Bone Marrow-derived Mesenchymal Stem Cells And Immunogenicity Of The Role Of Immune Tolerance Induced By The Clinical Application

Bone marrow-derived mesenchymal stem cells and immunogenicity of the role of immune tolerance induced by the clinical application Bone marrow-derived mesenchymal stem cell research in recent years the forefront of destination,as a result of bone marrow-derived mesenchymal stem cells proliferation and differentiation with its own potential,bone marrowderived mesenchymal stem cells,the immunogenicity and immune tolerance to the role of more and more of our attention,on the immune Immunogenicity and immune tolerance in rats and its clinical application research is all the more important.Objective:Of bone marrow-derived mesenchymal stem cells(MSCs) the role of immune tolerance of the initial research,the establishment of MSCs in vitro isolation,purification and identification of ways to in-depth study and wider application of MSCs provide a basis.At the same time,their immune regulation and the role of immune tolerance to a preliminary study in the transplantation of its wide application in the field,provide a theoretical basis and experimental data.Methods:1.MSCs isolated and cultured in vitro,purification and identification of(1) cells in primary culture:rat killed,removed the bone marrow to produce single-cell suspension,inoculated in culture bottles,discarded after 72 hours in addition to suspension cells by adding fresh culture medium,2-3 days for a semi-liquid to covered with single-cell amount of time,generally about 1 week.(2) passage:abandoned to the culture medium,using trypsin digestion, digestive juice poured,serum terminated poured culture medium,wind and percussion adherent cells,cell suspension made from the microscope to observe a day until the adherent cell fusion covered the bottom,the amplification of repeated subculture.Strict control of each passage and digestive enzymes,to achieve the purpose of purification of MSCs.(3) Determination of growth curves:1,3,5 check the growth of good cells, after trypsin digestion to produce single-cell suspension,inoculated into 96-well plates,24 hours later,after adding DMSO,microplate reader count calculation of the mean,for six days,every day count,the last time to cultivate Abscissa,cell number for the longitudinal coordinates of the growth curve mapping.(4) Determination of cell cycleBetter check the growth of three generations of cells,after trypsin digestion to produce single cell suspension using flow cytometry assay directly the cell cycle.Excitation light source used for the 15mW argon ion laser,a wavelength of 488nm.FACS software to collect cells,Lysis data analysis software to record the percentage of positive cells.(5) the state of cell growth observedIn the cell culture process,a day in the inverted microscope to observe cell growth,proliferation and morphological characteristics,and records at any time to take photographs.2.MSC growth curvesMSC surface expression of many adhesion molecules,including participation in the interaction of T lymphoid fine VCAM-1,CAM-1 and LFA3,MSC did not express MHCâ…¡molecules,Fasl.Did not express or express very low levels of MHCâ… .MSC can not easily be the host T cell recognition,and can escape the host immune system rejection.This experiment with immunohistochemical staining method.3.MSCs inhibitory effect on the immune responseMSC by inhibiting cell-to-cell contact with each other,hindering antigen-presenting cells and T cells in contact,thereby inhibiting T cell proliferation. Thymocytes of the experimental determination of spontaneous proliferative activity. 4.Thymocytes measured spontaneous proliferative activityRemove the single-cell suspension preparation of MSCs to regulate the cell concentration by adding 96-well plate in each of samples for 6-hole complex,the next day joined the MSCs in the culture plate 96 coupled with the good preparation of the above-mentioned thymus cell suspension culture box training, the collection of thymus cells by adding MTT,time to wait after adding DMSO, the machine detected.Results:1.MSC in vitro and morphological characteristics ofInoculation of bone marrow cells,primitive hematopoietic cells mainly, morphological diversity.Cultured for 4 days,adherent cells can be seen,after the passage,spindle or spindle started to fibroblast-like cells,large process.A week later,cell fusion into a single layer,spindle cell processes significantly large,the cells were arranged.2-3 on behalf of,the cell morphology became more homogeneous,the majority for the slender spindle cells,there are flat-shaped cells with processes,it is fibroblast-like cells,cells may be scattered in the growth, but also-like growth was cloned.Generation of cells after 6-7 passages,no significant changes in morphology,but the large flat-shaped cells increased and spindle cell reduction.6-7 on behalf of,the cell growth are aging.2.MSC growth curveCell division is relatively slow start,after the incubation period after entering the logarithmic phase,and then plateau after a few days.3.MSC cell cycleDirect fluorescence flow cytometry to detect cell cycle,see the percentage of positive cells recorded 73.18%in G0/G1 phase of cells,results showed that the detection of cells with the growth characteristics of MSCs.4.MSC to differentiate into osteoblasts:After induction of osteoblasts induced by fluid,detected using alkaline phosphatase staining and found that MSC differentiate into osteoblasts. Conclusion:Isolation and culture of1.MSCs,purification and identification of:initial stage of cell division is relatively slow,after a period of time after the incubation period and entered the logarithmic phase.And then into the plateau.MSCs differentiate into osteoblasts, MSCs no obvious morphological features and specific surface markers.2.MSCs immunogenicity studies:MSC with low immunogenicity: non-expression of MHCâ…¡,low expression of MHCâ… molecules that can escape immune rejection.Immunosuppression3.MSCs:rat MSCs to allogeneic rats inhibited the proliferation of T lymphocytes:With time,tolerance to the more obvious role.With the increase in the number of MSCS,tolerance,the more obvious role.