Sema3A Guiding Oligodendrocyte Progenitor Cells Migration And Its Signal Transduction Mechanism

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.