少突胶质系细胞的生物学特性及神经保护作用研究

发布时间：2018-08-24 16:16

【摘要】：少突胶质系细胞包括少突胶质细胞(oligodendrocyte OLGs)和少突胶质前体细胞(oligodendrocyte precursor cells OPCs)，是中枢神经系统尤其是白质内的重要细胞成分。此前较多研究表明，当中枢神经损伤时，少突胶质细胞的髓鞘成分对于轴突再生具有明显的抑制作用。但是，目前对于少突胶质系细胞在神经系统中发挥的营养与保护作用，尤其是对相对成熟神经元的保护作用，却研究甚少。近来的实验发现，体外培养的OPCs具有向神经干细胞(NSCs)逆向分化能力和无限增殖能力，而脑内的成年OPCs与神经元存在着突触联系现象，这提示少突胶质系细胞有可能在体内发挥着重要而复杂的功能。本课题从少突胶质系细胞的体外培养和体内移植入手，结合上丘逆行标记技术、视神经损伤技术和大鼠视网膜髓鞘形成模型，来研究少突胶质系细胞在体内外的发育、分化和营养因子表达情况，及其所具有的神经保护作用，以期进一步深入认识少突胶质系细胞与神经元之间的密切关系，并为中枢神经相关疾病的治疗提供新的思路。 为此，本研究采用改良的胶质细胞混合培养与差速贴壁方法获得大鼠OPCs，使用无血清培养基进行扩增、培养，用免疫组织化学和流式细胞技术对培养细胞的纯度进行鉴定，对少突胶质系细胞表达部分营养因子的情况进行检测；采用TUNEL、MTT等方法对少突胶质系细胞条件培养基对原代培养小脑颗粒神经元的保护作用进行检测；将OPCs移植入成年SD大鼠玻璃体内，利用上丘逆行荧光标记技术，观察眼内移植的OPCs对眶内视神经切断时的视网膜神经节细胞(RGCs)的保护作用及其持续时间；将OPCs或NSCs移植入新生和幼年SD大鼠玻璃体或视网膜内，观察不同时期视网膜内髓鞘形成与分布特点，分析髓鞘的超微结构，并观察眼内髓鞘形成对损伤神经节细胞的保护作用。主要结果及结论如下： 1．利用改良的混合胶质细胞原代培养方法，结合摇床振荡和差速贴壁，获得纯度大于93％的OPCs；扩增培养的OPCs可以表达多种标记物包括Nestin和GAP-43，因子撤除导致细胞自发向OLGs分化和成熟；短期的血清刺激使OPCs出现克隆样增殖现象；OPCs与OLGs可以在mRNA和蛋白水平表达BDNF和IGF-1；OPCs与OLGs的条件培养基能够促进原代培养的小脑颗粒神经元的存活。 2．大鼠玻璃体内移植的OPCs可在较长时间内存活，部分细胞变为多极形状；视神经切断后2周内，，OPCs移植组的RGCs存活数量大于对照组，表明少突胶质系细胞能够在体内发挥神经保护作用。 3．OPCs向新生大鼠玻璃体内移植后4周，多数视网膜内开始出现成束髓鞘，表明OPCs可在同种视网膜内向少突胶质细胞分化并成熟；髓鞘只分布于神经纤维层，提示视网膜神经纤维层具有促使髓鞘形成的作用；髓鞘束出现的比例、分布面积和形态变化与OPCs移植后大鼠的存活时间相关；纹状体NSCs也可以在视网膜内向OLGs分化并形成髓鞘。 4．OPCs向幼年大鼠视网膜内移植后7周，接近半数的视网膜内发现成束髓鞘，多分布于原移植象限内；透射电镜和免疫组织化学检测证实视网膜内无明显的RGCs退化现象及异位RGCs存在；形成的髓鞘具有正常的中枢神经髓鞘样特点，髓鞘化轴突的口径明显增大；视神经切断后10d内，分布于髓鞘形成扇形区域内的RGCs存活数量大于对照组，同时髓鞘束逐渐崩解消失，表明成熟的OLGs及其髓鞘有可能在体内发挥了神经保护作用。
[Abstract]:Oligodendrocyte oligodendrocyte (OLGs) and oligodendrocyte precursor cells OPCs are important cellular components in the central nervous system, especially in the white matter. Previous studies have shown that myelin sheath components of oligodendrocyte precursor cells play an important role in axonal regeneration in central nervous system injury. However, few studies have been done on the nutritional and protective effects of oligodendrocytes on the nervous system, especially on relatively mature neurons. Recent experiments have shown that OPCs cultured in vitro have the ability to differentiate into neural stem cells (NSCs) and proliferate indefinitely. The synaptic connection between adult OPCs and neurons suggests that oligodendrocytes may play an important and complex role in vivo. In this study, we started with the culture and transplantation of oligodendrocytes in vitro, combined with the retrograde labeling technique of superior colliculus, optic nerve injury technique and rat retinal myelin sheath formation model. To study the development and differentiation of oligodendrocyte in vitro and in vivo, the expression of nutritional factors and the neuroprotective effects of oligodendrocyte, so as to further understand the close relationship between oligodendrocyte and neurons, and to provide new ideas for the treatment of central nervous system related diseases.
In this study, rat OPCs were obtained by modified mixed culture of glial cells and differential adherence. The purity of cultured cells was identified by immunohistochemistry and flow cytometry. The expression of some nutrient factors in oligodendrocytes was detected by TUNEL. The protective effects of oligodendrocyte conditioned medium on primary cultured cerebellar granular neurons were examined by MTT, and OPCs were transplanted into the vitreous of adult SD rats to observe the protective effects of OPCs on retinal ganglion cells (RGCs) during intraocular optic nerve transection using retrograde labeling technique of superior colliculus. OPCs or NSCs were transplanted into the vitreous or retina of neonatal and juvenile SD rats to observe the formation and distribution of intraretinal myelin sheath, analyze the ultrastructure of myelin sheath, and observe the protective effect of intraocular myelin formation on injured ganglion cells.
1. OPCs with a purity of more than 93% were obtained by a modified mixed glial cell primary culture method combined with shaking table oscillation and differential adherence. The amplified OPCs could express a variety of markers including Nestin and GAP-43. Factor withdrawal led to spontaneous differentiation and maturation of cells into OLGs. Short-term serum stimulation resulted in clonal proliferation of OPCs. OPCs and OLGs can express BDNF and IGF-1 at mRNA and protein levels, and conditioned medium of OPCs and OLGs can promote the survival of primary cultured cerebellar granular neurons.
2. OPCs transplanted into rat vitreous can survive for a long time, and some cells become multipolar. Within 2 weeks after optic nerve transection, the number of RGCs in OPCs transplanted group was larger than that in control group, indicating that oligodendrocytes can play a neuroprotective role in vivo.
3. Four weeks after the transplantation of OPCs into the vitreous of neonatal rats, bundles of myelin appeared in most retinas, indicating that OPCs could differentiate and mature into oligodendrocytes in the same retina; myelin sheath was only distributed in the nerve fiber layer, suggesting that the nerve fiber layer of retina could promote the formation of myelin sheath bundles, and the distribution of myelin bundles. The changes of volume and morphology were related to the survival time of OPCs transplanted rats, and striatum NSCs could also differentiate into OLGs and form myelin sheath in retina.
4. Nearly half of the retinal bundles of myelin sheaths were found in the original transplantation quadrant 7 weeks after intraretinal transplantation of OPCs to juvenile rats. Transmission electron microscopy and immunohistochemistry showed that there were no obvious degeneration of RGCs and heterotopic RGCs in the retina. The myelin sheaths formed had normal myelin-like characteristics of central nervous system and myelin sheath. Within 10 days after optic nerve transection, the number of RGCs distributed in the sector of myelin sheath formation was larger than that in the control group, and the myelin bundle gradually disintegrated, suggesting that mature OLGs and their myelin sheath might play a neuroprotective role in vivo.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329

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