The Study Of Homing And Therapy Of Bone-marrowed Mesenchymal Stem Cells In A Mouse Model Of Immune-mediated Bone Marrow Failure

Background:In China, the bone marrow function disorders caused by a variety of reasons are more common. In recent years, with the increase in atmospheric pollution, the popularity of various types of electronic equipment and interior decoration of the complicated,these diseases with an increase in the number of patients has become a serious life-threatening diseases.Bone marrow failure syndrome (Bone marrow failure syndromes, BMFS) is a group derived from hematopoietic stem cell level damage of bone marrow failure diseases, involving a series of blood cells or the Department. Bone marrow failure diseases, including congenital and acquired two major categories. The inherited bone marrow failure syndromes due to bone marrow hematopoietic stem cell proliferation, differentiation, barriers and the hematopoietic microenvironment abnormality is bone marrow failure, cancer and congenital malformations tendency for the performance of a group of diseases, can be expressed as a reduction of whole blood cells, such as Fanconi anemia (FA), dyskeratosis congenita (DC), Shwachman-Diamond syndrome (SDS), but also can be expressed for the reduced single-lineage cells, such as congenital megakaryocytic cell dysplasia, thrombocytopenia (CAMT), Diamond-Blackfan anemia (DBA); acquired bone marrow failure diseases caused by the cellular immune disorder, which acquired aplastic anemia (Aplastic anemia, AA) is the most common.AA is by a variety of causes, various pathogenesis causes medullary hematopoiesis function exhaustion, caused by the decrease in the number of hematopoietic cells, to anemia, bleeding and infection as the main symptoms of a group of heterogeneous disease. Now think its incidence main proliferation with hematopoietic stem cell inner defects, hematopoietic microenvironment function defects and the body’s immune dysfunction related. However, the treatment of aplastic anemia is a major problem plaguing the medical profession, bone marrow transplantation is the preferred measures for the treatment of aplastic anemia.However, due to the age and source restrictions,immunosuppressants, and androgen drugs is still the primary means of treatment ofaplastic anemia.However, some patients recover incomplete or relapsed, and theinvalid in some patients, there are side effects, the high rate of recurrence. Immunosuppressive therapy or relapse is still a lack of effective treatment, Clinical need to explore new treatments.Mesenchymal stem cells (MSCs) exist in connective tissue and the interstitium of organ all over the body and maily reside in bone marrow, which are multipotential nonhematopoietic progenitor cells capable of differentiating into multiple lineages of the mesenchyme. MSCs are readily obtained, can be expanded rapidly in vitro, and have low immunogenicity. Resent studies indicated that BMSCs are the major components of the bone marrow microenvironment, and its main function is to participate in the survival and differentiation of hematopoietic stem cells,to maintain the hematopoietic microenvironment. BMSCs can be expressed not only a variety of hematopoietic cytokines. with the support of hematopoietic function, but also has immunomodulatory properties. The quality and quantity of BMSCs of AA patients are abnormal,which are defective hematopoietic support and immune regulation.BMSCs have low immunogenicity and portability.Heterogenic BMSCs can be implanted into the organs of the host and survive in a long term. BMSCs homing is defined as the arrest of BMSCs within the vasculature of a tissue followed by transmigration across the endothelium. It has been shown that BMSCs, when transplanted systemically, are able to homing to sites of injury in animals, suggesting that BMSCs possess homing capacity. Because of its multi-organ homing, involved in cell differentiation and immune regulation has been widely used in the prevention of allo-HSCT GVHD prevention and treatment, organ transplant rejection and tissue engineeringand treatment of various autoimmune diseases.In this study, we have established models of syngenic BALB/c mice and bone marrow failure model mice received BMSCs intravenously, and observed that BMSCs can improve the hematopoietic and immune function of the bone marrow failure model.BMSCs were labeled with the fluorescent membrane marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) in different organs was performed to assess whether the mechanisms of homing and distribution of BMSCs are different in recipients normal and with severe immunodeficiency and irradiation. levels of IFN-Y and IL-2in peripheral blood were also detected to investigate the immunemodulatory effects of BMSCs.The study includes three parts:1.Isolation, culture and identification of mouse BMSCs from bone marrow;2. BMSCs improve the hematopoietic and immune function in bone marrow failure model mice;3. The Study of homing of BMSCs of bone marrow failure model.Park1Isolation、culturation and identification of mouse mesenchymal stem cells from bone marrowObjective:To establish an efficient method for isolation and culture of mouse BMSCs, then proliferate and identify the cells in vitro. To study the biological characteristics and prepare for the animal experiments.Methods:The mononuclear cells were isolated from BALB/c mouse bone marrow and BMSCs were enriched and expanded by using bone marrow adherent culture. The phenotype was analyzed by Flow Cytometry (FCM) and morphology of the BMSCs was observed under a microscope. BMSCs were differentiated by the adipogenic,osteogenic and chondrogenic cluture medium.;the cell after induced along adipocytes.Results:The BMSCs were the adherent cells of similar fibroblastoid morphology. The expressions of CD29, CD44, CD90, CD105,CD34, HLA-DR and were95.45%,99.99%,1.06%, andl.20%.97.23%,99.67%.53.75%,29.83%,4.12%,5.40%. The BMSCs had been differentiation potential of osteogenic and adipocytes in vitro.Conclusions:The BMSCs can be isolated and expanded by using bone marrow adherent culture which is efficient and stable. The cells had been identified morphology、biological characteristics and differentiation potential in vitro. Isolated and cultured mouse BMSCs from bone marrow in vitro successfully, making it possible for further study. Park2Mesenchymal stem cells improve the hematopoietic and immune function in immune-mediated aplastic anemia mice.Objective:To observe the effect of MSCs on hematopoiesis and immune function in immune-mediated aplastic anemia mice.Methods:Thirty BALB/c mice were randomly divided into three groups (10mice in each group):the normal control group (group A), the aplastic anemia group (group B) and the MSCs group (group C). Aplasfic anemia was induced by immune-mediated method(Γ-ray radiation and lymphocytes injection).Balb/c mice were irradiated5.0Gy by γ rays, and then infused5×106lymph node(LN)cells from DBA/2mice in4hours to establish a mouse model of immune-mediated aplastic anemia.Group C was intravenously administered MSCs at3days after establishment of model. In group B, PBS of the equal volume was given instead of MSCs.On dying or day14after bone marrow failure induction, the average survival time, peripheral blood hemogram and the morphological features of bone marrow of mice in each group were determined. The average levels of IFN-γ、IL-2in peripheral blood were evaluated by enzyme linked immunosorbant assay (ELISA).The average levels of IFN-γ in peripheral blood in3groups were (176.19±29.36) ng/ml,(564.96±64.82) ng/ml),(238.92±28.66) ng/ml, respectively. The average levels of IL-2in3groups were (65.69±6.83) ng/ml,(199.45±8.05) ng/ml),(185.02±7.61) ng/ml, respectively.There comparisons of mean levels of cytokines were significant differences among group B and C(P=0.011; P=0.036).Statistical treatment:The software of SPSS version13.0for windows was used for statistical analysis. Continuous variables were expressed as χ±s. Independent-samples T test analysis the growth curve.One-way ANOVA test was performed for analyzing, pairwise comparisons using the Bonferroni method. Kaplan-Meier method for the question of survival. Differences were considered statistically significant when P was less than0.05.Conclusions:These findings suggest that the transplanted BMSCs can promote the recovery of hematopoietic function, alleviate the degree of bone marrow failure, suppress the activity of negative modulators such as IFN-γ, IL-2of peripheral blood and significantly prolong survival time. Park3The Study of homing of Bone-marrowed Mesenchymal Stem Cells of Immune-mediated Aplastic Anemia MiceObjective:To explore the differences homing of MSCs between immune-mediated aplastic anemia mice and normal mice.Methods:Bone marrow mesenchymal stem cells labeled with CM-Dil were considered experimental group. Bone marrow mesenchymal stem cells non-labeled with CM-Dil served as control group. Cell growth and proliferation were compared in both groups. Cell growth proliferation was measured by MTT. Twenty BALB/c mice were randomly divided into four groups (5mice in each group):the normal control group A, the normal control group B and the aplastic anemia group A, the aplastic anemia group B. Aplasfic anemia was induced by immune-mediated method(γ-ray radiation and lymphocytes injection). Base on normal mice and immune-mediated aplastic anemia mice.Twenty BALB/c mice was intravenously administered MSCs at3days after establishment of model.Statistical treatment:The software of SPSS version13.0for windows was used for statistical analysis. Continuous variables were expressed χ±s. Paired T test analysis the bone marrow mesenchymal stem cells labeled with CM-Dil in two groups. Differences were considered statistically significant when P was less than0.05.Results:In vitro culture result showed that growth, proliferation, splitting and morphology of bone marrow mesenchymal stem cells were similar between both groups. CM-Dil displayed red fluorescence. Fluorescence emerged by CM-Dil was visible obviously14days after labeling. Cell labeling rate was95%at24hours. In vivo culture result showed that bone marrow mesenchymal stem cells transplanted through the portal vein.Bone marrow mononuclear cell suspension were prepared at24h and days7following transplantation to detect the proportion of CM-Dil labeled cells by flow cytometry.Conclusions:These findings suggest that the transplanted BMSCs can promote the recovery of hematopoietic function, alleviate the degree of bone marrow failure, suppress the activity of negative modulators such as IFN-γ, IL-2of peripheral blood and significantly prolong survival time.