哮喘特异性免疫治疗诱导的卵蛋白致敏小鼠模型T细胞无能机制的探讨

目的:建立哮喘特异性免疫治疗小鼠模型,探讨特异性免疫治疗诱导T细胞无能的机制。方法:通过卵蛋白(OVA)皮下注射致敏,雾化吸入激发的方法,建立哮喘特异性免疫治疗的小鼠模型,并通过对肺组织病理切片及支气管肺泡灌洗液(BALF)中的细胞计数及分类,用ELISA法检测血清OVA特异性IgE(sIgE)及脾脏T细胞IL2和IL4的分泌等证实。分离培养脾脏T细胞,用3HTdR掺入法检测其增殖反应;用流式细胞仪(FACS)检测其表面CTLA4分子的表达。结果:与对照组小鼠相比较,哮喘特异性免疫治疗后,小鼠肺组织的炎症病理变化减轻;BALF中嗜酸性粒细胞(EOS)数、血清sIgE的水平、T细胞分泌IL2和IL4的水平以及小鼠对OVA刺激的反应性均显著降低(P<0.01);但T细胞表面CTLA4分子的表达明显上调。结论:建立了哮喘特异性免疫治疗的小鼠模型。T细胞表面CTLA4分子表达的上调可能是哮喘特异性免疫治疗诱导T细胞无能的机制之一。     

哮喘小鼠气道上皮TSLP表达及激活DCs加重气道炎症的研究

目的 研究支气管哮喘小鼠气道上皮中胸腺间质淋巴细胞生成素(TSLP)表达,探讨其对哮喘小鼠肺部炎症的影响.方法 BALB/c小鼠分为生理盐水对照组、哮喘模型组和TSLP中和抗体干预组.通过气道反应性和肺组织病理学评价哮喘模型;酶联免疫吸附试验(ELISA)检测支气管肺泡灌洗液(BALF)上清中IL-4、IL-5和IL13的含量;实时荧光定量PCR(qRT-PCR)测定肺组织中TSLP mRNA的表达;免疫组化及Western blot法测定肺组织中TSLP蛋白的表达;流式细胞术检测BALF中树突状细胞(OCs)表面CD40、CD80、CD86的表达水平.结果 小鼠气道反应性增高和肺组织病理学检查结果均符合哮喘的典型表现证实造模成功;哮喘组BALF中IL-4、IL-5和IL-13的水平显著高于正常组(P<0.05),且TSLP与其成正相关;与正常对照组相比,哮喘组气道上皮TSLPmRNA和蛋白高表达,两组间差异有统计学意义(P<0.05);哮喘组BALF中DCs表面CD40、CD80、CD86表达明显高于正常组(P<0.05).TSLP中和抗体干预后,BALF中DCs表面CD40、CD80、CD86表达明显减低,并进一步减少IL-4、IL-5和IL-13的表达.结论 哮喘气道上皮中TSLP表达增高,TSLP通过上调DCs表面CD40、CD80、CD86的表达,激活DCs诱导CD4~+T细胞向Th2分化发育,与加重哮喘的气道炎症有关;TSLP抗体干预可阻断DCs的活化,减少Th2细胞因子的分泌,这些因素可能与减轻哮喘炎症反应有关,为哮喘治疗途径提供新的思路.     

髓过氧化物酶、CD11b和IL-8在哮喘大鼠模型中表达增加

目的观察哮喘模型大鼠中性粒细胞(PMN)CD11bI、L-8和髓过氧化物酶(MPO)的表达,探讨PMN在哮喘中的作用及其可能的机制。方法复制哮喘大鼠模型,随机分成哮喘组和对照组,分离纯化血PMN;免疫组化法检测MPO表达,ELISA法测定IL-8蛋白,流式细胞术测定血PMN CD11b表达,支气管肺泡灌洗液(BALF)行细胞计数。结果哮喘组肺组织和血PMN中MPO、CD11b及血PMN和BALF中IL-8的水平显著高于对照组(P<0.01)。哮喘组BALF中PMN和嗜酸性粒细胞计数显著高于对照组(P<0.01)。结论BALF中PMN数量增加及血中PMN和肺组织中CD11bI、L-8和MPO在哮喘时表达增加,他们可能参与了哮喘的炎症过程。     

胸腺基质淋巴生成素受体及其抗体在哮喘小鼠气道炎症反应中的作用

目的 探讨胸腺基质淋巴生成素受体(TSLPR)及其抗体在实验性哮喘小鼠气道炎症反应中的作用以及对气道树突状细胞(DCs)成熟和活化的影响.方法 BALB/c小鼠随机分成A、B、C三组.B和C组小鼠腹腔注射卵清白蛋白(OVA)致敏,A组腹腔注射PBS作为正常对照.B和C组小鼠在OVA激发哮喘发作前分别吸入非特异性IgG和TSLPR IgG.采集各组小鼠支气管肺泡灌洗液(BALF)进行细胞分类计数,采用ELISA定量检测BALF中IL-4、IL-5、IFN-γ和IL-10浓度.采集各组小鼠肺组织标本进行病理学检查,采用流式细胞术分别检测各组小鼠淋巴结和肺组织中DCs数量和表型.结果 与A组小鼠比较,8和C组小鼠BALF中各种细胞因子水平均明显升高(P<0.01).C组小鼠BALF中IL-4和IL-5水平低于8组(P<0.05,P<0.01),IFN-γ和IL-10水平高于8组(P<0.05,P<0.01).C组小鼠BALF中细胞总数、嗜酸性粒细胞和淋巴细胞数也明显低于B组(P<0.01).B组小鼠支气管周围有大量炎性细胞浸润以及杯状细胞增生,黏液分泌增强,C组小鼠仅见微弱的炎性细胞浸润和杯状细胞增生.B组小鼠膈淋巴结中DCs数量以及肺组织中DCs的I-Ad、CD40、CD80和CD86表达水平均高于C组小鼠(P<0.05).结论 TSLP/TSLPR具有促哮喘效应并与其调节气道DCs活性的作用密切相关,TSLPR抗体干预可明显减弱TSLP/TSLPR上述作用,故有作为抗哮喘药物的前景.     

地塞米松抑制哮喘小鼠肺RANTES的表达

目的 研究地塞米松对哮喘小鼠调节激活正常T细胞表达和分泌细胞因子(RANTES)蛋白和mRNA表达的影响.方法 将30只雌性BALB/c小鼠随机分为3组(每组10只):对照组、哮喘组、激素干预组,收集支气管肺泡灌洗液(BALF),行白细胞和嗜酸粒细胞(EOS)计数;酶联免疫吸附法(ELISA)测定BALF和血清中RANTES含量;肺组织切片HE染色,光镜下计数细胞总数和EOS百分比;部分行免疫组化染色检测肺组织RANTES蛋白;用原位杂交法检测肺组织RANTES mRNA表达.结果 哮喘组白细胞总数、EOS百分比、RANTES浓度明显升高(P＜0.01),干预组明显低于哮喘组(P＜0.01);哮喘组肺组织RANTES蛋白及mRNA表达显著高于对照组(P＜0.01),主要表达于上皮细胞;干预组肺组织RANTES蛋白及mRNA表达显著低于哮喘组(P＜0.01).结论 地塞米松可抑制RANTES表达和活性而发挥抗EOS炎症作用,这可能是其控制哮喘发病的重要机制之一.     

Th17淋巴细胞及相关细胞因子加重哮喘小鼠气道炎症

目的:研究Th17淋巴细胞及其相关的细胞因子在加重哮喘小鼠气道炎症中的作用机制.方法:20只小鼠随机均分为哮喘组和正常对照组.哮喘组用卵白蛋白(OVA)致敏与激发建立小鼠哮喘模型.对照组致敏与激发均以生理盐水代替.HE染色观察小鼠气道及肺组织病理变化;光学显微镜下观察小鼠支气管肺泡灌洗液(BALF)中细胞分类及计数;酶联免疫吸附试验(ELISA)检测小鼠BALF上清中IL-4、IFN-γ及IL-17的含量,流式细胞技术(FCM)检测小鼠外周血Th1、Th2及Th17淋巴细胞占CD4+细胞百分率情况.将外周血各T淋巴细胞亚群与BALF的中性粒细胞数作相关性分析.结果:哮喘组小鼠BALF中细胞数及中性粒细胞、嗜酸性粒细胞、淋巴细胞百分率均显著高于对照组(P＜0.05),BALF上清中IL-4和IL-17的水平显著增高(P＜0.05),而IFN-γ的水平显著降低(P＜0.05),外周血Th2、Th17淋巴细胞占CD4+细胞百分比显著增高(P＜0.05),而Th1淋巴细胞占CD4+细胞百分比显著降低(P＜0.05).相关性分析显示Th1淋巴细胞与BALF的中性粒细胞数呈显著负相关(rThl=-0.446,P＜0.05),Th17淋巴细胞与BALF的中性粒细胞数呈显著正相关(rTh17=0.394,P＜0.05).结论:Th17淋巴细胞可促进中性粒细胞及炎性介质在哮喘小鼠气道内的聚集,加重气道炎症.     

地塞米松对哮喘小鼠肺组织中GPR43表达的调节作用

目的 研究G蛋白耦联受体43(GPR43)在哮喘小鼠肺组织内的表达,同时探讨地塞米松对GPR43表达的影响.方法 30只BALB/C6小鼠随机分为对照组、哮喘组、地塞米松组,每组10只;卵清白蛋白(OVA)致敏和激发建立哮喘小鼠模型;肺泡灌洗液(BALF)细胞计数;HE染色观察各组气道炎症发生及气道结构改变情况;RT-PCR测定各组小鼠肺组织GPR43mRNA的表达变化;免疫组化观察肺中GPR43的表达及定位.结果 哮喘组BALF嗜酸性粒细胞(EOS)与对照组相比明显增高,地塞米松组明显低于哮喘组(P＜0.01);HE染色提示哮喘组气道上皮出现EOS等大量炎性细胞浸润,管壁厚度较对照组明显增加(P＜0.01);RT-PCR结果提示GPR43受体mRNA的表达量在哮喘组最低,与对照、地塞米松组相比有统计学差异(P＜0.01);免疫组化图像分析提示GPR43蛋白表达哮喘组明显降低,与对照组、地塞米松组相比均有显著性差异(P＜0.01).结论 哮喘小鼠肺组织中GPR43表达下降,地塞米松能部分上调其表达,从而减轻气道炎症反应.     

金黄色葡萄球菌肠毒素对小鼠慢性哮喘模型的抗炎作用

目的：用金黄色葡萄球菌肠毒素A（SEA）和肠毒素B（SEB）干预幼年小鼠,观察它们对小鼠慢性哮喘气道炎症的影响以及对气道内相关细胞因子水平的调节作用。方法：实验组中BALB/c小鼠在出生后1周开始腹腔注射0.1 mL SEA或SEB（浓度1 mg/L）,隔天注射,共7次。其它小组注射相同剂量生理盐水对照。BALB/c 小鼠出生后4周建立哮喘模型,实验组和模型组分别在0、7和14 d用卵白蛋白（OVA）腹腔注射致敏,在28 d开始隔天OVA雾化激发哮喘,共7次,末次激发后24-48 h内取支气管肺泡灌洗液（BALF）,进行嗜酸性粒细胞和总白细胞计数,其余BALF离心-70 ℃冻存检测细胞因子,固定肺组织做病理切片分析。结果:SEA组和SEB组小鼠肺组织炎症反应轻于模型组,BALF中嗜酸性粒细胞数量少于模型组 (P＜0.05);SEA、SEB组BALF中Th1细胞因子干扰素-γ（IFN-γ）高于模型组(P＜0.05);而Th2细胞因子白细胞介素-4（IL-4）、白细胞介素-5（IL-5）和嗜酸性粒细胞趋化因子（eotaxin）均低于模型组(P＜0.01)。结论:早期感染 SEA、SEB可以减少小鼠慢性哮喘支气管肺泡灌洗液中嗜酸性粒细胞的数量,可能通过调节Th1/Th2细胞因子比值减轻气道中的哮喘炎症反应。     

IL-27对哮喘小鼠气道炎症的影响

目的研究IL-27对卵白蛋白(OVA)激发哮喘小鼠气道炎症的影响。方法 24只雌性BALB/c小鼠随机分为生理盐水组、哮喘组及IL-27组,每组8只。应用OVA建立哮喘模型,IL-27组小鼠应用1μgIL-27(溶于50μlPBS中)滴鼻给药,观察3组小鼠肺组织病理改变,计数支气管肺泡灌洗液(BALF)中嗜酸性粒细胞;ELISA法测定小鼠BALF中IL-4和IFN-γ浓度,RT-PCR测定肺组织T-bet mRNA的表达量。结果 IL-27组小鼠肺组织炎症反应明显轻于哮喘组小鼠;IL-27组小鼠BALF中嗜酸性粒细胞计数为(2.21±0.33)×107/L明显低于哮喘组的(12.82±2.17)×107/L(P0.01);IL-27组小鼠BALF中IL-4浓度为(20.4±3.2)μg/L,明显低于哮喘组的(61.3±13.1)μg/L(P0.05);IL-27组小鼠BALF中IFN-γ浓度为(50.3±6.3)μg/L,明显高于哮喘组的(11.1±3.3)μg/L(P0.05);IL-27组小鼠肺组织T-bet mRNA表达量(吸光度积分比值)为(0.268±0.048),明显高于哮喘组的(0.130±0.012)(P0.05)。结论 IL-27可能通过增强T-bet mRNA的表达增强Th1反应,减少BALF中嗜酸性粒细胞数量,进而减轻了哮喘小鼠肺组织炎症反应。     

IL-31在哮喘小鼠中的动态表达及对肺泡上皮细胞表达CCL11和CCL22的影响

目的通过观察IL-31在OVA诱导小鼠哮喘模型中的动态表达及IL-31对肺泡上皮细胞表达趋化因子CCL11和CCL22的影响,探讨IL-31在哮喘气道炎症中的作用。方法常规OVA法建立小鼠哮喘模型,于末次激发后取肺组织HE染色及AB-PAS染色,收集支气管肺泡灌洗液(BALF)进行白细胞和嗜酸性粒细胞(EOS)计数,ELISA法检测血浆中IgE、IL-4、IFN-γ、IL-31水平,荧光定量PCR检测肺组织IL-31R mRNA表达水平,同时体外培养小鼠肺泡上皮细胞,用IL-31处理24 h后检测CCL11和CCL22 mRNA的表达水平。结果成功构建哮喘小鼠模型,哮喘小鼠BALF中白细胞总数、嗜酸性粒细胞百分比和血浆中IgE水平明显增多。哮喘小鼠肺组织病理切片见中性粒细胞、EOS浸润。哮喘小鼠血浆中Th2类细胞因子IL-4水平明显高于对照组,Th1类细胞因子IFN-γ明显低于对照组。哮喘小鼠血浆IL-31水平和肺组织IL-31R mRNA表达水平明显增高,第2周至第8周虽略有降低但仍明显高于对照组。IL-31作用小鼠肺泡上皮细胞24h后,趋化因子CCL11、CCL22mRNA表达增高。结论IL-31通过刺激趋化因子表达募集炎性细胞,促进气道炎症的发生发展。     

Asymmetry of the internal connections of the striate cortex of the cat in the projection zone of the center of the field of vision

Studies were carried out on the organization of the internal connections of the striate cortex in cats in the projection zone of the center (0–5°) of the field of vision by microintophoretic application of horseradish peroxidase to electrophysiologically identified orientational columns. The area containing neurons showing retrograde labeling in most cases extended in the mediolateral direction. Labeled cells were located in the upper (II, III) and lower (V, VI) layers of the cortex, and the shapes and orientations of the areas containing labeled neurons in these layers coincided. Spatial asymmetry was detected in the distribution of labeled neurons relative to the orientational column studied. Labeled cells were located predominantly medial to the columns, regardless of the distance from the projection of the area centralis. Considering the visuotopical map of field 17, the asymmetry detected here provides evidence that neurons in orientational columns have more extensive connections with neurons of the peripheral part of the cortex. An asymmetrical distribution of “silent” zones around the receptive fields of neurons in orientational columns is suggested, and that these appear to receive influences from the periphery of the visual field. Laboratory of Visual Physiology and Laboratory of Central Nervous System Morphology, I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, 199034 St. Petersburg, Russia. Translated from Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 82, No. 12, pp. 23–29, December, 1996.     

Effects of the lesion of the postcommissural part of the septum on the behavior of the rat.

The effects of the lesion of the postcommissural part of the septum on behavior of the rat has been studied. Results may be summarized as follows. An increase in the exploratory behavior in the open field which decreases rapidly; a decrease in the number of defecations in this test and a decrease in time leaving a dark environment for exploration. In the shuttle box test, no facilitation of the acquisition, but a permanent and quite significant increase in the intertrial activity has been found. We conclude that the lesions tend to decrease the emotivity of the subjects. An interpretation on the basis of the species -- specific defensive reactions explains the transitory and permanent effects of the lesions on the spontaneous activity.     

The development of the ligament of the head of the femur

The hip joints of 30 human male and female fetuses and stillborns between 20 mm and 350 mm crown-rump length were studied by light microscopy. The ligament of the head of the femur developed in situ as a condensation of mesenchyme at the end of the second month of intra-uterine life (IUL), and was vascularized by branches of acetabular vessels early in the fourth month. In the majority of fetuses older than 5.5 months IUL, vessels in the ligament passed a short way into the femoral head within cartilage canals, to supply a small region around the fovea capitis. The remainder of the head was supplied by vessels in canals from around the upper part of the neck. The ligament changed from predominantly cellular to fibrous during the last 4 months of IUL. This increase in strength suggested significant mechanical functions in utero: limitation of adduction-flexion and opposition to postero-superior dislocation were the most likely.     

Counterpointing the functional role of the forebrain and of the brainstem in the control of the sleep-waking system

This paper reviews the lifetime contributions of the author to the field of sleep-wakefulness (S-W), reinterprets results of the early studies, and suggests new conclusions and perspectives. Long-term cats with mesencephalic transection show behavioral/polygraphic rapid eye movement sleep (REMS), including the typical oculo-pupillary behavior, even when the section is performed in kittens prior to S-W maturation. REMS can be induced as a reflex. Typical non-rapid eye movement S (NREMS) is absent and full W/arousal is present only after a precollicular section. The isolated forebrain (IF) rostral to the transection exhibits all features of W/arousal and NREMS [with electroencephalographic (EEG) spindles and delta waves], arousal to olfactory stimuli, and including the appropriate oculo-pupillary behaviors. These features also mature normally after neonatal transection. REMS is absent from the IF. After deprivation there is NREMS pressure and rebound in the IF, but the decerebrate cat only shows pressure for REMS. Most IF reactions to pharmacologic agents are within expectations, except for the tolerance/withdrawal effects of long-term morphine use which are absent. In contrast, these effects are supported by the brainstem (i.e. seen in the decerebrate cat). In cats with ablation of the telencephalon, or diencephalic cats, delta waves are absent in the thalamus. EEG thalamic spindle waves are seen triggering S for only 4-5 days after ablation. Therefore, true NREMS is absent in chronic diencephalic cats although pre- and postsomniac behaviors persist. These animals are hyperactive and show a pronounced, permanent insomnia; however, a low dose of barbiturate triggers a dramatic REMS/atypical NREMS rebound. Cats without the thalamus (athalamic cats), initially show a dissociation between behavioral hyperactivity/insomnia and the neocortical EEG, which for 15-20 days exhibits only delta and slower oscillations. Fast, low-voltage W rhythms appear later on, first during REMS, but spindle waves and S postures are absent from the start, such that these cats also display only atypical NREMS. Athalamic cats also show barbiturate-sensitive insomnia. Cats with ablation of the frontal cortices or the caudate nuclei remain permanently hyperactive. They also show a mild, but significant hyposomnia, which is permanent in afrontal cats, but lasts for about a month in acaudates. The polygraphic/behavioral features of their S-W states remain normal. We conclude and propose that: (a) the control of the S-W system is highly complex and distributed, but is organized hierarchically in a well-defined rostro-caudal manner; the rostral-most or highest level (telencephalon), is the most functionally complex/adaptative and regulates the lower levels; the diencephalic/basal forebrain, or middle level, has a pivotal role in inducing switching between S and W and in coordinating the lowest (brainstem) and highest levels; (b) W can occur independently in both the forebrain and brainstem, but true NREMS- and REMS-generating mechanisms exist exclusively in the forebrain and brainstem, respectively; (c) forebrain and brainstem S-W processes can operate independently from each other and are preprogrammed at birth; this helps understanding normal and abnormal polygraphic/behavioral dissociations in humans and normal dissociations/splitting in aquatic mammals; (d) NREMS homeostasis is present in the IF, but only REMS pressure after deprivation persists in the decerebrate cat; (e) the thalamus engages in both NREMS and W; (f) insomnia in diencephalic cats is the result of an imbalance between antagonistic W- and S-promoting cellular groups in the ventral brain (normally modulated by the telencephalon); (g) the EEG waves, which are signature for each S-W state, appear to truly drive the concomitant behaviors, e.g. a hypothetical human IF could alternate between behavioral NREMS and W/arousal/awareness; (h) a role for REMS is to keep the individual sleeping at the end of the self-limiting NREMS periods. The need for accelerating research on telencephaling NREMS periods. The need for accelerating research on telencephalic S-W processes and downstream control of the lower S-W system levels is emphasized.     

Topical organization of the projections of neurons of the substantia nigra and the ventral field of the tegmentum of the midbrain to the head of the caudate nucleus of the cat

Neuroscience and Behavioral Physiology -     

Involvement of the striatum in the central regulation of the hormonal functions of the gonads

Studies reported here show that intrastriatal administration of corticoliberin to rats decreases the blood testosterone level. However, in conditions of chemical deficiency of dopaminergic transmission in the dorsal striatum induced by injection of 6-hydroxydopamine, the effect of this neurohormone did not appear. It is concluded that extrahypothalamic corticoliberin is involved in regulating the hormonal reproductive system acting via dopaminergic mechanisms. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 594–597, April, 1999.     

Ultrastructure of the endothelium of the drainage system of the eye

The endothelium of the ocular drainage system (Schlemm’s canal, collector tubules, and aqueous veins) in primary juvenile glaucoma undergoes degenerative dystrophic changes with compensatory hypertrophy and proliferation at the initial stages of the glaucomatous process and atrophy and desquamation at advanced and terminal stages. Progressive decrease in the pinocytous function of endotheliocytes, reduction of the protein-synthesizing and mitochondrial compartments of the cytoplasm, and formation of autophagosomes reflect the process of endotheliocyte degeneration in general. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 574–577, May, 2008