Relativity Between Regulation Of MAP1B's Dephosphorylation By PP2A And BMSCs Migration After SCI

【objective】Axon guidance and upregulated of MAP1B after SCI are utilized as the topicresearch background. Based on fluorescent tags, western blotting, andimmunocytochemistry, we focus on the relativity between phosphorylated level ofMAP1B and migration of BMSCs towards injured spinal cord, especially on detectingand analyzing the signaling pathways and molecular mechanisms for the migrationregulated by MAP1B, which was designed to verify the correlation betweenMAP1B and BMSCs migration and tentatively elaborate the relevant signalingmechanisms which could provide an important theoretical basis to improve BMSCstherapy effect for SCI.【Methods】Part I: To establish an optimal culture system for cultivating the bone marrowmesenchymal stem cells (BMSCS) of rabbit in vitro and via microscopicmorphological observation, the best cell was chosen to analyze BMSCs activity. Todraw a cellular growth curve by MTT colorimetry, and analyze the expression of cellsurface antigens and cell cycle by flow cytometry and antigen-antibody reaction.Part II: Fluorescent dye CM-DIL to label rabbits BMSCs in vitro was used, afterwhich determinating the labelling rate by flow cytometry, analyzing and comparingBMSCs activity by MTT colorimetry and growth curve. The highest fluorescentlylabeling rate and the best growth activity of rabbit BMSCs were selected to pre-dealwith OA and C2-ceramide, and cells were divided into the inhibitor group, agonistgroupand control group, respectively. PP2A inhibitor OA and PP2A agonistC2-ceramide could regulate activity of PP2A directly and change phosphorylationlevels of MAP1B indirectly, after that analyzing and comparing BMSCs activity byMTT colorimetry again were proceeded. And PP2A activity via isotope-labeledenzyme activity assay and MAP1B phosphorylated levels via western blot weredetected. Part III: Animal models about experimental SCI via blocking segmental lumbarartery for25minutes was established. Based on modified Tarlov score, the behavioraldifference of animal models from the surgery group and the sham group wereobserved and compared before live sampling at2, and7days after surgery. Animalethology and pathological morphological observation could validate the model whichwas established successfully. The surgical group models are divided into three groupsand transplantated with BMSCs via the marginal ear vessels of the rabbits.Respectively, each group will be lively sampled at2,4and7days aftertransplantation and observed by using laser confocal microscope to detect fluorescentmarker of BMSCs in spinal cord.Part IV: To use inhibitors to block the PI3K and ERK pathway in rabbit BMSCsand detect MAP1B phosphorylation level of BMSCs via immunoblotting, themodification function of regulating the phosphorylation of MAP1B was analyzed.【Results】Part I: The rabbit BMSCs cultured system was established successfully.Comparing the different passages of BMSCs's morphology, there was no distinctdifference.The BMSCs harvested showed absence of CD34, CD45and the greatmajority of which were at the G0/G1point in the cycle. The detection of BMSCs'proliferation has shown that BMSCs accumulat greatly in the period of logarithmicphase for3-5days.Part II: The labeling yield of CM-Dil is more than99%on the condition that thecells' proliferation wasn't affected. Regulation of OA and C2-ceramide, however,made the activity of PP2A drop to39%and increases to178%, respectively, comparedwith that in the untreated group (p<0.05). The phosphorylation of BMSCs wasaffected accordingly by the C2-ceramide PP2A activity, according to immunoblotting.However, the changes above were back to normal at48hours after pretreatment(p>0.05). BMSCs' proliferation was affected by C2-ceramide and OA, the growthcurve of BMSCs showed that the BMSCs of AG group still kept the most of growingability.Part III: Rabbit ischemic spinal cord injury model was successfully established,and the assessment of hindlimb locomotor function have shown that surgery groupwas2.7~3.2points (P<0.01). After ischemic injury, pathomorphological observationsshowed that the majority of nuclear membrane is integral and available, and the nuclei was slightly swelling. Part of the nuclear was translocation, deeply stained;condensation, fragmentation and the neurite reduce or disappear. Via transmissionelectron microscopy(TEM), rough endoplasmic reticulum and mitochondrial werefound swelling, and part of the mitochondrial membrane ruptured. Wide gapsappeared between layers of myelin shea. The cavitation and other pathologicalchanges were found in nuclear matrix. By laser scanning confocal microscope, theBMSCs in the injured spinal cord was observed and the difference between inhibitiongroup and agonists group, comparing to untreated group, was recorded. At the3rddayafter transplantation, there was no BMSCs in the injured spinal cord in the inhibitiongroup and agonists group, which was different until the fifth day.Part IV: Compared with the control group, inhibition of PI3K resulted in higherphosphorylated levels of MAP1B, while inhibition of ERK led to lowerphosphorylated levels of MAP1B.【Conclusion】BMSCs were successfully cultured and maintained proliferation in theexperiment, which successfully established the rabbit segmental ischemic spinal cordinjury model. BMSCs that were transplanted have successfully migrated into theinjured spinal cord which could be affected by phosphorylated level of MAP1B. Asincreasing or reducing of P1-MAP1B could break the dynamic equilibrium ofMAP1B/P1-MAP1B, the number of BMSCs within the injured spinal cord will befallen off. All of these factors have shown that dynamic equilibrium of MAP1B playsan important role in the regulation of BMSCs migration. PI3K, ERK and PP2A werein charge of the dynamic equilibrium and participated in the signal transductionpathways which regulate MAP1B phosphorylation.【Innovation】The paper innovatively relates the axon guidance of MAP1B and itsphosphorylated I type with BMSCs migration. And it proves that the form ofMAP1B and its phosphorylated I type can play an important role in the process ofBMSCs migration by experiment. Maintaining homeostasis between MAP1B andP1-MAP1B is essential for BMSCs athletic ability, which provides a new theoreticalbasis for the MAP1B protein function. There has not been reported about relativitybetween MAP1B and BMSCs migration ability.In the past, MAP1B was mainly as a neural differentiation marker proteins of BMSCs. For the first time, we aims at studying the signal transduction pathways ofthe regulation of MAP1B phosphorylation in BMSCs, analyzing the signalmechanism of MAP1B in the cell migration-oriented function in BMSCs, enrichingand perfecting the migration signal transduction pathway of BMSCs migration.