【作者】 陈焕然；

【导师】 杨辉；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 中枢神经系统(central nervous system, CNS)髓鞘的形成是由少突胶质细胞(oligodendrocytes, OLs)完成的,是维持正常神经冲动传导的重要物质基础。但是,由于少突胶质细胞对损伤因子很敏感,当CNS损伤时,少突胶质细胞很容易受到损害而死亡或凋亡,导致脱髓鞘或再生的轴突不能髓鞘化。伴随CNS创伤出现的一种特征性病理变化就是轴突脱髓鞘,且多不能自发再髓鞘修复,其中一个重要原因,可能就是与OLs减少和不能及时得到补充有关。室管膜下区的少突胶质细胞祖细胞(oligodendrocyte progenitor cells, OPCs)定向迁移可能对OLs的补充具有重要意义。虽然目前对与少突胶质细胞系的发育有关的细胞因子(如GGF、PDGF、bFGF等)有了较深入了解,但是却对如何诱导OPCs迁移及其迁移机制知之甚少。由于神经元和少突胶质细胞之间存在密切的相互作用关系,因此,调控神经元轴突塌陷和定向的分子也可能对OPCs迁移产生影响。近年来发现的诱导分子Semaphorin3A(Sema3A)及其受体就是对轴突生长和神经元定向迁移发挥着重要作用。那么Sema3A是否对OPCs细胞迁移过程产生调控作用呢?在此过程中其信号传导机制如何呢?目前还不清楚。本文通过对传统的培养、纯化方法进行改良,从新生大鼠室管膜下区分离出高纯度的OPCs,转染增强型绿色荧光蛋白(EGFP)基因,使其携带绿色荧光;并利用DNA重组技术制备表达Sema3A-Fc融合蛋白的COS-7细胞,用millicell-PCF细胞迁移小室(孔径8um)进行细胞迁移试验,观察Sema3A-Fc对OPCs迁移的影响及并探讨可能的信号传导机制。研究目的:1、观察Sema3A-Fc对少突胶质细胞祖细胞迁移的影响;2、探讨cAMP-PKA、cGMP-PKG和RhoA-ROCK信号通路在Sema3A-Fc诱导少突胶质细胞祖细胞迁移中的作用;3、试图阐明Sema3A诱导少突胶质细胞祖细胞迁移中可能的信号机制。实验方法1、少突胶质细胞祖细胞的分离培养与鉴定在解剖显微镜下切取新生SD大鼠室管膜下区脑组织进行细胞培养,培养基为含10%小牛血清和10%胎牛血清的DMEM。细胞分层时由于OPCs生长在星形胶质细胞层上面,根据贴壁能力差异,采用差速振荡法分离上层的OPCs。OPCs采用DMEM/F12培养基(含2%B27,20ng/L bFGF和20ng/L PDGF-AA)进行扩增培养。分别用无血清培养基和血清培养基对OPCs进行促分化培养。分别选取少突胶质细胞系不同发育期标志性抗原(A2B5、GFAP和CNPase),采用间接免疫荧光染色技术对所培养的细胞进行类型鉴定。EGFP质粒转染体外培养的少突胶质细胞祖细胞,挑选成功转染GFP的少突胶质细胞祖细胞扩增培养备用。2、表达Sema3A-Fc的COS-7细胞的制备利用基因重组技术,构建Sema3A-Fc重组质粒,利用双酶切、基因测序进行重组质粒鉴定;脂质体法转染COS-7细胞,G418筛选抗性克隆,扩增培养,免疫组化和免疫印迹法鉴定重组蛋白表达情况,获得稳定表达Sema3A的COS-7细胞。用含有sema3A-Fc培养上清加入体外培养的OPCs培养瓶中,通过对比观察构建表达的sema3A-Fc是否具有生物学活性。并对细胞膜上NP-1受体进行了检测。3、Sema3A-Fc诱导少突胶质细胞祖细胞迁移信号机制的实验研究1)用millicell-PCF细胞迁移小室(孔径8um)进行细胞迁移试验。将稳定表达Sema3A-Fc的COS-7细胞培养在迁移仓下层,待细胞融合达70%以上后,将纯化培养的少突胶质细胞祖细胞按2×104/孔接种在迁移仓的上层,共培养12h后,固定细胞,甲苯胺蓝染色,计数迁移细胞数。设对照组:单纯培养基对照和转染pIG的COS-7对照组。2)PKA和PKG的激活实验:分别加入cAMP类似物8-溴-cAMP(浓度50μM),cGMP类似物8-溴-cGMP(浓度500μM)于分层迁移实验舱下层中,观察少突胶质细胞祖细胞的迁移情况,计数并比较迁移结果。3)PKA和PKG抑制实验:分别加入PKA的特异性抑制物KT5720(浓度10μM)和PKG的特异性抑制物KT5823(浓度1μM)于分层迁移实验舱下层中,观察少突胶质细胞祖细胞的迁移情况,计数并比较迁移结果。4)抑制Rock激酶试验:分别在上述实验组中加入有Rho活性的ROCK激酶抑制物Y27632(浓度10μM),分别观察少突胶质细胞祖细胞的迁移情况,计数并比较迁移结果。5)免疫荧光法检测PKA催化亚基和PKG1含量:分别用小鼠抗PKA催化亚基抗体和兔抗PKG1抗体做为一抗,PBS替代一抗作阴性对照,在相同条件下进行免疫荧光染色和照相。用Image-Pro Plus 5.1图像分析软件,对图像进行分析处理。6)观察PKG,PKA和ROCK活性对OPCs细胞迁移的直接作用。在没有sema3A-Fc存在的情况下,用8-Br-cAMP, 8-Br-cGMP, KT5720、KT5823和Y 27632直接作用于OPCs细胞(浓度同前),观察PKG,PKA和ROCK活性改变是否对体外培养的OPCs细胞迁移的产生直接影响。4、统计定量数据以均数±标准差( x±s)表示,用SPSS10.0统计软件对数据进行分析,使用独立样本t检验分析两个组间的差异,使用单因素方差分析比较多个组间统计学差异。以P < 0.05为有统计学差异,以P < 0.01为有显著差异。主要结果1、体外成功分离培养出少突胶质细胞祖细胞,A2B5标记阳性,纯度达到95%以上。并能进一步分化为CNPase标记阳性的少突胶质细胞和GFAP标记阳性Ⅱ型星形胶质细胞。2、成功构建了pIGsema3A-Fc重组质粒,经双酶切(HindⅢ和BamHⅠ)和基因测序鉴定正确,转染COS-7细胞后,经免疫组化和Western-blot检测到重组蛋白的表达。表达Sema3A-Fc的COS-7细胞的培养上清能够促使体外培养的OPCs细胞突起回缩,胞体变圆。用抗Fc抗体和抗NP-1抗体免疫荧光染色均呈膜性染色。3、sema3A-Fc诱导细胞迁移实验结果:Sema3A-Fc实验组OPCs迁移数为(24.3±6.7),单纯培养基对照组为(38.4±7.9),转染pIG的COS-7对照组为(37.7±8.3),实验组与两个对照组均存在明显差异(n=15, P <0.01)。4、OPCs细胞内PKG、PKA免疫组化检测:通过针对催化亚基的抗体用免疫荧光法对细胞内PKA和PKG进行检测,发现OPCs细胞内的PKA和PKG均有基础量的表达,主要分布在胞浆和突起内。当加入含sema3A-Fc的培养上清时,细胞内PKA催化亚基染色强度没有明显变化,而PKG1的染色强度则较对照组明显增高(P <0.01),表明PKG含量增加,活性增强。表明含sema3A-Fc激活了cGMP-PKG信号通路。5、PKA和PKG激活/抑制实验:OPCs迁移数分别为:8-溴-cAMP组OPCs迁移数为(32.6±7.3)较对照组(24.3±6.7)明显增加(P <0.01),8-溴-cGMP组为(19.3±5.5)较对照组(24.3±6.7)有减少明显(P <0.05)。PKA和PKG抑制实验:OPCs迁移数分别为:KT5720组(17.5±5.3)较对照组(24.3±6.7)明显减少(P <0.01)。KT5823组(29.9±6.2)较对照组(24.3±6.7)有明显增加(P <0.05)。表明PKG通路对sema3A信号具有正向调节作用,而PKA通路则具有负向调节作用。6、抑制ROCK激酶试验:Y27632干预前后的成对数据的统计结果为,对照组(37.7±8.3)对(38.4±8.2),sema3A-Fc组(24.3±6.7)对(35.3±8.7),8-溴-cAMP组(32.6±7.3)对(32.4±7.6) , 8-溴-cGMP组(19.3±5.5)对(29.9±6.2) , KT5720组(17.5±5.3)对(26.5±5.7),KT5823组(29.9±6.2)对(36.9±7.7)。单因素方差分析示组间有明显差异(P =0.012)。表明ROCK激酶抑制剂Y27632能明显抑制sema3A-Fc对OPCs细胞迁移的作用,间接证明Sema3A激活了OPCs细胞内的Rho-ROCK信号通路。7、在去除Sema3A-Fc情况,单独应用8-Br-cGMP、8-Br-cAMP、KT5720、KT5823和Y27632并不能改变OPCs原有的迁移能力。结果为:8-溴-cAMP组(36.8±7.1),8-溴-cGMP组(35.1±7.4),KT5720组(37.5±8.1),KT5823组(34.9±7.6),均与对照组(37.7±8.3)无明显差异(n=15, P>0.05)。但与sema3A-Fc存在时的数据进行配对比较则有明显差异(P<0.05)。全文结论1、通过二次接种和差速振荡法,成功分离出高纯度少突胶质细胞祖细胞,传代及分化培养,通过免疫组化法鉴定了细胞类型。脂质体法成功导入EGFP基因,成为可示踪的少突胶质细胞祖细胞。2、成功构建了pIGsema3A-Fc真核表达载体,通过脂质体法成功转染COS-7细胞,表达出具有生物活性的sema3A融合蛋白。3、Sema3A-Fc对体外培养的OPCs细胞迁移具有排斥作用;Sema3A-Fc对OPCs的排斥作用可能是通过激活cGMP-PKG和Rho-ROCK信号通路介导的,而cAMP-PKA信号通路没有直接参于该效应,但对该效应具有明显的负向调节作用。4、Sema3A-Fc诱导OPCs细胞迁移的信号转导机制是非常复杂的,cGMP-PKG和Rho-ROCK信号通路只是其中的一小部分,可能还存在其它的作用机制,尚需要更深入的研究。更多还原

【Abstract】 BACKGROUNDOligodendrocytes (OLs) are the myelinating cells of the central nervous system (CNS) that ensheath axonal projections, thereby facilitating saltatory conduction. However, OLs are particularly susceptible to damage factors resulting in dying apoptotic or necrotic deaths and decreased myelination and attenuated regenerative fiber tracts. Axonal demyelination is a consistent pathological characteristic of the traumatically injured spinal cord. Unfortunately, extensive remyelination does not occur spontaneously. This may be caused, at least in part, by loss of oligodendrocytes and a subsequent lack of oligodendrocyte progenitor cells migration to replace the lost cells.Growth factors and cytokines controlling OL development are well documented [e.g. glial growth factor (GGF), plateletderived growth factor(PDGF), basic fibroblast growth factor(bFGF), but much less is known about factors influencing the migration of progenitors into discrete regions of the brain and spinal cord. Because neurons and OLs interact intimately throughout their development, molecules controlling neural guidance and process collapse could possibly also affect OLs.One such candidate system is the Semaphorin3A(Sema3A) defined by an amino terminal‘sema’domain and its interacting partners, the neuropilin-1 (NP-1)/plexin receptors. Recently, several protien kinases (e.g. PKA, PKG, ROCK) were found to regulate the migration and process elongation of developing neural cells. Still, little is known about the role that these moleculars play in the process of OPCs migration guidanced by Sema3A .In this study, the OPCs were separated by improved agitation method and purified by differential adhesion. We construct an eukaryotic expression recombinant plasmid named pIGsema3A-Fc which were transfected to COS-7 cells in order to gain Sema3A-Fc fusion protein. Then, use millicell-PCF cell chamber to observe influence of sema3A-Fc moleculars on the migration of OPCs and to study its signal transduction pathway.OBJECTIVE1. To observe influence of sema3A-Fc moleculars on the migration of oligodendrocyte progenitor cells.2. To study the role of cAMP-PKA, cGMP-PKG and RhoA-ROCK signal pathway play in the process of OPCs migration guidanced by Sema3A-Fc.3. To explore the effects of Sema3A-Fc molecule on guiding oligodendrocyte progenitor cells migration and its signal transduction pathway.METHODS1. Culture and identification of Oligodendrocyte progenitor cells.The tissue of subventricular zone( SVZ) was disconnected from neonatal rats under anatomical microscope and was culture in high-glucose DMEM medium(with 10% calf serum and 10% fetal bovine serum). The OPCs which grew on the surface of astrocytes monolayer, were isolated by a technique of differential revolutions shaking skill. Then the OPCs were further purified by differential adhesion and cultured in the DMEM/F12 medium adding growth factors (bFGF, B27 and PDGF-AA). In order to promote the cells differentiate, the OPCs were moved into serum medium or serum-free medium, respectively. The oligodendrocyte lineage cells were identified by indirect immunofluorescent staining with different cell surface markers (A2B5, GFAP or CNPase). The gene of enhanced green fluorescent protein(EGFP) was transfected into the OPCs with lipofectamine reagent.2. Prepare COS-7 cells expressing sema3A-Fc fusion protenThe pIGsema3A-Fc recombinant vector was constructed by gene cloning technique And transfected into COS-7cells with lipofectamine reagent. The plasmids were identified by incision enzyme HindⅢand BamHⅠand DNA sequencing. The expression of Sema3A-Fc fusion proten was assessed by immunohistochemistry and Western blotting with the anti-IgG1Fc antibody. Then, detect NP-1 on the OPCs by Immunofluorescent staining.3. To study the effects of Sema3A-Fc on OPCs migrationUse millicell-PCF cell culture chamber to observe influence of sema3A-Fc moleculars on the migration of oligodendrocyte progenitor cells. First, we cultured COS-7 cells which secreting sema3A-Fc fusion proten on the underlayer of the chamber in high-glucose DMEM medium(with 10% calf serum and 10% fetal bovine serum). Then, the OPCs(2×10~4/well)were inoculated culture on the up-layer of the chamber for 12 hours. Finally, the cells had migrated through the PCF membrane were fixed, stained and counted. The pure medium and COS-7 culture supernatant served as control groups.4. Detect the Catalytic subunit of PKA and PKG: use anti-PKA alpha Catalytic subunit antibody and anti-PKG1 beta antibody to assay the contents of PKA and PKG by indirect immunofluorescent staining in the same condition. The photos to be analysised with Image-Pro Plus 5.1 software system.5. Activation essay of PKA and PKG: The OPCs were treated with 8-Br-cAMP (final concentration: 50μM) or 8-Br-cGMP (final concentration: 500μM) in the above migration chamber system (step 3) alternatively, to observe the variance of OPCs migration status.6. Inhibition essay of PKA and PKG: The OPCs were treated with KT5823 (final concentration:1μM) or KT5720 (final concentration:10μM) in the above migration chamber system (step 3) alternatively, to observe the variance of OPCs migration status.7. Inhibition essay of ROCK: The OPCs were treated with the ROCK inhibitor Y27632 (final concentration: 10μM) in the above migration chamber system (step 3) alternatively, to observe the variance of OPCs migration status.RESULTS1. OPCs were successfully isolated and cultured in vitro. The purified OPCs were identified by immunofluorescence of A2B5 and the purity quotient was up to 99%. These cells could differentiated into GFAP and CNPase marker positive cells.The reproduction active of OPCs keep over one month later in serial subcultivation. The cells modified by EGFP gene showed green fluorescence and reproductive activity.2. The pIGsema3A-Fc recombinant vector was successfully constructed by gene cloning technique. The outcome of DNA sequencing indicated that the open-reading frame of Sema3A-Fc gene was coincident with what we had expected. And transfected into COS-7 cells with lipofectamine reagent. The Sema3A-Fc fusion protein expression in COS-7 cells was confirmed by immunohistochemistry and Western blotting and the fusion protein--Sema3A-Fc was proved to have the biologic activity and could combinate to the membrane receptor (NP-1) on the OPCs.3. Effects of Sema3A-Fc on OPCs migrationThe cell number of Sema3A-Fc guiding OPCs migration was (24.3±6.7). Contrasting with the two control groups: pure medium control group (38.4±7.9) and COS-7 cells’culture supernatant control group(37.7±8.3), the outcome indicated that Sema3A-Fc significantly reduced the OPCs migration number (n=15, p<0.01) .4. Activation essay of PKA and PKG: The migrated cell number of 8-Br-cAMP group was (32.6±7.2), that of 8-Br-cGMP group was (19.3±5.5). (n=15, p<0.05 vs control).5. Inhibition essay of PKA and PKG: The migrated cell number of KT5823 group was (36.9±7.7), that of KT5720 group was (17.5±5.3). (n=15, p<0.05 vs control).6. Inhibition essay of ROCK: Under the interference of ROCK inhibitor Y27632, the migrated cell number of 8-Br-cAMP group was (32.4±7.6), that of 8-Br-cGMP group, KT5823 group and KT5720 was (29.9±6.2, 36.9±7.7, 26.5±5.7) respectively.7. assay the contents of PKA and PKG: The sema3A-Fc could significantly promote the PKG contents in the kytoplasm and growth cone of OPCs (p<0.05 vs control); but there was no significant change of PKA activity when sema3A-Fc was administered to OPCs compared with the control group.CONCLUSIONS1. The high purified OPC line can be successfully isolated, cultured and identified by immunofluorescence in vitro, then be successfully modified by EGFP gene showed green fluorescence.2. The pIGsema3A-Fc recombinant vector was successfully constructed by gene cloning technique. After it was transfected into COS-7cells, the Sema3A-Fc fusion protein expression was confirmed by immunohistochemistry and Western blotting and the fusion Sema3A-Fc protein has shown the biologic activity.3. The Sema3A-Fc moleculars acts as a repellent for oligodendrocyte progenitor cells (OPCs) in vitro. Not the cAMP-PKA but the cGMP-PKG and Rho-ROCK signal transduction pathway in OPCs acts as a mediator of semaphorin signals.4. As a guidance clue for OPCs migration, the Sema3A-Fc signal transduction pathway is very complicated. It is relative specificity that Sema3A-Fc moleculars repel oligodendrocyte progenitor cells (OPCs) in vitro. The cGMP-PKG and Rho-ROCK signal pathway maybe is a small part of it. Thus further research would be demanded.更多还原