Study On The Effect Of Beta2-adrenergic Receptor On Th1/Th2 Cell Immune Imbalance In Aplastic Anemia

Bone marrow failure syndromes is a common syndrome of hematopoietic system diseases, which includes an area of reduced bone marrow, hyperplasia of the lower, peripheral whole blood cells to reduce a variety of diseases. With the basic and clinical hematology research in depth primary aplastic anemia as an independent disease has been gradually separated. It is mediated by T lymphocytes in the hematopoietic stem cells for the destruction of target cells in autoimmune diseases, its pathogenesis has been mainly positive: Th1/Th2 ratio increased,â… -type cytokine secretion excessive immunosuppressive therapy effective . But what causes the imbalance between helper T cells, Th1 cells appear to divide the direction of the trend? The initial trigger factor is not clear. In addition to unknown factors of the physical and chemical biological properties of host cell antigen changes, the host antigen-presenting cells, T lymphocytes and other immune response in the course of AA itself is the cause of the defects in important aspects of research.Immune pathogenesis of AA due to the establishment of an effective clinical application of immunosuppressive agents before androgen has also been found, securinine base, anisodamine, propranolol and other drugs have some efficacy, such treatment may be suggested that neuroendocrine factors involved in the pathogenesis of AA. There are many auto-immune features in AA: HLA - DR2 frequency increased,CD3+ cells increased in local bone marrow, TCR selection was restrictive, increased expression of TLRs receptor, T cells activated in the pre-state performance. Therefore AA is a special kind of bone marrow failure syndrome, are excluded from the past that is not the typical AA diseases. AA reclassified in accordance with the pathogenesis of autoimmune characteristics and awareness of research for the AA has provided a new platform. Bone marrow has a rich innervation, some nerve endings extend to the surrounding bone marrow cells, bone marrow cells in neurotransmitter receptor analysis is to examine important aspects of neurological function of bone marrow.A variety of immune cells and non-immune cells with theβ₂-AR surface distribution, the performance of its functions in addition to heart and lung organs, but also on the immune system, immune cells, inflammatory mediators in a wide range of regulation, and ultimately affect cell proliferation, differentiation, apoptosis and homing. The excitedβ₂-AR on immune cells can inhibit the secretion of IL-12 of APC cells , play an inhibitor ofâ… type cytokines, impact of Th1/Th2 balance. There is evidence thatβ₂-AR on immune cells of rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis and other autoimmune diseases expressed abnormally.The expression ofβ₂-AR on immune cells of aplastic anemia is yet unknow.In order to study the expression ofβ₂-AR in patients with aplastic anemia bone marrow cells and the relationship between Th1/Th2 balance andβ₂-AR, we have adopted a patient's bone marrow cells and immune-mediated mouse model of aplastic anemia to study in vitro and vivo.(1) In the first chapter, from 11 cases of acute and chronic bone marrow cells in patients with AA line real-time quantitative fluorescence PCR, the level of detection in theβ₂ receptor mRNA expression, and megaloblastic anemia (MA) compared. The results of real-time fluorescence quantitative PCR detection of bone marrow cells in patients with AA prompted the expression ofβ₂-ARmRNA decrease compared with the MA group were significantly different (P <0.05). 7 cases were detected by flow cytometry also revealed that bone marrow cells in patients with AA adrenergicβ₂ - AR expression was reduced compared with the MA group were significantly different (P <0.05).(2) We use the ELISA assay of AA bone marrow immune cells after activation of INF-γ, IL-4 capabilities. Activation of bone marrow immune cells were incubated with ISO after 48h, when the ISO concentration of 10-7M patients when the AA cell culture supernatant of INF-γand not ISO than the control group no significant difference, and then the performance of MA for the INF group -γproduced significantly inhibited, and no ISO control group P <0.05. when the ISO concentration of 10-5M, when the two groups of patients with non-ISO than in the control group have significant difference, but the MA group inhibited more significantly P <0.01; visible ISO model of AA generated by INF-γwas dose-dependent inhibitory effect, when the inhibitory effect of higher concentration. MA group more vulnerable to INF-γinhibited ISO easily Th1/Th2 reduced, Th0 tend to Th2 differentiation. When the ISO concentration of 10-5M at the same time joined theβ₂-AR antagonist ICI118551 cells after INF-γwere produced by inhibition of the phenomenon are to be dismantled.(3) We used the MTT assay of isoproterenol on normal human bone marrow CFU-GM, the proliferation of stromal cells, l0-5mol / L with the high concentration of ISO to promote the role of bone marrow stromal cell proliferation compared with control group, P <0.01. This proliferative effect can be blocked byβ₂-AR antagonist.(4) We adopted the method of immunohistochemical detection of bone marrow cells the expression ofβ₂-AR, of which 48% of mononuclear cells strongly positive, 15% medium-positive, 12% weak positive, 25% negative. Bone marrow CFU-GM cells 55%middle-positive, 45% weakly positive .Bone marrow stromal cells were strongly positive in 68%, 32% medium positive. Paraffin sections of bone marrow cells can be seen all kinds of different shades of brown, bone marrow support cells were strongly positive, medullary sinus endothelial medium was positive, a small number of lymphocytes were negative, other supporting cells were medium-positive, medullary sinus negative red blood cells. Smears of bone marrow cells in a variety of different cells were positive. Bone marrow cells confirmed the expression ofβ2-AR, while other cells in bone marrow is also the expression ofβ2-AR.(5) We have successfully reproduced the immune-mediated AA mouse model, AA was found that mice IFN-γ/IL-4 ratio accompanied by increased apoptosis and the rate of bone marrow cells with a positive correlation, correlation coefficient r = 0.9289. The AA mice was also found that through a certain dose (2.0mg/Kg/d) ofβ2-AR subtype-specific antagonist ICI118551 in advance more than 7 days by intraperitoneal injection of medication for 15 days, bone marrow cells increased expression ofβ2-AR, expressed as the phenomenon of receptor regulation of the reverse by westernblotting. Further experiments found that the reverse regulation ofβ2-AR expression increased accompany with IFN-γ/IL-4 ratio decreased significantly.(6) To explore the AA mouse bone marrow T cells with reducedβ2-AR, we used immunomagnetic bead to purify of CD3+ cells, Pathway Array using high-throughput screening method was to analyzeβ2-AR signal transduction pathway protein . Six kinds of proteinβ-arrestin 1,2, p-p38, p-Stat3, p-PKAa, iNOS expression more than 2 times,but the expression of GRK2 is reduced about 2-fold.All above the experimental results, we believe that bone marrow cells expressed a variety ofβ2-AR, in addition to regulation of synthesis of immune factors may also piay the role of promotting cells proliferation, aplastic anemia bone marrow cells in patients with decreased expression ofβ2-AR, and an obstacle occurred in Th1 shift to Th2; When theβ2-AR of AA mouse up-regulate, the Th1 to Th2 shift was promoted;β2-AR antagonist can increase the express ofβ2-AR in AA patient. to increaseβ2-AR or change the state of receptor desensitization may be give a new target for treatment of aplastic anemia.Innovation: (1) This experiment based on the pathogenesis of aplastic anemia immune. Idea starting from the neuroimmunoendocrine network that regulates organ function in the overall ,to explore aplastic anemia causes bone marrow failure.This subject has a fully theoretical basis and a novel design. The results showed that AA patients with bone marrow cells decreased expression ofβ₂-AR and has existence of obstacles Th1 to Th2 shift.Put forward a new thesis that increaseβ₂-AR in patients with AA may be a new target for the treatment. (2) Nerve fibers in bone marrow through the role of neurotransmitters in immune cells, which release a variety of hematopoietic regulatory factors to indirectly participate in the mode of regulation of hematopoiesis.This subject has enriched the theory of hematopoietic regulation.