘤A172和鼠源胶质母细胞瘤D1A细胞中TNF-α表达的影响,同时应用免疫荧光的实验方法,进一步检测Aβ_(1-42)寡聚体诱导TNF-α表达的具体调控机制。结果发现,A172和D1A细胞经Aβ_(1-42)寡聚体处理后,Aβ_(1-42)寡聚体可上调炎症因子TNF-α的表达,并且免疫荧光结果显示,此诱导作用是通过PI3K-AKT信号通路实现的。基于本实验研究,说明Aβ_(1-42)寡聚体参与AD患者脑内炎症反应的发生,提示抗炎及抗Aβ形成在阿尔茨海默病的临床治疗方面具有重要的理论意义。     

黄曲霉素对裸鼹鼠肺泡上皮Ⅱ型细胞基因表达的影响

目的:观察不同浓度黄曲霉素对裸鼹鼠肺泡上皮Ⅱ型细胞(AEC II)相关因子m RNA表达的影响,探讨裸鼹鼠抵御肺癌的分子机制。方法:通过酶消化法分离裸鼹鼠肺组织细胞并用免疫黏附法进行纯化得到裸鼹鼠肺泡上皮Ⅱ型细胞进行原代培养,40小时后,实验组分别给予不同浓度(0.25、0.5、1.0、2.0和4.0 mg/L)的黄曲霉素处理,溶剂对照组给予DMSO(0.4 m L/L)处理。黄曲霉素作用48小时后收集细胞,用实时定量PCR技术检测裸鼹鼠AECII细胞中的SP-C基因及TNF-α、IL-1、IL-6、IL-8、IL-12等炎症因子的m RNA表达变化。结果:原代分离的裸鼹鼠AEC II细胞纯度70%,活性90%,可用于体外实验。定量PCR结果显示黄曲霉素使裸鼹鼠AEC II细胞SP-C表达水平显著下降,TNF-α、IL-1、IL-6、IL-8、IL-12等炎症因子的表达水平在黄曲霉低于2 mg/L浓度的条件下没有显著变化。结论:裸鼹鼠AEC II细胞在黄曲霉素导致细胞受损的情况下,仍然保持较低水平的炎症因子表达,这可能是其抵抗肺癌的机制之一。     

聚合度7-15的壳寡糖抑制脂多糖刺激的单核细胞产生TNF-α和IL-8的作用研究

本文通过建立脂多糖刺激的单核细胞炎症损伤模型,观察聚合度7-15的壳寡糖对炎性单核细胞白细胞介素-8(IL-8)和肿瘤坏死因子-α(TNF-α)表达的影响,及对p38丝裂原激活的蛋白激酶(p38 mitogen-activated protein kinases,p38MAPK)信号通路磷酸化的影响。采用p38信号通路抑制剂(SB203580)验证抑制p38信号通路对脂多糖诱导的单核细胞表达IL-8和TNF-α的作用,从而探索壳寡糖抑制单核细胞炎性损伤的分子机制。结果表明壳寡糖可抑制脂多糖诱导的单核细胞表达IL-8和TNF-α,并且抑制p38信号蛋白的磷酸化水平。因此,初步认为壳寡糖可能通过抑制炎性U937细胞中p38MAPK信号通路抑制IL-8和TNF-α的表达。     

Aβ-Cu复合物与Aβ纤丝对小胶质细胞激活作用比较研究

目的:比较具有氧化还原活性的过渡金属离子Cu~(2+)(Cu)诱导形成的Aβ聚集物(Aβ-Cu复合物)与Aβ自聚集形成的纤丝(Fibrillar Aβ,f Aβ)对小胶质细胞激活作用的差异。方法:制备Aβ-Cu复合物和fAβ,利用小鼠BV-2小胶质细胞株,分别以不同浓度的Aβ-Cu复合物和fAβ于37℃刺激24 h,检测细胞上清液中的TNF-α(Tumor necrosis factor-α)、NO(Nitric oxide)以及H_2O_2(Hydrogen Peroxide)的含量。分别收集Aβ-Cu复合物和f Aβ作用24 h后的大鼠原代小胶质细胞条件培养液,通过观察该条件培养液对大鼠原代海马神经元细胞活力的影响,评价小胶质细胞介导的间接神经元毒性。结果:(1)在不引起直接神经毒性剂量(2.5μM)下,Aβ-Cu复合物激活小胶质细胞释放TNF-α(P0.01)、NO(P0.05)以及H_2O_2(P0.05)的作用强于f Aβ。(2)在此剂量下,Aβ-Cu复合物通过激活小胶质细胞引起的间接神经元毒性强于fAβ(P0.05)。结论:与fAβ相比,非神经毒性剂量的Aβ-Cu复合物对于小胶质细胞具有更强的激活作用,并由此引发更为明显的神经元毒性。     

安子合剂对抗磷脂抗体阳性流产小鼠TLR2、TLR4及TNF-α的影响

探讨安子合剂对抗磷脂抗体阳性流产小鼠母胎界面TLR2、TLR4及炎症因子TNF-α的影响。以人β2GPⅠ为免疫原建立抗磷脂抗体阳性流产小鼠模型,计算胚胎吸收率,采用ELISA法检测小鼠外周血清抗β2GPⅠ抗体浓度、TNF-α含量,实时定量PCR测定胎盘组织TLR2、TLR4mRNA水平,免疫组织化学法检测胎盘组织TLR2、TLR4蛋白表达。结果显示,与空白组相比,模型组TLR2、TLR4mRNA水平显著升高,蛋白均呈弥漫性高表达;与模型组相比,安子合剂组能显著减少胚胎吸收率,降低抗β2GPⅠ抗体及TNF-α浓度,可同时下调模型小鼠TLR2及TLR4mRNA水平,使TLR2、TLR4蛋白呈中低表达。综上提示安子合剂抑制母胎界面TLR2、TLR4信号转导通路中的上游关键分子,减少炎症因子释放,这可能是其安胎作用机制之一。     

亚高山绣球菌多糖的提取优化、结构表征和抗炎作用CSCD

研究亚高山绣球菌多糖的提取纯化、结构表征及抗炎作用。采用响应曲面法优化提取工艺,柱层析等方法纯化亚高山绣球菌多糖(Sparassis subalpina polysaccharide,SSP);采用气相色谱、核磁共振谱和电子显微镜等表征结构,实时荧光定量PCR等分析抗炎活性。结果显示,在最佳提取条件(液料比80 mL/g,提取时间60 min,提取温度70℃)下,总糖提取率为(18.21±0.68)%。SSP重均分子质量是50 kDa,由葡萄糖、甘露糖、半乳糖和阿拉伯糖组成(6.5∶1.3∶1∶1),重复结构单位是→3)-α-D-Galp-(1→2)-β-D-Glup-(1→3)-β-L-Araf-(1→3)-α-D-Manp-(1→,呈无分支的网状结构。SSP对炎症细胞中TNF-α、COX-2和iNOS的表达均有显著抑制作用(P<0.05)。结果表明无分支网状结构SSP具有抗炎作用。     

Vitrification of human monocytes

Human monocytes purified from peripheral blood by counterflow centrifugal elutriation were cryopreserved in a vitreous state at 1 atm pressure. The vitrification solution was Hanks' balanced salt solution (HBSS) containing (w/v) 20.5% Me2SO, 15.5% acetamide, 10% propylene glycol, and 6% polyethylene glycol. Fifteen milliliters of this solution was added dropwise to 1 ml of a concentrated monocyte suspension at 0 degrees C. Of this, 0.8 ml was drawn into silicone tubing and rapidly cooled to liquid nitrogen temperature, stored for various periods, and rapidly warmed in an ice bath. The vitrification solution was removed by slow addition of HBSS containing 20% fetal calf serum. The numerical cell recovery was about 92% and most of these retained normal phagocytic and chemotactic ability. Differential scanning calorimeter records of the solution show a glass transition at -115 degrees C during cooling and warming, but no evidence of ice formation during cooling. Devitrification occurs at about -70 degrees C during warming at rates as rapid as 80 degrees C/min. The amount of devitrification is dependent upon the warming rate. Freeze-fracture freeze-etch electron microscope observations revealed no ice either intra- or extracellularly in samples rapidly cooled to liquid nitrogen temperatures except for small amounts in some cellular organelles. However, if these cell suspensions were warmed rapidly to -70 degrees C and then held for 5 min, allowing devitrification to occur, the preparation contained significant amounts of both intra- and extracellular ice. Biological data showed that this devitrification was associated with severe loss of cell function.     

Cryopreservation of human monocytes

Monocytes were isolated from human peripheral blood by Ficoll-Isopaque density-gradient centrifugation and adherence to glass. These cells were then frozen according to an automatically controlled cooling program and stored in liquid nitrogen.After the freezing, thawing and washing, 63% of the cells present before cryopreservation were recovered. Over 95% of the recovered cells excluded trypan blue. Storage at ?196 °C did not alter the percentage of monocytes (70–80%) in the supensions.Although the percentage of cells that formed rosettes with erythrocytes sensitized with IgG antibodies (EAIgG) was unaltered after freezing, formation of EA rosettes was more readily inhibited by free IgG. The capacity of monocytes to lyse EAIgG was not influenced by cryopreservation, in contrast with their potency to phagocytize zymosan particles, which was decreased. The chemotactic response toward casein was also diminished after freezing. There was no significant difference in reactivity between monocytes frozen for a short time (2–15 hr) and those frozen for a longer period (more than 3 months).Electron microscopic examination showed alterations in the mitochondrial structure of the frozen cells.     

Adhesion-mediating molecules of human monocytes

Adhesion of monocytes to each other and to T cells and substrates is increased by phorbol esters. In the presence of these compounds monocyte aggregation was almost completely inhibited (greater than 90%) by monoclonal antibody 60.3. This antibody recognizes GP90 (CD18), a leukocyte surface glycoprotein which is separately and noncovalently associated to either GP160 (CD11a), GP155 (CD11b), or GP130 (CD11c). Anti-LFA-1 antibody (CD11a) was only partially inhibitory (35%) while antibodies 60.1 (CD11b) and anti-Leu-M5 (CD11c) had a minimal inhibitory effect (10%). Antibody LB-2 recognizing a single glycoprotein distinct from the GP90-GP160 complex and expressed on activated B and T cells, monocytes, and vascular endothelial cells was partially inhibitory (22%). Monoclonal antibodies anti-C3bR (CD35), T29/33 (CD45, leukocyte common antigen 200). TA-1 (CD11a), OKM1 (CD11b), F10-44-2 (brain-leukocyte antigen), OKM5 (monocyte-endothelial cell antigen) and to class I or class II molecules exerted no inhibition on the monocyte aggregation. Fab fragments of antibody 60.3 efficiently inhibited not only monocyte aggregation in the absence or presence of phorbol esters but also adhesion of these cells to autologous or allogeneic T lymphocytes and, to a lesser extent, to plastic surfaces. It is thus concluded that GP90, either alone or associated to the larger glycoproteins, and LB-2 antigen mediate monocyte adhesion.     

Proliferation of mature and immature subpopulations of bronchoalveolar monocytes/macrophages and peripheral blood monocytes

A continuous influx of peripheral blood monocytes (PBM) to the lung is thought to maintain the local population of alveolar macrophages (AM). However, local proliferation of a small subpopulation of AM has been demonstrated in animal studies and in humans. AM exhibit a great heterogeneity with regard to their morphology (cell size, shape of nucleus), immunophenotype (expression of CD14 and RFD9 antigen), and function. Part of this heterogeneity may be explained by the presence of different maturation stages of AM, ranging from small immature, CD14⁺ RFD9^? PBM-like cells to large, CD14^? RFD9⁺ mature AM. These findings prompted us to study whether proliferation of PBM and AM is related to their stage of maturation. The expression of the proliferation marker Ki-67 was studied in AM from both healthy volunteers and patients suffering from sarcoidosis. Using double immunofluorescence staining, we studied proliferation of immature, CD14⁺ AM, and mature, RFD9⁺ AM in sarcoidosis, and we compared this with PBM. A significantly larger percentage of AM in general expressed Ki-67 antigen in sarcoidosis (3.0 (median); range 1.1–5.5) as compared with healthy volunteers (0.8; 0.2-1.3). In sarcoidosis, proliferation was observed in both the immature and the mature subpopulation of AM. Proliferating PBM were rarely observed [less than 0.2% of the CD14⁺ mononuclear cells (MNC)] both in healthy volunteers and sarcoidosis patients. A small subpopulation of PBM showed a weak expression of RFD9 antigen (less than 1% of MNC). Interestingly, proliferation of PBM was concentrated in this subpopulation (15% of the RFD9⁺ MNC). These data show that even mature AM, which are generally thought to be terminally differentiated cells with little capacity to replicate, are able to proliferate, whereas a relatively very low percentage of their precursors in the blood circulation proliferates. Furthermore, the findings suggest that lung tissue in sarcoidosis creates an environment which promotes proliferation of monocytic cells.     

Morphometry of monocytes of human blood

Morphometric methods have been used to analyse the ultrastructural characteristics of human blood monocytes in 20 normal adults. Data were obtained relating to whole cell, nucleus and mitochondria and, using a method not previously applied to blood cells, for the distribution of heterochromatin and euchromatin. The results provide a normal baseline against which monocyte changes in disease can be assessed.     

Chemiluminescent determination of esterases in monocytes

Esterase from monocytes promotes the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) yielding 2-methyl-1-propenol, which is oxidized by horseradish peroxidase/H₂O₂ producing triplet acetone. The chemiluminescence of this reaction can be enhanced by the addition of 9,10-dibromoanthracene-2-sulphonate. The non-specific esterase present in monocytes is responsible for MPB hydrolysis, since (a) the chemiluminescence of the reaction was inhibited by fluoride, and (b) cells that do not contain a significant amount of non-specific esterases, e.g. lymphocytes and neutrophils, did not trigger light emission. The analytical application of this reaction is considered. © 1998 John Wiley & Sons, Ltd.     

Cytotoxicity of human peripheral blood monocytes

Native tumoricidal activity of human peripheral blood mononuclear cells was examined before and after their separation by counterflow centrifugation elutriation (CCE). Tumoricidal activity was found in the subpopulation of small mononuclear cells but not within the relatively pure subpopulation of large monocytes. Addition of lymphokine and/or lipopolysaccharide demonstrated that large monocytes were resistant to activation for tumor killing, in contrast to small mononuclear cells. However, cryopreservation or simply exposure to dimethyl sulfoxide (DMSO) rendered the large monocytes sensitive to activating agents without altering their unstimulated tumoricidal activity. Cryopreservation was not detrimental to small or large monocytes either in number or tumoricidal function but did decrease the number of large granular lymphocytes (LGL). The small mononuclear cell fraction was enriched for small monocytes to 80% by combining CCE with Percoll gradient separation. HNK-1 mouse monoclonal antibody against human LGL was used with complement to remove virtually all LGL from cryopreserved cells as judged by morphology and tumoricidal activity against K-562 human lymphoblastoid cells. Such treatment actually augmented rather than suppressed tumoricidal activity against P-815 mastocytoma cells. Therefore, we conclude that small monocytes but not large monocytes possess native tumoricidal activity distinct from that attributed to LGL or natural killer lymphocytes. Further, small monocytes are readily activated for tumor killing and can be cryopreserved without loss of tumoricidal activity.