| BackgroundMyasthenia gravis(MG)is an autoimmune disease(AID)mediated by antibodies against acetylcholine receptor(ACh R),muscle-specific kinase(Mu SK),low-density lipoprotein receptor-related protein 4(LRP4)or other ACh R-related protein,T cell dependence,complement system involvement,mainly involving neuromuscular junction post-synaptic membrane.The exact pathogenesis is not yet clear.Studies found that the ability of autoantibodies to inhibit colony forming units of granulocyte-megakaryocyte(CFU-GM)and burst forming units of erythroid(BFU-E)in patients with Systemic lupus erythematosus(SLE).Bone marrow stromal cells in active SLE patients have a low ability to support growth of hematopoietic stem cells.Stromal cell function is restored after autologous hematopoietic stem cell transplantation(AHSCT),suggesting that hematopoietic defects in SLE patients may be at least partly due to autoreactive T lymphocytes in the bone marrow.T lymphocyte mediated growth inhibition of stem cells,even at multipotent progenitor cells and antibodies against fibronectin can affect the interaction of stem cells with stromal cells.Therefore,researches suggest that AID may be a clonal disorder of hematopoietic stem and progenitor cells(HSPCs).Defects in the bone marrow hematopoietic microenvironment may affect the growth of stem and progenitor cell.However,there are few reports on bone marrow stem cells of patients with MG at home and abroad,mostly focusing on the isolation and purification of hematopoietic stem cells and clinical transplantation applications as well as animal model of bone marrow mesenchymal stem cell transplantation.Proliferation,differentiation,and the ability of bone marrow stromal cells to support hematopoiesis are poorly understood and require further study.PurposeTo study the immunephenotype,the proliferation and differentiation characteristics of HSPCs in patients with MG in different disease states,and the the biological characteristics and hematopoietic support function of bone marrow stromal cells in MG patients.MethodsPart one.Immunotyping of bone marrow hematopoietic stem and progenitor cells in patients with MG1 Bone marrow mononuclear cells(BMMNCs)were obtained by separating bone marrow from Ficoll lymphocyte separation fluid.2 After incubating BMMNCs with PE-CD34 and FITC-CD38 antibodies in the dark,stem cell subsets were detected by flow cytometry.Part two.Culture of bone marrow hematopoietic stem and progenitor cells in patients with MG1 Incubate the isolated BMMNCs with CD34 immunomagnetic bead antibody and then use midi MACS to obtain purified CD34~+HSPCs.2 CD34~+cells were cultured in semi-solid medium for a short period of 2weeks to observe the formation of BFU-E,colony forming units-megakaryocyte(CFU-Meg)and CFU-GM.3 Observe the formation of non-adherent nucleated cells(NACs)and colony forming cells(CFCs)by long-term culture of BMMNCs.To identification of CFU-GM,cells were stained with PE-CD15,Per Cy5.5-CD14and APC-33 and then were checked by flow cytometry.Part three.The regulation mechanism of bone marrow stromal cells on CD34~+cells in patients with MG1 After separating BMMNCs,use midi MACS to sort CD34~+hematopoietic stem and progenitor cells.2 Observe the growth and doubling time of bone marrow fibroblasts and the formation of colony forming units-fibroblast(CFU-F)in BMMNCs.3 Passage and purification culture of BMMNCs to obtain bone marrow fibroblasts,co-culture with CD34~+cells of the same healthy person and observe the formation of BFU-E,CFU-Meg and CFU-GM by semi-solid culture and the numbers of NACs and CFCs in long-term culture.4 After co-cultivating bone marrow fibroblasts with CD34~+cells,collect the cell-free supernatant of the culture,and use enzyme-linked immunosorbent assay(ELISA)and western blot(WB)to test stem cell factor(SCF),stromal cell derived factor-1?(SDF-1?),FMS-like tyrosine kinase 3 ligand(FL).ResultsPart one.Immunotyping of bone marrow hematopoietic stem and progenitor cells in patients with MG1 The numbers of CD34~+,CD34~+CD38~+and CD34~+CD38~-in group MG were significantly lower than those in group Controls(P<0.05).The proportions of CD34~+and CD34~+CD38~+in group ocular MG(OMG)were significantly higher than those in group generalized MG(GMG)and lower than those in group Controls(P<0.05).The number of CD34~+CD38~-was not significantly different between group OMG and GMG(P>0.05),but lower than that in group Controls(P<0.05).2 The numbers of CD34~+,CD34~+CD38~+and CD34~+CD38~-in group thymoma-associated MG(TAMG)were significantly higher than those in group MG without thymus abnormalities(MGWT),but significantly lower than those in group Controls(P<0.05).The proportions of the three cells population between MG before immunomodulatory therapy(MGBI)and MG after immunomodulatory therapy(MGAI),early-onset MG(EOMG)and late-onset MG(MG)Late-onset MG,LOMG)showed no significant difference(P>0.05),but each group was significantly lower than that group Controls(P<0.05).Part two.Culture of bone marrow hematopoietic stem and progenitor cells in patients with MG1 The number of CFU-Meg in group MG was significantly higher than that in group Controls,while CFU-GM was significantly lower than that in group Controls(P<0.05).CFU-Meg in group OMG and TAMG were significantly lower than those in group GMG and MGWT,respectively,and CFU-GM was significantly higher than group GMG and MGWT.CFU-Meg in each group was higher than group Controls,and CFU-GM was lower than group Controls(P<0.05).There was no significant difference in CFU-Meg,CFU-GM and BFU-E between group MGBI and MGAI,as well as group EOMG and LOMG(P>0.05).2 The number of CFCs in long-term culture of BMMNCs in group MG was significantly lower than that in group Controls(P<0.05).After four weeks of co-culture,the number of NACs in group MG began to decrease significantly(P<0.05).The flow cytology identification of the culture supernatant cell immunophenotype revealed that the number of CD33~+CD14~+and CD33~+CD15~+in group MG was significantly lower than those in group Controls(P<0.05).The proportions of CD33~+CD14~+and CD33~+CD15~+in group OMG and TAMG were significantly higher than those in group GMG and MGWT,respectively,and lower than those in group Controls(P<0.05).The number of the two cells population were not significantly different between group MGBI and MGAI,as well as group EOMG and LOMG(P>0.05),but both were significantly lower than that of group Controls(P<0.05).Part three.The regulation mechanism of bone marrow stromal cells on CD34~+cells in patients with MG1 There were no significant difference in the number of days required for bone marrow fibroblasts to fill the bottom of the bottle and the ability to form CFU-F between group MG and Controls(P>0.05).The growth curve of bone marrow fibroblasts is S-line,but the doubling time of group MG is slower than that of group Controls.2 Whether it is short-term or long-term BMMNCs cultivation,the colonies of CFU-Meg and CFU-GM and the numbers of NACs and CFCs in group MG were lower than those in group Controls(P<0.05).3 The expression levels of SCF and SDF-1?in group MG were significantly lower than those in group Controls by ELISA and WB(P=0.000),while the expression of FL was no significant difference between the two groups(P>0.05).Conclusions1.The proliferation and differentiation dysfunction of BMMNCs,the lack of bone marrow CD34~+cell reserves and abnormal differentiation into CFU-GM and CFU-Meg in patients with MG are related to the severity of the disease and thymoma,suggesting that the pathogenesis of MG may be related to abnormal hematopoietic stem and progenitor cells.2.In the analysis of subtypes,the number of CD34~+cell reserves and the degree of abnormal differentiation into CFU-GM and CFU-Meg in patients with MGWT are significantly more deficient than those in patients with TAMG,and further research is required.3.The growth of bone marrow stromal cells in patients with MG is delayed,and the secretion of hematopoietic factors SCF and SDF-1?is impaired,suggesting that there may be abnormalities in the function of MG bone marrow hematopoietic microenvironment. |
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