Plexin A3介导少突胶质前体细胞迁移的实验研究
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摘要
少突胶质前体细胞（Oligodendrocyte precursor cells，OPCs）是中枢神经系统（central nervous system，CNS）中存在并广泛分布的一类胶质细胞，它可以分化成熟为少突胶质细胞和星形胶质细胞，其迁移活动对CNS的发育、损伤修复与功能重建，以及中枢神经系统脱髓鞘疾病如多发性硬化（multiple sclerosis，MS）等的发生、发展发挥着非常重要的作用。近年来的研究认为CNS损伤修复过程中神经纤维的脱髓鞘反应以及髓鞘的修复与再生障碍是制约神经损伤修复结局与功能重建的重要原因，而成功的髓鞘修复与再生则极有可能成为改善这一困难局面的重要环节。因此，研究髓鞘的形成机制与相关的调节因素成为CNS损伤修复研究领域的热点问题。众所周知，CNS髓鞘的形成在胚胎时期主要是由少突胶质细胞完成，然而在机体成熟以后，少突胶质细胞是否具有修复与再生髓鞘的能力尚无确切的结论。近来的研究发现，由OPCs分化形成的少突胶质细胞（Oligodendrocyte, OL）具有再生和形成髓鞘的能力。体外实验发现，将OPCs与胚胎脊髓共组织培养，OPCs能够分化形成OL并为新生的神经纤维形成髓鞘；在脊髓损伤的研究中发现，OPCs能够从脊髓的腹侧迁移到背侧，部分细胞能在损伤灶周围分化形成OL；同样，在对MS的研究中也发现，脱髓鞘病灶的周围有大量NG2染色阳性的OPCs，进一步的研究发现，这些NG2阳性细胞主要是从室管膜下区迁移而来；这些研究都有力的提示OPCs的确参与了CNS损伤与修复的过程，并且很可能在这个过程中扮演了十分重要的角色。为此，研究OPCs的发育成熟规律、迁移机制以及相关因素成为阐明上述问题的重要内容。
众所周知，细胞的迁移不仅取决于细胞本身的特性，而且受众多环境因素的影响，如细胞基质蛋白、层粘连蛋白等，环境中的离子含量变化、甚至pH变化也可能对细胞的迁移产生影响。近年来尤其引人关注的是各种细胞因子对细胞迁移的作用。因为，环境因素可以通过多种措施加以控制和调节，并能在特定的条件下得到稳定，而细胞因子通常需要与特定的细胞受体结合，并通过一系列的细胞内过程才能实现，作用的机制更加复杂而多变；更为重要的是，细胞因子的迁移诱导作用具有定向性，信号过程的传递、浓度梯度的变化等均可以造成细胞的定向迁移。课题组在前期的研究中发现，轴突诱导因子Semaphorins家族成员不仅对再生轴突的生长具有诱导作用，而且对OPCs的迁移也有诱导作用。陈焕然博士的研究发现Semaphorins家族成员Sema3A对OPCs迁移具有诱导作用，其作用机制与RhoG信号通路相关分子的变化有关；而对轴突定向延伸作用更明显的Sema3F是否也有类似的作用？尚无相关的文献报道。新近的研究发现sema3与多种肿瘤细胞的迁移有关，而此诱导迁移的功能的实现需要与其受体分子NRP2/plexin A3复合体共同参与，方能激活相应的细胞内信号传导链，从而诱导细胞迁移。既然Plexin A3及神经菌毛素2（neuropilin2，NRP2）作为sema3F的特异性受体，介导sema3F的在多种细胞内信号传导，在细胞的迁移活动中发挥重要的调节作用，Sema3F对OPCs是否也具有类似的诱导作用？其作用是否同样是通过NRP2/plexin A3介导，以上问题尚未见类似报道。作者在前期预实验中通过Transwell迁移仓实验研究发现，Sema3F的确对OPCs的定向迁移具有诱导作用。如果能够在OPCs上发现plexin A3及其NRP2的表达，无疑对解释上述现象并认识OPCs的迁移机制具有重要意义。因此，我们设计实验对以上问题进行了研究：首先于体外分离培养及纯化OPCs，并诱导其向成熟少突胶质细胞分化，通过RT-PCR、Western blot、免疫组化、免疫荧光染色等手段，研究plexin A3在OPCs不同分化阶段上的表达与特点；其次，在此基础上，通过分子生物学手段调控plexin A3在OPCs中的表达，观察plexin A3表达变化对Sema3F诱导OPCs迁移的影响，最后从上述研究结果中，阐明plexin A3在Sema3F诱导OPCs迁移中的作用，为深入研究OPCs定向迁移的机制奠定实验基础。研究结果如下：
第一部分（第二章内容）：Plexin A3在OPCs不同分化阶段的表达
采用振荡分离法体外分离培养新生大鼠OPCs，并观察了bFGF及PDGF-AA诱导OPCs增殖与分化的作用，采用NG2、O4及MBP抗体检测OPCs不同分化阶段细胞的表达；在此基础上，通过RT-PCR，Western blot、免疫荧光双标检测并分析plexin A3及NRP2在OPCs不同分化阶段细胞中的表达与特点，主要结果如下：
1.振荡分离法分离新生大鼠OPCs，方法简单、稳定、可靠，获得的OPCs纯度高，通过二次接种培养即可达到满足实验所需纯度及数量的OPCs；bFGF及PDGF-AA能诱导OPCs的增殖与分化，NG2，O4及MBP免疫组化染色可识别到OPCs不同分化的细胞阶段；
2.在OPCs阶段，即（NG2+染色阳性阶段）及未成熟阶段（即O4+染色阳性阶段）,plexin A3高表达，当OPCs分化成熟为OL后（即MBP+染色阳性阶段）其表达明显下调。作为协同受体的NRP2在OPCs分化过程中的表达特点与plexin A3类似；但从Westernblot及RT-PCR等方法的检测结果观察，在OPCs各分化阶段， NRP2的表达水平较plexinA3稍高，推测可能与其具有结合多种协同受体的特殊功能有关；
3. Plexin A3及NRP2均主要表达于OPCs及未成熟的少突胶质细胞的细胞膜及突起上，提示二者可能OPCs的诱导迁移有关。
第二部分（第三章内容）：plexin A3对sema3F诱导OPCs迁移的影响
第一部分研究证实plexin A3及NRP2表达在OPCs的细胞膜及细胞突起上，并均出现在OPCs阶段及未成熟阶段，故推测其可能参与了Sema3F介导的OPCs迁移的调控。为此，本部分实验拟通过siRNA干扰技术调节plexin A3及NRP2在OPCs中的表达水平，通过干扰其表达后观察Sema3F诱导OPCs迁移的改变，从而证实plexin A3在sema3F诱导OPCs迁移中的作用。首先分别设计了plexin A3及NRP2的siRNA，利用MTT实验检测plexin A3siRNA及NRP2siRNA对OPCs生存能力的影响，确认其无细胞毒性后，采用RT－PCR技术分别检测两种siRNA的干扰效果，达到实验要求后，利用脂质体介导基因转染技术，将plexin A3siRNA及NRP2siRNA分别转染OPCs，通过Transwell迁移仓实验，比较下调plexin A3及NRP2表达后，sema3F诱导OPCs迁移的变化，藉此阐明plexin A3及NRP2在OPCs迁移中的作用。主要实验结果如下：
1. MTT试验结果证实，使用合成的plexin A3siRNA及NRP2siRNA浓度在0.125~2.00μg/μl范围内对OPCs没有明显的细胞毒性作用；
2.脂质体介导plexin A3siRNA及NRP2siRNA转染OPCs后，通过TR-PCR检测，二者能分别有效地抑制OPCs表达plexin A3及NRP2，其抑制效率可达64~79%；
3. Trenswell迁移仓实验证实Sema3F对OPCs具有诱导迁移作用，加入plexin A3siRNA后，OPCs的迁移率明显降低，加入NRP2siRNA后，OPCs的迁移率下降更加明显，两者相比具有统计学差异，提示NRP2-siRNA的抑制效果更加明显。
以上结果说明，sema3F对于OPCs具有诱导迁移作用，plexin A3及NRP2作为sema3F诱导作用的协同受体共同参与了其对OPCs迁移过程的调控，相对于plexin A3而言，NRP2的调节作用似乎更加明显，推测可能与其具有多种协同受体功能有关。
Oligodendrocyte precursor cells (OPCs) are special glial cells which widely distributedin the central nervous system (CNS). OPCs can differentiate and mature intooligodendrocytes (OLs) and astrocytes. The migration and the differentiation of OPCs playa very important role during CNS development, injury repair and functional reconstruction,as well as the occurrence and development of demyelinating disease (such as multiplesclerosis, MS). Recent studies suggested that the demyelination of nerve fibers, as well asinsufficient of myelin repairment and regeneration in CNS are important reasons forrestricting the outcome of damage repair and functional reconstruction after CNS injury,and it was wildly accepted that the successful myelin repair and regeneration may be a keylink to improve this situation. Therefore, studying the mechanism of myelin formation andits regulate factors became a hot issue about injury and repair of CNS. As we all know, themyelin formation in the embryonic period in CNS was mainly by oligodendrocytes, butafter mature, whether oligodendrocytes have the ability to repair and regeneration of myelinor not is not clear. Recent studies found that oligodendrocytes from OPCs differentiationhad the capacity of regeneration and forming myelination. In vitro experiment found thatOPCs can differentiate into OL and form myelin for new nerve fibers in the co-culturesystem of OPCs with embryonic spinal cord tissue; and it was observed that OPCs canmigrate from the spinal cord ventral to dorsal,while some of those cells can differentiateinto OL around the injury lesions in the study of spinal cord injury. Similarly, in the studyof MS, demyelinating lesions surrounded by a large number of NG2positive staining OPCs,and further study found that these NG2-positive cells came from subependymal zone.These studies are the powerful evidence that the OPCs indeed involved in CNS injury andrepair process, and OPCs are likely to play a very important role in this process. Therefore,to study the development regularity, migration mechanisms of OPCs and their relatedfactors becomes a very important content to illustrate the above problems.
Cell migration depends not only on the characteristics of the cells themselves, but alsoon many environmental factors, such as cell matrix proteins, laminin, ion content in theenvironment, and even pH value. In recent years, the particular concern is the cytokineseffect on cell migration, because environmental factors can be controlled and regulated by avariety of measures, and can be stabilized under certain conditions. Cytokines often need tobind with their cellular receptors, and through the series process of intracells, so that theeffect can be accomplished, the mechanisms are more complex and varied; and moreimportantly, the induced migration effect of cytokins have a directivity, the transmission ofthe signal process, the concentration gradient changes of cytokins, etc. can cause thedirectional migration of cells. In early studies, we found that axon guiding factor,Semaphorins family members, can not only induce regenerative axon growth, but alsoinduce the migration of OPCs. Dr. Chen Huanran observed the Sema3A, one of theSemaphorins family members, can induce the migration of OPCs, the mechanism involed insome changes of RhoG signaling pathway molecules. However, does Sema3F, which hasmore obvious effect of axon guidance, have similar effect on OPCs? There was no relatedreport yet. Recent studies found sema3s is involed in the migration of several tumor cells,and when combined with its receptor molecule composite of NP2/plexin A3, sema3F canmediated intracellular signal processes, and induce cell migration. During the process,plexin A3is an important molecule. The plexin A3and neuropilin2(NRP2), as the specificreceptor for sema3F, mediated intracellular signal transduction induced by sema3F, play animportant regulatory role in various cells migration activity. Does Sema3F also inducedOPCs migration? Is its inducing effect also mediated by NRP2/plexin A3receptorcomposite? These questions are still not clear. In the early experiments, authors foundSema3F really induce OPCs directional migration. If the expression of co-operationreceptor composed of plexin A3and NRP2on OPCs is confirmed, undoubtedly, it will be avery important significance to explain the effect of sema3F on OPCs migration andrecognize its mechanism. For this purpose, the experiment was designed and divided intotwo parts: firstly, OPCs were isolated and cultured, and then OPCs differentiation wereinduced, the expression and characteristics of plexin A3on cells during the various stagesof OLs differentiation were detected by immunohistochemistry, immunofluo-rescencestaining, RT-PCR, Western blot, etc. On this basis, the expression level of plexin A3in OPCs was regulated by means of molecular biology technology, and then the changes ofOPCs migration induced by Sema3F were observed. Through the above study, we not onlyclarified the role of plexin A3on OPCs migration induced by Sema3F, but also made someexperimental basis for further studying of the OPCs migration mechanism. The results areas follows:
The first part (chapter2): The expression of plexin A3on differentdifferentiation stages of OPCs.
The neonatal rat OPCs was isolated and cultured in vitro by oscillation separationmethod, then the role of bFGF and PDGF-AA induced OPCs proliferation anddifferentiation was observed, while different differentiation stages of OLs were identifiedby NG2, O4and MBP antibodies; based on this, the expression and characteristics of plexinA3and NRP2in the different differentiation stages of OPCs was detected and analyzed byimmunofluorescence double-labeled staning, RT-PCR, Western blot etc.. The main resultsare as follows:
1. The oscillation separation method is a simple, stable, reliable way to isolatenewborn rats OPCs, high purity OPCs can be obtained by this culture method. The purityand quantity of OPCs can meet the experiment requirement completely through the secondinoculation. bFGF and PDGF-AA can induce and regulate the proliferation anddifferentiation of OPCs, while NG2, O4and MBP immunohistochemical staining canindentify the different differentiation stages of OPCs.
2. In the stages of OPCs, namely NG2+staining positive stage and immature stages(ie, O4+staining positive stage), plexin A3was high expressed, when OPCs differentiatedinto mature OL (MBP+staining positive stage), its expression was significantly lowered.As a co-receptor, NRP2expression characteristics was similar to plexin A3. However,according to the results of Western blot and RT-PCR test，the expression levels of NRP2was slightly higher than those of plexin A3in the OPCs and immature OLs stages ofdifferentiation, presumably it is related that NRP2can combined with a variety ofco-receptors.
3. Plexin A3and NRP2are mainly expressed in NG2-positive cells and O4-positive cells, and located on the cell membrane and protruding of OPCs, suggesting that plexin A3and NRP2may be related with OPCs migration.
The second part (chapter3): The effect of plexin A3on OPCs migration inducedby sema3F
In the first part, our studies confirmed that the plexin A3and NRP2both express onOPCs or immature OLs stage and located on cell membrane and protrusions of OPCs,suggesting their possible involvement in the sema3F mediated OPCs migration. In this part,we proposed to regulate plexin A3and NRP2expression level in OPCs by siRNAinterference technology, and observe OPCs migration changes induced by sema3F, toexplore the role of plexin A3during sema3F induce OPCs migration.
Firstly, the plexin A3siRNA and NRP2siRNA were designed, and then their toxiceffect on the survival of OPCs was assessed by MTT test. After confirming neither ofsiRNAs has cytotoxic on OPCs, siRNA interference effect was detected by RT-PCR. Toachieve the experimental requirements, the plexin A3siRNA and NRP2siRNA wererespectively transfected into OPCs by liposome-mediated gene transfection technology, andOPCs migration induced by sema3F through transwell chamber was observed to clarify theeffect of plexin A3and NRP2on OPCs migration. The main results were as follows:
1. MTT test results confirmed that the concentration of plexin A3siRNA and NRP2siRNA ranged from0.125μg/μl to2.00μg/μl, which have not cell toxic effects on OPCssurvival;
2. When liposome-mediated the plexin A3siRNA and NRP2siRNA transfected intoOPCs, both siRNAs can effectively inhibit plexin A3and NRP2expression in the OPCs byRT-PCR, and their inhibition efficiency can be up to64~79%;
3. Transwell chamber confirmed the sema3F can induced migration of OPCs, andafter plexin A3siRNA was added, the migration rate of OPCs was significantly reduced,when NRP2siRNA was added, the migration rate of OPCs went down more obviously,which have a statistically significant difference with that of plexin A3siRNA. This resultsimpled the inhibitory effect of NRP2siRNA was more obvious than plexin A3.
These results confirmed that sema3F indeed can induce OPCs migration. As co-receptor of sema3F, both of plexin A3and NRP2take part in the regulation of OPCsmigration process, and it seems that NRP2play more important role than plexin A3,speculate the difference may be related with NPR2has a variety of co-receptor function.
众所周知，细胞的迁移不仅取决于细胞本身的特性，而且受众多环境因素的影响，如细胞基质蛋白、层粘连蛋白等，环境中的离子含量变化、甚至pH变化也可能对细胞的迁移产生影响。近年来尤其引人关注的是各种细胞因子对细胞迁移的作用。因为，环境因素可以通过多种措施加以控制和调节，并能在特定的条件下得到稳定，而细胞因子通常需要与特定的细胞受体结合，并通过一系列的细胞内过程才能实现，作用的机制更加复杂而多变；更为重要的是，细胞因子的迁移诱导作用具有定向性，信号过程的传递、浓度梯度的变化等均可以造成细胞的定向迁移。课题组在前期的研究中发现，轴突诱导因子Semaphorins家族成员不仅对再生轴突的生长具有诱导作用，而且对OPCs的迁移也有诱导作用。陈焕然博士的研究发现Semaphorins家族成员Sema3A对OPCs迁移具有诱导作用，其作用机制与RhoG信号通路相关分子的变化有关；而对轴突定向延伸作用更明显的Sema3F是否也有类似的作用？尚无相关的文献报道。新近的研究发现sema3与多种肿瘤细胞的迁移有关，而此诱导迁移的功能的实现需要与其受体分子NRP2/plexin A3复合体共同参与，方能激活相应的细胞内信号传导链，从而诱导细胞迁移。既然Plexin A3及神经菌毛素2（neuropilin2，NRP2）作为sema3F的特异性受体，介导sema3F的在多种细胞内信号传导，在细胞的迁移活动中发挥重要的调节作用，Sema3F对OPCs是否也具有类似的诱导作用？其作用是否同样是通过NRP2/plexin A3介导，以上问题尚未见类似报道。作者在前期预实验中通过Transwell迁移仓实验研究发现，Sema3F的确对OPCs的定向迁移具有诱导作用。如果能够在OPCs上发现plexin A3及其NRP2的表达，无疑对解释上述现象并认识OPCs的迁移机制具有重要意义。因此，我们设计实验对以上问题进行了研究：首先于体外分离培养及纯化OPCs，并诱导其向成熟少突胶质细胞分化，通过RT-PCR、Western blot、免疫组化、免疫荧光染色等手段，研究plexin A3在OPCs不同分化阶段上的表达与特点；其次，在此基础上，通过分子生物学手段调控plexin A3在OPCs中的表达，观察plexin A3表达变化对Sema3F诱导OPCs迁移的影响，最后从上述研究结果中，阐明plexin A3在Sema3F诱导OPCs迁移中的作用，为深入研究OPCs定向迁移的机制奠定实验基础。研究结果如下：
第一部分（第二章内容）：Plexin A3在OPCs不同分化阶段的表达
采用振荡分离法体外分离培养新生大鼠OPCs，并观察了bFGF及PDGF-AA诱导OPCs增殖与分化的作用，采用NG2、O4及MBP抗体检测OPCs不同分化阶段细胞的表达；在此基础上，通过RT-PCR，Western blot、免疫荧光双标检测并分析plexin A3及NRP2在OPCs不同分化阶段细胞中的表达与特点，主要结果如下：
1.振荡分离法分离新生大鼠OPCs，方法简单、稳定、可靠，获得的OPCs纯度高，通过二次接种培养即可达到满足实验所需纯度及数量的OPCs；bFGF及PDGF-AA能诱导OPCs的增殖与分化，NG2，O4及MBP免疫组化染色可识别到OPCs不同分化的细胞阶段；
2.在OPCs阶段，即（NG2+染色阳性阶段）及未成熟阶段（即O4+染色阳性阶段）,plexin A3高表达，当OPCs分化成熟为OL后（即MBP+染色阳性阶段）其表达明显下调。作为协同受体的NRP2在OPCs分化过程中的表达特点与plexin A3类似；但从Westernblot及RT-PCR等方法的检测结果观察，在OPCs各分化阶段， NRP2的表达水平较plexinA3稍高，推测可能与其具有结合多种协同受体的特殊功能有关；
3. Plexin A3及NRP2均主要表达于OPCs及未成熟的少突胶质细胞的细胞膜及突起上，提示二者可能OPCs的诱导迁移有关。
第二部分（第三章内容）：plexin A3对sema3F诱导OPCs迁移的影响
第一部分研究证实plexin A3及NRP2表达在OPCs的细胞膜及细胞突起上，并均出现在OPCs阶段及未成熟阶段，故推测其可能参与了Sema3F介导的OPCs迁移的调控。为此，本部分实验拟通过siRNA干扰技术调节plexin A3及NRP2在OPCs中的表达水平，通过干扰其表达后观察Sema3F诱导OPCs迁移的改变，从而证实plexin A3在sema3F诱导OPCs迁移中的作用。首先分别设计了plexin A3及NRP2的siRNA，利用MTT实验检测plexin A3siRNA及NRP2siRNA对OPCs生存能力的影响，确认其无细胞毒性后，采用RT－PCR技术分别检测两种siRNA的干扰效果，达到实验要求后，利用脂质体介导基因转染技术，将plexin A3siRNA及NRP2siRNA分别转染OPCs，通过Transwell迁移仓实验，比较下调plexin A3及NRP2表达后，sema3F诱导OPCs迁移的变化，藉此阐明plexin A3及NRP2在OPCs迁移中的作用。主要实验结果如下：
1. MTT试验结果证实，使用合成的plexin A3siRNA及NRP2siRNA浓度在0.125~2.00μg/μl范围内对OPCs没有明显的细胞毒性作用；
2.脂质体介导plexin A3siRNA及NRP2siRNA转染OPCs后，通过TR-PCR检测，二者能分别有效地抑制OPCs表达plexin A3及NRP2，其抑制效率可达64~79%；
3. Trenswell迁移仓实验证实Sema3F对OPCs具有诱导迁移作用，加入plexin A3siRNA后，OPCs的迁移率明显降低，加入NRP2siRNA后，OPCs的迁移率下降更加明显，两者相比具有统计学差异，提示NRP2-siRNA的抑制效果更加明显。
以上结果说明，sema3F对于OPCs具有诱导迁移作用，plexin A3及NRP2作为sema3F诱导作用的协同受体共同参与了其对OPCs迁移过程的调控，相对于plexin A3而言，NRP2的调节作用似乎更加明显，推测可能与其具有多种协同受体功能有关。
Oligodendrocyte precursor cells (OPCs) are special glial cells which widely distributedin the central nervous system (CNS). OPCs can differentiate and mature intooligodendrocytes (OLs) and astrocytes. The migration and the differentiation of OPCs playa very important role during CNS development, injury repair and functional reconstruction,as well as the occurrence and development of demyelinating disease (such as multiplesclerosis, MS). Recent studies suggested that the demyelination of nerve fibers, as well asinsufficient of myelin repairment and regeneration in CNS are important reasons forrestricting the outcome of damage repair and functional reconstruction after CNS injury,and it was wildly accepted that the successful myelin repair and regeneration may be a keylink to improve this situation. Therefore, studying the mechanism of myelin formation andits regulate factors became a hot issue about injury and repair of CNS. As we all know, themyelin formation in the embryonic period in CNS was mainly by oligodendrocytes, butafter mature, whether oligodendrocytes have the ability to repair and regeneration of myelinor not is not clear. Recent studies found that oligodendrocytes from OPCs differentiationhad the capacity of regeneration and forming myelination. In vitro experiment found thatOPCs can differentiate into OL and form myelin for new nerve fibers in the co-culturesystem of OPCs with embryonic spinal cord tissue; and it was observed that OPCs canmigrate from the spinal cord ventral to dorsal,while some of those cells can differentiateinto OL around the injury lesions in the study of spinal cord injury. Similarly, in the studyof MS, demyelinating lesions surrounded by a large number of NG2positive staining OPCs,and further study found that these NG2-positive cells came from subependymal zone.These studies are the powerful evidence that the OPCs indeed involved in CNS injury andrepair process, and OPCs are likely to play a very important role in this process. Therefore,to study the development regularity, migration mechanisms of OPCs and their relatedfactors becomes a very important content to illustrate the above problems.
Cell migration depends not only on the characteristics of the cells themselves, but alsoon many environmental factors, such as cell matrix proteins, laminin, ion content in theenvironment, and even pH value. In recent years, the particular concern is the cytokineseffect on cell migration, because environmental factors can be controlled and regulated by avariety of measures, and can be stabilized under certain conditions. Cytokines often need tobind with their cellular receptors, and through the series process of intracells, so that theeffect can be accomplished, the mechanisms are more complex and varied; and moreimportantly, the induced migration effect of cytokins have a directivity, the transmission ofthe signal process, the concentration gradient changes of cytokins, etc. can cause thedirectional migration of cells. In early studies, we found that axon guiding factor,Semaphorins family members, can not only induce regenerative axon growth, but alsoinduce the migration of OPCs. Dr. Chen Huanran observed the Sema3A, one of theSemaphorins family members, can induce the migration of OPCs, the mechanism involed insome changes of RhoG signaling pathway molecules. However, does Sema3F, which hasmore obvious effect of axon guidance, have similar effect on OPCs? There was no relatedreport yet. Recent studies found sema3s is involed in the migration of several tumor cells,and when combined with its receptor molecule composite of NP2/plexin A3, sema3F canmediated intracellular signal processes, and induce cell migration. During the process,plexin A3is an important molecule. The plexin A3and neuropilin2(NRP2), as the specificreceptor for sema3F, mediated intracellular signal transduction induced by sema3F, play animportant regulatory role in various cells migration activity. Does Sema3F also inducedOPCs migration? Is its inducing effect also mediated by NRP2/plexin A3receptorcomposite? These questions are still not clear. In the early experiments, authors foundSema3F really induce OPCs directional migration. If the expression of co-operationreceptor composed of plexin A3and NRP2on OPCs is confirmed, undoubtedly, it will be avery important significance to explain the effect of sema3F on OPCs migration andrecognize its mechanism. For this purpose, the experiment was designed and divided intotwo parts: firstly, OPCs were isolated and cultured, and then OPCs differentiation wereinduced, the expression and characteristics of plexin A3on cells during the various stagesof OLs differentiation were detected by immunohistochemistry, immunofluo-rescencestaining, RT-PCR, Western blot, etc. On this basis, the expression level of plexin A3in OPCs was regulated by means of molecular biology technology, and then the changes ofOPCs migration induced by Sema3F were observed. Through the above study, we not onlyclarified the role of plexin A3on OPCs migration induced by Sema3F, but also made someexperimental basis for further studying of the OPCs migration mechanism. The results areas follows:
The first part (chapter2): The expression of plexin A3on differentdifferentiation stages of OPCs.
The neonatal rat OPCs was isolated and cultured in vitro by oscillation separationmethod, then the role of bFGF and PDGF-AA induced OPCs proliferation anddifferentiation was observed, while different differentiation stages of OLs were identifiedby NG2, O4and MBP antibodies; based on this, the expression and characteristics of plexinA3and NRP2in the different differentiation stages of OPCs was detected and analyzed byimmunofluorescence double-labeled staning, RT-PCR, Western blot etc.. The main resultsare as follows:
1. The oscillation separation method is a simple, stable, reliable way to isolatenewborn rats OPCs, high purity OPCs can be obtained by this culture method. The purityand quantity of OPCs can meet the experiment requirement completely through the secondinoculation. bFGF and PDGF-AA can induce and regulate the proliferation anddifferentiation of OPCs, while NG2, O4and MBP immunohistochemical staining canindentify the different differentiation stages of OPCs.
2. In the stages of OPCs, namely NG2+staining positive stage and immature stages(ie, O4+staining positive stage), plexin A3was high expressed, when OPCs differentiatedinto mature OL (MBP+staining positive stage), its expression was significantly lowered.As a co-receptor, NRP2expression characteristics was similar to plexin A3. However,according to the results of Western blot and RT-PCR test，the expression levels of NRP2was slightly higher than those of plexin A3in the OPCs and immature OLs stages ofdifferentiation, presumably it is related that NRP2can combined with a variety ofco-receptors.
3. Plexin A3and NRP2are mainly expressed in NG2-positive cells and O4-positive cells, and located on the cell membrane and protruding of OPCs, suggesting that plexin A3and NRP2may be related with OPCs migration.
The second part (chapter3): The effect of plexin A3on OPCs migration inducedby sema3F
In the first part, our studies confirmed that the plexin A3and NRP2both express onOPCs or immature OLs stage and located on cell membrane and protrusions of OPCs,suggesting their possible involvement in the sema3F mediated OPCs migration. In this part,we proposed to regulate plexin A3and NRP2expression level in OPCs by siRNAinterference technology, and observe OPCs migration changes induced by sema3F, toexplore the role of plexin A3during sema3F induce OPCs migration.
Firstly, the plexin A3siRNA and NRP2siRNA were designed, and then their toxiceffect on the survival of OPCs was assessed by MTT test. After confirming neither ofsiRNAs has cytotoxic on OPCs, siRNA interference effect was detected by RT-PCR. Toachieve the experimental requirements, the plexin A3siRNA and NRP2siRNA wererespectively transfected into OPCs by liposome-mediated gene transfection technology, andOPCs migration induced by sema3F through transwell chamber was observed to clarify theeffect of plexin A3and NRP2on OPCs migration. The main results were as follows:
1. MTT test results confirmed that the concentration of plexin A3siRNA and NRP2siRNA ranged from0.125μg/μl to2.00μg/μl, which have not cell toxic effects on OPCssurvival;
2. When liposome-mediated the plexin A3siRNA and NRP2siRNA transfected intoOPCs, both siRNAs can effectively inhibit plexin A3and NRP2expression in the OPCs byRT-PCR, and their inhibition efficiency can be up to64~79%;
3. Transwell chamber confirmed the sema3F can induced migration of OPCs, andafter plexin A3siRNA was added, the migration rate of OPCs was significantly reduced,when NRP2siRNA was added, the migration rate of OPCs went down more obviously,which have a statistically significant difference with that of plexin A3siRNA. This resultsimpled the inhibitory effect of NRP2siRNA was more obvious than plexin A3.
These results confirmed that sema3F indeed can induce OPCs migration. As co-receptor of sema3F, both of plexin A3and NRP2take part in the regulation of OPCsmigration process, and it seems that NRP2play more important role than plexin A3,speculate the difference may be related with NPR2has a variety of co-receptor function.
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96. Geretti E, Shimizu A, Klagsbrun M. Neuropilin structure governs VEGF and semaphorinbinding and regulates angiogenesis. Angiogenesis,2008,11(1):31-9.
97. Begum R, Nur-E-Kamal MS, Zaman MA. The role of Rho GTPases in the regulation ofthe rearrangement of actin cytoskeleton and cell movement. Exp Mol Med,2004,36(4):358-66.
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101.Wang H, Hota PK, Tong Y, et al. Structural Basis of Rnd1Binding to Plexin Rho GTPaseBinding Domains (RBDs). J Biol Chem,2011,286(29):26093-106.
102.Janssen BJ, Robinson RA, Perez-Branguli F, et al. Structural basis of semaphorin-plexinsignalling. Nature,2010,467(7319):1118-22.
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4. Melendez-Herrera E, Colin-Castelan D, Varela-Echavarria A, et al. Semaphorin-3Aand its receptor neuropilin-1are predominantly expressed in endothelial cells along therostral migratory stream of young and adult mice. Cell Tissue Res,2008,333(2):175-184.
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9. Tong Y, Chugha P, Hota PK, et al. Binding of Rac1, Rnd1, and RhoD to a novel RhoGTPase interaction motif destabilizes dimerization of the plexin-B1effector domain. JBiol Chem,2007,282(51):37215-37224.
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1. Tran TS, Kolodkin AL, Bharadwaj R. Semaphorin regulation of cellular morphology.Annu Rev Cell Dev Biol,2007,23:263-292.
2. Rolny C, Capparuccia L, Casazza A, et al. The tumor suppressor semaphorin3Btriggers a prometastatic program mediated by interleukin8and the tumormicroenvironment. J Exp Med,2008,205(5):1155-1171.
3. Basile JR, Holmbeck K, Bugge TH, et al. MT1-MMP controls tumor-inducedangiogenesis through the release of semaphorin4D. J Biol Chem,2007,282(9):6899-6905.
4. Melendez-Herrera E, Colin-Castelan D, Varela-Echavarria A, et al. Semaphorin-3Aand its receptor neuropilin-1are predominantly expressed in endothelial cells along therostral migratory stream of young and adult mice. Cell Tissue Res,2008,333(2):175-184.
5. Serini G, Valdembri D, Zanivan S, et al. Class3semaphorins control vascularmorphogenesis by inhibiting integrin function. Nature,2003,424(6947):391-397.
6. Kruger RP, Aurandt J, Guan KL. Semaphorins command cells to move. Nat Rev MolCell Biol,2005,6(10):789-800.
7. Antipenko A, Himanen JP, van Leyen K, et al. Structure of the semaphorin-3A receptorbinding module. Neuron,2003,39(4):589-598.
8. Yaron A, Huang PH, Cheng HJ, et al. Differential requirement for Plexin-A3and-A4in mediating responses of sensory and sympathetic neurons to distinct class3Semaphorins. Neuron,2005,45(4):513-523.
9. Tong Y, Chugha P, Hota PK, et al. Binding of Rac1, Rnd1, and RhoD to a novel RhoGTPase interaction motif destabilizes dimerization of the plexin-B1effector domain. JBiol Chem,2007,282(51):37215-37224.
10. Toyofuku T, Yoshida J, Sugimoto T, et al. FARP2triggers signals for Sema3A-mediated axonal repulsion. Nat Neurosci,2005,8(12):1712-1719.
11. Wu KY, Hengst U, Cox LJ, et al. Local translation of RhoA regulates growth conecollapse. Nature,2005,436(7053):1020-1024.
12. Schmidt EF, Strittmatter SM. The CRMP family of proteins and their role in Sema3Asignaling. Adv Exp Med Biol,2007,600:1-11.
13. Oinuma I, Katoh H, Negishi M. Semaphorin4D/Plexin-B1-mediated R-Ras GAPactivity inhibits cell migration by regulating beta(1) integrin activity. J Cell Biol,2006,173(4):601-613.
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16. Swiercz JM, Worzfeld T, Offermanns S. ErbB-2and met reciprocally regulate cellularsignaling via plexin-B1. J Biol Chem,2008,283(4):1893-1901.
17. Potiron VA, Sharma G, Nasarre P, et al. Semaphorin SEMA3F affects multiplesignaling pathways in lung cancer cells. Cancer Res,2007,67(18):8708-8715.
18. Renaud J, Kerjan G, Sumita I, et al. Plexin-A2and its ligand, Sema6A, controlnucleus-centrosome coupling in migrating granule cells. Nat Neurosci,2008,11(4):440-449.