The Influence Of CXCL12/CXCR4 On Rat Oligodendrocyte Precursor Cells Migration And Its Mechanism

Background and AimsDuring development and the repair of spinal cord injury, Oligodendrocyte Precursor Cells(OPCs) which are the Major sources of oligodendrocytes are guided by several cues(such as PDGF and netrin-1) as they follow distinct migratory routes to target axons of the CNS, where they differentiate into myelinating oligodendrocytes. A large number of studies have shown that CXCL12 may regulate survival and outward chemotactic migration of OPCs during embryonic and postnatal CNS development and the repair of spinal cord injury by regulating CXCR4, and other studies have found that CXCL12/CXCR4 do not regulate the migration of OPCs. But it has reported that CXCL12/CXCR4 may control chemotaxis and survival in human liver cancer cells and Mature Dendritic Cells through AKT and MEK1/2 signal. Therefore, the influence of CXCL12/CXCR4 on rat oligodendrocyte precursor cells migration and its mechanism is far from clear. In this experiment, we explored the influence of CXCL12/CXCR4 on rat oligodendrocyte precursor cells migration and the important role of AKT and MEK1/2 pathway.Methods1. OPCs were seperated from the cerebral cortices of 48-hour neonate Sprague-Dawle rats by the shaking process and differential adhesion,and then the separated OPCs were identified and analyzed by the immunocytochemical technique. OPCs were positively identified by immunostaining for PDGFR-α. OPCs were differentiated to oligodendrocytes, and then oligodendrocytes were positively identified by immunostaining for O4 and MBP.2. The migration of rat OPCs which were cultured under different concentrations of CXCL12(0 ng/ml、5 ng/ml、10 ng/ml、20 ng/ml)for 48 hours was assayed using boyden chamber, and the expressions of CXCR4, ERK, p-ERK, AKT and p-AKT proteins were investigated by Western blotting.3. The expression of CXCR4 protein was inhibited by CXCR4 sh RNA in rat OPCs, and the expressions of CXCR4, ERK, p-ERK, AKT and p-AKT proteins were investigated by Western blotting, then the migration of rat OPCs was studied by boyden chamber.Respectively through LY294002 blocking PI3 K pathway, U0126 locking MEK1/2pathway, and LY294002 and U0126 blocking both PI3 K and MEK1/2 pathways at the same time, the expression of ERK, p-ERK, AKT and p-AKT protein was detected by Western blotting, and The effect of CXCL12 on the migration of rat OPCs was detected by Boyden chamber assay.Results1. A large number of OPCs were isolated through the shaking separation and differential adhesion. The body of OPCs is nearly circular. And there was two or three slender bipolar processes around the body of OPCs. The PDGFR-α positive OPCs accounted for 95 % of the isolated cells. After 4-5 days in the condition medium, most of the OPCs were defined by O4+ and characterized by more slender bipolar processes. After 6-7 days in the condition medium, most of the OPCs gradually differentiated into mature oligodendrocytes, which were characterized by the complex profile of “ramificated” or “cobweb-like” processes reticulating in their periphery and defined by MBP+.2. With the increase of the concentration of CXCL12, the migration of OPCs was ever-increasing. And the migration of OPCs was increased by CXCL12 with a maximum at 20 ng/ml CXCL12(p<0.05), and increased about 3.64 times compared with control group. With the increase of the concentration of CXCL12, the expression of CXCR4、p-ERK and p-AKT proteins was ever-increasing. And the expression of those proteins was increased by CXCL12 with a maximum at 20 ng/m L CXCL12(p<0.05), but the expression of ERK and AKT proteins was not increased(p >0.05).3. Compared with the control group, the influence of CXCL12(20ng/ml) on rat OPCs migration was suppressed after the expression of CXCR4 protein was obviously inhibited by CXCR4 sh RNA(P<0.05), and the expression of p-ERK and p-AKT proteins was also obviously inhibited(P<0.05, P<0.05).4. The effect of CXCL12 on migration of rat OPCs was significantly inhibited after PI3 K pathway blocked by LY294002 or MEK1/2 signaling pathway blocked by U0126. When using LY294002 and U0126 blocking both PI3 K and MEK1/2 pathways at the same time, the effect of CXCL12 on migration of rat OPCs was more obviously inhibited(P<0.01).Conclusion1. A large number of OPCs were isolated through the shaking separation and differential adhesion.2. CXCL12 could promote the migration of rat OPCs and the expression of CXCR4, p-ERK and p-AKT proteins in concentration dependence manner.3. The influence of CXCL12/CXCR4 on rat OPCs migration and the expression of CXCR4, p-ERK and p-AKT proteins could be suppressed by CXCR4 sh RNA.4. The influence of CXCL12/CXCR4 on rat OPCs migration and the expression of CXCR4, p-ERK and p-AKT proteins could be suppressed by PI3 K pathway blocked by LY294002 or MEK1/2 signaling pathway blocked by U0126. CXCL12/CXCR4 may regulate the migration of the OPCs through MEK1/2 and PI3 K pathway.