【作者】 唐军；

【导师】 姚裕家；

【作者基本信息】 四川大学 ， 儿科学， 2007， 博士

【摘要】 目的：获取高纯度的少突胶质前体细胞系，并作鉴定，为进一步研究作细胞准备。方法：出生2日龄的SD大鼠，无菌条件下断头取脑，剔除脑膜及血管。根据星形胶质细胞和少突胶质前体细胞系生长的时间差异，细胞的粘附特性不同，利用振荡分离纯化法获得纯化的大鼠少突胶质前体细胞，再用添加了N2、PDGF、bFGF的无血清培养基传代培养。分别用少突胶质前体细胞系不同发育阶段的特异性抗体：A285、O4、O1，采用免疫荧光法对表面抗原进行细胞鉴定。结果：获得纯度95％以上的未成熟SD大鼠少突胶质前体细胞，少突胶质祖细胞A285、O4抗体阳性；未成熟少突胶质细胞O4、O1阳性。结论：通过振荡分离纯化法及结合少突胶质细胞定向培养基是培养少突胶质前体细胞的可靠方法；N2、PDGF、bFGF的添加可以显著提高细胞的产量，并使细胞保持在未成熟阶段。目的：建立未成熟大鼠少突胶质前体系细胞缺氧缺糖损伤模型，并观察缺氧不同时间点细胞的变化。方法：体外分离纯化培养未成熟SD大鼠少突胶质前体细胞系（OLPs）并鉴定（同第一部分）。缺氧缺糖模型组（oxygen&glucosedeprivation，OGD）选择A285或O4或O1表面抗体标记阳性的细胞（即晚期OL前体和未成熟OL，OLPs），使用耗氧剂连二亚硫酸钠（Na₂S₂O₄）及无糖Earle’s液制造OGD环境，细胞分别在此环境下培养10min、30min、60min及90min，并设立成熟OL OGD对照组和OLPs正常（无OGD）对照组。观察各组细胞在不同时间点光镜、电镜下形态的改变；MTT法检测不同时间点各组细胞存活率；DAPI染色，荧光显微镜下观察各组少突胶质细胞凋亡百分率。结果：（1）加入Na₂S₂O₄浓度为10mmol／L时，无糖培养基中氧分压在90min内保持无氧或低氧，故选择该浓度为制作OGD模型的适当浓度。（2）光镜下，模型组OGD10min出现胞体水肿；30min即出现了明显的细胞死亡和凋亡，胞体水肿，胞膜破裂，轴突肿胀，断裂；正常对照组和成熟OL组细胞形态变化不明显。（3）MTT结果显示模型组OGD30min,细胞存活率降至75％；OGD90min，细胞存活率降至约50％。明显低于正常对照组和成熟OL组（ρ均＜0．05）。（4）DAPI染色显示，模型组0GD10min即出现细胞凋亡，随着OGD时间的延长，凋亡细胞百分率逐渐增加，OGD60min，凋亡细胞百分率接近50％，模型组细胞凋亡百分率明显高于正常对照组和成熟OL组（ρ均＜0．05）。结论：OGD导致OLPs形态学损伤，细胞存活率下降及凋亡率增加，同时表明OLPsOGD模型（OGD模型）建立成功，证实了OLPs对缺氧损伤的成熟依赖性。目的：观察Nogo-A、NgR及RhoA在未成熟大鼠少突胶质前体细胞系（OLPs）的表达和细胞定位，以及缺氧缺糖损伤后的表达，探讨它们在OLPs系损伤后再生抑制中的作用。方法：采用改良振荡分离纯化法体外培养OLPs（同第一部分）；用含连二亚硫酸钠的无糖培养基模拟造成OLPsOGD模型（同第二部分）。用OLPs特异性抗体A285、O4、O1和MBP进行免疫荧光法作细胞鉴定。然后在相应的OLPs分化阶段和分别在OGD不同时段：10min、30min、60min用免疫荧光法和免疫印迹法观察Nogo-A、NgR及RhoA的表达。结果：Nogo-A、NgR、RhoA在OLPs分离纯化培养后的第1、2和4天均有阳性表达，且随细胞成熟度增加而表达增强，Nogo-A、NgR的阳性信号位于胞体及突起；RhoA的阳性信号位于胞浆及突起。Nogo-A在OLPs缺氧缺糖10min表达较正常对照组增强，30min继续增高，60min最高，差异有统计学意义（ρ＜0．05）；NgR在OLPs缺氧缺糖后10min，表达较正常对照明显增强，30min达最高，差异有统计学意义（ρ<0．05），60min降低，与对照组比较差异无统计学意义（ρ＜0．05）：RhoA在OGD后10min，表达较正常显著增强，且迅速达最高峰，OGD后30min，RhoA表达逐渐减弱，60min继续下降，但RhoA表达均正常对照组显著增强，差异有统计学意义（ρ＜0．05）。结论：在OLPs胞体及突起上均表达Nogo-A和NgR；胞浆和突起上表达有RhoA。它们在OGD损伤后表达明显增强，与此同时，OLPs细胞在形态学上出现明显损伤，细胞存活率显著降低，凋亡率明显增加。提示Nogo-A和NgR表达增强可能影响OLPs损伤后的修复过程，其通路可能通过激活下游RhoA的表达而发挥作用。该结果为研究神经再生抑制因子Nogo-A、NgR，及其信号传导蛋白RhoA在以OLPs为主要成分的早产儿脑室周围白质软化再生抑制机制中的作用提供了基础资料。更多还原

【Abstract】 Object: To obtain highly purified oligodendrocyte precursor lineage cells of newborn immature SD rats in vitro for the needs of further study.Method: The 2 day-old SD rats were sacrificed by disconnecting neck under aseptic conditions and the brain was taken out. The meninges and blood vessels on the brain were removed. The oligodendrocyte precursors of the brain were separated from astrocyte by orbital shaker and further purified by differential adhesion and finally cultured in chemically defined serum-free medium with appended N2, PDGF, bFGF. Immunofluorescence assay was applied to identify the separated cells with A2B5,O4 and O1 antibodies which represent different phases of differentiation of oligodendrocyte precursors.Result: Over 95％of cultured oligodendrocyte precursor cells were obtained. The oligodendrocyte progenitors are A2B5 and O4 positive, while immature oligodendrocytes are O4 and O1 positive.Conclusion: Separation and purification by shaking and differential adhesion and chemically defined medium are suitable and effective to obtain highly purified oligodendrocyte precursor cells. The output of cells increases notably and keep in immature phase by Object: To set up the model of oligodendrocyte precursors （OLPs） of immature SD rat deprived of oxygen & glucose and observe the changes of OLPs cells morphologically and in survival rate and apoptosis rate in different time of deletion of oxygen and glucose.Method: separate and culture highly purified oligodendrocyte precursor lineage cells and make identification of immature SD rat in vitro. Choose the cells that surface antibody A2B5 or O4 or O1 positive to culture in absence of oxygen and glucose conditions by using Na₂S₂O₄ and Earle’s fluid in the medium for 10min, 30min, 60min and 90min, respectively to set up the oxygen & glucose deletion injured model（OGD） of OLPs . Meanwhile, the both of mature oligodendrocytes （OL） with deletion of oxygen & glucose and normal OLPs without deletion of oxygen & glucose are set up for the control groups. The morphologic changes of cells are observed by light microscope and electron microscope in different hypoxic duration and the livability of cells in each group is detected by MTT and the cell apoptosis rate is measured by DAPI dye. Results: （1） When Na₂S₂O₄’S concentration is 10mmol/L in culture medium, the oxygen pressure in culture medium without glucose keeps in very low or zero, so we choose 10mmol/L Na₂S₂O₄ as the property concentration for making the hypoxia model. （2） Compared with the two control groups, OLPs in OGD model was significantly damnified OLPs cells became markedly swelling and cell prominences reduced and the cell membrance ruptured. The nuclei were large and chromatin was condensed, and OLPs were collapsing and floating in culture medium. （3） MTT’s results showed that OLPs livability rate in OGD model was significantly lower than those of the both control groups （P＜0. 05） . （4） The apoptosis rate by DAPI in OGD model group was significantly higher than those in the both control groups.Conclusion: The OGD model of oligodendrocyte precursors damage was successfully established, approving that hypoxic lesions of OLPs is maturity- dependent.Objective: To detect the expressions of Nogo-A, NgR & RhoA in OLPs and in OGD model in vitro to discuss their functions in restraining OLPs regeneration after the OLPs damage.Method: The OLPs were separated by improved separation and purification through agitation and then cultured in chemically defined medium. Set up OGD model of OLPs by N₂S₂O₄ in vitro. Immunofluorescence assay is applied to identify the separated cells with A2B5, O4, O1 antibodies and western blotting to observe the expressions ofNogo-A, NgR and RhoA in OLPs of different duration （10min, 30minand 60min, respectively）of OGD model and normal OLPs groups.Results: Nogo-A, NgR & RhoA were detected in purified OLPs and the positive signals of Nogo-A, NgR were located in the cell’s body and the prominence whereas RhoA in cell plasm and prominence of OLPs. Compared with the control,the expressions of NogoA, increased significantly at 10min , 30min and 60min in OGD model（P＜0.05）. The expressions of NgR increased significantly at 10min and 30min（P＜0.05）,whereas decreased at 60min in OGD model（P＞0.05）. The expressions of RhoA increased significantly at 10min ,30min and 60min of OGD compared with the control（P＜0.05）.Conclusion: The expressions of Nogo-A and NgR proteins locate both in prominence and cell body of OLPs, and the expression of RhoA protein in the cell plasm and promincence of OLPs.After OGD injury, their expression all increased. Meanwhile, OLPs damage was consistant with the expression of Nogo-A,NgR and RhoA.suggesting that Nogo-A and NgR may play a role inOLPs damage, which might be due to the activation of RhoApassway. These findings may be useful for further experimental research in the understanding of pathogenesis ofrestraining OLPs’ regeneration in premature periventricularleukomalacia （PVL）.更多还原