Experimental Study Of Sympathetic Nerve Neurotransmitter On BMSCs Regulative Function And Its Mechanism

The sympathetic nervous system (SNS) has been shown to histologically innervate bone marrow (BM) cells. NA nerves regulated bone marrow cell's function. Sympathetic nerve induced vessel contraction, increased red blood cell, leucocyte, normoblast into blood circulation under electrical stimulation. The SNS regulate bone marrow cells function through adrenergic receptor. Functional assays and radioligand binding studies indicate that bone marrow cells express functionalα-adrenergic receptor (AR).Adding isoproterenol to murine bone marrow cultures increases cellular proliferation and granulopoiesis dose-dependently in vitro and in vivo. The SNS regulate hematopoietic stem and progenitor cell mobilization (HSPC) through AR.NE and dopamine (DA) improved efficiency of CD34+ migration. Granulocyte colony-stimulating factor (G-CSF) induced HSPC mobilization throughβ-AR agonist isoproterenol.Katayama et al. reported a new regulatory axis for the mobilization of hematopoietic stem cells that links these cells to the nervous system and bone in an unanticipated way. This suggested an even more complex perspective of stem cells mobilization and homing. There are close relation between SNS, skeletal system and hematopoietic system. These systems must be in coordination with each other to keep haematopoiesis function.Katayama et al. indicated G-CSF dependent mobilization of HSPC through adrenergic stimulation is likely to require other cell communication mechanisms and given the newly identified non-OB stem cell niches and soluble factors. Stem cell niche is a microenvironment that maintains its self-renewal but not differentiation. Stem cell niche include niche cell, extracellular matrix (ECM), and soluble factors.They provide a protective environment through soluble factors and stem cells interaction directly and indirectly to regulate stem cells. Growth factors and cytokines are crucial cellular components of the stem cell proliferation. Zipori and Sasson et al. reported that stem cell microenvironment regulated progenitor cell differentiation. These findings support the presence of a ''brain-bone-blood triad''. Within the brain-bone-blood axis, some signals are delivered to the HSPC pool directly, while others are exerted indirectly via an impact on niche-supporting stromal cells. We hypothesis that NE regulate HSPC mobilization and homing through BMSCs.Bone marrow mesenchymal stem cells (BMSCs) located in stem cell niche and are one of components of the BM microenvironment. BMSCs are a vital source of growth factors and regulatory adhesion molecules and are thought to be the key in maintaining HSPC proliferation, homing and apoptosis. BMSCs highly expressed extracellular matrix protein such as collogen, proteoglycans, chemokine receptor which play an important role in constructon of extracellular matrix. If our hypothesis were demonstrated, our result were important to illuminate the indirectly mechanism of HSPC mobilization and homing.Our result provide a new method to improve bone marrow microenvironment an rebuild function of haemopoiesis.Adding isoproterenol to irradiated murine bone marrow cultures increases cellular proliferation and granulopoiesis dose-dependently, this suggest that sympathetic nerve neurotransmitter provide protection through bone marrow cells proliferation from irradiation injury. Whether norepinephrine relatedα1-AR activation exists in BMSCs and what biological effect it may cause remains unknown. Explores the sympathetic nervous system regulation on BMSCs function and its mechanism, provides the new treatment and the method for the clinical wound tissue repair and the BMSCs transplantation.Isolated and cultured rat BMSCs is the premise which we carry on our research. First, we established the method of isolated and cultured rat BMSCs and identified them from the cell appearance, the symbolic CD member and the differentiation potency in first part. Neurotransmitter plays its biological function through adrenergic receptor. Second, we examined the expression of of AR in rat BMSCs in vitro. Our result suggested thatα1A-,α1B-, andα1D-adrenergic receptor mRNA notα2-ARs,β1,β2,β3-AR mRNA were expressed in BMSCs in vitro. Whether NE relatedα1-AR activation exists in BMSCs and what biological effect it may cause remains unknown. In second part we examined the effect ofα-AR agonist NE andβ-AR agonist Iso on BMSCs proliferation to demonstrate regulatory mechanism. NE treatment increased [3H] thymidine incorporation in a dose-dependent manner. Iso didn't have the stimulatory effect on cell proliferation. NE stimulated DNA synthesis viaα1-adrenergic receptors and downstream Ca2+/PKC and ERK1/2 activation in BMSCs.Whether NE regulated BMSCs differentiation and cytokines and chemokines secretion as a vital source of growth factors and regulatory adhesion molecules? In third part we examined the effect of NE on BMSCs differentiation in cell differentiation rate. Then we used RT-PCR and ELISA methods to examine the change of growth factors and chemotatic factor in transcriptional level and protein level. Our result showed that there were no effct of NE on BMSCs differentiated into adipocyte and osteoblast .NE upregulated cytokines secretion in BMSCs.Main results were as follow:1. In our experiment we used density gradient centrifugation and natural adherence methods to isolate BMSCs. In order to identify cultured BMSCs, the immunophenotype and differentiation potential of the cells was determined. When maintained in Stromal Medium, the BMSCs showed fibroblast-like morphology and formed typical colony-forming-units (CFU). Flow cytometry analysis demonstrated that rBMSCs expressed stromal-associated surface antigens CD29, CD44, CD73 and CD90; however, they did not express CD31 or CD45. Cells had multipotential differentiation (to differentiate into osteocytes, adipocytes). Our result demonstrated that cells we isolated were BMSCs.2. We examined the expression of of AR in BMSCs. Our result suggested thatα1A-,α1B-, andα1D-adrenergic receptor mRNA notα2-ARs,β1,β2,β3-AR mRNA were expressed in BMSCs in vitro. Our result was consistent with early report.3. To investigate whether norepinephrine induced BMSCs proliferation, [3H] thymidine incorporation into DNA of BMSCs was tested in the presence of various concentrations of norepinephrine (10-7-10-4M) for 8h. Norepinephrine treatment (10-7-10-4M) for 8 h increased 5.12%,10.06%,37.3%,25.28% than control group. The maximal stimulatory effect was observed at 10-5M of norepinephrine. To determine whether the effect of norepinephrine on cell proliferation was mediated throughα-AR dependent signaling, BMSCs were pretreated with phentolamine,anα-adrenergic receptor antagonist. This stimulatory effect of norepinephrine on cell proliferation was blocked by co-incubation with phentolamine.4. To investigate whether Iso induced, [3H] thymidine incorporation into DNA of BMSCs was tested in the presence of. We observed the effects of various concentrations of Iso (10-7-10-4M) on BMSCs proliferation for 8h.Our result showed that there were no promoted effect on BMSCs proliferation.5. Our result showed that BMSCs maintained in basal medium had low levels ofα1A-,α1B-, andα1D-AR mRNA expression as assessed by real-time RT-PCR. NE upregulatedα1A-,α1B-, andα1D-AR mRNA expression 76.54%,59.8%,113.32% than control group.of different AR subtypes in BMSCs.α1D-AR mRNA expression upregulated significantly. Our result showed that NE stimulates DNA synthesis viaα1-adrenergic receptors.6. To investigate whether NE induced BMSCs proliferation viaα1-adrenergic receptors and downstream Ca2+/PKC, Ca2+/PKC were tested by confocal microscopy. NE significantly increased [Ca2+]i concentration. The NE-induced increase in [Ca2+]i was completely blocked by pretreatment with phentolamine. The elevation of intracellular Ca2+ is necessary for PKC activation. We investigated the effect of NE on PKC activation using immunofluorescent staining. NE stimulation for 10 min induced PKC translocation to the cell membrane, which was blocked by pretreatment with phentolamine for 30 min. These results were consistent with the results of Western blot analysis. In addition, we examined the effect of staurosporine (10-6M) on NE-induced increases in [3H] thymidine incorporation in BMSCs. As shown in our result, pretreatment with staurosporine inhibited the increase in [3H] thymidine incorporation induced by norepinephrine. These findings suggest that the PKC pathway is involved in NE-induced increases in [3H] thymidine incorporation.7. We then examined the effect ofα1-adrenoceptor stimulation on ERK1/2 by Western blot, using an antibody specific to phosphorylated ERK1/2 at various time points. In BMSCs, ERK1/2 phosphorylation peaked 10 min following NE treatment, and phentolamine pretreatment inhibited NE-induced ERK1/2 phosphorylation. In order to determine whether PKC is involved in ERK1/2 activation, Western blot analysis was performed. Staurosporine (10-6M) inhibited NE-induced phosphorylation of ERK1/2. In cells subjected to pharmacological inhibition of PD98059 (10-5M), cell proliferation induced by norepinephrine was decreased.8. We examined the effect of NE on BMSCs differentiation in cell differentiation rate.Our result showed that there was no statistics difference in cell differentiation rate between control group and NE group in adipocyte and osteoblast.9. We examined the effect of NE on cytokines secretion by RT-PCR and ELISA. Our result showed that IL-6, SCF, LIF, VEGF mRNA expression increased 5.79, 3.08, 1.75 and 5.99 times in 10-5M NE group compared with control group. CXCL12 mRNA expression had no significant difference between NE group and control group.Our result showed NE regulated cytokines secretion and no effect on CXCL12 mRNA expression in BMSCs. Protein level of IL-6, SCF, VEGF upregulated 1.24, 1.3 and 2.04 times in NE group.Our result suggested NE promoted cytokines secretion.Conclusion:1. The method of isolated and cultured rat BMSCs were established successfully. Our result suggested isolated and cultured rat BMSCs were multipotential cells by identified them from the cell appearance, the symbolic CD member and the differentiation potency.2. BMSCs expressed functionalα1-ARs mRNA and regulated NE induced BMSCs proliferation.3. Iso had no effect on BMSCs proliferation and didn't expressedβ-ARs mRNA.These result demonstrate that BMSCs didn't express functionalβ-ARs mRNA.4. NE regultated BMSCs proliferation but not differentiation.NE stimulated DNA synthesis viaα1-adrenergic receptors and downstream Ca2+/PKC and ERK1/2 activation in BMSCs. There were no effect of NE on BMSCs differentiate into osteocytes and adipocytes.5. NE upregulated IL-6, SCF, LIF, VEGF mRNA and protein level expression in BMSCs.NE had no effect on CXCL12 mRNA expression. Our research demonstrated that NE stimulated BMSCs proliferation viaα1-ARs and downstream Ca2+/PKC and ERK1/2 activation .We discoverd the effect of NE on cytokines secretion first. Our result demonstrated signals from SNS are delivered to the HSPC pool directly, while others are exerted indirectly via an impact on niche-supporting BMSCs.These results provide a new recognition of sympathetic nerve neutotransmitter in bone marrow cells proliferation.