卵巢切除后大鼠垂体前叶生长相关蛋白-43免疫反应性及其与促性腺激素细胞的关系

目的:探讨卵巢切除后大鼠垂体前叶生长相关蛋白-43(growth-associated protein 43,GAP-43)免疫反应神经纤维的数量变化及其与腺细胞的关系.方法:大鼠经双侧卵巢切除,分别于术后4d和15d取垂体,应用免疫荧光双重标记技术,进行GAP-43和卵泡刺激素(follicle stimulating hormone,FSH),GAP-43和P物质(substance P,SP)免疫荧光双标,激光共聚焦显微镜观察垂体前叶GAP-43免疫反应神经纤维的数量变化、与促性腺激素细胞及SP免疫反应神经纤维的关系.结果:正常大鼠垂体前叶很少有GAP-43免疫反应神经纤维.卵巢切除后4d垂体前叶出现了数量较多的GAP-43免疫反应神经纤维,术后15d神经纤维明显增多,多走行于腺细胞间,少量神经纤维与FSH细胞密切接触.GAP-43免疫反应神经纤维与SP共存.结论:外周内分泌干预后,垂体前叶的神经纤维可通过芽生增加纤维数量,以对机体内分泌状态改变作出积极的应答.神经纤维可能直接作用于腺细胞,但更大的可能性是通过旁分泌方式实现对腺细胞的调节.     

大鼠生后垂体前叶P物质免疫反应阳性神经纤维的发育变化

研究大鼠出生后不同发育阶段,其脑下垂体前叶P物质(substance P,SP)免疫反应阳性神经纤维的变化.SD大鼠按其出生后年龄分为5个实验组:P1～2,P8,P15,P30,P45.采用免疫组织化学法观察大鼠垂体前叶SP阳性神经纤维的发育变化.P1～2期,大鼠垂体前叶中央分布有少量SP阳性神经纤维,膨体呈长梭形.P8期,阳性神经纤维可分为短粗类纤维和屈曲样膨体型纤维,前者主要分布于垂体前叶的浅表,后者主要分布于前叶的吻侧部及中央部,数量比P1～2期多,且膨体较P1～2期大,呈串珠状.P15期,阳性神经纤维分布较P8期广泛,呈片状,短粗类纤维较P8期长,屈曲样膨体型纤维分布于前叶中央.P30期,阳性神经纤维以屈曲样膨体型纤维为主,数量较P15期增多.P45期,垂体前叶SP纤维类型、分布同成年大鼠,其屈曲样膨体型纤维广泛分布于整个前叶.结果表明随出生后年龄的增长,大鼠垂体前叶SP阳性纤维的类型、数量、分布逐渐由少到多,由分布于周边部到广泛分布于整个垂体前叶,呈现一种动态变化,说明出生后大鼠垂体前叶中SP阳性纤维发育迅速.     

γ—氨基丁酸,谷氨酸能神经纤维在大鼠垂体前叶的分布

本文报告大鼠垂体前叶γ－氨基丁酸（ＧＡＢＡ），谷氨酸（Ｇｌｕ）能神经纤维的直接支配。ＧＡＢＡ和Ｇｌｕ免疫反应神经纤维呈斑片状分布在大鼠垂体前叶远侧部，虽然部分沿血管走行，但大部分神经纤维迂曲于腺细胞周，并与腺细胞接触。神经纤维可见膨体型、细和中等粗细三种类型，其上有大量的膨体。在垂体前叶也发现有ＧＡＢＡ、Ｇｌｕ免疫反应细胞分布。     

大鼠垂体前叶GABA能神经纤维支配的电镜研究(英文)

近年来的研究表明哺乳动物的垂体前叶接受SP、CGRP等肽能神经支配。免疫电镜研究发现 ,在SP、CGRP阳性末梢中 ,除了含有阳性的大致密芯囊泡外 ,还含有大量的清亮小泡 ,提示经典递质的存在 ,其性质目前尚不清楚。GABA参与垂体前叶腺细胞分泌活动的调节 ,是可能的递质候选者之一。应用免疫组化技术发现 ,在大鼠垂体前叶两翼的外侧部及浅表部位 ,分布有GABA免疫阳性末梢。应用包埋前免疫电镜技术 ,发现 85%的GABA免疫阳性末梢与前叶腺细胞直接接触 ,由多到少依次为生长激素细胞、泌乳素细胞和促肾上腺皮质激素细胞 ,并且与上述细胞形成典型的突触结构。GABA免疫阳性末梢与生长激素细胞形成非对称型突触结构 ,与促肾上腺皮质激素细胞形成对称型突触结构 ,而与泌乳素细胞间则既有对称型突触结构 ,又有非对称型突触结构。GABA免疫阳性末梢极少与促性腺激素细胞接触。结果表明大鼠垂体前叶接受GABA能神经纤维的支配 ,GABA能末梢可直接调控垂体前叶腺细胞的分泌。大鼠垂体前叶GABA能纤维的来源则有待进一步研究     

大鼠生后垂体前叶P物质免疫反应阳性神经纤维的发育变化

研究大鼠出生后不同发育阶段,其脑下垂体前叶Ｐ物质（ｓｕｂｓｔａｎｃｅ Ｐ,ＳＰ）免疫反应阳性神经纤维的变化,ＳＤ大鼠按其出生后年龄分为５个实验组：Ｐ１－２,Ｐ８,Ｐ１５,Ｐ３０,Ｐ４５。采用免疫组织化学法观察大鼠垂体前叶ＳＰ阳性神经纤维的发育变化。Ｐ１－２期,大鼠垂丛前叶中央分布有少量ＳＰ阳性性神经纤维,膨体呈长投形。Ｐ８期,阳性神经纤维可分为短粗类纤维和屈曲样膨体型纤维,前者主要分布于垂体前叶的     

大鼠垂体前叶ＧＡＢＡ能神经纤维支配的电镜研究

近年来的研究表明哺乳动物的垂体前叶接受ＳＰ、ＣＧＲＰ等肽能神经支配。免疫电镜研究发现,在ＳＰ、ＣＧＲＰ阳性末梢中,除了含有阳性的大致密芯囊泡外,还含有大理的清亮小泡,提示经典递质的存在,其性质目前尚不清楚。ＧＡＢＡ参与垂体前叶腺细胞分泌活动的调节,是可能的递质候选者之一。应用免疫组化技术发现,在大鼠垂体前叶两翼的外侧部及浅表部位,分布有ＧＡＢＡ免疫阳性末梢。应用包埋前免疫电镜技术,发现８５％的ＧＡＢＡ免疫阳性末梢与前叶腺细胞直接接触,由多到少依次为生长激素细胞、泌乳素细胞和促肾上腺皮质激素细胞,并且与上述细胞形成典型的突触结构。ＧＡＢＡ免疫阳性末梢与生长激素细胞形成非对称型突触结构,与促肾上腺皮质激素细胞形成对称型突触结构,而与泌乳素细胞间则既有对称型突触结构,又有非对称型突触结构。ＧＡＢＡ免疫阳性末梢极少与促性腺激素细胞接触。结果表明大鼠垂体前叶接受ＧＡＢＡ能神经纤维的支配,ＧＡＢＡ能末梢可直接调控垂体前叶腺细胞的分泌。大鼠垂体前叶ＧＡＢＡ能纤维的来源则有待进一步研究。     

经咽旁入路选择性切除大鼠垂体前叶的研究

目的研究经咽旁入路选择性的切除大鼠垂体前叶，以制备大鼠垂体前叶激素缺乏模型如生长激素（GH）缺乏模型。方法经腹侧咽旁入路，运用显微神经外科技术经基蝶骨底选择性地切除垂体前叶。然后采用竞争免疫沉淀法检测大鼠生长激素的含量，术后统计死亡率、成功率。结果切除垂体前叶后大鼠的死亡率为26．7％，成活率为73．3％，全切率为91％；成功制备大鼠模型的GH显著低于假手术组和对照组。结论运用显微外科技术可以成功的选择性的切除大鼠垂体前叶，制备出垂体前叶激素如生长激素缺乏的动物模型。     

生长相关蛋白-43影响神经细胞分裂方向作用的研究

目的研究生长相关蛋白-43(growth associated protein-43,GAP-43)与G蛋白对神经细胞分裂方向的调控,探讨GAP-43参与神经发生的机制。方法 6只C57BL/6J GAP-43高表达的转基因孕鼠(E13.5 d和E17.5 d的胎鼠均为24只)为实验组,6只野生型孕鼠(E13.5 d和E17.5 d的胎鼠均为24只)为对照组。分别取E13.5 d的3只实验组及对照组胎鼠,脑室区进行免疫荧光染色测定GAP-43的表达位置;再分别取E13.5 d的12只实验组及对照组胎鼠,向脑中加入裂解液分别提取膜蛋白和细胞质蛋白,通过Western blot测定GAP-43的表达位置及表达量;取E17.5 d的3只GAP-43高表达的转基因胎鼠的鼠脑进行GAP-43和G蛋白的免疫荧光共染色,观察二者的共定位情况;取E17.5 d的9只GAP-43高表达的转基因胎鼠的鼠脑进行免疫共沉淀(coimmunoprecipitation,co-IP)检测GAP-43与Gαi间的相互作用。取E13.5 d和E17.5 d的9只实验组胎鼠计数转基因胚胎鼠脑中神经前体细胞(neuro progenitor cell,NPC)中沿水平和垂直不同方向分裂的细胞数量,并与对照组小鼠进行对照。结果 E13.5 d实验组胎鼠的GAP-43蛋白的表达量高于对照组(P0.05);GAP-43特异性表达于细胞膜上;免疫荧光共染色结果表明GAP-43与Gαi共定位于细胞膜上,进一步通过co-IP证明GAP-43和Gαi相互结合;E13.5 d时,转基因组与对照组相比,细胞沿水平、中间和垂直角度分裂的细胞数量差异有显著性(P0.05)。结论 GAP-43可调控神经细胞分裂方向,这可能是GAP-43参与神经发生的机制。本研究为进一步研究GAP-43对卒中后神经损伤修复的作用机制提供基础。     

海藻酸钠-多聚赖氨酸-海藻酸钠微囊化大鼠卵巢激素分泌细胞异体移植对垂体形态学的影响

目的：已有研究证实海藻酸钠-多聚赖氨酸-海藻酸钠微囊对机体无明显毒副作用且生物相容性好，应用这一技术包裹细胞，可明显延长异体内移植物的存活时间。将海藻酸钠-多聚赖氨酸-海藻酸钠微囊化大鼠卵巢细胞体外培养后进行异体移植，观察其治疗卵巢功能丧失模型大鼠的可行性及垂体形态学变化。 方法：实验于2006-03/2007-09在首都医科大学生殖医学研究中心完成。①实验动物：30只Wistar雌性大鼠,随机分为正常对照组，卵巢摘除组，微囊移植组，每组10只。实验过程中对动物处置符合动物伦理学要求。②实验方法：卵巢摘除组：无菌条件下切除双侧卵巢；微囊移植组：海藻酸钠-多聚赖氨酸-海藻酸钠生物膜包裹传至P2代的大鼠卵巢细胞，制成微囊体外培养，收集24 h培养液以检测雌二醇分泌状态；将微囊化的卵巢细胞移植入已行双侧卵巢切除术的大鼠腹腔。用阴道涂片观察大鼠动情周期恢复情况。术后40 d麻醉后处死动物，放射免疫法检测血清雌二醇质量浓度，垂体组织常规石蜡包埋，连续切片，分别进行苏木精-伊红染色和雌、孕激素受体免疫组织化学染色。LEICA Q500IW自动图像分析仪对垂体细胞的面积，雌、孕激素受体吸光度值进行定量测定。 结果：①放射免疫法检测培养液显示，微囊化的卵巢激素分泌细胞具有继续合成和分泌雌二醇的功能。②检测血清中雌二醇质量浓度结果显示，卵巢摘除组明显低于正常对照组和微囊移植组，差异具有显著性意义，正常对照组和微囊移植组之间差异无显著性。③动情周期未恢复。④苏木精－伊红染色可见卵巢摘除组大鼠垂体中有大量阉割细胞,平均面积>180 μm2,与卵巢摘除组比较，微囊移植组大鼠垂体中细胞平均面积和阉割细胞数目均显著减少。⑤垂体雌、孕激素受体免疫组织化学吸光度值结果显示，卵巢摘除组明显低于正常对照组和微囊移植组，差异具有显著性意义。 结论：微囊化大鼠卵巢细胞异体移植后，可在受体大鼠体内继续合成和分泌雌二醇，并可以减少垂体内阉割细胞的形态和数目，提高垂体细胞雌激素受体和孕激素受体的表达，提示移植后细胞对垂体有反馈调节作用。     

从Nogo-A-NgR信号通路探讨MCAO大鼠皮脊髓束重塑的启动及复健片干预机制

目的探讨缺血性脑梗死颈髓水平皮质脊髓束重塑的启动机制及复健片的脑外作用靶点。方法制备大脑中动脉闭塞(MACO)大鼠模型。采用免疫组化自由漂片染色法进行颈髓切片BDA染色;横木行走实验(BWT)评估大鼠运动功能; Western blot法检测颈髓GAP-43蛋白表达;免疫荧光法检测颈髓Nogo-A、NgR蛋白表达; RT-qPCR检测颈髓GAP-43 m RNA。结果术后1 w末3组大鼠GAP-43蛋白表达并无差异,但术后2 w末模型组与药物组GAP-43表达及梗死侧阳性CST神经纤维数均高于假手术组;术后1 w、2 w末,较之假手术组,模型组和药物组大鼠Nogo-A、NgR蛋白表达均减少,且BWT评分也有显著改善,其中药物组趋势均更为明显。结论脑梗死后颈髓由于失神经支配自发完成一定程度的皮质脊髓束重塑,而复健片可更大程度地促进CST重塑,从而促进缺损神经功能的恢复。     

Axons Containing the Growth Associated Protein GAP-43 Specifically Target Rat Corticotrophs Following Adrenalectomy

An extensive network of nerve fibers immunoreactive for the neuronal growth associated protein GAP-43 (GAP-43-IR) is present within the anterior pituitary (AP) of the rat, and the density of these fibers has been reported to increase 4 days after adrenalectomy (ADX). In the present study, we employed confocal dual-label immunofluorescence microscopy to determine whether GAP-43-IR fibers are specifically associated with corticotrophs at various intervals after ADX. A dramatic increase in the density of GAP-43-IR was apparent 4 days after ADX, and this increase was sustained at 7 and 14 days post-ADX. The percentage of corticotrophs in apparent contact with GAP-43-IR axons was 87% at 4 days after ADX and 92% at 14 days. In addition, fewer than 15% of GAP-43-IR terminals were associated with cells other than corticotrophs in either group. This highly specific targeting of corticotrophs during a period in which these cells are undergoing both hypertrophy and hyperplasia indicates that axonal sprouting is occurring in response to ADX. While the less intense GAP-43-IR in the AP of intact rats precluded precise quantitative analysis, the majority of corticotrophs also appeared to be selectively innervated in these animals. The observations that GAP-43-IR axons selectively contact corticotrophs, and that both the specificity and thoroughness of innervation are maintained by targeted growth of GAP-43-IR axons following ADX, strongly suggest that these fibers are of functional significance.     

Galanin immunoreactive innervations of the anterior pituitary in the monkey and the dog

As we know, the anterior pituitary is regulated by hypothalamic hormones via the portal system. However, our recent studies have demonstrated the presence of a substantial amount of substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing nerve fibers in the anterior pituitary. In the present paper, the existence of a considerable amount of galanin (GAL)like immunoreactive (ir) nerve fibers with numerous of varicosities in the anterior pituitaries of the Macaca mulatta monkey and the dog were reported. In the monkey, GAL-ir nerve fibers with a large amount of varicosities were mainly located in the medial part of the gland, dominantly in its dorso-posterior regions. A great majority of varicosities were found to be closely related to the glandular tissue although some were located along the walls of blood sinus. GAL-ir nerve fibers were traced to enter the pars distalis of the anterior pituitary from the stalk as well as from the meningeal sheath covering the upper part of the anterior pituitary. Numerous GAL-ir cells presented in the pars distalis and the pars intermedia of the adenohypophysis. In the dog pituitary, GAL-ir nerve fibers were mainly located in the tail part and some in oral region. A majority of GAL-ir nerve fibers were at the periphery of the gland, especially in the medial planes, although some could be found deep in the gland. They appeared in individual fibers or in patches. Many GAL-ir nerve fibers and fiber fascicles existed in the median eminence and the sheath covering the tail and the oral part of the pituitary.     

Immunohistochemical localization of gamma-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution of gamma-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigated using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Expression of growth-associated protein 43 in the skin nerve fibers of patients with type 2 diabetes mellitus

The growth-associated protein 43 (GAP-43) is known as a marker of regenerating nerve fibers and their continuous remodeling in the adult human skin. The purpose of this pilot study was to investigate a possible role for GAP-43 in the detection of the early stages of small-fiber neuropathy in patients with type 2 diabetes mellitus (DM2) as compared with a well- established and validated parameter - intra-epidermal nerve fiber density (IENFD) of protein gene product 9.5 (PGP 9.5) immunoreactive intra-epidermal C fibers. In a group of 21 patients with DM2 within three years of diagnosis (13 men, 8 women; mean age 53.9±12.8; range 30-74) and a group of 17 healthy volunteers (8 men, 9 women; mean age 55.8±8.5; range 45-70 years), skin punch biopsies were taken from a distal calf and double immunostained with both PGP 9.5 and GAP-43. In healthy controls, 96.8% of 629 PGP 9.5 immunoreactive fibers were immunostained with GAP-43; the proportion of PGP 9.5 intra-epidermal nerve fibers immunoreactive for GAP-43 in control subjects ranged from 86.5 to 100%. In DM2 patients, IENFD was significantly lower compared to controls (median, 1.5 vs. 11.2/mm; p<0.001). The proportion of GAP-43 immunoreactive intraepidermal nerve fibers was significantly lower in DM2 patients compared to healthy controls (73.6% of 337 PGP 9.5 positive fibers; p<0.001); ranged from 0 to 98.1%. In conclusion, these results show that impaired regeneration of intra-epidermal C fibers in the early stages of type 2 diabetes mellitus, as indicated by GAP-43, might be a marker of incipient diabetic neuropathy.     

Immunohistochemical localization of γ-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution on γ-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigaed using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Peptidergic innervation in pars distalis of the human anterior pituitary

It has been previously shown that the peptidergic nerve fibers are present in the anterior pituitary of monkeys, dogs and rats. In our study, which is reported here, thick nerve fiber bundles, large numbers of peptidergic nerve fibers and their varicosities, which are substance P (SP)-, calcitonin gene-related peptide (CGRP)- and galanin (GAL)-immunoreactive (ir), are found in the human pituitary stalk. All these peptidergic nerve fibers run along the pituitary stalk and enter the pars distalis, and some GAL-ir nerve fibers even reach the center of the human anterior pituitary as well as in parenchyma of adenohypophysis. The number of SP-ir nerve fibers is much more than that of other kinds of peptidergic nerve fibers. All these peptidergic nerve fibers are mainly located in the medial part of the gland and distributed in its dorsal-posterior region. A substantial amount of these peptidergic nerve fibers with numerous varicosities are found to be close to the glandular tissue in the pars distalis of the human anterior pituitary. Furthermore, same SP-ir and CGRP-ir cells have been demonstrated in the pars distalis of the anterior pituitary. More or less, these peptidergic nerve fibers came also from the meningeal shell and enter the parenchyma of the anterior pituitary. Whatever the function of peptidergic nerve fibers in the human anterior pituitary might be, the concept that the adenohypophysis is regulated only hormonally by way of the portal system could be challenged.     

Quantitative Evaluation of Synaptophysin-Like Immunoreactive Nerve Terminals or Varicosities in Anterior Pituitary of Normal and Adrenalectomized Rats

Our previous studies have demonstrated the presence of substantial amounts of peptidergic nerve fibres in the anterior pituitary of monkeys, dogs and rats. The nerve fibres increase in number following adrenalectomy and ovariectomy. Furthermore, typical synapses between nerve fibres and anterior pituitary glandular cells have been demonstrated in the dog and rat, strongly suggesting the existence of some direct neural regulation. However, it is difficult to evaluate the functional importance of synapses or nerve terminals in general, unless quantitative data can be provided. The present study therefore aimed to quantify the number of terminals or varicosities in the anterior pituitary of the rat and its changes following adrenalectomy. Nerve terminals or varicosities were identified by their synaptophysin (SYN)-like immunoreactivity. Under the light microscope, SYN-like immunoreactivity appeared mainly as dots, mostly among glandular cells, and some were found distributed along blood vessels. SYN-like immunoreactive dots were found under the electron microscope to be localized in the nerve terminals or varicosities. Synapse-like contracts between the nerve terminals or varicosities and the glandular cells could be identified. The number of SYN-like immunoreactive nerve terminals or varicosities was then counted under the light microscope. There were as many as 12424.2 +/- 2941.5 and 20700.4 +/- 6128 (mean +/- SD) nerve terminals or varicosities per anterior pituitary of normal and adrenalectomized rats, respectively. The increase in number following adrenalectomy was statistically significant. It is concluded that nerve fibres may play an important role in the regulation of the anterior pituitary.     

Response of Substance P-Immunoreactive Nerve Fibres in the Anterior Pituitary to Plasma Oestrogen Levels in the Rat

The mammalian anterior pituitary has recently been shown to be innervated by substantial amounts of peptidergic nerve fibres. It has also been found that adrenalectomy increases the amount of calcitonin gene-related peptide-like immunoreactive nerve fibres in the anterior pituitary of the rat, and that stimulation of the nerve fibres in vitro can enhance release of ACTH. In the present study, the density of substance P-like immunoreactive nerve fibres in the anterior pituitary of the rat were examined in relation to variations in plasma oestrogen status. The amount of substance P-like immunoreactive nerve fibres increased significantly (P<0.001) after ovariectomy, and decreased significantly (P<0.01) under oestrogen replacement, in a dose-dependent manner. The results suggest the possibility of the direct neural involvement of oestrogen secretion in anterior pituitary regulation, which further substantiates the hypothesis of neural-humoral dual regulation of the mammalian anterior pituitary.