检测体外中性粒细胞自发性激活的方法学研究

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的自发性激活,研究了正常人中性粒细胞体外自发激活的时相特征,对这两处方法进行了对比分析。发现：（１）检测体外中性粒细胞自发性激活,伪足法较ＮＢＴ法更灵敏、可靠、且可实时观察;（２）体外中性粒细胞自发性激活的百分率呈现由低升高、再逐渐降低的时相过程,最高激活百分率出现在采血后第３小时,研究结果为今后深入研究中性粒细胞激活与多     

中性粒细胞自发性凋亡的研究概况

中性粒细胞是血细胞中寿命最短的终末分化细胞，也是机体最主要的炎症细胞。同其它的血细胞和组织细胞在凋亡的发生机制上有所不同，中性粒细胞从分化成熟开始，就启动它的自发性凋亡程序(spontaneous apoptosis program)。机体的炎症反应是一把双刃剑，在清除异物的同时也带来损伤。中性粒细胞这一最主要的炎症细胞则可以通过自发性凋亡的机制调控和减轻炎症损伤，构成炎症反应的主要收敛机制之一。中性粒细胞的自发性凋亡是一个受自身多基因调控和众多细胞因子及其它多种因素调节的复杂过程。     

激活素A对小鼠中性粒细胞活性的调控作用

目的:通过检测中性粒细胞激活素受体表达以及细胞活性,阐述激活素A调控中性粒细胞的作用。方法:分离小鼠腹腔中性粒细胞;通过免疫荧光细胞化学染色及流式细胞术检测中性粒细胞激活素ⅡA型受体(ActRⅡA)表达;激活素A刺激后,Western blot检测中性粒细胞Smad3表达;检测呼吸爆发、NO分泌及吞噬能力的变化以确定中性粒细胞活性。结果:分离的小鼠腹腔中性粒细胞纯度大于90%;免疫荧光细胞化学染色显示中性粒细胞可以表达ActRⅡA,流式细胞术检测结果显示Gr-1与ActRⅡA双阳性细胞约41.1%;激活素A刺激后,中性粒细胞p-Smad3表达上调,ROS及O2-产生升高(P0.05),NO分泌增加(P0.01),流式细胞术检测结果显示激活素A还可以明显促进中性粒细胞对荧光微球的吞噬(P0.01)。结论:中性粒细胞可以表达激活素受体及其信号传导蛋白,激活素A对中性粒细胞活化及功能具有重要的调节作用。     

抗类风湿病药金诺芬对中性粒细胞自发性凋亡的影响

目的 研究抗类风湿病药金诺芬(AF)对中性粒细胞自发性凋亡的影响。方法 运用流式细胞技术和MTS细胞活性测定技术对不同浓度的金诺芬影响下的中性粒细胞活性及其凋亡进行观察，并用低分子质量DNA“梯形条带”(DNA ladder)检测技术对其凋亡进行鉴定。结果 低浓度金诺芬(1μmol／L)可通过延迟中性粒细胞的自发性凋亡而增加其生命期限；但当金诺芬浓度大于5μmol／L时，中性粒细胞的坏死则明显增加，因而缩短了其生命期限。结论 金诺芬临床治疗量时的血清浓度一般在0．7μmol／L左右，在此浓度下，中性粒细胞通过延迟自发性凋亡来延长其功能性生命期限，所以金诺芬对类风湿关节炎患者的抗炎、抗风湿作用不可能通过直接清除炎症局部的中性粒细胞来完成；金诺芬的抗炎、抗风湿作用可能存在更为复杂的作用机制。     

结核杆菌抗原激活MEK-ERK信号通路延迟中性粒细胞自发性凋亡的作用

探讨结核杆菌抗原(Mtb-Ag)对中性粒细胞自发性凋亡的影响。取健康成人外周血分离中性粒细胞,加Mtb-Ag培养24 h,或用Mek抑制剂PD98059预处理30 min,Annexin V染色流式细胞术观察细胞凋亡;并用Western blot印迹法检测Mek活化情况。与中性粒细胞体外培养24 h后自发凋亡(55%±6%)相比,加入Mtb-Ag(1.125 mg/ml)后,中性粒细胞凋亡率(31%±3%)明显降低;并且Mtb-Ag可诱导Mek的激活;而PD98059(50μmol/L)预处理可阻断Mtb-Ag的抑制凋亡作用。Mtb-Ag对中性粒细胞自发凋亡有抑制作用,这一作用涉及Mek-Erk途径。     

槲皮素对LPS延迟中性粒细胞自发性凋亡效应的抑制作用

目的：研究槲皮素对接受细菌脂多糖(LPS)刺激的中性粒细胞(PMN)自发性凋亡的影响，探讨槲皮素的抗炎作用机制。方法：以人外周血PMN为研究对象，选择光镜和流式细胞术检测细胞凋亡情况，对裂解的片段化DNA进行定量(二苯胺测定法)和定性(琼脂糖凝胶电泳)分析。结果：槲皮素(1～100μmol／L)对PMN的自发性凋亡率并无影响，但却能部分恢复由LFS所延迟的PMN自发性凋亡进程。当槲皮素的浓度为40μmol／L时，其恢复作用达到最大。结论：槲皮素对LPS延迟PMN自发性凋亡的效应产生了抑制作用，减轻了因预激因子活化PMN而加重的炎症反应，部分揭示了槲皮素的抗炎作用机制。     

中性粒细胞激活及其与内皮细胞粘附的关系

本文利用不同浓度的趋化物质N-甲酰甲硫亮氨酰苯丙氨酸(fMLP)处理中性粒细胞,并采用硝基蓝四唑(NBT)还原法检测中性粒细胞激活率,结合流室系统定量地研究了在不同切应力作用下不同激活状态的中性粒细胞与人脐静脉内皮细胞(HUVEC)粘附的关系。通过显微镜观察、计数及统计分析,结果表明:随着fMLP浓度的增加,中性粒细胞激活率显著增加,二者有着明显的量效关系。同时本实验还表明:中性粒细胞经fMLP处理后,与对照组一样,随着切应力增加,中性粒细胞与内皮细胞粘附率按指数曲线下降;中性粒细胞激活率的增加可以引起中性粒细胞-内皮细胞粘附显著增加,当切应力由小变大时,这种作用有减缓的趋势。以上结果揭示:(1)体内血流产生的切应力可能是阻止中性粒细胞—内皮细胞粘附的重要因素,中性粒细胞粘附随切应力增加而减少有利于体内正常血液循环的维持;(2)中性粒细胞激活可以增加其对内皮细胞的粘附,心脑血管疾病过程中,中性粒细胞激活率增加的病理生理意义可能在于其对内皮细胞粘附的增加,从而引发一系列病理过程。     

山莨菪碱对中性粒细胞糖皮质激素受体的影响

以~3H地塞米松(Dex)为特异性配基,以一点分析法测得大白鼠腹腔中性粒细胞(PMNs)的糖皮质激素受体(GCR)位点数为5270±216/个PMN(n=18,X±SE下同);体外实验,PMNs直接与山莨菪碱(654-23×10~(-5)M)温育,对GCR无明显影响,测得位点数为4896±360/个PMNs(n=20,P>0.4);体内实验表明,给大鼠肌肉注射654-2(10mg/kg)每日两次共二天,则GCR位点数降至2740±101/个PMNs(n=18,P<0.001)。此时还测得血浆皮质酮含量明显增高(P<0.01)。提示654-2对GCR的影响可能与增加皮质激素所致的“降调节”有关。     

高脂血症循环白细胞自发活化作用的研究

高血脂是心脑血管病的重要危险因素。活化白细胞在缺血过程中微血管嵌塞和组织损伤中起重要作用。为了研究血清胆固醇对循环白细胞活化作用的影响，采用硝基蓝四唑试验测量了高脂血症家兔循环白细胞自发活化率（SAR）。结果指出，高脂血症动物循环白细胞SAR（％）显著高于对照组（P＜0.01）。SAR升高与血清胆固醇水平有关。循环白细胞SAR升高导致白细胞粘附分子CD11／CD18表达增加和微循环障碍。因此循环白细胞SAR升高是心脑血管病重要危险因素之一。     

Effects of Anticoagulants and Temperature on Expression of Activation Markers CD11b and HLA-DR on Human Leukocytes

A whole-blood model was used to evaluate the effects of temperature and anticoagulant on the expression of activation markers HLA-DR and CD11b on peripheral leukocytes. Venous blood, anticoagulated with either EDTA or heparin, was obtained from six healthy blood donors and 13 hospitalized patients (8 human immunodeficiency virus type 1-seropositive individuals with concurrent pulmonary tuberculosis and 5 patients with pneumonia). A preliminary evaluation was carried out with whole blood from two of the normal donors, and cells were stained immediately for HLA-DR and CD11b markers or stained after incubation at room temperature or 37°C for 18 h with or without the addition of the cytokines gamma interferon (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ plus GM-CSF, tumor necrosis factor beta, or interleukin-6. Of the cytokines tested, the combination of IFN-γ and GM-CSF had the most pronounced modulation of marker expression on polymorphonuclear neutrophils (PMN), in particular, HLA-DR expression, which required induction for its detection. These cytokines were therefore used in further evaluations that considered the above-mentioned effects in the presence of disease. Results indicated that the expression of activation markers on PMN and lymphocytes in whole blood are influenced by the temperature of incubation and the choice of anticoagulant and the effects noted were dependent on (i) the particular cell surface marker, (ii) the cell type being studied, and (iii) the presence or absence of disease. It is therefore recommended that ex vivo whole-blood models for evaluating phenotype or immune function be carefully evaluated for the above-mentioned effects.     

氯喹对体外培养星形胶质细胞激活的抑制作用

  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,经纯化鉴定后分组：a 对照组;b 戊四氮（PTZ）组;c 氯喹干预组（25mg、50mg、75mg/L）,经相应处理后,分别运用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,并分析比较各组星形胶质细胞激活情况。结果 与b组比较,MTT法显示氯喹阻滞了PTZ激活的星形胶质细胞的增殖（P<0.05）;免疫荧光结果显示,氯喹可抑制PTZ激活星形胶质细胞异常增加的GFAP的表达;western-blot结果显示氯喹可抑制PTZ激活星形胶质细胞的CyclinD1的表达量（P<0.05）;与a组比较,3种结果均显示75mg/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。     

The Effects of Sex Protein Receptors and Sex Steroid Hormone Gene Polymorphisms on Breast Cancer Risk

Breast cancer is a common disease and a major cause of death among women throughout the world. Various genes are believed to be involved in the initiation and progression of the disease. Some polymorphisms of these genes increase susceptibility to breast cancer in particular ethnicities. This study used electronic literature search to review the effects of different sex steroid hormone gene polymorphisms on breast cancer risk. Our findings indicated that some polymorphisms in estrogen receptor alpha (ER-α), ER-β, progesterone receptor (PGR), pregnane X receptor (PXR), and cytochrome P450 enzymes (CYPs) affected breast cancer susceptibility, especially in African American women.     

Effect of [Pyridylmethanimine]/[CuI] Ratio,Ligand, Solvent and Temperature on the Activation Rate Constants in Atom Transfer Radical Polymerization

Summary: The effect of [L]/[Cu^I] ratio, ligand, the choice of solvent and temperature on the activation rate constants in ATRP with catalyst complexes formed with two pyridylmethanimine ligands [PMI, i.e., N‐propyl‐pyridylmethanimine (NPPMI) and N‐octyl‐pyridylmethanimine (NOPMI)] were studied. Maximal values for the apparent rate constants were observed at an [L]/[Cu^I] ratio of ∼2/1 and 1/1 in polar and nonpolar solvents, respectively. This is similar to the results obtained with the Cu^I/bpy system and was attributed to the formation of [Cu^I/L₂]⁺Br⁻ and [Cu^I/L₂]⁺Cu^IBr species. The Cu^I/NPPMI system was only soluble in polar reaction media; however, the Cu^I/NOPMI complex was soluble in both polar and less polar reaction media. The activation rate constants increased with increasing temperature and the value of activation energy (E_a) for Cu^I/NPPMI in the activation process was 58.1 kJ · M ⁻¹ · K⁻¹.

Arrhenius plot of ln k_a versus 1/T for the activation process for NPPMI and bpy in acetonitrile; [Cu^IBr/NPPMI₂]₀ ([Cu^IBr/bpy₂]₀)/[EtBriB]₀/[TEMPO]₀/[TCB]₀ = 20/1/10/2 × 10⁻³ M .     

温度对去甲肾上腺素引起的大鼠主动脉收缩张力的影响

研究温度对去甲肾上腺素（ＮＥ）引起的动脉血管收缩张力变化的影响及原因。测量３９．５、３６．５、３３．０℃时，在ＮＥ作用下的张力－时间曲线，拟合出两者的函数关系式。并对关系常数进行Ｆ检验。实验结果表明：在三种不同温度下，函数式中的常数Ｘ１间比较有显著性差异，Ｐ〈０．０１；常数Ｘ２间比较无显著性差异，Ｐ〉０．０５。认为ＮＥ引起的血管收缩张力的大小，显然也处于温度的影响之下；随着温度的升高，血管收缩张力     

正常人和精神分裂症患者脑灰质体积分析的性别效应

目的:通过比较男性和女性正常人、男性和女性精神分裂症患者之间的脑灰质体积差异,探讨脑结构分析中的性别效应。方法:采集60例正常人(30例男性)和96例精神分裂症患者(48例男性)的高分辨率三维脑结构磁共振图像,运用基于体素的形态测量法(VBM)进行分析,获得每一个体的脑灰质体积图像。采用两样本t检验分别比较男性和女性正常人,男性和女性精神分裂症患者之间的脑灰质体积差异。结果:与女性正常人相比,男性正常人左侧的颞中回和额中回、右侧的边缘叶和颞中回的灰质体积较大(P<0.001),而左侧尾状核和右侧海马的灰质体积较小(P<0.001)。与女性精神分裂症患者相比,男性患者双侧的颞下回和海马、左侧的颞上回和颞中回的灰质体积较大(P<0.001),而未发现灰质体积较小的脑区。结论:性别效应是影响正常人或精神分裂症患者脑结构分析的重要因素。     

Comparison of the Effects of Selective and Non-selective Beta-adrenoreceptor Agonists on the Pro-inflammatory Activities of Human Neutrophils in Vitro

The objective of this study was to measure and compare the effects of 4 selective (fenoterol, formoterol, salbutamol, salmeterol) and 3 non-selective (epinephrine, norepinephrine, isoproterenol), -adrenoreceptor agonists, at a fixed, final concentration of 1 M, on intracellular cyclic AMP levels in human neutrophils in vitro and to relate alterations in these to the effects of the test agents on the production of superoxide and release of elastase following activation of the cells with the chemoattractant, FMLP. Intracellular cAMP was measured by radioimmunoassay, while superoxide and elastase were assayed using lucigenin-enhanced chemiluminescence and colorimetric procedures respectively. Of the 7 agents tested, fenoterol, formoterol, epinephrine and isoproterenol caused a substantial increase in neutrophil cAMP levels, which correlated well with the inhibitory effects of these agents on superoxide production and elastase release. The other agents were either inactive (salmeterol), or weakly active (norepinephrine, salbutamol). Pretreatment of neutrophils with the non-selective -adrenoreceptor blockading agent, propranolol (2 M), attenuated the cAMP-mediated, anti-inflammatory interactions of formoterol, epinephrine and isoproterenol with neutrophils, while atenolol, a selective ₁-blockading agent, as well as ₁- and ₂-adrenoreceptor antagonists were ineffective. These findings demonstrate that some, but not all, currently used -agonists suppress the proinflammatory activity of human neutrophils through interaction with ₂-adrenoreceptors on these cells and activation of adenylyl cyclase. If operative in vivo, these anti-inflammatory properties may contribute, albeit in a secondary manner, to the therapeutic activity of fenoterol, formoterol, epinephrine and isoproterenol.