Study On The Role Of Mesenchymal Stem Cells Transplantation To Rats With Experimental Autoimmune Enphalomyelitis

Objective:Acute disseminated encephalomyelitis?ADEM?is a commonly inflammatory demyelinating disease of the central nervous system?CNS?in the period of children.Experimental autoimmune encephalomyelitis?EAE?is a demyelinating disease of the central nervous system mediated by cell immunity and humoral immunity.At present,the exact etiopathogenisis and pathogenesy still are not understood.ADEM can induce permanent sequelae of CNS.In the focus of infection of the ADEM,oligodendrocytes----the main affected cells show lost differently degree.The chief function of the oligodendrocyte is to make of myelin.The reparation of the impaired myelin is the reparation of oligodendrocyte basically.These are our treatment strategy that how to subdue the damage of the oligodendrocyte,how to promote endogenic oligodendrocyte to different to functional oligodendrocyte and how to precipitate the recovery of the damage in the CNS by cellular transplant.The anti-two treatment strategy have some clinical therapeutic effect,but still not satisfactory.Nearly decade years,followed with the deeply study on the nerve stem cells,cell transplantion suffered more and more reconstruction.The clinical application of the nerve stem cells from embryo or adult was restricted,the reason were difficult drawing materials,scarcely tissue source,immunological rejection during the process of the transplantion,implantation technique,the dispute of ethics,and law confinement.Bone marrow mesenchymal stem cells become the most outlookly treatment strategy in the damage recovery of the CNS due to generally resource,facility for drawing the materials,easy to autoplastic transplantation,holding the latent energy of self replication and multi-directional differentiation during the process of cultivation all the time,poorly immunogenicity,and it can create active effect by transplantation through regional or intravenous route.The main source of MSCs is the bone marrow.Apart from the bone marrow,MSCs are also located in other tissues of the human body.MSCs posses an extensive proliferation potential and ability to differentiate into various cell types,including:neurons,nervous glial cell,osteocytes,adipocytes,chondrocytes,matrix cell,skeletal muscle cell,vascular endothelial cell,endodermal denticells.At the condition of no inducing factor,MSCs remain same morphous and phenotype after 70 dissociates,and still possess extensive proliferation potential.MSCs'source is adequacy,easy to amplification in vitro,unexpress major histocompatibility complex?,can be tolerance by receptor.Moreover,it can come from self,avoiding immunological rejection.All of those make MSCs become hot spot in the domain of stem cells research.MSCs transplantation show effect on the treatment of myocardial damage,caitilage and bone damage and so on.In the experimentation treatment of neurological diseases such as spinal cord injury,cerebral infarction,those findings show clinical symptoms have be improved.MSCs facilitate axon growth,different into nervous cells,enhance the generation and differentiation of endogenous nervous precursor cells.At the treatment of MSCs transplantation to experimental autoimmune encephalomyelitis?EAE?,the clinical symptom of the rat in the treatment group was improved,but the mechanism is still not known.The aim of the present research is to deploy the classic animal model for ADEM,at the base of stating oligodendrocyte distribution and myelination in normal and EAE rat,transplanting MSCs in culture to EAE rat through lateral cerebral ventrical injection,is to explain initially the treatment machine of MSCs transplantation to EAE by means of clinical symptom,pathological,the change of oligodendrocyte,the expression of myelin transcription factor and myelin basic protein.Methods:1.Choosing the 6⁸ weeks Wistar rats were employed.Wistar rats were inoculated intradermal injection with the emulsion homogenate contained spinal cord of guinea pig and complete Freund's adjuvant in feet.2.Wistar rat weighed 100g was anesthesiaed,femoral bone were drawn asepsisly.Cavitas medullaris were bathed by DMEM-F12 culture fluid.Cell suspention was inoculation to culture flask,cultured at37??5%CO₂.Unattached cells were deserted at 24 hours.Culture fluid was changed every 2³days.Cells were passaged when cells coalesce achieve 80%.Cells were identified by flow cytometry sorting.3.All the animals were divided 4 groups:normal control,EAE,notho-treatment,MSCs treatment groups.4.Infusing and affixing through aortic ventrical of heart,conservation in 4%paraform for myelin staining,conservation in-80?icebox after 20%cane sugar 24 hour after 4%paraform 24 hour for immunofluorescence,conservation in-80?icebox for western-blot and real-time PCR.5.Thechange of clinical symptom was observed by praxiology score,pathological change of myelin was observed by Bielschowsky silver staining,the change of oligodendrocyte lineage cells by immunofluorescence,the express of MBP mRNA,MyT1 mRNA by Real-time PCR,the express of MBP,MyT1 by Western-Blot.Results:1.Changes in clinical behavior:The clinical behavioral score of EAE group was the highest on 1d,3d and 7d,and decreased on 14d,and the lowest on 21d and28d.The changes of the sham treatment group and EAE model group were basically the same.The clinical behavior score of the MSCs treatment group was highest at 1d,decreased significantly from 3d,and returned to normal at 7d?the score dropped to 0?.2.Pathological changes of myelin sheath:The structure of cerebrospinal sheath was intact in normal group.On day 1 of EAE group,the destruction of myelin was worse,and the worst was on day 3 and 7,but on day 14,21 and 28,it recovered somewhat,it did not reach the level of the normal group.The change trend of myelin damage in the sham treatment group was the same as that in the normal group.The myelin sheath was significantly damaged on 1d in the MSCs treatment group,and gradually recovered from 3d.The subsequent time points were significantly improved compared with EAE group?p<0.05?.3.Changes in oligodendrocytes lineage:The progenitor cells of oligodendrocytes?A2B5?and immature oligodendrocytes?O4?can be seen in the cerebral cortex,white matter and cerebellum.White matter is the most abundant,especially in the hippocampus.Mature oligodendrocytes?CNPase?are mainly seen in white matter.At 1d of EAE model group,the number of positive cells of A2B5 and O4 was not significantly changed,while the number of positive cells of CNPase was significantly reduced.The number of A2B5,O4 and CNPase positive cells was significantly decreased at 3d and 7d,compared with the 1d group.The number of A2B5,O4 and CNPase positive cells increased at EAE14d,21d and 28d.The variation trend of the sham treatment group was the same as that of the EAE model group?p>0.05?.There was no significant change in the number of positive cells of A2B5,O4and CNPase at 1d in the MSCs treatment group,and the number of positive cells of A2B5,O4 and CNPase gradually increased after 3d,reaching a peak at 14d.4.Changes of MBP mRNA and MyT1 mRNA expression levels:The mRNA expression of MBP and MyT1 in the EAE group at 1d had no significant change compared with the normal group.MBP and MyT1 mRNA expression increased at 3d and 7d,compared with 1d group.On EAE 14d,21d and 28d,MBP and MyT1 mRNA expression gradually increased.There was no significant difference in the expression of MBP and MyT1 mRNA between the sham treatment group and the EAE group?p>0.05?.MBP and MyT1 mRNA expression increased at 1d in the MSCs treatment group compared with EAE group,and MBP and MyT1 mRNA expression increased significantly at 3d,7d,14d,21d and 28d after treatment?p<0.05?.5.Changes in MBP and MyT1 protein expression levels:At 1d of EAE group,the MBP and MyT1 protein expression in rats was not significantly different from that in the normal group,while at 3d and 7d,the MBP and MyT1 protein expression gradually increased compared with that in 1d group.At EAE14d,21d and 28d,MBP and MyT1 expression gradually increased.There was no significant difference in MBP and MyT1 protein expression between the sham treatment group and the EAE group?p>0.05?.At 1d of MSCs treatment group,MBP and MyT1 protein expression increased compared with EAE group.The expression of MBP and MyT1 was significantly increased on 3d,7d,14d,21d and 28d?p<0.05?.Conclusion:1.The structure of myelin sheath in the brain was intact at normal 6 to 8weeks,and more oligodendrocytes were distributed more than in the brain.Under normal conditions,MBP mRNA and MyT1 mRNA were less expressed,MBP content was higher,and MyT1 expression was less.2.The preparation of EAE model is relatively stable.At the early stage of EAE,the destruction of myelin in the brain is heavy.In the middle and late stage of disease course,the myelin damage is restored to a certain degree.There were different degrees of proliferation and differentiation of oligodendrocytes in the brain.MyT1 mRNA and MBP mRNA expression increased,while MyT1 and MBP protein expression increased.3.Bone marrow mesenchymal stem cell transplantation between clinical behavior of EAE rats have improved significantly,the myelin sheath of EAE rats damage repair effect significantly,between bone marrow mesenchymal stem cell transplantation,less glial progenitor cells increased significantly,between bone marrow mesenchymal stem cell transplantation promotes the MBP mRNA,MyT1 mRNA expression of bone marrow mesenchymal stem cell transplantation promotes the MBP,MyT1 protein expression.