| Despite the progress that has been made in bone regeneration, fracture caused by trauma, infection and a variety of reasons and bone nonunion still represents the medical and socioeconomic challenge. In order to improve the treatment outcome fundamentally, clarify the pathophysiology and molecular mechanisms of boneformation, 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 bonemicro-environment, local bloody supply and neural factors between bone and nerve system, are all important in regulating bone metabolism. Early researchers conducted experiments showed that congenital craniofacial developmental defects usually associated with neurological diseases, this phenomenon indicated that the nervous system may play an important role in controlling and regulating the bone development. In addition, numerous studies have demonstrated neural factors play an important role in bone repair and fracture healing. However, there is less research regarding the biological mechanisms of neural factors in bone tissue regeneration. 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. Recent years, role of neuropeptides in fracture healing and bone tissue regeneration attracts people’s attention and becomes one of the hot spots. Neuropeptide is a peptide-like substances, produced by the activity of the nervous system and has the role of neuromodulator, SP, CGRP, NPY is the representative substance. SP and CGRP are the two main neuropeptide secreted by sensory nerve fibers; NPY is the most widely distributed in the central nervous system and one of the highest levels of neuropeptides in this area, which is an important factor mediating the peripheral nervous system and central nervous systems. Neuropeptide SP, as an important neurotransmitter secreted by bone sensory neurons, plays an important role in the regulation of bone physiology, neuropeptide SP belongs to the tachykinin family, five kinds of tachykinin subtypes and3SP receptors has been found,(SP, tachykinin A, tachykinin B, tachykinin C, tachykinin K) and (NK1,2and3receptors). The regulation of neuropeptide SP in many physiological processes is via NK1receptors.The current studies mainly focus on the effects of neuropeptide in osteoblasts, but the research regarding the osteoblast precursor cells-BMSCs and MC3T3E1cells is less. BMSCs have a strong potential of proliferation and differentiation, plays an important role in bone physiology, and can be induced to differentiate into bone cells, stromal cells, cartilage cells and hematopoietic cells and other kinds of cells in vitro or in vivo in a suitable environment, is an ideal seeded cells in bone tissue engineering research; MC3T3E1cells is pre-osteoblasts, has certain characteristics of osteoblast and can differentiate into osteoblasts in a suitable environment, so usually used as the seed cells, used in bone tissue engineering. Therefore, to clarify the specific role of neuropeptide in BMSCs and MC3T3E1Cells can help us understand the role of neuropeptides in the process of bone physiology, to further revealed the mechanism of neurobiology in bone regeneration.From the perspective of the molecular mechanisms of signal transduction, exploration of which signal transduction pathway involved in the process of SP exert its role of regulating the biological effects in BMSCs and MC3T3E1cells is equal important which is less studied. Wnt/β-catenin signaling pathway is a hot study point in bone metabolism studies and diseases of pathogenesis-related in skeletal system. Wnts protein binds to low-density lipoprotein receptor-related protein (LDLreceptor-relatedprotein, Lrp) and Frizzleds receptor (Fzd) receptor complex, through a series of action by cytoplasmic proteins (Dsh, Gsk3β, APC, Axin) leading to stabilization of β-catenin and accumulated in the cytoplasm, and then β-catenin transferred into the nucleus and interacts with the transcription factor:T cell factor (T cell factor, Tcf) and lymphoid enhancer factor-1(lymphoid enhancer factor1, Lef-1), exert its role by activating the target genes. This path is called the classic Wnt/β-catenin pathway.Therefore, investigation the effect of SP on the proliferation and differentiation of BMSCs, MC3T3E1cells, and exploration of the underlying mechanism is very necessary.lt can provide certain foundation to further explore regarding the bone regeneration, bone metabolism and neurobiological mechanisms of Wnt/β-catenin signaling pathway and provide the experimental evidence for clinical application in fracture.Objective1. To explore the relationship between the effect of SP on the proliferation of BMSCs and Wnt/β-catenin signaling pathway2. To explore the relationship between the effect of SP on the differentiation of BMSCs and Wnt/β-catenin signaling pathway3. To investigate the relationship between the effect of SP on of the differentiation of MC3T3E1cells and Wnt/β-catenin signaling pathwayPart1The effect of SP on the proliferation of BMSCs and Wnt/β-catenin signaling pathwayObjective:To acquire BMSCs for experiments and test its surface markers, and explore the effect of SP on proliferation of BMSCs and Wnt/β-catenin signaling pathway Methods:1. Taking28～35d,130-150g SD rats and then they were sacrificed, BMSCs were isolated using established techniques, adding medium of DMEM containing10%FBS, culture medium was changed to remove the suspension of blood cells after72h, culture was inoculated in25cm2plastic flasks using DMEM culture medium containing10%fetal bovine serum. The medium was changed after2-3d, the cells can be passaged by1:2on7-9d. Light microscope and inverted microscope were used to observe the cell growth, proliferation and cell morphology. P3-generation BMSCs were digested using Trypsin, washed3times with PBS, cells were counted, and detected the CD29, CD34, CD44, CD45expression using the Flow cytometry method.2. Take3passage cells, the cells were divided into four groups, SP (10-8mol/L) group, SP+SP receptor antagonist (1μmol/1), SP+DKK1group (0.2mg/mL) and the control group (the same amount of PBS). Cells were seeded in96-well plates, the cell density is3000/cm2, the medium was changed daily, and the replace stimuli until1,3,5,7,9d, each experimental group are located three holes, after stimulation, direct the operation of Alamar Blue following the instructions, each well at each time point were measured to acquire fluorescence values and were measured on wavelength: excitation wavelength (570nm), emission wavelength (585nm).3. Take the3rd generation of cells, cells were divided into four groups, SP (10-mol/L) group, SP+SP receptor antagonist (1μmol/1), SP+DKK1group (0.2mg/mL) the control group (the same amount of PBS). Q-PCR and Western blot methods were used, respectively, gene and protein expression of Wnt/β-catenin signaling pathway was detected in each group at the time point of1,3,5,7d, and the location of (3-catenin in cells of each group is detected using the method of immunofluorescence at the time point of4d.Results:1. Flow cytometry showed that the cultured cells were CD29(93.5%±2.3) and CD44(87.5%±3.9) positive, and CD34(5.5%±1.6) and CD45(11.2%±3.4) negative, indicating that the cells were BMSCs 2.10-8mol/l SP enhanced the proliferation of BMSCs, and the number of viable cells was reduced by pretreatment with1μM NK1antagonist or0.2μg/ml DKK1at each time-point (Figure2). SP significantly increased the cell number at each time point compared to the other3groups3. Q-PCR showed that SP acts on BMSCs can promote the expression of C-myc mRNA, Lefl but reduce expression of Tcf7, SP receptor antagonist, DKK1inhibited these effects of SP; whereas β-catenin mRNA, Cyclin D1mRNA expression were unaffected. Western blot showed that SP can improve expression of C-myc and β-catenin protein, reduced expression of p-β-catenin, SP receptor antagonist, DKK1inhibited these effects of SP. Immunofluorescence showed that SP can promote the distribution of β-catenin in the nucleus, SP receptor antagonist, DKK1can inhibit these effects of SP.Conclusion:BMSCs can be obtained by the established method, SP can promote the proliferation of BMSCs, and improve the expression of genes and protein of Wnt/β-catenin signaling pathway which is associated with the activation. SP can promote β-catenin transfer into the nucleus, and ultimately activate Wnt/p-catenin signaling pathway, while the SP receptor antagonist, DKK1inhibited these effects of SP. These results indicate that SP can promote BMSCs proliferation; Wnt/β-catenin signaling pathway may be involved in these processes.Part2The effect of SP the differentiation of BMSCs, and activation of Wnt/p-catenin signaling pathwayObjective:To investigate the effect of SP on the differentiation of BMSCs, and explore the underlying mechanism by studying Wnt/β-catenin signaling pathway.Methods:BMSCs can be obtained by the established method, take the3rd generation of cells, the cells were divided into three groups, SP (10-8mol/L) group, SP (10-12mol/L) group and control group, the cells in each group were cultured in osteogenic environment (L-DMEM+10mM β-glycerol phosphate+100nm dexamethasone+50μg/mL of vitamin C+10%FBS).The expression of bone markers (alkaline phosphatase mRNA, collagen type I mRNA, osteocalcin mRNA, Runx2mRNA) were detected by Q-PCR method at the time point of7,14d to determine the optimum concentration of SP promoting the differentiation of BMSCs. According to the results of the first step, cells were divided into four groups, SP group, SP+SP receptor antagonist (1μmol/1), SP+DKK1group (0.2mg/mL) and control group (the same amount of PBS), each group were cultured in the osteogenic environment, the expression of bone markers and Wnt/β-catenin signaling pathway related genes (C-myc mRNA, cyclin D1mRNA, Lefl, Tcf7and P-catenin mRNA) were detected using the method of Q-PCR.Results:10"12mol/L SP can significantly promote the expression of bone markers, while high concentrations SP (10-8mol/L) does not have this effect, and10-12mol/L SP can significantly improve the expression of C-myc mRNA, cyclin D1mRNA, Lefl but reduce expression of Tcf7, without affecting the expression of β-catenin mRNA, and these effects can be inhibited by SP receptor antagonist and DKK1.Conclusion:Low concentrations of SP (10-12mol/L) can significantly promote osteogenic differentiation of BMSCs in osteogenic environment, and can activate Wnt/β-catenin signaling pathway, so we hypothesized that SP (10-12mol/L) can promote osteogenic differentiation of BMSCs in osteogenic environment, and Wnt/β-catenin signaling pathway may be involved in its process.Part3The effect of SP on the differentiation of pre-osteoblasts (MC3T3E1cells), and activation of Wnt/β-catenin signaling pathwayObjective:To investigate the effect of SP on the differentiation of pre-osteoblasts (MC3T3E1cells), and to explore the underlying mechanism by testing Wnt/β-catenin signaling pathway Methods:1. MC3T3E1cells were purchased from USA Cell Bank, culture condition was a-MEM+10%FBS, when the cells passaged to the6generation,Using optical microscope and inverted microscopeto observe the growth of cells, cell morphology, and cells were stained with Alizarin Red method and alkaline phosphatase staining method.2. Take6th generation cells, the cells were divided into four groups, SP (10"8mol/L) group, SP (10-9mol/L) group, SP (10-10mol/L) group and control group, each group were cultured under osteogenic environment (a-MEM+10%FBS+10mMβ-glycerol phosphate+50μg/mL vitamin C), expression of bone markers (alkaline phosphatase mRNA, collagen type I mRNA, osteocalcin mRNA and Runx2mRNA) were detected by Q-PCR to determine the optimum SP concentrations promoting the differentiation of BMSCs.3. According to SP concentration determined in the previous step, cells were divided into four groups, SP group, SP+SP receptor antagonist (1μmol/1), SP+DKK1group (0.2mg/mL) and the control group (the same amount of PBS), each group were cultured in osteogenic environment, expression of bone markers and Wnt/β-catenin signaling pathway related gene (C-myc mRNA, cyclin D1mRNA, Lefl, Tcf7and p-catenin mRNA) were detected by Q-PCR. Meanwhile expression of C-myc and β-catenin protein were detected by Western blot in each group at the time points of4,7,14d, the location of β-catenin in cells of each group were detected by immunofluorescence at the time point of4d.Results:1. To show the osteoblastic features of the MC3T3-E1line, cells before exposure to the differentiation medium were used for ALP and Alizarin red staining. ALP activity and presence of Ca deposits in extracellular matrix indicate that the MC3T3-E1cells possess osteogenic properties.2. Q-PCR results showed that SP (10-8mol/L,10-9mol/L,10-10mol/L) in the osteogenic conditions can significantly promote the expression of osteogenic markers in MC3T3E1cells, while the effect of10-8mol/L SP is the most significant. 3.10-8mol/L SP can significantly promote the expression of osteogenic markers in MC3T3E1, and can promote the expression of genes in Wnt/β-catenin signaling pathway, improve the expression of C-myc and β-catenin protein, promote distribution of β-catenin protein in the nucleus, SP receptor antagonist and DKK1could inhibit these effects of SP.Conclusion:MC3T3E1cells possess osteogenic properties of osteoblast, and therefore called the pre-osteoblasts.10-8mol/L SP can significantly promote differentiation of MC3T3E1in osteogenic environment, Wnt/β-catenin signaling pathway may be the potential mechanism. |
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