Cx43介导的星形与少突胶质细胞间连接调控少突胶质前体细胞发育分化的作用研究

发布时间：2018-03-02 12:40

本文选题：少突胶质细胞　切入点：少突胶质前体细胞　出处：《第三军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：少突胶质细胞(oligodendrocyte，OL)是中枢神经系统的髓鞘形成细胞，髓鞘发育的完整性保证了神经系统信息准确有效的传递。OL起源于室周区及室下区的神经上皮细胞，迁移并发育为少突胶质前体细胞(oligodendroglia precursor cell，OPC)，表达PDGFαR、NG2等标记物；而后OPC进一步发育分化为CNPase、O4阳性的幼稚OL(imamature oligodendrocyte)，，并最终发育为MBP、PLP阳性的成熟OL(matureoligodendrocyte)。了解OL分化调控机制对OL成熟分化、髓鞘修复再生及功能维持有重要意义，也是神经科学研究中一个难点和热点课题。作为OL生存微环境中的主体，AST对维持OL谱系发育、分化和功能稳定具有重要的作用也是不争的事实，而通过细胞-细胞的直接接触/相互作用，AST是如何调控OL谱系的发育和髓鞘的形成，其具体机制和途径目前尚不清楚。胶质细胞间的相互作用区别于神经元活动的显著特点之一就是细胞高表达各种连接蛋白，并以此形成缝隙连接(Gap junction,GJ)将细胞相互连接起来，形成胶质网络，而钙波在其信号传导中发挥了重要的作用。研究发现OL与AST通过缝隙连接相连并形成功能合胞体，钙波可通过O/A缝隙连接在这两种细胞间传播。作为细胞内重要的第二信使，钙参与了OL谱系发育分化的整个过程，但其具体调控途径仍不清楚。过去对OL谱系钙信号的研究多集中于胞膜上的电压依赖钙通道、G-蛋白偶联的代谢型钙通道等，研究证实增加细胞内钙水平可促进OL的发育分化。内质网是OL细胞内钙储存库，雷喏啶碱受体(ryanodinereceptor，RyR)钙通道是近年来发现位于内质网上重要的钙通道之一，该通道开放可迅速释放内质网钙提高细胞内钙的水平。而迄今未见有报道研究RyR通道在OPC发育过程中的作用。在本研究中我们先建立OPC-AST共培养体系，通过将Cx43沉默后观察其是否影响OPC的发育分化，再进一步观察RyR钙信号对OPC分化的影响。本研究分为两部分：第一部分：星形胶质细胞连接蛋白Cx43对少突胶质前体细胞分化的影响体外培养OPC，利用免疫荧光染色比较OPC-AST共培养后与单独培养OPC的分化差异，阐明细胞间接触作用的重要性。构建Cx43干扰质粒，运用免疫荧光及Westernblot方法比较Cx43干扰后共培养的OPC发育至成熟OL各阶段特定蛋白的表达情况。主要结果如下： 1、OPC与AST共培养后，AST促进OPC增殖并抑制其分化，且AST对OPC的作用更依赖于细胞的接触。 2、成功构建Cx43干扰质粒，转染Cx43干扰质粒后AST Cx43蛋白表达明显下降(P0.05)，AST谷氨酸释放明显减少(P0.05)。 3、OPC与转染Cx43干扰质粒的AST共培养后，与转染对照质粒组相比，OPC早期及中期标记物NG2、CNPase无明显变化，而晚期MBP蛋白表达明显下降(P0.05)。上述结果说明AST对OPC分化的影响更多地是通过细胞间的直接接触发挥作用；Cx43与OL谱系连接蛋白形成的O/A缝隙连接可能对OPC晚期分化起重要作用。第二部分：RyR介导的钙信号对少突胶质前体细胞分化的影响应用RT-PCR检测了RyR mRNA在OL谱系中的分布，同时用钙成像的方法观察OPC不同发育阶段的钙波特性。将RyR阻断剂加入培养的OPC,利用免疫荧光染色及Western blot等技术观察加入阻断剂后对OL谱系发育各个时期特定蛋白表达的影响。此外，加入RyR通道激动剂咖啡因进一步验证RyR通道对OPC发育分化的作用。主要结果如下： 1、RyR钙通道的三种亚型mRNA均在大鼠脑内表达，而仅RyR3mRNA在体外培养的OL谱系检测到，且随着细胞成熟即MBP表达增加，其表达反而下降。 2、将OL谱系按不同发育时期形态分为OPC、Immature OL、Mature OL三个阶段。用咖啡因刺激不同发育阶段的OPC,观察到RyR通道介导的钙释放在OPC不同发育阶段存在差异，早期的OPC为较活跃的尖波，而分化晚期则为低平波。 3、用高浓度Ryanodine阻断RyR通道后OPC分化明显延迟，MBP表达明显减少(P0.01)；而在培养OPC中加入RyR通道阻滞剂并同时加入咖啡因后，MBP表达与对照无明显差异(p0.05)。以上结果表明RyR介导的钙信号在OPC分化过程中起重要作用。
[Abstract]:Oligodendrocytes (oligodendrocyte, OL) is the myelin forming cells of the CNS, integrity of myelin development that neural epithelial cells of neural system is accurate and effective transmission of.OL originated in the periventricular area and the subventricular zone, migration and development of oligodendrocyte precursor cells (oligodendroglia precursor cell, OPC) expression of PDGF, R, NG2 and OPC markers; further development and differentiation of CNPase, O4 positive naive OL (imamature oligodendrocyte), and eventually developed into MBP, PLP positive mature OL (matureoligodendrocyte). The solution of OL differentiation mechanism of OL differentiation, myelin regeneration and functional recovery has important significance also, neuroscience research a hot and difficult topic.
As the main body of OL survival in the micro environment, AST to maintain OL lineage development, differentiation and function of stability has an important role is also an indisputable fact, and through cell to cell contact / direct interaction, AST is formed to control the OL lineage development and myelin, the specific mechanism and the way it is not sure. One of the outstanding characteristics of high expression in neuronal activity of the interaction between glial cells is cell junction proteins, and thus the formation of gap junction (Gap junction, GJ) were connected to each other, forming glial network, while calcium wave in signal transduction plays an important role.
Study found that OL and AST connected by gap junctions and the formation of a functional syncytium, calcium wave through the O/A gap junction communication in these two kinds of cells. As an important intracellular second messenger, calcium in the process of OL lineage differentiation, but its specific regulatory pathways remains unclear. The past research of voltage dependent calcium channel on the spectrum of OL calcium signals are more concentrated in the cell membrane, G- protein coupled metabolic type calcium channel, studies have confirmed that increased intracellular calcium levels can promote the differentiation and development of OL. The endoplasmic reticulum is OL intracellular calcium stores, Lei Nuo Ding (ryanodinereceptor, RyR) ryanodine receptor calcium channel is found in recent years is located in one of the important endoplasmic reticulum calcium channel, the channel can open the rapid release of endoplasmic reticulum calcium increase intracellular calcium levels. But so far no report on role of RyR channels in the OPC development process. In this study, we first establish In OPC-AST co culture system, the effect of RyR calcium signal on the differentiation of OPC was further observed by observing whether or not it affected the development and differentiation of OPC after Cx43 silencing.
This study is divided into two parts:
The first part: the effect of astrocyte connexin Cx43 on the differentiation of oligodendrocyte precursor cells
OPC were cultured in vitro, differentiation of co cultured and cultured alone OPC OPC-AST by immunofluorescence, importance cell-cell contacts. Cx43 plasmid was constructed and the expression by immunofluorescence and Westernblot method after Cx43 interference of co cultured OPC development and maturation of specific protein OL in each stage.
The main results are as follows:
1, after co culture of OPC and AST, AST promotes OPC proliferation and inhibits its differentiation, and the effect of AST on OPC is more dependent on cell contact.
2, Cx43 interference plasmids were successfully constructed. The expression of AST Cx43 protein decreased significantly (P0.05), and the release of AST glutamic acid decreased significantly (P0.05) after transfection of Cx43 interference plasmid.
3, OPC co cultured with AST transfected with Cx43 interference plasmid, compared with the transfected control plasmid group, there was no significant change in OPC and NG2 markers, but the expression of late MBP protein decreased significantly (P0.05).
The above results indicate that the effect of AST on OPC differentiation is more likely to play a role through direct contact between cells. The gap junctions between Cx43 and OL lineage proteins may play an important role in the late differentiation of OPC.
The second part: the effect of RyR mediated calcium signal on the differentiation of oligodendrocyte precursor cells
RT-PCR was used to detect the distribution of RyR mRNA in the OL lineage, while observing the calcium wave characteristics of OPC in different developmental stages by calcium imaging. The RyR inhibitor is added into the culture of OPC, to observe the blockade of OL lineage specific protein expression influence development in each period by using immunofluorescence staining and Western blot technology. In addition, adding RyR channel agonist caffeine to validate the RyR channel on OPC development and differentiation.
The main results are as follows:
1, the three subtypes of RyR calcium channel mRNA were expressed in rat brain, while only RyR3mRNA was detected in OL pedigree in vitro, and the expression decreased with the increase of cell maturation, namely the expression of MBP.
2, according to the different developmental stages of OL pedigree were divided into OPC, Immature OL, Mature OL three stages. With caffeine to stimulate the different development stages of OPC, observed that RyR channel mediated calcium release are different in different developmental stages OPC, early OPC wave is active, and the differentiation is advanced low and flat wave.
3, after blocking the RyR channel with high concentration of Ryanodine, the OPC differentiation was significantly delayed, and the MBP expression was significantly reduced (P0.01). However, after adding RyR channel blocker and adding caffeine in OPC, there was no significant difference between MBP expression and control (P0.05).
These results suggest that RyR mediated calcium signals play an important role in the process of OPC differentiation.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【共引文献】