The Role Of T Cells, MSC And FSH In The Pathogenesis Of Patients With Aplastic Anemia And The Mechanism Of Ginsenoside Rb1 In The Treatment Of Aplastic Anemia

Part I Study on the functions of T cell and MSC and FSH in aplastic anemiaObjective To investigate the T cell and mesenchymal stem cells immune function and follicle-stimulating hormone in the role of aplastic anemia.Methods Peripheral blood with90cases of aplastic anemia patients and37cases of control group was collected and some tests were performed by using FCM or Luminex or the methods of transmission turbidimetric and chemiluminescence, including of CD3+CD4T cells, CD3+CD8T cells, CD25+/CD3+CD4+T cells, CD69+/CD3+CD4+T cells, HLA-DR/CD3+CD4+T cells, CD25+/CD3+CD8+T cells, CD69+/CD3+CD8+T cells, HLA-DR/CD3+CD8+T cells), regulatory T cells, B cells, NK cells, gamma delta T cells, CD55and CD59protein, IL-2, IL-4, IL-6, IL-10, IFN-gamma, TNF alpha cytokines C3, C4and FSH. Bone marrow was used to culture primary mesenchymal stem cells, and it was identifed by phenotype through FCM and induced differentiation in vitro and drawed the growth curve.Results Compared with the control group, there were more significantly reduced in the numbers of helper T cells, regulatory T cell, the expression of CD55, CD59, the levels of C3, C4in patients with aplastic anemia, conversely, there were more increased with the numbers of CD3+CD8+T cells,CD25+/CD3+CD4+T cells, HLA-DR/CD3+CD8+T cells, gamma delta T cells, and the levels of IFN-y, TNF-α, IL-4and FSH. It is higer of the levels of IL-4, IL-6, IL-10in patients with acute aplastic anemia than those of chronic aplastic anemia. There were more significantly reduced in MSC proliferation ability of patients with aplastic anemia.Conclusion T cells are abnormal in patients with aplastic anemia, including of T cells numbers, subgroup phenotype, distribution, and activation state. It is related to the occurrence and processes of diseases with cytokines mediated by T cells. MSC and FSH may be involved in the onset of aplastic anemia. Part Ⅱ Study on the Follicle Stimulating Hormone in regulation of the Immunomodulatory functions of bone marrow mesenchymal stein cellsObjective To explore the effects of Follicle Stimulating Hormone regulating the inhibition derived bone marrow mesenchymal stem cells lymphocyte proliferation and cytokine secretionMethods①bone marrow was used to culture primary mesenchymal stem cells, and identifed by phenotype through FCM and induced differentiation in vitro.2Co-culture system was established, consisted of mesenchymal stem cells pretreated with mitomycin and in vivo fluorescent dyestuff CFSE processing lymphocytes. Lymphocyte proliferation response was observed by using FCM to detect fluorescent, and the changes of lymphocyte immune functions were observed by using luminex to detect a variety of cytokines.(?)The expression of follicle-stimulating hormone receptor was detected using RT-PCR in bone marrow mesenchymal stem cells. Following acted with follicle-stimulating hormone with0,3,10,30,100ng/mL for48h, Co-culture system was observed the changes of lymphocyte proliferation and cytokine secretionResults①Bone marrow mesenchymal stem cells express CD44, CD90, CD105, do not express CD34,CD45, CD14, CD11b, HLA-DR, and can be induced to be divided into fat cells in a concomitant conditions.(?) Fluorescent intensity and IL-6and IL-10were significantly higher in co-culture of Mesenchymal stem cell and lymphocyte culture compared with lymphocyte culture lonely, relatively, TNF alpha secretion has dropped significantly (P<0.05).(?) There were more significant reductions in the secretion of IL-6and fluorescent intensity of the groups with30,100ng/mL FSH Compared with no FSH group(P<0.05).Conclusion Bone marrow mesenchymal stem cells express follicle-stimulating hormone receptor, promote the secretion of lymphocyte IL-6, can restrain the function of lymphocyte proliferation. follicle-stimulating hormone can down regulate the function with dose dependent. Part III Study on Ginsenoside Rb1Regulating Hematopoietic and Immune Functions of Aplastic Anemia Mice ModelObjective:To investigate the effect of different doses of Chinese medicine Ginsenoside Rbl on model mice with AA, observing the indicators of peripheral blood WBC、Ret、 CD4+/CD8+、IL-4、IFN-y and bone marrow CD34+. To explore the pathogenesis of AA and the research mentality of Ginsenoside Rbl cure mechanism and ensure the reasonable clinical application in later.Methods:40in48female adult Balb/c mice in a cleaning grade were irradiated by X-ray and injected allogeneic mouse DBA/2thymocytes to establish immune-mediated aplastic amenia model mice, which were respectively represent the low, medium and high dose of treatment of Ginsenoside Rbl, positive control group of treatment of CSA, model control group. And the other eight untreated mice were represented normal control group. At the end of days12, the mice were killed, then observing the change of the peripheral blood WBC, Ret, and detecting the level of peripheral blood CD4+/CD8+and bone marrow CD34+with FCM, and detecting the expression of plasma IL-4and IFN-γ with ELISA.Results:Compared with the model group:(1) Ret were significantly increased in the low, medium, high dose of treatment of GRb1group and CSA group (P<0.05);(2) the proportion of CD34+in the high dose of treatment of GRbl group and CSA group was significantly elevated(P<0.05),(3) the ratio of CD4+/CD8+in the medium, high dose of treatment of GRbl group and CSA group was significantly increased (P<0.05),(4) the expression level of IFN-y was declined in the medium, high dose of treatment of GRbl group and CSA group(P<0.05).Conclusion:GRb1can improve hematopoietic function that is related to regulate T cells...