【作者】 郑华；

【导师】 周国民； 钟翠平； 左伋；

【作者基本信息】 复旦大学 ， 人体解剖与组织胚胎学， 2006， 博士

【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。更多还原

【Abstract】 Oligodendrocyte lineage cells including both oligodendrocytes and oligodendrocyte precursor cells, are the important cellular parts of CNS, especially in the white matter. A lot of reports confirmed that the myelin of oligodendrocyte could inhibit the regeneration of injured axons in the CNS, but less attention on the trophic and protective roles of oligodendrocyte lineage cells in the CNS, especially in the mature CNS. On the other hand, recent experiments indicated perinatal OPCs have the ability to proliferate indefinitely in appropriate culture conditions, reprogrammed to neural stem cells, and the adult OPCs in the hippocampus make synaptic junctions with the axons terminals of neurons. These phenomena indicated that the oligodendrocyte lineage cells maybe act as a more important and complicated role in vivo. Taking advantage of ’cell culture and transplantation, retrograde fluorescent labeling the superior colliculus, optic nerve transection and myelinformation in the retina, we studied the development, differentiation and neuroprotective effects of oligodendrocyte lineage cells in vitro and in vivo, to further clarify the relationship between oligodendrocyte lineage cells and neurons, and seek for a new approach for the therapy of correlated diseases.We introduced improved primary mixed glial culture and different-attachment method to isolate and purify the OPCs, the cells were proliferated in serum-free medium, flow cytometry and immunohischemistry methods were employed to estimate the purity of cultured OPCs. Their abilities of differentiation and expression of trophic factors were identified by RT-PCR and immunostaining. Several methods including TUNEL and MTT were adopted to estimate the protective effects of conditioned culture medium from oligodendrocyte lineage cells on the primary cultured cerebellar granule neurons. Intravitreal transplant of OPCs, combined with retrograde fluorescent labeling the superior colliculus (SC) and intraorbital optic nerve transection, were used to investigate the protective effects of OPCs on the axotomized RGCs in vivo. Intravitreal transplantof OPCs or NSCs on the newborn rats, and retinal transplant of OPCs on the young rats were performed, to observe the myelin formation in the retina at different stages after cellular transplantation. Optic nerve transection was carried out on some rats with myelinated retinae, to study the influence of myelination on the injuried RGCs. The results and conclusions are as follows:1. Using the modified mixed glial culture and different-attachment method, we harvested OPCs with purity more than 93%. The cultured OPCs could express Nestin and Gap-43. OPCs automatically differentiated into mature oligodendrocytes after PDGF withdrawn. Short-term exposed to serum could stimulated the proliferation of OPCs. Both OPCs and OLGs could express BDNF and IGF-1 at mRNA and protein levels. The conditioned culture medium promoted the survival of primary cultured cerebellar granule neurons.2. The intravitreal transplanted OPCs could survive for a long time, the morphology of partial cells became multi-polar. The density of survival RGCs in the OPCs transplantation group is higher than in the control groups within 2 weeks after optic nerve transection, indicating that the oligodendrocyte lineage cells have the ability to save the injured neurons.3. 4 weeks after OPCs transplanted into the vitreous space of newborn rats, bundles of myelinated axons were observable, indicating that OPCs could differentiate and mature in the homological retina. The myelination limited within the NFL but the distributed areas of myelinated bundles in the retina is expanded with prolonged survival time after cellular transplantation. Striatal neural precursor cells could differentiate into oligodendrocytes and produce myelin after transplanted into the vitreous space.4. Bundles of myelinated axons were found in nearly half of the retinae of young rats received retinal OPCs transplantation 7 weeks later. Most of the bundles distributed within the superonasal quarter of retina where transplantation were performed. Electron microscope and immunohistochemistry inspection confirmed no obvious RGCs degeneration within the retina. The myelin sheaths revealed normal CNS morphology and the thickness were diverse. The myelinated axons had larger calibers comparing with unmyelinated axons. The amount of survival RGCs within the myelinated sectors were higher than the control groups within 10 daysafter optic transection. At the same time, the myelinated axon bundles suffered gradual disintegration. The results indicated that mature oligodendrocytes with their myelin sheats maybe play protective roles in vivo.更多还原