约束应激对大鼠5-HT1A和5-HT2A受体表达的影响

慢性应激如长期压抑 ,环境不适等易导致身心健康损害 ,出现学习记忆、情绪行为等多方面的改变[1,2 ] ,目前对其发生机制的研究多集中在下丘脑 -垂体 -肾上腺 (HPA)轴、自主神经系统和免疫系统等 ,而对 5 -羟色胺 (5 -HT)的研究较少。 5 -HT是一种重要的中枢神经递质 ,参与多种行为、情绪活动的调节 ,迄今已发现了 5 -HT受体的 7种类型 13个亚型。近来的研究报道认为 5 -HT1A 和 5 -HT2A 可能与精神机能和学习记忆有关[3 ,4] 。为探讨慢性应激与 5 -HT1A、5 -HT2A 受体的关系 ,我们采用PCR观察大鼠经 30天慢性约束应激后不同脑区 5…     

人淋巴组织中5-HT1A受体的表达

目的检测5-HT1A受体蛋白及其mRNA 在人淋巴组织中的表达情况,寻求神经免疫内分泌网络间功能双向调节的形态学依据.方法应用免疫组织化学Picture法及核酸分子原位杂交的方法,检测5-HT1A受体蛋白及其mRNA 在100例人淋巴结、脾脏及肠道淋巴组织中的表达.结果免疫表型显示5-HT1A受体蛋白在人各种淋巴组织中表达的阳性率为86.25%,与原位杂交(阳性率为75.0%)之间的差异无统计学意义(χ2=0.570, P》0.05).结论人的淋巴组织不但可以表达5-HT1A受体蛋白还可以合成其mRNA, 5-HT1A受体为神经免疫内分泌网络共有的生物学语言媒介;神经源性与免疫源性的5-HT都可以通过免疫细胞膜上的5-HT1A受体对免疫系统发挥调节作用.     

大鼠前扣带回皮质5-HT_(1A,1B,2A,2C)受体亚型蛋白表达的细胞定位

目的:观察5-HT_(1A、1B)和5HT_(2A、2C)等受体在大鼠前扣带回皮质(anterior cingulated cortex,ACC)的分布及细胞定位。方法:将20只成年雄性Sprague-Dawley大鼠平均分成四组,每组5只分别进行四种亚型5-HT的免疫组织化学反应。动物经过灌注固定、脑冠状切片(25μm)后,以卵白素-生物素复合物法(ABC法)对切片进行染色、显微镜观察。结果:四种5-HT受体亚型在ACC都有分布。ACC内II-VI层均有5-HT_(1A)免疫阳性反应神经元的胞体和树突。5-HT_(1B)受体亚型定位在神经元胞体上,表达较弱。I-III层5-HT_(2A)免疫阳性反应较强,主要为标记的锥体神经元树突(Cg1区III层观察到部分阳性反应的神经元胞体);而V-VI层可见到明显的免疫阳性神经元胞体。5-HT2C受体定位于ACC之II-VI层神经元胞体上。结论:5-HT_(1A)、5-HT_(1B)、5-HT_(2A)和5-HT_(2C)等不同受体亚型在ACC内细胞上的定位有一定差异,提示它们介导5-HT对ACC神经元发生调节反应的细胞作用部位有选择性,因之它们介导的5-HT对ACC神经元的作用效果也可能不同。     

海马CA1区5-HT_(1A)受体调控PTSD大鼠空间记忆的作用

目的:观察创伤后应激障碍(PTSD)大鼠海马长时程增强(LTP)的变化以及5-羟色胺1A受体(5-HT_(1A)受体)和突触后致密物蛋白95(PSD-95)的表达,探讨5-HT_(1A)受体调控PTSD大鼠空间记忆的机制。方法:健康成年SD大鼠36只,随机分为正常对照组和模型组,每组18只。模型组采用连续单一刺激构建PTSD大鼠模型。Morris水迷宫实验检测2组大鼠的学习和记忆能力,电生理实验检测强直性高频刺激对海马LTP的影响Western blot法和免疫荧光实验检测海马5-HT_(1A)受体和PSD-95蛋白的表达。结果:Morris水迷宫实验结果显示在各实验日模型组大鼠逃避平台的潜伏期较对照组显著延长(P0.05)。电生理实验结果显示在强直性高频刺激后,2组大鼠海马诱发电位的幅值明显升高,模型组诱发电位的幅值显著低于对照组(P0.01)。Westem blot实验和免疫荧光实验结果显示,与对照组比较,模型组大鼠海马CA1区5-HT_(1A)受体的表达显著增加(P0.05),但PSD-95的表达明显减少(P0.05)。结论:PTSD大鼠空间记忆能力减退,可能与海马CA1区5-HT_(1A)受体的表达增加和PSD-95的表达减少有关。     

大鼠神经系统内5-羟色胺_(1A)受体亚型的定位分布(英文)

应用免疫组织化学技术观察了大鼠神经系统内 5 -羟色胺 1A受体亚型 ( 5 -HT1 AR)免疫阳性结构的分布。结果显示 :5 -HT1 AR免疫阳性结构主要分布于梨状皮质、隔核、丘脑腹后核、丘脑网状核、杏仁基外侧核、Purkinje细胞层、红核、面神经核、斜方体核等 ;在海马、额叶皮质、丘脑背内侧核、脚间核、三叉神经中脑核、中缝背核、三叉神经脊束核、脊髓背角浅层、背根神经节和三叉神经等结构内有中等强度的分布 ;在嗅球、尾壳核、苍白球、斜角带核、终纹床核、缰核、黑质、上橄榄等部位有弱的分布。本文的结果提示 5 -HT1 AR阳性结构广泛地分布在大鼠神经系统 ,它们可能介导 5 -HT在神经系统中的多种生理功能     

连续单一应激后大鼠海马5-HT_(1A)受体活性与糖皮质激素受体表达的关系

目的:观察连续单一应激(SPS)大鼠海马糖皮质激素受体(GR)变化与5-HT1A受体的关系,探讨创伤后应激障碍的发病机制。方法:选用雄性成年Wistar大鼠45只,将大鼠随机分为对照组、模型组和阻断组,每组15只。模型组和阻断组给予SPS应激,其中阻断组大鼠在接受SPS前用55-HT1A受体阻断剂WAY100635预处理。采用免疫组织化学和免疫印迹技术测定海马GR水平,采用逆转录-聚合酶链式反应检测GRmRNA表达变化。结果:(1)免疫组织化学结果显示,模型组大鼠海马GR表达高于对照组(P0.01),阻断组则低于模型组(P0.05);(2)WesternBlot结果显示,模型组大鼠海马GR相对表达高于对照组(P0.01),阻断组低于模型组(P0.01);(3)RT-PCR结果表明,与对照组比较,模型组大鼠海马GRmRNA表达增强(P0.01);与模型组比较,阻断组表达量降低(P0.05)。结论:SPS海马GR表达变化与5-HT1A受体有关。     

雌二醇对去卵巢大鼠下丘脑5-HT_(1A)和5-HT_(2A)受体亚型mRNA表达的影响

观察17β-雌二醇(17β-estradioI,E2)对去卵巢大鼠下丘脑5-HT1A和5-HT2A受体mRNA表达的影响的时间效应。实验动物分为假去卵巢对照组(OVX+S组)和去卵巢组(OVX)。去卵巢组大鼠又分为雌激素处理组(OVX+E+组)和无雌激素处理组(OVX+E-组),分别在处理后3、6、9和12 d时,采用反转录-聚合酶链式反应(RT-PCR)方法观察5-HT1A和5-HT2A受体mR-NA在大鼠下丘脑的表达。结果显示:5-HT1A和5-HT2A受体mRNA在所有大鼠下丘脑均有表达;5-HT1A受体mRNA在3、6和9 d的OVX+E-组大鼠下丘脑的表达水平明显高于同时间点的OVX-E+组,而5-HT2A受体mRNA在6、9和12 d的OVX+E-组下丘脑的表达量也显著高于同时间点的OVX+E+组。本文结果表明去卵巢后大鼠下丘脑5-HT1A和5-HT2A受体亚型mRNA表达水平升高,17β-雌二醇可下调两种受体的表达。     

慢性应激抑郁模型大鼠脑内5-HT1A和5-HT2A受体的变化

为了在5-羟色胺受体水平研究抑郁症的机制和三环类抗抑郁药物(TCAs)阿米替林的药理学机理,将24只SD雄性大鼠随机均分为三组,即对照组、抑郁组、阿米替林治疗组.应用[3H]8-OH-DPAT、[3H]Ketanserin作为标记配基,采用放射性配体受体结合法,分别测定大鼠海马5-HT1A受体、大脑皮层5-HT2A受体结合.结果显示抑郁大鼠海马 [3H]8-OH-DPAT 特异性结合(18.78±5.62 fmol/mg prot),较正常对照组(26.12±5.52fmol/mg prot )明显下降(P＜0.05).抑郁大鼠大脑皮层[3H]Ketanserin特异性结合(112.58±4.21fmol/mg prot),较正常对照组(86.28±4.24fmol/mg prot)明显增加( P＜0.05).阿米替林治疗3周后,可使抑郁大鼠海马5-HT1A受体与大脑皮层5-HT2A受体结合恢复正常.提示 海马5-HT1A受体结合下降、大脑皮层5-HT2A受体结合增加可能与抑郁症病因有关;海马5-HT1A受体、大脑皮层5-HT2A受体是阿米替林发挥抗抑郁作用的环节.     

大鼠前庭神经核复合体内5-羟色胺1A受体的分布

为了研究5-羟色胺1A受体(5-HT1AR)亚型在大鼠前庭神经核复合体(VNC)内的分布情况,本文采用免疫组织化学方法,在光学显微镜下对5-HT1AR亚型免疫阳性结构进行了观察。结果显示:5-HT1AR免疫阳性产物在VNC各个核团全长均有分布,主要定位于VNC神经元的胞体和近侧端树突,呈弥散分布,但在胞浆中也观察到许多染色深浅不同、分布不均匀的点状阳性结构。其中5-HT1AR样阳性神经元在前庭内侧核的全长呈密集分布,在前庭下核的尾段呈中等密度分布,在前庭上核、前庭外侧核和X核的全长、前庭下核的吻段和中段以及Y核的中、尾段均呈低密度分布,Y核的吻侧呈稀疏分布。本文结果提示,5-HT1AR阳性结构广泛地分布于大鼠VNC内,它们可能在介导5-HT对神经元活动的调节,参与前庭信息的整合与加工方面发挥重要作用。     

低氧性肺动脉高压大鼠肺内5-HT_(1B)受体的分布和表达变化

目的:观察低氧性肺动脉高压(HPH)大鼠体内5-羟色胺(5-HT)水平及其肺内5-羟色胺1B(5-HT1B)受体的分布和表达变化,探讨低氧性肺动脉高压的形成机制。方法:40只健康雄性SD大鼠随机分为正常组(control)、低氧3周组、低氧4周组和低氧5周组。除正常组外,其余3组大鼠分别在低氧环境中饲养3周、4周和5周。测定各组大鼠的平均肺动脉压力(mPAP)、右心室收缩压(RVSP)、右心室肥厚度[RV/(LV+S)%]、血浆和肺组织中5-HT含量。应用免疫组织化学法观察大鼠肺组织中5-HT1B受体的分布和表达,Western blotting法测定大鼠肺组织中5-HT1B受体的蛋白含量。结果:和正常组相比,低氧3周组大鼠的mPAP、RVSP和右心室肥厚度均显著升高(均P0.05),并且随着低氧时间的延长而持续升高(均P0.05)。低氧大鼠血浆和肺组织中5-HT的含量均显著高于正常组大鼠(均P0.05),并随着低氧时间的延长而持续升高(均P0.05)。免疫组织化学结果显示:5-HT1B受体主要分布在正常大鼠肺动脉的内膜层,而平滑肌层中仅有少量表达;和正常组相比,低氧3周组大鼠肺动脉平滑肌层中5-HT1B受体的表达显著增多;随着低氧时间的延长,大鼠肺动脉平滑肌层中5-HT1B受体表达持续增多。Western blotting结果表明,大鼠肺组织中5-HT1B受体的蛋白含量变化和免疫组织化学结果相一致。结论:低氧性肺动脉高压大鼠体内5-HT水平显著升高,其肺动脉中5-HT1B受体呈过度表达,这可能是低氧性肺动脉高压形成的分子机制之一。     

绿色荧光蛋白转基因小鼠骨髓基质细胞体外诱导分化为神经元样细胞

目的 研究绿色荧光蛋白转基因小鼠骨髓基质细胞(GFP-GM-BMSCs)在体外无血清培养基+神经细胞因子诱导条件下,向神经细胞分化的能力。方法 用贴壁法体外培养GFP-GM-BMSCs,取第3代GFP-GM-BMSCs,用含浓度均为20μg/L的表皮生长因子(EGF)和碱性成纤维细胞生长因子（bFGF）的无血清培养基（neurobasal-A+2%B27）诱导分化。第5天用免疫细胞荧光方法检测巢蛋白(nestin)的表达,第10天用神经元烯醇化酶 (NSE)、神经胶质纤维酸性蛋白(GFAP)免疫细胞化学方法鉴定阳性细胞。结果 GFP-GM-BMSCs经无血清培养基+神经细胞因子诱导后,细胞胞体变圆,伸出细长突起, 有的突起连接成网,呈神经元样形态。诱导第5天,nestin阳性表达的细胞为40.24%+5.09%;第10天,NSE阳性、GFAP阳性的细胞分别为36.43%+5.27%和49.73%+6.28%。 结论 GFP-GM-BMSCs在体外含EGF、bFGF的无血清培养基中,能分化成神经元样细胞。     

Parathyroid hormone(1-34)-induced apoptosis in neuronal rat PC12 cells: implications for neurotoxicity

Based on accumulated evidence, we speculate that a high concentration of parathyroid hormone (PTH) may cause neurotoxicity in patients with uremia through apoptosis-induced neuropathy. In this study, we demonstrated that in vitro stimulation with PTH(1-34) induced a significant decrease in PC12 cell numbers in both dosage- and time-dependent fashions when these cells were treated with PTH(1-34) at concentrations of 0.01, 0.1 or 1.0 μM for 24, 48, 72, and 96 h, respectively, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Decreased numbers of PC12 cells were caused by PTH(1-34)-induced apoptotic and cytotoxic processes, as determined by DNA fragmentation, flow cytometry, and lactate dehydrogenase (LDH)-leakage assays. Upregulation of the extracellular signal-regulated kinase (ERK) and p38 signaling pathway was the underlying mechanism responsible for 1.0 μM PTH(1-34)-induced apoptosis in PC12 cells, as elucidated by Western blotting analysis and confirmed with ERK and p38 inhibitors. Furthermore, 1.0 μM PTH(1-34)-induced apoptosis was accompanied by a release of cytochrome c and subsequent caspase-3 activation. These data suggest that a high concentration of PTH(1-34)-induced cytotoxicity and apoptosis in PC12 cells was associated with upregulation of ERK and p38 through a mitochondria-mediated apoptotic pathway.     

A synergic simulation-optimization approach for analyzing biomolecular dynamics in living organisms

A synergic duo simulation–optimization approach was developed and implemented to study protein–substrate dynamics and binding kinetics in living organisms. The forward problem is a system of several coupled nonlinear partial differential equations which, with a given set of kinetics and diffusion parameters, can provide not only the commonly used bleached area-averaged time series in fluorescence microscopy experiments but more informative full biomolecular/drug space–time series and can be successfully used to study dynamics of both Dirac and Gaussian fluorescence-labeled biomacromolecules in vivo. The incomplete Cholesky preconditioner was coupled with the finite difference discretization scheme and an adaptive time-stepping strategy to solve the forward problem. The proposed approach was validated with analytical as well as reference solutions and used to simulate dynamics of GFP-tagged glucocorticoid receptor (GFP-GR) in mouse cancer cell during a fluorescence recovery after photobleaching experiment. Model analysis indicates that the commonly practiced bleach spot-averaged time series is not an efficient approach to extract physiological information from the fluorescence microscopy protocols. It was recommended that experimental biophysicists should use full space–time series, resulting from experimental protocols, to study dynamics of biomacromolecules and drugs in living organisms. It was also concluded that in parameterization of biological mass transfer processes, setting the norm of the gradient of the penalty function at the solution to zero is not an efficient stopping rule to end the inverse algorithm. Theoreticians should use multi-criteria stopping rules to quantify model parameters by optimization.     

Differential distribution of MAP1A isoforms in the adult mouse barrel cortex and comparison with the serotonin 5-HT2A receptor

Microtubule-associated protein 1A (MAP1A) is essential during the late differentiation phase of neuronal development. Here, we demonstrated the presence of two MAP1A isoforms with a differential spatial distribution in the adult mouse barrel cortex. Antibody A stained MAP1A in pyramidal and stellate cells, including dendrites that crossed layer IV in the septa between barrels. The other antibody, BW6 recognized a MAP1A isoform that was mainly confined to the barrel hollow and identified smaller caliber dendrites. Previously, an interaction of MAP1A and the serotonin 5-hydroxytryptamine 2A (5-HT(2A)) receptor was shown in the rat cortex. Here, we identified, by double-immunofluorescent labeling, MAP1A isoform and serotonin 5-HT(2A) receptor distribution. MAP1A co-localized mainly with 5-HT(2A) receptor in larger apical dendrites situated in septa. This differential staining of MAP1A and a serotonin receptor in defined barrel compartments may be due to changes in the expression or processing of MAP1A during dendritic transport as a consequence of functional differences in processing of whisker-related sensory input.     

Mapping of the binding sites for the OX1 orexin receptor antagonist, SB-334867, using orexin/hypocretin receptor chimaeras

The binding sites for agonists and antagonist of orexin receptors are not know, hampering progressive drug design approaches. In the current study, we utilized chimaeric orexin receptor approach to map the receptor areas contributing to the selectivity of the classical antagonist, SB-334867, for OX₁ receptors. Altogether ten chimaeras between OX₁ and OX₂ orexin receptors were utilized. The receptors were transiently expressed in HEK-293 cells. The ability (K_B) of SB-334867 to inhibit orexin-A-induced inositol phosphate release (phospholipase C activity) was measured. The results, in synthesis, suggest that there are several possible interactions contributing to the high affinity binding, all of which are not required simultaneously. This is indicated by the fact that most of the chimaeras display affinity (at least somewhat) higher than OX₂. As previously shown for the agonist distinction, the second quarter of the receptor, from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 seems to be most central also for SB-334867 binding, but also the third quarter, from the transmembrane helix 4 to the transmembrane helix 6 is able to contribute (and compensate for loss of other sites). A previous study has suggested that amino acids conserved between OX₁ and OX₂ receptors would somehow confer selectivity for subtype-selective antagonists. In contrast to previous findings, our results indicate that the amino acids distinct between the receptor subtypes are in key position.     

Efficient non-viral transfection of THP-1 cells

Macrophages are an important part of the cellular immune system and play a key role during immune responses. Thus, macrophages are interesting targets in basic and clinical research. Primary monocytes or monocyte-derived macrophages do not proliferate on a suitable scale so that their use for functional studies invitro is limited. Immortal proliferating cell lines, such as the human THP-1 monocytic leukemia cell line, are therefore often used instead of primary cells. Transfection is a useful tool to study the function of gene products, but transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages with subsequent differentiation into mature THP-1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. In this study, we describe a simple and rapid approach for efficient transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages using a modified Nucleofection-based approach. The protocol maintains cell viability and functionality, thus allowing efficient transfection of THP-1 cells combined with subsequent differentiation of transfected THP-1 cells into mature macrophages.     

Erythroblast differentiation at spleen in Q137E mutant ribosomal protein S19 gene knock-in C57BL/6J mice

We recently found that erythroblast-like cells derived from human leukaemia K562 cells express C5a receptor (C5aR) and produce its antagonistic and agonistic ligand ribosomal protein S19 (RP S19) polymer, which is cross-linked between K122 and Q137 by tissue transglutaminases. RP S19 polymer binds to the reciprocal C5aRs on erythroblast-like cells and macrophage-like cells derived from human monocytic THP-1 cells and promotes differentiation into reticulocyte-like cells through enucleation in vitro. To examine the roles of RP S19 polymer in mouse erythropoiesis, we prepared Q137E mutant RP S19 gene knock-in C57BL/6J mice. In contrast to wild-type mice, erythroblast numbers at the preliminary stage (CD71^high/TER119^low) in spleen based on transferrin receptor (CD71) and glycophorin A (TER119) values and erythrocyte numbers in orbital artery bloods were not largely changed in knock-in mice. Conversely, erythroblast numbers at the early stage (CD71^high/TER119^high) were significantly decreased in spleen by knock-in mice. The reduction of early erythroblast numbers in spleen was enhanced by the phenylhydrazine-induced pernicious anemia model knock-in mice and was rescued by a functional analogue of RP S19 dimer S-tagged C5a/RP S19. These data indicated that RP S19 polymer plays the roles in the early erythroblast differentiation of C57BL/6J mouse spleen.