發(fā)布時(shí)間：2018-03-02 12:40

  本文選題：少突膠質(zhì)細(xì)胞　切入點(diǎn)：少突膠質(zhì)前體細(xì)胞　出處：《第三軍醫(yī)大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：少突膠質(zhì)細(xì)胞(oligodendrocyte，OL)是中樞神經(jīng)系統(tǒng)的髓鞘形成細(xì)胞，髓鞘發(fā)育的完整性保證了神經(jīng)系統(tǒng)信息準(zhǔn)確有效的傳遞。OL起源于室周區(qū)及室下區(qū)的神經(jīng)上皮細(xì)胞，遷移并發(fā)育為少突膠質(zhì)前體細(xì)胞(oligodendroglia precursor cell，OPC)，表達(dá)PDGFαR、NG2等標(biāo)記物；而后OPC進(jìn)一步發(fā)育分化為CNPase、O4陽性的幼稚OL(imamature oligodendrocyte)，，并最終發(fā)育為MBP、PLP陽性的成熟OL(matureoligodendrocyte)。了解OL分化調(diào)控機(jī)制對(duì)OL成熟分化、髓鞘修復(fù)再生及功能維持有重要意義，也是神經(jīng)科學(xué)研究中一個(gè)難點(diǎn)和熱點(diǎn)課題。 作為OL生存微環(huán)境中的主體，AST對(duì)維持OL譜系發(fā)育、分化和功能穩(wěn)定具有重要的作用也是不爭(zhēng)的事實(shí)，而通過細(xì)胞-細(xì)胞的直接接觸/相互作用，AST是如何調(diào)控OL譜系的發(fā)育和髓鞘的形成，其具體機(jī)制和途徑目前尚不清楚。膠質(zhì)細(xì)胞間的相互作用區(qū)別于神經(jīng)元活動(dòng)的顯著特點(diǎn)之一就是細(xì)胞高表達(dá)各種連接蛋白，并以此形成縫隙連接(Gap junction,GJ)將細(xì)胞相互連接起來，形成膠質(zhì)網(wǎng)絡(luò)，而鈣波在其信號(hào)傳導(dǎo)中發(fā)揮了重要的作用。 研究發(fā)現(xiàn)OL與AST通過縫隙連接相連并形成功能合胞體，鈣波可通過O/A縫隙連接在這兩種細(xì)胞間傳播。作為細(xì)胞內(nèi)重要的第二信使，鈣參與了OL譜系發(fā)育分化的整個(gè)過程，但其具體調(diào)控途徑仍不清楚。過去對(duì)OL譜系鈣信號(hào)的研究多集中于胞膜上的電壓依賴鈣通道、G-蛋白偶聯(lián)的代謝型鈣通道等，研究證實(shí)增加細(xì)胞內(nèi)鈣水平可促進(jìn)OL的發(fā)育分化。內(nèi)質(zhì)網(wǎng)是OL細(xì)胞內(nèi)鈣儲(chǔ)存庫，雷喏啶堿受體(ryanodinereceptor，RyR)鈣通道是近年來發(fā)現(xiàn)位于內(nèi)質(zhì)網(wǎng)上重要的鈣通道之一，該通道開放可迅速釋放內(nèi)質(zhì)網(wǎng)鈣提高細(xì)胞內(nèi)鈣的水平。而迄今未見有報(bào)道研究RyR通道在OPC發(fā)育過程中的作用。在本研究中我們先建立OPC-AST共培養(yǎng)體系，通過將Cx43沉默后觀察其是否影響OPC的發(fā)育分化，再進(jìn)一步觀察RyR鈣信號(hào)對(duì)OPC分化的影響。 本研究分為兩部分： 第一部分：星形膠質(zhì)細(xì)胞連接蛋白Cx43對(duì)少突膠質(zhì)前體細(xì)胞分化的影響 體外培養(yǎng)OPC，利用免疫熒光染色比較OPC-AST共培養(yǎng)后與單獨(dú)培養(yǎng)OPC的分化差異，闡明細(xì)胞間接觸作用的重要性。構(gòu)建Cx43干擾質(zhì)粒，運(yùn)用免疫熒光及Westernblot方法比較Cx43干擾后共培養(yǎng)的OPC發(fā)育至成熟OL各階段特定蛋白的表達(dá)情況。 主要結(jié)果如下： 1、OPC與AST共培養(yǎng)后，AST促進(jìn)OPC增殖并抑制其分化，且AST對(duì)OPC的作用更依賴于細(xì)胞的接觸。 2、成功構(gòu)建Cx43干擾質(zhì)粒，轉(zhuǎn)染Cx43干擾質(zhì)粒后AST Cx43蛋白表達(dá)明顯下降(P0.05)，AST谷氨酸釋放明顯減少(P0.05)。 3、OPC與轉(zhuǎn)染Cx43干擾質(zhì)粒的AST共培養(yǎng)后，與轉(zhuǎn)染對(duì)照質(zhì)粒組相比，OPC早期及中期標(biāo)記物NG2、CNPase無明顯變化，而晚期MBP蛋白表達(dá)明顯下降(P0.05)。 上述結(jié)果說明AST對(duì)OPC分化的影響更多地是通過細(xì)胞間的直接接觸發(fā)揮作用；Cx43與OL譜系連接蛋白形成的O/A縫隙連接可能對(duì)OPC晚期分化起重要作用。 第二部分：RyR介導(dǎo)的鈣信號(hào)對(duì)少突膠質(zhì)前體細(xì)胞分化的影響 應(yīng)用RT-PCR檢測(cè)了RyR mRNA在OL譜系中的分布，同時(shí)用鈣成像的方法觀察OPC不同發(fā)育階段的鈣波特性。將RyR阻斷劑加入培養(yǎng)的OPC,利用免疫熒光染色及Western blot等技術(shù)觀察加入阻斷劑后對(duì)OL譜系發(fā)育各個(gè)時(shí)期特定蛋白表達(dá)的影響。此外，加入RyR通道激動(dòng)劑咖啡因進(jìn)一步驗(yàn)證RyR通道對(duì)OPC發(fā)育分化的作用。 主要結(jié)果如下： 1、RyR鈣通道的三種亞型mRNA均在大鼠腦內(nèi)表達(dá)，而僅RyR3mRNA在體外培養(yǎng)的OL譜系檢測(cè)到，且隨著細(xì)胞成熟即MBP表達(dá)增加，其表達(dá)反而下降。 2、將OL譜系按不同發(fā)育時(shí)期形態(tài)分為OPC、Immature OL、Mature OL三個(gè)階段。用咖啡因刺激不同發(fā)育階段的OPC,觀察到RyR通道介導(dǎo)的鈣釋放在OPC不同發(fā)育階段存在差異，早期的OPC為較活躍的尖波，而分化晚期則為低平波。 3、用高濃度Ryanodine阻斷RyR通道后OPC分化明顯延遲，MBP表達(dá)明顯減少(P0.01)；而在培養(yǎng)OPC中加入RyR通道阻滯劑并同時(shí)加入咖啡因后，MBP表達(dá)與對(duì)照無明顯差異(p0.05)。 以上結(jié)果表明RyR介導(dǎo)的鈣信號(hào)在OPC分化過程中起重要作用。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張立志;馮俊強(qiáng);陳晶;宋宏杉;陳業(yè)鵬;;難治性癲癇患者致癇灶中CX43的表達(dá)[J];中國(guó)實(shí)用醫(yī)藥;2012年10期

相關(guān)碩士學(xué)位論文 前2條

1 張立志;難治性癲癇患者臨床及大腦皮層CX32、CX43的表達(dá)[D];吉林大學(xué);2011年

2 沙晶;抑制AMPK活性對(duì)小鼠腦缺血/再灌注損傷后星型膠質(zhì)細(xì)胞形態(tài)及功能的影響[D];新疆醫(yī)科大學(xué);2012年

本文編號(hào)：1556563