摘要
传统观点认为胶质细胞是一种支持细胞，起支持、营养和保护神经元的作用。近20多年来随着研究手段的进步，对神经胶质细胞的研究不断深入，越来越多的实验显示，胶质细胞与神经元之间存在双向通讯，并能主动调控神经元的活动，参与多种信号的整合。现已发现星形胶质细胞在对低渗刺激的反应中起关键性的作用：星形胶质细胞感受渗透压的变化，通过大量释放牛磺酸(taurine)，作用于神经元上的甘氨酸(glycine)受体，从而抑制神经元释放血管加压素(vasopressin，VP)。但是，关于胶质细胞在高渗刺激状况下，是否参与渗透压的调节及其与神经元的关系的研究尚未见报道。
因此在以往研究的基础上，本课题通过观察视上核神经元和胶质细胞(星形胶质细胞和小胶质细胞)对高渗刺激的反应及其相互关系，探讨视上核胶质细胞在高渗刺激后调节过程中的作用。
应用免疫组织化学方法光镜下观察高渗刺激后，大鼠视上核胶质细胞(星形胶质细胞和小胶质细胞)受体(NMDAR2)、信号分子、骨架蛋白及缝隙连接蛋白的表达的时空变化；应用放免测定检测高渗刺激前后血浆中VP含量。
应用免疫电镜观察高渗刺激后被激活的视上核星形胶质细胞和神经元之间的超微结构的变化。
侧脑室注射缝隙连接阻断剂甘珀酸(carbenoxolone，CBX)后，再给予高
第四军医大学博士学位论文
渗刺激，观察视上核胶质细胞和神经元免疫组化反应的改变，并检测血浆VP
含量的变化。
应用 western blot技术检测高渗刺激后视上核缝隙连接蛋白（connexin，Cx）
32和 43含量。
原代培养神经元和星形胶质细胞，给予高渗刺激，免疫荧光化学法检测缝
隙连接蛋白（CX32和 CX43）在神经元和星形胶质细胞上的表达；应用 FllJO－3八M
荧光探针，激光扫描共聚焦显微镜检测培养的星形胶质细胞和小胶质细胞内
Ca‘”瞬间 时空变化。
通过实验得到主要研究结果包括：
（l）高渗刺激后，大鼠 SON内出现 PLC、NMDARZ、FOS、CX43、GFAP
样兔疫反应阳性星形胶质细胞，PLC。NMDARZ、F。S、CX32样免疫反应阳性
的神经元和 OX42样免疫反应阳性的小胶质细胞，反应性星形胶质细胞和小胶
质细胞的出现要早于反应性神经元。反应性星形胶质细胞和小胶质细胞与反应
性神经元之间有密切的时空关系。
（2）免疫电镜观察，SON内星形胶质细胞与神经元接触部位可以观察到
膜增厚的结构——电子致密区（EDAS），在神经元一侧可见Cx32阳性金颗粒，
而在星形胶质细胞一侧可见CX43阳性物质分布。高渗刺激后，EDAS的数量明
显增多。
门）高渗刺激后扔dn，血浆中＊P含量明显升高，若事先经侧脑室注入
CBX后再给高渗刺激，血浆中VP含量不升高，视上核内星形胶质细胞GFAP
阳性反应与单纯高渗组无差别，而FoS阳性神经元明显减少。
（4）Western blot结果显示高渗刺激后，SON内 Cx43和 Cx32表达增加，
并从胞浆向胞膜转移。而对培养的神经元和星形胶质细胞施予高渗刺激，星形
胶质细胞膜上CX43样阳性颗粒迅速增多，神经元CX32表达明显增强。
门）高渗刺激后，培养的星形胶质细胞和小胶质细胞胞内游离 Cay水平迅
4
第四军医大学博士学位论文
速出现先升高随之降低的变化，星形胶质细胞的 CaV水平的下降比小胶质细胞
缓慢。
根据实验结果，得出以下结论：
l、高渗刺激可以引起大鼠SON内的星形胶质细胞和小胶质细胞快速的反
应，星形胶质细胞的反应要早于神经元。
2、反应性星形胶质细胞和小胶质细胞与反应性神经兀之问有密切关系。星
形胶质细胞可能通过类似缝隙连接的结构－EDAS的信息通道主动调节神经元的
反应，共同参与对高渗刺激的调节。
Glias are traditionally thought to assume a structural, trophic, and protective role to neurons. With the development of research techniques and the further study on glias, mounting evidences have suggested the bidirectional signaling between neurons and glias. Glias may be an integral part of the communication network within the central nervous system and regulate neuronal activity. It is known that astrocytes take a key role in the response to hypoosmotic stimulation: astrocytes can perceive the change of osmotic pressure and release taurine which then activates glycine receptors on the neurons, and inhibits the release of VP from the neurons in SON. While it is not clear that whether SON astrocytes respond to hyperosmotic stimuli and what's the relationship between them and neurons?
Based on the previous researches, the present study investigated the response of SON neurons and glias (astrocytes and microglias) to hyperosmotic stimulation and their relationship to find out the roles of glias in regulating hyperosmotic stimulation.
Immunohistochemistry method was used to observe the temporal and spatial
expression of NMDAR2, signal molecules, skeleton proteins and connexins in SON neurons and glias (astrocytes and microglias). Radioimmunoassay was used to detect vasopressin (VP) content in plasma before and after hyperosmotic stimulation.
Ultrastructure between activated SON astrocytes and neurons was observed by double immune-electron-microscopic staining method.
We injected carbennoxolone, a gap junction blocker, into the lateral ventricle, which was followed by hyperosmotic stimulation, the immunohistochemical staining of neurons and astrocytes and VP content in plasm were studied.
Western blot was performed to detect the content of Cx43 and Cx32 in SON following hyperosmotic stimulation.
Treat primary cultured neurons and astrocytes with hyperosmotic stimulation. Immunofluorescence was used to study the expression of Cx43 and Cx32 in SON neurons and astrocytes. By means of Fluo-3/AM and laser scanning confocal microscopy, we also observed intracellular calcium transient in astrocytes and microglias.
The main results included:
1, After hyperosmotic stimulation, there emerged in rat SON the activated astrocytes which showed NMDAR2, Fos/GFAP and Cx43 positive immuno-histochemical staining, the activated neurons which showed PLC, NMDAR2, Fos and Cx32 staining, and also OX42 positive stained microglias. The activated astrocytes and microglias emerged earlier than the activated neurons. These activated cells formed intimate temporal and spatial relationship.
2, The electron dense area (EDA) consisting of the astrocytic process on one side and the neuron (dendrite) on the other side was observed in immune-electron-microscopic staining studies, and the EDA was characterized with
double layers thickening and dark staining cytomembranes with a narrow cleft between them. Cx32-LI appeared on the neuron side or and Cx43-LI on the astrocyte side respectively. This structure increased obviously following hyperosmotic stimulation.
3, VP content in plasma increased significantly 45min after hyperosmotic stimulation. When pre-injected carbennoxolone, a gap junction blocker, into the lateral ventricle, followed by hyperosmotic stimulation, VP content remained the base line. The expression of GFAP-LI astrocytes showed no difference, while that of Fos-LI neurons decreased significantly.
4, Western blot showed increase of Cx43 and Cx32 in SON and also a translocation of them from plasm to membrane. After hyperosmotic treatment, Cx43-LI granules increased quickly on the plasm of the cultured astrocytes and Cx32-LI increased apparently in the cultured neurons.
5, After being treated with hyperosmotic stimulation, the cultured astrocytes and microglias showed a fast increase of intracellular calcium concentration followed by a decrease. The [Ca2+]j of astrocytes decreased slower than that of microglia
引文
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