【作者】 钟琳；

【导师】 姚裕家；

【作者基本信息】 四川大学 ， 儿科学， 2007， 博士

【摘要】 第一部分新生未成熟大鼠少突胶质前体细胞系细胞的分离、纯化、培养和鉴定目的确定新生未成熟SD大鼠少突胶质前体细胞系细胞的培养体系，获取大量高纯度的少突胶质前体细胞（OPCs），为研究大鼠少突胶质前体细胞缺氧缺糖损伤模型中RhoA和细胞骨架蛋白的表达情况提供足够数量的处于特定分化阶段的少突胶质细胞，以满足实验的需要。方法将出生72h以内的SD大鼠在无菌条件下断颈处死，取出大脑，去除脑干和海马，剔除脑膜和血管后，将胰酶消化制备的单细胞悬液接种于培养瓶中混合培养7-9d，再利用改良振荡分离纯化法使少突胶质前体细胞与星形胶质细胞分离，得到纯化的少突胶质前体细胞，并用添加了N2的无血清化学限定培养基进行传代培养，促进细胞分化。运用免疫荧光法，在体外培养1d、2d、4d时，对少突胶质细胞不同发育时期特异性表达的细胞表面抗原A2B5、O4、O1以及星形胶质细胞特异性抗原GFAP进行检测，评估所得少突胶质前体细胞的纯度。结果1．得到了纯度大于95％的少突胶质前体细胞系细胞。2．各发育阶段的特异性细胞表面抗原依次表达。3．星形胶质细胞的沾染率小于5％。结论利用改良振荡分离纯化法和添加N2的无血清化学限定培养基可获得大量高纯度的少突胶质前体细胞系细胞，能满足实验对特定分化阶段少突胶质细胞的需求。第二部分新生未成熟大鼠少突胶质前体细胞缺氧缺糖模型的建立及评价目的联合应用耗氧剂和无糖培养基在常氧培养条件下建立新生未成熟大鼠少突胶质前体细胞缺氧缺糖模型，并观察缺氧缺糖对细胞形态和存活率的影响以及超微结构的改变，评估模型是否有效。方法常氧培养条件下在无糖培养基中加入10mM连二亚硫酸钠，检测加入10min、30min和60min后培养基的PO₂和PCO₂，确定可形成无氧液相环境。将培养2d得到的、以O4阳性为主的少突胶质前体细胞分为正常对照组和缺氧缺糖组，分别在DMEM-F12培养基和含10mM连二亚硫酸钠的无糖DMEM培养基中培养。倒置显微镜下观察细胞形态的改变，并利用MTT法检测处理后即刻、10min、30min、60min和90min的细胞存活率，DAPI荧光染色和透射电镜观察细胞在缺氧缺糖60min后以及复氧1d后的改变，对缺氧缺糖模型进行评价。结果1．连二亚硫酸钠迅速清除了培养基中的氧，并使培养基在常氧培养条件下能够保持无氧状态至少60min。2．联合应用无糖DMEM培养基和连二亚硫酸钠后，细胞出现明显的形态学改变，细胞突起肿胀、变形、断裂，细胞水肿，并随时间延长而加剧。3．少突胶质前体细胞存活率随缺氧缺糖时间的延长而逐渐下降，30min、60min和90min细胞存活率均明显低于正常对照组，差异有统计学意义（P均＜0.05）。4．在缺氧缺糖60min后，少突胶质前体细胞表现为坏死；复氧1d后，细胞出现凋亡。结论1．连二亚硫酸钠可迅速清除培养基中的氧并使其在短时间内维持在无氧状态。2．利用无糖培养基和连二亚硫酸钠可以在常氧培养条件下建立体外培养的少突胶质前体细胞缺氧缺糖模型。3．应用此模型，可模拟缺氧缺血对体内细胞造成的损伤，引起体外培养的少突胶质前体细胞出现类似体内的缺氧缺血损伤的改变。第三部分大鼠少突胶质前体细胞缺氧缺糖模型中RhoA、ezrin和F-actin的表达目的检测RhoA、膜细胞骨架连接蛋白ezrin和F-actin在正常少突胶质前体细胞和缺氧缺糖模型中的表达和分布，了解其表达在缺氧缺糖损伤中的变化，探讨三者在缺氧缺糖损伤发生中的作用及相互之间的关系。方法利用荧光显微镜和激光共聚焦显微镜观察RhoA、磷酸化ezrin和F-actin在纯化培养1d、2d、4d大鼠少突胶质前体细胞中的表达与分布情况。将纯化培养2d，O4阳性为主的少突胶质前体细胞分为正常对照组和缺氧缺糖组，利用无糖DMEM和连二亚硫酸钠建立缺氧缺糖模型，分别运用免疫荧光法、激光共聚焦显微镜和免疫印迹法检测在缺氧缺糖损伤即刻、10min、30min和60min时RhoA、磷酸化ezrin（Thr567）和F-actin的表达情况，观察其荧光强度或灰度值，并与正常对照组相比较。运用SPSS11.0软件进行统计学分析，P＜0.05有统计学意义。结果1．RhoA分布在OPCs细胞浆内，主要在细胞体，细胞突起的分支处可见，在OPCs的发育过程中其分布无明显改变。2．磷酸化ezrin（Thr567）在OPCs发育中由遍布整个胞体、细胞膜下逐渐变为仅局限在OPCs延伸的突起的前端。3．F-actin在发育中OPCs的分布由最初的整个胞体可见逐渐向细胞膜下、细胞突起部位转移，并逐渐集中于突起的前端，其改变趋势与磷酸化ezrin一致。4．OPCs缺氧缺糖损伤后，RhoA的分布变化不明显，表达水平升高，10min达高峰，此后下降但仍维持在较正常对照组高的水平，各时点与对照组相比，差异有统计学意义（P均＜0.05）。5．磷酸化ezrin（Thr567）在OPCs缺氧缺糖损伤后，10min时表达水平升高至高于正常对照组水平（P＜0.05），分布变得不均匀，随后表达水平迅速、持续下降，30min、60min时的表达水平低于对照组（P均＜0.05），其变化趋势与RhoA不完全一致。6．F-actin在缺氧缺糖后表达持续下降并处于较正常对照组低的水平（P均＜0.05），其分布变得不均匀，变化趋势与磷酸化ezrin基本一致。结论1．在大鼠OPCs的正常发育中，RhoA、磷酸化ezrin和F-actin的表达和分布影响着细胞的形态，三者均参与了少突胶质前体细胞的分化与成熟。2．在大鼠OPCs缺氧缺糖模型中，缺氧缺糖引起RhoA、磷酸化ezrin和F-actin的表达和分布明显改变，并最终导致细胞结构的变化。3．缺氧缺糖导致大鼠OPCs中RhoA表达增加、激活，引起ezrin和F-actin的相应改变，三者之间相互联系，但是可能还存在其他的机制影响三者的表达和分布。更多还原

【Abstract】 ObjectivesTo establish a culture system of rat’s oligodendrocyte precursor lineage cells and obtain highly purified oligodendrocyte precursor lineage cells from neonatal and immature SD rat’s brain in vitro, so that we can get enough oligodendrocyte precursor cells（OPCs） which are in the given stage of differentiation to investigate the expression of RhoA and cytoskeleton proteins in an oxygen/glucose deprived model.MethodsNeonatal SD rats born less than 72 hours of age were sacrificed by disconnecting neck under aseptic conditions, brains were taken out the brain and brain stems and hippocampus were discarded, then meninges and vessels were removed. The unicellular suspension was prepared by trypsogen and then the cells were mixed cultured for 7 to 9 days. The oligodendrocyte precursors were separated from astrocyte by orbital shaker, further purified by differential adhesion, and finally cultured in chemically defined serum-free medium with N2 supplement. Immunofluorescence assay was applied to identify the specific antigens A2B5、04 and 01 which expressed in order during the development of oligodendrocyte precursors. GFAP which is the astrocyte-specific antigen was also detected by immunofluorescence assay to evaluate the purity of oligodendrocyte precursors obtained.Results1. The percentage of cultured oligodendrocyte precursor cells was obtained above 95%.2. The ratio of astrocyte-stained cells was less than 5%.3. The stage-specific antigens expressed in order in OPCs cells.ConclusionsSeparation and purification by developed shaking and differential adhesion and chemically defined medium with N2 are suitable and effective to obtain oligodendrocyte precursor cells, and the purity of oligodendrocyte precursor cells can be high. The need for oligodendrocyte precursors at specific stage can be met. ObjectivesTo establish an oxygen/glucose deprived（OGD） model of SD rat’s oligodendrocyte precursor cells（OPCs） in a nomaxic environment in vitro with dithionite sodium and DMEM without glucose, and evaluate the model by investigating to the changes of cellular shapes, survival and ultrastructure caused by OGD.MethodsDithionite was applied into DMEM without glucose in a normaxic environment and the pressure of O₂ and CO₂ was detected. To establish the OGD model of neonatal SD rat’s oligodendrocyte precursors which are predominently O4 positive with 10mM sodium dithionite and DMEM without glucose. The persistence of 04 positive oligodendrocyte precursors in OGD model and normal control which were cultured in DMEM-F12 in normoxic environment was detected with MTT at 0min, 10min, 30min, 60min and 90min after the oxygen/glucose deprivation. The degree of injury was evaluated by observing the morphologic changes of oligodendrocyte precursors at different time spots under inverted phasecontrast microscope, survival, nuclear changes by DAPI staining, and ultrastructural changes by transmission electron microscope.Results 1. Sodium dithionite eliminated the oxygen in culture medium rapidly and keep the medium in anoxia for 60minutes at least when the medium is placed in a normoxic incubator.2. When the O4 positive OPCs were cultured with sodium dithionite and DMEM without glucose, the morphologic changes were obvious: cell body swelling and the processes swelling, deformed and collapsed gradually.3. When the O4 positive oligodendrocyte precursor cells were cultured with sodium dithionite and DMEM without glucose, the survival rate decreases gradually with the time and the rate was about 50% at 60min while it decreased to a level lower than 50% at 90min.4. Necrosis occured in the cells which had been oxygen/glucose deprived for 60 min while apoptosis took place one day later after reoxygenation.Conclusions1. Sodium dithionite eliminated the oxygen in culture medium rapidly and kept it in anoxia for 60minutes at least when the medium was placed in a normoxic incubator.2. DMEM without glucose and sodium dithionite can be applied to the construction of OGD model in O4 positive oligodendrocytes in a normoxic incubator in vitro.3. The damages of oligodendrocyte precursor cells caused by hypoxia-ischemia in vivo can be analogized with this model, and similar damages occur in the oligodendrocyte precursor cells in vitro. ObjectivesTo observe the expressions and locations of RhoA, phosphorylated ezrin（p-ezrin） and F-actin in the oxygen/glucose deprived model of immature neonatal SD rat’s oligodendrocyte precursor cells and normally developing OPCs, and investigate their changes induced by OGD damage ,the roles of the three proteins and the relationship among them during the damage process.MethodsFirst, immunofluorescence assay and laser scanning Confocal microscope were used to observe the expressions and locations of RhoA, p-ezrin（Thr567） and F-actin in normally developing OPCs when they had been cultured for 1d, 2d and 4d. Then the OPCs which had been cultured for 2d and were mostly O4 positive were divided into two groups, normal control and oxygen/glucose deprived（OGD）. The OGD model was esstablished by sodium dithionite and DMEM without glucose first, then the expressions and locations of RhoA, p-ezrin（Thr567） and F-actin were observed at 0min, 10min, 30min and 60min after oxygen/glucose deprivation by immunofluorescence assay, laser scanning Confocal microscope and western blotting. The mean fluorescence densities were analyzed by SPSS 11.0.Results1. In developing OPCs, RhoA located in cytoplasm and processes which branches stretch out, and the location didn’t change obviously.2. P-ezrin（Thr567） in OPCs were in cell body first, then transfered to submembrane and processes, and concentrated in tips of process at last during developing.3. The distribution and alteration of F-actin coincided with p-ezrin.4. After OGD, the expressions of RhoA increased and reached to the peak at 10min, then decreased gradually, but the levels were higher than the control group’s at all the time spots（P<0.05）. But the distribution didn’t change obviously.5. The fluorescence density of p-ezrin（Thr567） increased to the peak and was higher than the control’s level, then descentd rapidly, kept decreasing to a level persistently lower than the control group’s（P<0.05）, but the tendency didn’t coincide with RhoA’s. And the distribution of p-ezrin changed to a nonuniform state after OGD damage.6. F-actin’s expression decreased and was always lower than the control group’s too（P<0.05）, and its distribution also becomes nonuniform which is similar to p-ezrin’s.Conclusions1. Expressions and locations of RhoA, p-ezrin and F-actin regulate the morphology of OPCs during developing, and all of them play an important role in differentiation and maturation of OPCs.2. Oxygen/glucose deprivation in OGD model of rats’ OPCs induces the changes of expression and distribution of RhoA, p-ezrin（Thr567） and F-actin significantly, and change the cytoskeleton and morphology of OPCs ultimately.3. OGD increase expression and activity of RhoA which cause changes of p-ezrin and F-actin in their expressions and distributions sequently. But there may be some other mechanisms which affect the expressions and distributions of RhoA, p-ezrin and F-actin in rat’s OPCs during the OGD damage.更多还原