SDF-1/CXCR4 Induced Migration Of Bone Marrow Mensenchymal Stem Cells To Injured Spinal Cord Via PI3K/Akt Pathway

ObjectsTo investigate the effect of SDF-1/CXCR4 induced migration of bone marrow mensenchymal stem cells (BMSCs) to injured spinal cord via PI3K/Akt pathway in rats.Methods1. Isolated BMSCs from bone marrow of rat by density gradient centrifugation with percoll, and transfered these cells from generation to generation. Identified these cells with immunohistochemistry (Sreptavidin-biotin complex, SABC)2. Built model of rat with acute spinal cord injury by Allen’s method.3. Detected gene of CXCR4 and PI3K in BMSCs with Real-time PCR.4. Detected expression of CXCR4 and PI3K in BMSCs with Western Bolt5. Took Transwell migration experiment as the method of studying migration of BMSCs induced by SDF-1/CXCR4 in vitro, and got the peak concentration of SDF-1. Groups were divided into negative control group (cell suspension of BMSCs in upper room, serum culture medium without SDF-1 in lower room), experimental group (cell suspension of BMSCs in upper room, serum culture medium with SDF-1 of different concentration in lower room), CXCR4 blocked group (cell suspension of BMSCs pre-treated with AMD3100 in upper room, serum culture medium with SDF-1 of different concentration in lower room), and reagent control group (cell suspension of BMSCs pre-treated with AMD3100 in upper room, serum culture medium without SDF-1 in lower room). Studied the migration of BMSCs to injured spinal cord in vivo by compared the recoveries of spinal cord function of model rats with different treatments. Groups were divided into blank control group (no treatment given), negative control group (injected normal saline through caudal vein), AMD3100 control group (injected AMD3100 through caudal vein), BMSCs transplanted group (injected BMSCs through caudal vein), and CXCR4 blocked group (injected BMSCs pre-treated with AMD3100 through caudal vein).6. Took Transwell migration experiment as the method of studying the effect of PI3K/Akt pathway in the migrating of BMSCs induced by SDF-1/CXCR4 in vitro. Groups were divided into negative control group (cell suspension of BMSCs in upper room, serum culture medium without SDF-1 in lower room), AMD3100 control group (cell suspension of BMSCs pre-treated with AMD3100 in upper room, serum culture medium without SDF-1 in lower room), LY294002 control gourp (cell suspension of BMSCs pre-treated with LY294002 in upper room, serum culture medium without SDF-1 in lower room), CXCR4 blocked group (cell suspension of BMSCs pre-treated with AMD3100 in upper room, serum culture medium with SDF-1 in lower room), PI3K blocked group (cell suspension of BMSCs pre-treated with LY294002 in upper room, serum culture medium with SDF-1 in lower room), and exogenous PIP3 added group (cell suspension of BMSCs pre-treated with AMD3100 and exogenous PIP3 in upper room, serum culture medium with SDF-1 in lower room). Studied the migration of BMSCs to injured spinal cord by compared the recoveries of spinal cord function of model rats with different treatments. Groups were divided into blank control group (no treatment given), negative control group (injected normal saline through caudal vein), AMD3100 control group (injected AMD3100 through caudal vein), LY294002 control group (injected LY294002 through caudal vein), BMSCs transplanted group (injected BMSCs through caudal vein), CXCR4 blocked group (injected BMSCs pre-treated with AMD3100 through caudal vein), PI3K blocked group (injected BMSCs pre-treated with LY294002 through caudal vein), and exogenous PIP3 added group (injected BMSCs pre-treated with AMD3100 and exogenous PIP3 through caudal vein).7. Means were showed with x±s in this study. Analysis of variance (ANOV) was used to compare difference among more than two groups, q-test was used to compare difference between two groups. All data were treated with Statistic Package for Social Science (SPSS) 17.0. Results were accepted wiht P<0.05.Results1. Cytomorphology and results of immunohistochemistry of BMSCs:typical cytomorphology of BMSCs was similar with fibrocyte, long spindle or polygon with long or short cytoplasm protuberant. Cells gathered to colonies, which were radial or gyrate, in early growth period. Cell colonies became different to identify with the increase of cell number. CD34-、CD44+、CD54+、CD99+ were the results of immunohistochemistry.2. Rat model of acute spinal cord injury built by Allen’s method:spinal cord of rat was hit with the energy of 70g-cm. When its spinal cord was hit, limbs and body of the rat shocked and its tail wiggled. Spinal cord changed to dark red with bleeding and edema 3 to 5 minutes later. One week later, the BBB scores of model rats declined from 18.73±0.85 pre-operation to 3.04±0.65 post-operation, the difference was significant (P<0.05). 3. The existences and expressions of CXCR4 and PI3K genes in BMSCs:the existence of CXCR4 and PI3K genes were detected with real-time PCR, changes of cycle threshold (ΔCT) were 16.47 and 13.43. Expressions of CXCR4 and PI3K were detected with western blot.4. Migration of BMSCs to injured spinal cord induced by SDF-1/CXCR4:AMD3100 and was proved to have no influence to experiment (P>0.05). In vitro experiment, more cells migrated to lower room in experimental group compared with negative control group (P<0.05), and cell number increased with concentration of SDF-1. Best effect was observed when SDF-1 reached the concentration of 144.5±4.4ng/ml. SDF-1 didn’t induce the migration of BMSCs when CXCR4 was blocked with AMD3100.5. Influence of PI3K/Akt pathway to the migration of BMSCs to injured spinal cord induced by SDF-1/CXCR4:AMD3100 and LY294002 were proved to have no influences to experiment (P>0.05). In vitro experiment, less cells migrated to lower room in CXCR4 blocked group and PI3K blocked group compared with SDF-1 control group (P<0.05). However, there was no difference between exogenous PIP3 added group and SDF-1 group (P>0.05). In vivo experiment, recoveries of spinal cord function model rats in CXCR4 blocked group and PI3K blocked group was similar with each other (P>0.05), both were between that in BMSCs transplanted group and negative group (P<0.05). Recoveries of spinal cord function model rats in exogenous PIP3 added group were the same as that in BMSCs transplanted group (P>0.05).Conclusion1. BMSCs of higher purity can be isolated from bone marrow of rat by density gradient centrifugation with percoll.2. Rat model of acute spinal cord injury can be build with Allen’s method.3. SDF-1/CXCR4 can induce the migration of BMSCs to injured spinal cord.4. Best effect of migration can be obtained when the concentration of SDF-1 reach 144.5ng/ml. 5. PI3K/Akt pathway is the downstream mechanism of migration of BMSCs to injured spinal cord induced by SDF-1/CXCR4.