| BACKGROUNDDespite the progress that has been made in bone regeneration, fracture caused by trauma infection and a variety of reasons and Bone nonunion still represent the medical and socioeconomic challenge.In order to improve the treatment outcome fundamentally, clarify the pathophysiology and molecular mechanisms of bone formation reconstruction and remodeling are the base and starting point. Bone is a complex tissue with a dynamic processional of extracellular matrix mineralizing and the ability to adjust to its functional demands and self-healing.It is known that humoral factors, as well as communication amongst cells of the bone micro-environment, local bloody supply and neural factors between bone and nerve system, are all important in regulating bone metabolism.During the initial research period, our research group described the implantation of sensory nerve bundles for prefabrication of neurotized bone tissues, had greater osteogenic potential than grafting of bone alone. Additionally, several neuropeptides released from the sensory nerves, such as calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY), may also play important roles in these experimental results.CGRP and SP are two neuropeptides distributed in the sensory nerve fibers that innervate the medullar tissues of bone, as well as the periosteum.NPY is a novel neuropeptide transmitter that is co-localized with the classic neurotransmitter noradrenaline (NA) in the sympathetic nervous system, and its signaling though the Y1 receptor sub-type (NPY1R) has been implicated in the neurobiology of bone formation. Bone Marrow Stem Cells (BMSCs) are multipotent stem cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes and adipocytes, considering as the ideal seed cells in the field of tiissue engineering. Consequently, elucidate the effects of neuropeptides on BMSCs can greatly help to interpret the role of neuropeptides on bone metabolism, further reveal the neurobiology mechanism in the process of bone regeneration.However, little research has been done in the area of the biological biological effect and action mechanism of CGRP,SP and NPY on BMSCs. Therefore, in this study, we investigated the direct effects of neuropeptides on BMSCs.The objectives of our study were to 1) determine whether neuropeptides receptors are expressed in BMSCs culture,2) determine whether neuropeptides stimulates cell proliferation and osteogenic differentiation in BMSCs,3 investigate the mechanisms of neuropeptides on proliferation of BMSCs from the perspective of the cell cycle,4) explore the relationship among neuropeptides,BMP and VEGF in the process of osteogenic differentiation,5)explore the effects of neuropeptides in transmigration and adhesion of BMSCs.OBJECTIVE1. Clarify the trend of the receptors of CGRP. SP and NPY during the osteogenic differentiation of BMSCs.2. To investigate the effect and mechanism of CGRP,SP and NPY on proliferation of BMSCs.3. To investigate the effects and mechanisms of CGRP, SP and NPY on osteogenic differentiation of rat BMSCs.4. To explore the effects of CGRP,SP and NPY on the migration of BMSCs.METHODS1. Expression of neuropeptides receptors in BMSCs during osteogenic differentiationThe rBMSCs were isolated in vitro using whole bone marrow adherence method, Cells was subculture at 1:3 ratio when cells achieved 80% confluence. rBMSCs (P2) were used for flow cytometric analysis. Surface marker expression contained CD29,CD34,CD44 and CD45 was detected by flow cytometric analysesThen rBMSCs were classified as osteoblast-induced group and non-induced group.In the different periods of culturing (one week,two week,three week), identification of osteoblasts was measured by Immunocytochemistry and Real Time Reserve Transcriptase-Polymerase Chain Reaction (Real Time RT-PCR), expression of the neuropeptides receptors was detected by using Western Blot and Real Time RT-PCR.2. Mechanism of the effects of neuropeptides on proliferation of rat BMSCsThe rBMSCs were isolated using whole bone marrow adherence method.In the different periods of culturing (1,2, and 3 weeks), expressions of the neuropeptide receptors were detected by Western Blot and Real Time RT-PCR. The BMSCs were treated with CGRI,SP and NPY at different concentrations at different time points (1,3,5,7 days), proliferation was detected with MTT assay, the protein expressions of cyclin D1,cyclin E and p53 were examined using Western Blot.3. Mechanism of the effects of neuropeptides on osteogenic differentiation of rat BMSCs The rBMSCs were isolated using whole bone marrow adherence method. The BMSCs were treated with CGRf,SP and NPY at different concentrations at different time points (1,7,14,21 days), the protein expressions of ALP,BGP and COL I were measured by using Western Blot, expressions of BMP-2 and VEGF were detected by Immunocytochemistry and Western Blot.4. neuropeptides promote the migration of rat BMSCs and stimulates the expression of VCAM-1The rBMSCs were isolated using whole bone marrow adherence method. The BMSCs were treated with neuropeptides at different concentrations, at different time points (1,3,5,7 days), the efficacy of BMSCs migration was analyzed by Transwell chamber assay, the mRNA expressions of VCAM-1 were examined by Real Time RT-PCR.5. statistical analysisStatistical anaysis was performed using SPSS 13.0. Repeated measures was used to compare the overall two-factorial analysis of factor.One-way ANOVA tests were used to evaluate differences between the sample of interest and its respective control.Two-tailed asymptote parametric values being considered. The values were expressed as mean±standard deviation (SD).P-value<0.05 was considered statistically significant.RESULTS1. Isolation and culture of BMSCsThere has been a lot of disc-shALPed red blood cells suspended in the bottom of the medium delay when three groups of primary cell culture in serum medium was changed after 4d. But the bottom flask can be seen in several clusters of adherent cell clones growing colony, cells become spindle. After 7-9d culture, adherent cells significantly increased the number of cloned colonies; after 12-14d culture, the cells can reach 80 to 90% confluence. After passage, cells were completely adhered, spread, once again become a long spindle cells strong after 24h. After passage 3, hBMSCs were more homogeneous, hybrid cells gradually disALPpeared.Phenotypic analysis of BMSCs in the second generation showed that CD29 (96 %),CD44 (95.9%),CD45 (2.9%),CD34 (2.6%). Immunohistochemistry results showed positive Diaminobenzidine (DAB) stain with ALP,BGP and COLⅠwe could see the obvious brown on the cytoplast and cell nucleus. BMSCs can be induced into osteoblasts by induction of osteogenesis successfully.2. Expression of neuropeptides receptors in BMSCs during osteogenic differentiationRT-PCR test demonstrated that osteoblast-induced group had a higher expression of CGRP receptor than non-induced group at the same time point,expression of CGRP receptor was increased in a time-dependent manner in the osteoblast-induced groups. Western Blot test demonstrated that osteoblast-induced group had a higher expression of CGRP receptor than non-induced group at the same time point.Expression of CGRP receptor was increased in a time-dependent manner in osteoblast-induced groups.There was SP receptor expression on the cultured BMSCs and osteoblasts. RT-PCR test demonstrated that osteoblast-induced group had a lower expression of SP receptor than non-induced group at week one(P<0.05),but was significantly increased at week three(P<0.05).osteoblast-induced group had a higher expression of SP receptor than non-induced group at the same time point(P<0.05),expression of SP receptor was increased strongly in a time-dependent manner at week three(P< 0.05).RT-PCR test demonstrated that osteoblast-induced group had a lower expression of Y1 Recptor than non-induced group at the same time point,expression of Y1 Recptor increased in a time-dependent manner among osteoblast-induced groups and non-induced groups. Western Blot test demonstrated that osteoblast-induced group had a higher expression of Y1 Recptor than non-induced group at the same time point,expression of Y1 Recptor decreased in a time-dependent manner among osteoblast-induced groups and non-induced groups.3. Effects of neuropeptides on proliferation of rat bone marrow mesenchymal stem cells and its mechanismAll the neuropeptides stimulated the proliferation of BMSCs significantly at 9 days.In this process, the protein expressions of cyclinD1 and cyclinE were up-regulated,the protein expression of p53 was down-regulated.4. Effects of neuropeptides on osteogenic differentiation of BMSCs and its mechanismWestern blot results showed that the protein expression of ALP and BGP had a distinct increase in BMSCs after simulated with different neuropeptides at 7,14,21 days.There were significant differences between control group and experimental groups at the same time point(P<0.05).Immunohistochemistry results showed positive DAB stain with BMP2 and VEGF, we could see the obvious brown on the cytoplast and cell nucleus.Western blot results showed that the protein expressions of BMP2 and VEGF both increased significantly at 5,7days after stimulated by neuropeptides, VEGF reached a peak in the 7 days.There were statistical differences between control group and experimental groups at the same time point(P<0.05).5. Neuropeptides promotes the migration of rat bone marrow mesenchymal stem cells and stimulates the expression of VEGFCompared with control group, neuropeptides had a significantly enhanced role in promoting cell migration.From control group to CGRP,SP,NPY group, the number of cell migration are (1.11±0.49),(3.20±1.77),(4.78±1.77),(4.86±0.96) cells/High power field.The expressions of VCAM-1 were up-regulated in this process, increased in a time-dependent manner and peaked at 7 days. There were significant differences between control group and experimental groups at the same time point(P<0.05).CONCLUSION1. During the process of osteoblastic differentiation of rat BMSCs, CGRP,SP and NPY Recptors mRNA and protein expression showed different trends, CGRP,SP and NPY may mediated the different effects on the osteoblastic differentiation of BMSCs through various Recptor pathways.2. CGRP,SP and NPY had direct effects on the proliferation of BMSCs, the regulation of cell cycle proteins is one of the mechanisms.3. CGRP,SP and NPY had direct effects on the osteogenic differentiation of BMSCs, the regulation of osteogenic proteins is one of the mechanisms.4. CGRP,SP and NPY can increase the expressions of BMP2 and VEGF.5. CGRP,SP and NPY can promote the migration of BMSCs.6. CGRP,SP and NPY may play an important role in regulating bone regeneration by increasing the proliferation,osteogenic differentiation and migration on BMSCs. |
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