【摘要】：少突膠質(zhì)系細(xì)胞包括少突膠質(zhì)細(xì)胞(oligodendrocyte OLGs)和少突膠質(zhì)前體細(xì)胞(oligodendrocyte precursor cells OPCs)，是中樞神經(jīng)系統(tǒng)尤其是白質(zhì)內(nèi)的重要細(xì)胞成分。此前較多研究表明，當(dāng)中樞神經(jīng)損傷時，少突膠質(zhì)細(xì)胞的髓鞘成分對于軸突再生具有明顯的抑制作用。但是，目前對于少突膠質(zhì)系細(xì)胞在神經(jīng)系統(tǒng)中發(fā)揮的營養(yǎng)與保護作用，尤其是對相對成熟神經(jīng)元的保護作用，卻研究甚少。近來的實驗發(fā)現(xiàn)，體外培養(yǎng)的OPCs具有向神經(jīng)干細(xì)胞(NSCs)逆向分化能力和無限增殖能力，而腦內(nèi)的成年OPCs與神經(jīng)元存在著突觸聯(lián)系現(xiàn)象，這提示少突膠質(zhì)系細(xì)胞有可能在體內(nèi)發(fā)揮著重要而復(fù)雜的功能。本課題從少突膠質(zhì)系細(xì)胞的體外培養(yǎng)和體內(nèi)移植入手，結(jié)合上丘逆行標(biāo)記技術(shù)、視神經(jīng)損傷技術(shù)和大鼠視網(wǎng)膜髓鞘形成模型，來研究少突膠質(zhì)系細(xì)胞在體內(nèi)外的發(fā)育、分化和營養(yǎng)因子表達(dá)情況，及其所具有的神經(jīng)保護作用，以期進一步深入認(rèn)識少突膠質(zhì)系細(xì)胞與神經(jīng)元之間的密切關(guān)系，并為中樞神經(jīng)相關(guān)疾病的治療提供新的思路。 為此，本研究采用改良的膠質(zhì)細(xì)胞混合培養(yǎng)與差速貼壁方法獲得大鼠OPCs，使用無血清培養(yǎng)基進行擴增、培養(yǎng)，用免疫組織化學(xué)和流式細(xì)胞技術(shù)對培養(yǎng)細(xì)胞的純度進行鑒定，對少突膠質(zhì)系細(xì)胞表達(dá)部分營養(yǎng)因子的情況進行檢測；采用TUNEL、MTT等方法對少突膠質(zhì)系細(xì)胞條件培養(yǎng)基對原代培養(yǎng)小腦顆粒神經(jīng)元的保護作用進行檢測；將OPCs移植入成年SD大鼠玻璃體內(nèi)，利用上丘逆行熒光標(biāo)記技術(shù)，觀察眼內(nèi)移植的OPCs對眶內(nèi)視神經(jīng)切斷時的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(RGCs)的保護作用及其持續(xù)時間；將OPCs或NSCs移植入新生和幼年SD大鼠玻璃體或視網(wǎng)膜內(nèi)，觀察不同時期視網(wǎng)膜內(nèi)髓鞘形成與分布特點，分析髓鞘的超微結(jié)構(gòu)，并觀察眼內(nèi)髓鞘形成對損傷神經(jīng)節(jié)細(xì)胞的保護作用。主要結(jié)果及結(jié)論如下： 1．利用改良的混合膠質(zhì)細(xì)胞原代培養(yǎng)方法，結(jié)合搖床振蕩和差速貼壁，獲得純度大于93％的OPCs；擴增培養(yǎng)的OPCs可以表達(dá)多種標(biāo)記物包括Nestin和GAP-43，因子撤除導(dǎo)致細(xì)胞自發(fā)向OLGs分化和成熟；短期的血清刺激使OPCs出現(xiàn)克隆樣增殖現(xiàn)象；OPCs與OLGs可以在mRNA和蛋白水平表達(dá)BDNF和IGF-1；OPCs與OLGs的條件培養(yǎng)基能夠促進原代培養(yǎng)的小腦顆粒神經(jīng)元的存活。 2．大鼠玻璃體內(nèi)移植的OPCs可在較長時間內(nèi)存活，部分細(xì)胞變?yōu)槎鄻O形狀；視神經(jīng)切斷后2周內(nèi)，，OPCs移植組的RGCs存活數(shù)量大于對照組，表明少突膠質(zhì)系細(xì)胞能夠在體內(nèi)發(fā)揮神經(jīng)保護作用。 3．OPCs向新生大鼠玻璃體內(nèi)移植后4周，多數(shù)視網(wǎng)膜內(nèi)開始出現(xiàn)成束髓鞘，表明OPCs可在同種視網(wǎng)膜內(nèi)向少突膠質(zhì)細(xì)胞分化并成熟；髓鞘只分布于神經(jīng)纖維層，提示視網(wǎng)膜神經(jīng)纖維層具有促使髓鞘形成的作用；髓鞘束出現(xiàn)的比例、分布面積和形態(tài)變化與OPCs移植后大鼠的存活時間相關(guān)；紋狀體NSCs也可以在視網(wǎng)膜內(nèi)向OLGs分化并形成髓鞘。 4．OPCs向幼年大鼠視網(wǎng)膜內(nèi)移植后7周，接近半數(shù)的視網(wǎng)膜內(nèi)發(fā)現(xiàn)成束髓鞘，多分布于原移植象限內(nèi)；透射電鏡和免疫組織化學(xué)檢測證實視網(wǎng)膜內(nèi)無明顯的RGCs退化現(xiàn)象及異位RGCs存在；形成的髓鞘具有正常的中樞神經(jīng)髓鞘樣特點，髓鞘化軸突的口徑明顯增大；視神經(jīng)切斷后10d內(nèi)，分布于髓鞘形成扇形區(qū)域內(nèi)的RGCs存活數(shù)量大于對照組，同時髓鞘束逐漸崩解消失，表明成熟的OLGs及其髓鞘有可能在體內(nèi)發(fā)揮了神經(jīng)保護作用。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R329

【共引文獻(xiàn)】

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本文編號：2201350