The Study Of Myelin Repair And Emotional Changes Of Demyelination Model Promoted By BM-MSCs

Background:The myelin sheath of central nervous system(CNS)was defined as the helical membranous structure with effects of protecting neuraxon,conducting nerve impulses and insulation,which was formed by lamellar processes of oligodendrocytes around neuron axon.Multiple sclerosis(MS)is a kind of autoimmune diseases,the main pathological features of which is the demyelination of the white matter,was more likely to happen at young and middle-aged people.MS had multiple distribution in both space and time,caused severe nerve functional defects,different extent of physical and psychological disorders and high level of disability rate,which brought both physical and mental harm to patients.MS patients were likely to have mental symptoms such as depression,anxiety and irritability,the living quality and compliance of treatment of which were severely damaged,so the MS patients with mental symptoms(depression,anxiety)brought more and more attention of academic and doctors.Plenty of researches suggested that the mechanism of MS combined with depression and anxiety included encephalatrophy,inflammatory cytokines drugs,cognitive impairment and the social psychological factors.At present,the clinical treatment of MS were mostly glucocorticoids,high doses of immunoglobulin,beta interferon and immunosuppressants,and the treatment of MS combined with anxiety or depression were Anti-anxiety medication and psychological treatment,therefore how to promote myelin regeneration and repairing nervous function,improve emotional changes and reverse the progress of the disease was crucial for the treatment of MS combiend with of anxiety and depression.Nowadays,stem cell transplantation was one of the most promising approaches of regenerative medical researches,so bone marrowed-mesenchymal stem cells(BM-MSCs)has become the research focus because of its biological characteristics and clinical potential.BM-MSCs,known as a pluripotent stem cell population,was different from bone marrow hematopoietic stem cells.BM-MSCs was easy to get in vitro,had stronger proliferation ability,multiple differentiation potential and less obvious surface marker antigen,could remain stem cell properties after several times of subcultivation and weak immune rejection after transplataiton,these characteristics made BM-MSCs applicable for treating autoimmune diseases,diseases of the nervous system,blood,bone and cartilage diseases,as well as liver failure.Large amount of studies have shown that BM-MSCs could treat MS,but the mechanism was still controversial,mainly by regulating the immune response.Presently,one of the most heated issue about BM-MSCs-transplantation therapy was which one's therapeutical effect was better,autologous transplantation or allograft transplantation? The autologous transplantation of BM-MSCs after amplification in vitro could avoid tissue matching,immunological rejection and medical ethical problems,but it was important to consider whether the BM-MSCs of patient itself was affected by the pathological conditions,thus reducing the effect for the treatment of disease.Theoretically,BM-MSCs had low immunogenicity and immune negative regulatory effect,as well as induction of allograft immune tolerance,which played important role in tissue regeneration and repairing functions by allograft transplantation,but there were few report concerning contrast research of effects of different sources of BM-MSCs on myelin repair.Cuprizone(CPZ)was a kind of selective copper ion chelating agent,it could damage mitochondria of oligodendrocytes targetedly,made oligodendrocytes dead and induced demyelination of CNS,which could be used as induced factor to set up demyelination animal model.The biggest difference between CPZ induced demyelination model and Experimental allergic encephalopalomyelitis(EAE)was that in the process of induction the complicated reactions of immune system were not involved.Moreover,some academics chose CPZ induced chronic demyelination model as a relevant model of mental disorders.Therefore,we could use CPZ induced demyelination model and some immunohistochemical,molecular biological,pathological and behavior research toobserve the effect of normal BM-MSCs on myelin repair and emotional changes of CPZ-induced model mice,the changes of neuro-transmitters,and the differences of repair effects between different sources of BM-MSCs,so as to find out the better one.As well,we disscuss the mechanism by the way of secretome to provide theoretical and practical bases for clinical cell therapy.Part One: The experimental study of myelin repair and emotional changes of CPZ-induced demyelination model promoted by BM-MSCsMethods and results1.materials and methods:1.1 Seventy 8 week-old C57 BL /6 mice were divided into 2 groups: the normal control group and experimental group(the chronic demyelination group,the remyelination group,cell therapy group).The normal group was given normal diet,while the experimental group was given 0.2% Cuprizone mixed diet for 12 weeks,afterwards,chronic demyelination group was given the same diet continully,the remyelination group and the therapy was given normal diet,all for 2 weeks,meanwhile the therapy group was injected BM-MSCs 13 th week.1.2 Extract BM-MSCs from bone marrow of juvenile C57 BL /6 mice by the whole bone marrow culture method and density gradient centrifugation,cultivate,identify and transplant the cells.1.3 Measure the bodyweights 2 times every week to observe and record the changes.1.4 Detect the behavioral changes by open field test,elevated plus maze and tail suspension test.1.5 Observe the demyelination of corpus callosum areas by Lucas fast blue staining,MBP immunofluorescence,and electron microscope;measure the changes of astrocytes in corpus callosum and cortex areas by GFAP immunofluorescence.1.6 Measure the concentration of the monoamine neurotransmitters by enzyme-linked immuno sorbent assay(ELISA)and high-performance liquid chromatography-electrochemical detection(HPLC-ECD).2.Results2.1 BM-MSCs could be attached after primary cultivation for 24-48 hours,the cell phenotypic markers CD29,CD90 of BM-MSCs were immunofluorescence stained after the 2nd generation with the positive rate could reach more than 90%.After transplantation BM-MSCs could migrated towards demyelination areas.2.2 Compared with the normal group,the bodyweight of the experimental group increased slowly at earlier stage,while the bodyweight of the remyelination(29.93±2.74)g and the therapy group(32.33±2.96)g increased abruptly after 15 th week.Compared with the demyelination group(27.98±3.31)g,the bodyweight differences of the normal group(33.85±1.70)g and the therapy group showed statistically significance(P<0.05).2.3 Behavioral experiment results: the ratio of central distance(CD)in open field test,the percentage of open arm time(OT)and open arm entries(OE)represented the degree of anxiety,while the tail hanging inactive time(HT)in tail suspension test represented the degree of depression.We found that CD of the demyelination group:(0.07±0.03);OT:(15.50±5.03)%;OE(14.10±6.54)%;HT(170.08±21.69)s,as controls,CD of the normal group:(0.12±0.03);OT:(25.92±5.43)%;OE(31.33±5.69)%;HT(121.33±38.66)s,after treatment,CD of the therapy group(0.11±0.04);OT:(21.64±3.42)%;OE(26.54±6.49)%;HT(134.67±37.49)s.These data suggested that demyelination group present obvious depressed or anxious behaviors(P<0.05),which could be improved in therapy group(P<0.05).2.4 MBP and LFB staining and electron microscope results showed that the loss of myelin sheath in the corpus callosum area of the experimental group(OD value:0.14±0.07)were significant(P<0.01)compared with the normal group(OD value:0.29±0.09),while the remyelination group(OD value:0.21±0.09)and the therapy group(OD value:0.25±0.08)were improved than the demyelination group(P<0.05),of which the therapy group was better(P<0.05);GFAP immunofluorescence results showed that the number of astrocytes in corpus callosum area(78.12±15.47)V.S(21.56±10.63)and cortex area(101.15±23.30)V.S(34.67±12.25)of experimental group increased compared with the normal group(P<0.05),the expression level of GFAP in therapy group was declined compared with the demyelination and remyelination group.2.5 HPLC-ECD and results showed that the concentration of NE,5-HT and 5-HIAAof the normal group were 1.32±0.92ng/L,126.04±22.43ng/L,27.26±3.17ng/L,the concentration of the demyelination group were 2.82±1.01ng/L,159.68±23.06ng/L,43.89±6.59ng/L,which had obvious difference(P < 0.05).The concentration of 5-HT(27.77±5.81ng/L),5-HIAA(1.10±0.76ng/L)of therapy group was lower than that of the demyelination group(P<0.05).ELISA results showed that the concentration of NE(23.82±19.65 V.S50.83±17.64)and 5-HT(39.72±19.16 V.S99.30±29.11)of the normal group and the demyelination group were significantly different(P < 0.05),the concentration of NE(25.58±19.33)and 5-HT(54.83±18.41)of the therapy group were lower than that of demyelination group.3.ConclusionFrom the aspects of behavior and pathological changes,confirmed cuprizone-induced demyelination model is feasible and successful.The demyelination model mice induced by CPZ has behavior changes such as anxiety and depression,which maybe connected with the demyelination of central nervous system and the changes of neurotransmitters.This model is suitable as the study carrier of MS with anxiety and depression,transplatation of BM-MSCs can promote remyelination and emotionl improvements of demyelination model.Part Two: The comparative study of myelin repair of different sources of BM-MSCs Methods and results1.materials and methods:1.1 Forty 8 week-old C57 BL /6 mice were divided into 2 groups: the normal control group and experimental group(the normal cell therapy group(human or mice),the sick cell therapy group(CPZ cells or MS cells),the demyelination group),the method of model induction and cell therapy as above.1.2 Extract BM-MSCs from bone marrow of human by the density gradient centrifugation,cultivate,identify and transplant the cells.1.3 Detect whether CPZ medication had toxic on MSCs of mice with cell proliferation-toxicity kit(CCK-8);evaluate whether there was difference between the proliferation ability of MSCs from healthy volunteers and MS patients.1.4 Take behavioral and pathological experiment 2 weeks after transplatation to investigate whether there was difference between the remyelination and funcion recovery ability of MSCs from different sources.1.5 Analyze the HGF level of cell culture supernatant and cells quantitatively by western blotting(WB).2.Results2.1 Human BM-MSCs could be attached after primary cultivation for 24-48 hours,detect the phenotype by flow cytometry in the 2nd generation,the CD90 positive rate was 99.12%,the CD105 positive rate was 82.41%,the CD73 rate was 99.94%,while the CD34,CD11 b,CD19,CD45,HLA-DR positive rate was only 0.34%.2.2 CCK-8 test results showed that the proliferation ability of demyelination mice BM-MSCs decreased significantly compared with that of the normoal group(P<0.01),CPZ medication had no effect on the proliferation ability of the normal mice BM-MSCs,there was also significant difference between the abilities of MSCs from healthy volunteers and MS patients(P<0.01).2.3 Behavioral test(2 weeks after transplantation)results showed that the volunteerstem cell therapy group(8304.91±1188.34cm)have longer distance than the MS stem cell therapy group(6883.99±1246.70),(P<0.05).Pathological staining results showed that the volunteer stem cell therapy group(0.19±0.08)have better repair effect than the MS stem cell group(0.27±0.06),(P<0.05).The numbers of astrocytes in CC area and CTX area in normal mice stem cell group were 44.34±10.27 and 52.02±12.02;the number of astrocytes in CC area and CTX area in sick mice stem cell group were 67.34±11.74 and 72.02±17.53,which were significantly different between 2 groups(P<0.05).The numbers of astrocytes of the volunteer stem cell group in CC area and CTX area(38.34±12.46 and 47.36±9.69)were significantly slower than that of the MS stem cell group(65.61±12.58 and 86.02±16.14),(P<0.05).2.4 WB test results of HGF showed that the OD value of normal stem cell groups(mice and human)were 0.40±0.11 and 0.37±0.06,which were higher than the OD value of sick stem cell groups(mice and human,0.33±0.08 and 0.28±0.10),(P<0.05).The HGF expression level of the normal mice group(0.28±0.05)was higher than that of the CPZ mice group(0.21±0.05)(P<0.05).3.ConclusionThe healthy mice MSCs are more effective than the sick mice MSCs in remyelination and functional recovery,the mechanism may be related to the difference on proliferation and secretion of nutritional factors.