The Experimental Study Of Plexin A3Involved In Oligodendrocytes Precursor Cells Migration

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 sema3FIn 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.