MHC-?b Participates In Immune Rejection After Allogenic Bone Marrow Mesenchymal Stem Cells Transplantation

Objective:Bone marrow mesenchymal stem cells(BMSCs)are pluripotent stem cells that can differentiate into a variety of terminal cells under specific conditions,due to its characterized by easy access,immunomodulatory,multi-differentiation functions,excellent regeneration capabilities.They are currently widely used in transplantation and tissue regeneration.Studies have shown that,stem cell transplantation can protect the heart after acute myocardial infarction(AMI).However,after allogeneic stem cell transplantation,host versus graft reaction is still unavoidable.The main reason for immune rejection is that the major histocompability complex(MHC)activates T lymphocytes to produce a specific immune response.Related studies have found that transplantation of allogeneic BMSCs in the treatment of AMI rats can increase the expression of MHC-II and decrease the expression of MHC-Ib,during 3 to 6 months.This change of the immunogenic antigen on the surface of the stem cell results in an immunological rejection of the transplant by the recipient,which ultimately results in the BMSCs can not survive permanently in the recipient.Therefore,this experiment aims to investigate:1.Immune response after allogeneic transplantation of BMSCs;2.The expression of subtypes of MHC-Ib gene cluste after BMSCs were differentiated into terminal cells.Methods:1.BMSCs were recoveried and cultured in vitro,digested with 0.25% trypsin after BMSCs fused to 80%-90%.After cell counting,the cells were passaged and plated for induction experiments.2.In vitro,experiment was divided into 3 groups: shamp,AMI,and BMSCs.Model of myocardial infarction was established by ligation of anterior descending coronary artery.The cells were transplanted in the marginal area of myocardial infarction after 1week.3.Four weeks after stem cell transplantation,Echocardiography was performed to assess heart function.Determine the expression of CD8,by Immunofluorescence,with myocardial infarction tissue.4.BMSCs were induced to differentiate into vascular endothelium like cells by 50ng/ml VEGF and 10 ng/ml b-FGF.The morphological changes of the cells were observed under microscope after 2 weeks,3 weeks and 4 weeks of induction.Detected the gene expression of KDR,ICAM-1,v WF by RT-PCR and the protein expression of ICAM-1,v WF by Western Blotting,at the same time,so that we can assess the effect of differentiation.After that we detected the expression of MHC-Ib gene,including RT1-S3,RT1-E,RT1-E2,with RT-PCR and agarose gel electrophoresis.5.BMSCs were induced to differentiate into cardiomyocyte like cells by10ummol/L 5-aza.The morphological changes of the cells were observed under microscope after 2 weeks and 3 weeks induction.Detected the gene expression of GATA4,MHC and c Tn I by RT-PCR and the protein expression of MHC and c Tn I by Western Blotting,at the same time,so that we can assess the effect of differentiation.After that we detected the expression of MHC-Ib gene,including RT1-S3,RT1-E,RT1-E2,with RT-PCR.6.BMSCs were induced to differentiate into osteoblasts by ascorbic acid,?-sodium glycerol and dexamethasone.The morphological changes of the cells were observed under microscope after 2 weeks induction.Eosin staining was used to identify osteogenic differentiation.At last detected the expression of MHC-Ib gene,including RT1-S3,RT1-E,RT1-E2,with RT-PCR.Results:1.The BMSCs grew into “S”shape,looks like spindle-shaped cells and triangular-shaped cells under microscope.With the prolongation of the culture time and passage,the cells gradually appeared aging,and showed a “rag like” change under microscope.2.The rat myocardial infarction model were successfully constructed.After ligation of the anterior descending coronary artery,the myocardial infarction area was whitened and ventricular motion was reduced.Also successful transplantation of stem cells by myocardial injection.3.After four weeks stem cell transplantation,echocardiography suggested that the heart function of the BMSCs group was improved compared with the AMI group.After took out the heart from the rat,we can find that the infarction regional tissue collapse and hyperplasia.Later immunofluorescence showed that CD8 expression in BMSC group was significantly higher than that in AMI and Sham groups(p<0.01).4.After induced by VEGF and b-FGF,BMSCs were appeared morphological changes gradually.It became small,round,oval,spindle,full shape,enhanced three-dimensional sense.Three weeks induced,some of cells showed “island like”or“paving stone like”appearances,which were endothelial cells characteristics.With the prolongation of the induction time,more and more of the original long spindle cells were changed into a round cells,also the “paving stone like”cells were more typical.After 2,3,4 weeks induce differentiated,RT-PCR showed that the expression of KDR,ICAM-1,and v WF genes were progressively increased with the prolonged induction time(p<0.05).Western Blotting analysis also showed that the expression of ICAM-1 and v WF proteins in the induction group had a progressive growth trend with the induction time(p<0.01).However,the expression of v WF m RNA and protein increased in 3 weeks but decline after 3 weeks(p<0.01).5.Overall,after 2,3,and 4 weeks of induction with VEGF and b-FGF,the MHC-Ib gene had differert change.RT1-S3 was slighttly increase of 1.32-fold after 1 week induction(p>0.05),no statistical significance.But with the prolonged induction time,the expression was progressively decreased(p < 0.01),while RT1-E,RT1-E2 gene expression was progressively increased with the prolonged induction time(p <0.05).The band size also conformde to the size of the primer design in later agarose gel electrophoresis.6.After 2 weeks and 3 weeks of 5-aza induction,BMSCs were appeared morphological changes gradually.The cells showed different kind of shape including irregular,extended,heterogeneity changed.The cells then aggregated and gradually increased in size and the adherent cells were completely in contact with adjoining cells,arranged in a uniform direction,and formed a“muscle island”like structure.But,no typical myotube like structure was seen,and no spontaneous myocardial beats were observed.Then deteced gene expression of GATA4,MHC,and c Tn I by RT-PCR.GATA4 was increased at the 2 week of induction,7.14-fold higher than uninduced group(p<0.01).But decreased later,2.68-fold after 3 weeks(p<0.05).The others were gradually increased with the prolonged induction time(p<0.01).By Western Blotting assay,the expression of heavy chain protein also progressively increased with the induction time(p<0.01),but no expression of c Tn I protein was observed.7.Overall,after 2 and 3 weeks of induction with 5-aza,the expression of RT1-S3 decreased progressively with the induction time(p < 0.001).RT1-E transiently decreased after 2 weeks induction,0.52-fold compair with uninduced group(p >0.05),no statistical significance.In third week,RT1-E gene expression was increased,2.87-fold compair with uninduced group(p<0.05).RT1-E2 gene expression was progressively increased with the prolonged induction time(p<0.05).8.Two weeks after the BMSCs were induced by the osteogenic inducer,the morphology of the cells was changed by microscopy.Bone nodules,calcium nodules was appeared.And calcium nodules deposition after alizarin red staining.The expression of RT1-S3,E and E2 genes in the induced group was increased detected by RT-PCR,compared with the control group(p<0.05).Conclusions:1.MHC-I involved in immune rejection after BMSCs transplantation.2.MHC-Ib gene cluster changes after BMSCs are differentiated.The decline of RT1-S3 gene expression may be one of the cause of transplant rejection.