淀粉样β蛋白对小胶质细胞活化作用的体外观察

目的:观察淀粉样β蛋白对小胶质细胞活性、形态学以及分泌炎性细胞因子的作用,为阿尔茨海默病的炎性反应机制及抗炎治疗提供体外依据。方法:实验于2003-03/2004-03在解放军总医院老年医学研究所进行。应用BV-2细胞作为体外小胶质细胞模型,加入不同浓度淀粉样β蛋白25～35或1～42(5,10,20,40,60μmol/L)及20μmol/L不同孵育状态(37℃孵育3,6,12,24h、3,5和7d)的淀粉样β蛋白25～35或1～42分别培养2～24h。四唑盐法检测淀粉样β蛋白对细胞活性的影响,倒置相差显微镜下观察细胞形态学改变,并应用放射免疫法测定加入淀粉样β蛋白后BV-2细胞上清白细胞介素1β及白细胞介素6水平。结果:①细胞活性:淀粉样β蛋白对BV-2细胞的生存活性没有影响(P均>0.05)。②细胞形态学改变:在较低浓度时(5～10μmol/L)淀粉样β蛋白1～42就可以使BV-2细胞出现明显的形态学改变(多数细胞聚集成团,有的胞体变得肥大,胞核大而圆,核仁明显;有的胞体变为梭形,由胞体伸出一个或多个较长的枝状突起),与淀粉样β蛋白1～42孵育状态有关(37℃孵育5～7d时最为明显),而淀粉样β蛋白25～35无此作用。③白细胞介素1β水平:淀粉样β蛋白1～42与25～35均可以刺激BV-2细胞分泌,二者作用类似。20μmol/L淀粉样β蛋白作用6h后其水平升高,至12h达到高峰,约为对照组的3倍,此后逐渐下降(P<0.01)。当不同浓度淀粉样β蛋白作用12h时,淀粉样β蛋白为20μmol/L时在上清中可以检测到白细胞介素1β明显增加,此后随着浓度的增加其分泌也逐渐增加,至60μmol/L时为对照组的5倍(P<0.01)。④白细胞介素6水平:各淀粉样β蛋白处理组细胞上清液均不能检测到其水平有明显变化。结论:淀粉样β蛋白对BV-2细胞的生存活性没有影响,淀粉样β蛋白可以激活BV-2细胞发生形态学改变并释放炎性细胞因子,但二者的作用途径不同,形态学改变与淀粉样β蛋白片段及聚集状态均有关;而分泌白细胞介素1β与淀粉样β蛋白片段及聚集状态均无关,并且这种刺激作用具有时间以及浓度依赖性。     

姜黄素对β淀粉样蛋白诱导的老年性痴呆大鼠小胶质细胞活化的影响

目的探讨姜黄素对β淀粉样蛋白(Aβ)诱导的老年性痴呆(AD)大鼠学习记忆障碍及小胶质细胞活化的影响。方法将Aβ1-40微量注射至大鼠右侧海马,制作AD大鼠模型,干预组给予腹腔注射姜黄素连续7d,造模1个月后进行Morris水迷宫试验测定大鼠学习记忆能力,分为空白对照组、对照组和姜黄素给药组。免疫组织化学方法检测小胶质细胞。结果 AD对照组大鼠较空白对照组逃避潜伏期延长,而跨越原平台位置次数明显减少;姜黄素给药组大鼠较AD对照组第2～5天平均逃避潜伏期明显缩短,与空白对照组比较无明显差异(P>0.05),跨越原平台位置次数明显增多,与空白对照组无明显差异(P>0.05),表明姜黄素干预后AD大鼠空间学习、记忆能力明显改善。与空白对照组相比,AD对照组大鼠脑组织海马区可见小胶质细胞表达明显增多(P<0.05);姜黄素组大鼠脑组织海马区小胶质细胞较AD对照组明显减少(P<0.05)。结论姜黄素能够改善Aβ1-40诱导的AD模型大鼠空间学习记忆障碍,其机制可能与抑制Aβ所致小胶质细胞活化有关。     

线粒体分裂蛋白抑制剂对β淀粉样蛋白诱导小胶质细胞凋亡的作用及机制

目的:研究线粒体分裂蛋白抑制剂对β淀粉样蛋白(Aβ)诱导小胶质细胞凋亡的作用及其机制。方法:随机将BV-2小胶质细胞分为con组、Aβ组、mdi组和Aβ+mdi组,con组不做特殊处理,mdi组培养基中加入10μmol/L mdivi-1,Aβ组培养基中加入20μmol/L Aβ,Aβ+mdi组培养基分别加入2、5、10、20μmol/L mdivi-1和20μmol/L Aβ。采用MTT法检测细胞存活率,TUNEL染色检测细胞凋亡,Western blot法检测Drp1、CytC和Caspase-3蛋白水平变化,RT-PCR法检测CA11b mRNA表达变化。结果:与con组相比,Aβ组的细胞存活率明显下降,凋亡显著增加,CA11b mRNA上升,线粒体Drp1、细胞浆CytC和激活的Caspase-3增加,差异有统计学意义(P<0.05);与Aβ组相比,Aβ+mdi组的细胞存活率明显上升,凋亡显著减少,CA11b mRNA下降,线粒体Drp1、细胞浆CytC和激活的Caspase-3减少,差异有统计学意义(P<0.05)。结论:线粒体分裂蛋白抑制剂对Aβ诱导小胶质细胞凋亡有保护作用,其机制可能为抑制线粒体/CytC/Caspase-3凋亡途径。     

β淀粉样蛋白对小胶质细胞促分裂原活化蛋白激酶激酶2和肿瘤坏死因子-α mRNA水平影响

目的:探讨β淀粉样蛋白谤导的炎症反应在阿尔茨海默病发病机制中的作用.方法:采用不同浓度(0.62.5,125.0,250.0nmol/L)的β淀粉样蛋白1-42孵育原代培养的新生大鼠海马小胶质细胞2 h,建立β淀粉样蛋白诱导损伤的神经细胞模型,半定量RT-PCR反应方法观察体外条件下β淀粉样蛋白1-42对小胶质细胞中促分裂原活化蛋白激酶激酶2和肿瘤坏死因子-α mRNA水平的影响.结果:随着β淀粉样蛋白1-42浓度增加,促分裂原活化蛋白激酶激酶2和肿瘤坏死因子-α mRNA水平升高,与β淀粉样蛋白1-42浓度呈正相关.结论:β淀粉样蛋白谤导的炎症反应在阿尔茨海默病的发病机制中具有重要作用.     

小胶质细胞与阿尔茨海默病炎性反应的相关研究

阿尔茨海默病(Alzheimer’s disease,AD)是老年人常见的神经系统变性疾病,临床特征为隐袭起病,进行性智能衰退,多伴有人格障碍,其病因迄今不明。目前认为,β淀粉样蛋白(amyloidβ-protein,Aβ)沉积引起的炎性反应是AD的病理机制核心。小胶质细胞(microglial,MG)是AD炎性反应中最主要的炎性细胞,介导并贯穿AD病理发展全程。本文就MG及AD的炎性反应的相关研究作一概述。     

线粒体分裂蛋白抑制剂对小胶质细胞氧化损伤的保护作用

目的：研究线粒体分裂蛋白抑制剂(mdivi-1)能否减轻β淀粉样蛋白（Aβ）诱导的体外原代培养小鼠小胶质细胞的氧化应激损伤。方法：随机将体外原代培养BALB/C小鼠小胶质细胞分为con组、Aβ组、mdi组和 Aβ+mdi 组,con 组不予处理,Aβ组中加入 Aβ,mdi 组中加入 mdivi-1,Aβ+mdi 组分别加入2、5、10、20μmol/L mdivi-1后1 h加入Aβ。分别检测小胶质细胞存活率及凋亡、线粒体膜电位、丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、8-羟基脱氧鸟苷（8-OHdG）含量。结果：与con组相比,Aβ组小胶质细胞存活率下降,凋亡增加,线粒体膜电位下降,MDA和8-OHdG含量升高,SOD活性下降,差异有统计学意义（＜0.05）;与Aβ组相比,Aβ+mdi组小胶质细胞存活率上升,凋亡减少,线粒体膜电位增加,细胞内MDA和8-OHdG水平下降,SOD活性上升,差异有统计学意义（＜0.05）。结论：mdivi-1对Aβ诱导的体外原代培养小胶质细胞氧化应激损伤具有保护作用。     

栝楼桂枝汤对活化小胶质细胞抗炎作用的研究

目的：探讨栝楼桂枝汤对活化小胶质细胞抗炎因子的表达水平以及上游炎症信号通路的活化情况。方法：体外LPS刺激制备小胶质细胞的细胞炎症模型,用MTT实验筛选药物最佳干预浓度,经栝楼桂枝汤水提液处理后,应用ELISA法、RT-PCR法、Western-blotting法检测细胞抗炎因子的表达及相关炎症信号通路蛋白的表达水平。结果：栝楼桂枝汤水提液可显著促进LPS诱导的小胶质细胞中抗炎因子的表达,同时可上调相应通路蛋白的表达水平。结论：栝楼桂枝汤可通过TRIF信号通路发挥抗神经炎症的作用,为栝楼桂枝汤的临床应用提供基础实验依据。     

小胶质细胞与Aβ研究进展

Alzheimer病(Alzheimer’s disease,AD)是以记忆力丧失和认知功能损害为特点的神经元退行性疾病。病理特征为β淀粉样蛋白(β-amyloid,Aβ)沉积及反应性胶质增生(re-active gliosis)等组成的老年斑(senile plaques,SP)、过度磷酸化tau蛋白形成的神经原纤维缠结(neuro fibrillar     

脊髓小胶质细胞活化状态与前列腺的炎性疼痛

目的：观察化学性刺激大鼠前列腺后相应脊髓节段小胶质细胞活化的状况。 方法：实验于2004-05／2005—11在第三军医大学神经生物学教研室实验室完成。①分组：健康雄性SD大鼠24只，随机分为4组：对照组6只；完全弗氏佐剂刺激3d组6只；完全弗氏佐剂刺激10d组6只；完全弗氏佐剂刺激28d组6只。②模型制备：对照组：取下腹部正中直切口，于前列腺腹侧叶注入生理盐水0．1mL，可吸收线缝合伤口；完全弗氏佐剂刺激组：取下腹部正中直切口，于前列腺腹侧叶注入完全弗氏佐剂0．1mL，可吸收线缝合伤口。③用免疫组织化学技术对不同时间段大鼠相应脊髓节段（L6和S1）的小胶质细胞活化进行检测。 结果：24只大鼠均进入结果分析。完全弗氏佐剂刺激大鼠前列腺后3，10，28d，L6～S1脊髓背角浅层和深层小胶质细胞的活化与对照组相比均显著增加，其中3d时已显著增强，10d时增强最显著，28d时较10d时活化有所减少，但仍显著高于对照组。 结论：完全弗氏佐剂刺激大鼠前列腺引起前列腺炎性疼痛，导致了相应节段L6-S1脊髓背角小胶质细胞的激活，提示小胶质细胞活化在前列腺炎性疼痛的神经病理过程可能发挥了重要作用。     

活化小胶质细胞对骨髓间充质细胞分化方向影响的研究

[目的]为探讨小胶质细胞激活后对骨髓间充质细胞(MSC)转化方向的影响.[方法]采用原代培养法分别培养小胶质细胞及骨髓基质干细胞,不同浓度脂多糖(LPS)激活小胶质细胞,免疫荧光法检测神经胶原纤维酸性蛋白(GFAP)阳性细胞及神经元特异性烯醇化酶(NSE)阳性细胞.[结果]高浓度LPS组激活的小胶质细胞显著提高了MSC的GFAP阳性细胞数,同对照组相比差异有显著性(P＜0.05),而NSE阳性细胞在各组之间均差异无显著性.[结论]激活小胶质细胞能够促进干细胞向胶质细胞的转化.     

Juzen-taiho-to, an herbal medicine, activates and enhances phagocytosis in microglia/macrophages

Microglia are the main resident immunocompetent and phagocytic cells in the central nervous system (CNS). Activated microglia could play phagocytic roles as well as mediate inflammatory processes in the CNS. Involvement of activated microglia in the pathogenesis has been demonstrated in several neurological diseases including Alzheimer's disease (AD). Juzen-taiho-to (JTT), a traditional herbal medicine, has been reported to have effects on activating immune responses and phagocytosis. So far, little is known about the effects of this Kampo formulation JTT on microglia and in AD. In this report, we studied the effects of JTT on the activation and phagocytic functions of mouse microglia and bone marrow-derived macrophages (BMM). JTT could activate microglia, which was confirmed by the prominent morphological change and increased surface expression of an activation marker CD11b. In addition, JTT was revealed to induce microglial proliferation, and enhance microglial phagocytosis of, without eliciting an excessive production of nitric oxide. Furthermore, when mice were administrated with JTT in vivo, their BMM showed more effective phagocytosis of fibrillar Abeta(1-42). These findings implicate the therapeutic potential of JTT in AD and other neurological diseases accompanied by microglial activation.     

构建一种高产量小胶质细胞体外纯化培养的方法

背景小胶质细胞在一些神经系统疾病如帕金森病、阿尔茨海默病中有重要作用.体外原代培养是进行小胶质细胞功能研究的基础,但目前常用的一些经典培养方法存在步骤繁琐、产量过低的问题.目的建立一种简易高产量的原代小胶质细胞体外纯化培养方法.设计以细胞为单一样本的探索性研究.单位华中科技大学同济医学院附属协和医院神经内科.材料实验于2004-04/10在协和医院中心实验室完成,新生(出生1 d)昆明小鼠10只,雄性.方法改进的培养方法以McCarthy等的经典培养方法为基础,通过提高初次接种密度,减少细胞离心过滤过程,进行不换液的营养缺失培养等重要改进,并使用低浓度胰酶-乙二胺四乙酸消化法分离纯化小胶质细胞,经MAC-1抗体免疫化学染色标记,计算MAC-1抗原阳性细胞,观察小胶质细胞培养的产量和纯度.主要观察指标①倒置显微镜观察小胶质细胞形态.②免疫组织化学鉴定比较两种方法下小胶质细胞纯度、激活状态.结果经典的McCarthy培养方法小胶质细胞培养周期为20 d,产量为2×105个/瓶,细胞纯度为95%～97%;改进的方法小胶质细胞培养周期为15 d,产量为1×106个/瓶,得到的小胶质细胞纯度为96%～98%.与经典的McCarthy培养方法比较,改进的方法周期短,产量提高了3～10倍.两种方法培养的小胶质细胞纯度和细胞激活状态没有明显差别.结论新方法得到的小胶质细胞纯度与激活状态均与经典方法相似,但由于其步骤简单,周期短和有效提高了培养产量,为进行小胶质细胞功能特性以及在神经修复中作用的深入研究提供了良好的方法学基础.     

Apolipoprotein C-III-1 activates lysosomal sphingomyelinase in vitro.

Apolipoprotein (apo)C-III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphingomyelinase from human fibroblasts. At the sphingomyelin concentrations tested, maximal stimulation was obtained with 5 microM apoC-III-1 or apoC mixture. Apolipoproteins A-I, A-II, B, and C-I conferred little or no stimulation. Sphingomyelinase was stimulated 20-fold by lysophosphatidylcholine with an optimum concentration of 70 microM using 0.3 mM substrate. Sphingomyelinase activity was inhibited by concentrations of apoC-III-1 and lysophosphatidylcholine three- to fivefold above stimulatory levels. Triton X-100 activated sphingomyelinase 300-fold with a pH optimum of 5.0, while the pH optimum with the biological activators was 4.0. These results raise the possibility of an in vivo activity for the biological activators. The proteins that enter lysosomes as part of a lipoprotein complex may activate lysosomal enzymes that degrade the lipid components.     

H60/TNT-3 fusion protein activates NK cells in vitro and improves immunotherapeutic outcome in murine syngeneic tumor models

H60 is a murine minor histocompatibility antigen that binds to NKG2D and activates an effector phenotype in NK and T cells. In the present study, H60 was genetically fused to the tumor-targeting murine MAb TNT-3. The resultant fusion protein, named H60/TNT-3, was produced in NS0 cells and determined by ELISA to possess an H60 epitope. The Ka of H60/TNT-3 (2.43 x 10(9) M(-1)) was nearly identical to that of the parental Ab (2.22 x 10(9) M(-1)), demonstrating that addition of the H60 moiety to the N-terminus of TNT-3 heavy chain did not affect antigen affinity. In vitro, H60/TNT-3 bound and activated murine NK cells, eliciting IFN-gamma production in a higher percentage of cells than the activating NKG2D Ab A10. In vivo, H60/TNT-3 possessed a half-life of approximately 12 hours and effectively targeted tumor tissue versus control organs, with nearly 2% injected dose per gram of tumor retained after 48 hours. Finally, H60/TNT-3 was tested for antitumor efficacy in BALB/c and C57BL/6 mice bearing subcutaneous syngeneic carcinomas. Tumor volume reduction was observed in both CT26 and Lewis Lung models (53% and 52%, respectively) relative to untreated control mice. Further, Lewis Lung carcinoma-bearing mice treated with H60/TNT-3 experienced a statistically significant survival advantage. Taken together, these data characterize a new immunotherapeutic MAb with antitumor efficacy that prolonged overall survival in a resistant solid tumor model.     

Acute hypertension activates mitogen-activated protein kinases in arterial wall.

Mitogen-activated protein (MAP) kinases are rapidly activated in cells stimulated with various extracellular signals by dual phosphorylation of tyrosine and threonine residues. They are thought to play a pivotal role in transmitting transmembrane signals required for cell growth and differentiation. Herein we provide evidence that two distinct classes of MAP kinases, the extracellular signal-regulated kinases (ERK) and the c-Jun NH2-terminal kinases (JNK), are transiently activated in rat arteries (aorta, carotid and femoral arteries) in response to an acute elevation in blood pressure induced by either restraint or administration of hypertensive agents (i.e., phenylephrine and angiotensin II). Kinase activation is followed by an increase in c-fos and c-jun gene expression and enhanced activating protein 1 (AP-1) DNA-binding activity. Activation of ERK and JNK could contribute to smooth muscle cell hypertrophy/hyperplasia during arterial remodeling due to frequent and/or persistent elevations in blood pressure.     

Interleukin 2 activates extracellular signal-regulated protein kinase 2

Interleukin 2 (IL-2) stimulated activation of the 42-kD extracellular signal-regulated kinase 2 (Erk2) in murine IL-3-dependent cells, expressing either high or intermediate affinity IL-2 receptors. Activation was both rapid, occurring within 5 min of IL-2 addition, and prolonged, remaining elevated for 30 min. Activation of Erk2 appeared to be necessary for IL-2 stimulation of proliferation, as deletion of a region of the cytoplasmic domain of the IL-2 receptor beta chain, essential for IL-2 stimulation of proliferation, abolished Erk2 activation by IL-2. Furthermore, cells that had been deprived of cytokine for 24 h were then refractory to IL-2 stimulation of both Erk2 activity and proliferation. However, elevation of Erk2 activity was not sufficient to stimulate proliferation, as protein kinase C activation stimulated Erk2 activity but not DNA synthesis. Also, cells exposed to IL-2 in the presence of rapamycin showed full Erk2 activation but not DNA synthesis. These data suggest that IL-2 must stimulate both Erk2 activity and a further pathway(s) to trigger cell proliferation.