| The treatment of clinical fractures and possiblely combined with nonunion as well as other common orthopedic diseases is always the hot issue in addition to difficult problem in the field of orthopedic research, which causes the severe impact of functional outcome in patients.In recent years, with the rapid development of orthopedic trauma treatment, other new external fixation and bone tissue engineering technology research, patients were benefited from the in-depth mechanism study of the fracture regeneration and reconstruction. Thus, fracture regeneration and reconstruction process and its regulatory mechanism of the biological effects relative to variety of factors in extensive research, will provide a new treatment idea and possible techniques in clinical treatment.Neural factors in bone tissue regeneration and reconstruction process have an important role, also it is nerve neuropeptide factors that play a major role in regulating substances. Substance P (SP) is a neuropeptide secreted by sensory neurons widely distributed in the bone marrow and periosteum, and its biochemical structure consists of 11 amino acids, which involved in the regulation of bone tissue cells such as bone marrow stromal cells and osteoblasts. SP has been shown to participate in the cellular inflammation, proliferation, osteogenic differentiation, apoptosis, and the mobilization process. Bone regeneration and reconstruction process are revealed with the neuropeptides SP-positive nerve fibers, suggesting that SP may be directly involved in the local regulation of bone formation. Many studies in vivo and in vitro have shown that, SP can promote bone marrow stromal cell proliferation, differentiation and mineralization of osteoblasts thus promoting bone formation and regulation the physiological process of fracture healing.Bone formation bone marrow stromal cells requires a certain amount of foundation, and neuropeptide SP may be utilized to maintaining the basis of the number of bone marrow stromal stem cells, working as regulatory factors, namely the regulation of cell proliferation and apoptosis by controlling the number of cells. Studies have shown that, SP may act on human colon cancer cells by inhibiting apoptosis signaling pathway and inhibiting radiation damage in rat bone marrow stromal cell initially shows the protective effect of substance P. However, whether SP owns the regulation of apoptosis induced by serum in rat bone marrow stromal cells, has not yet been studied. Therefore, further research to explore the regulatory effect of neuropeptide substance P on bone marrow stromal cells to apoptosis induced by serum helps to understand the protective effect of neuropeptides in the fracture healing process.Regulation effect of neuropeptide SP is usually realized through related receptor, SP receptor mainly neurokinin-1 receptor (NK-1 receptor), and this receptor mediates the biological effects of SP to promote the process of bone formation. Studies have confirmed that bone marrow stromal cells and osteoblasts MC3T3-E1 cells stably expressing NK-1 receptor mRNA, and the expression of the site is located within the cytoplasm and cell membrane, suggesting the feasibility of the relevant regulatory role of SP. NK-1 receptor may mediate some of the anti-apoptotic response, such as NK-1 receptor antagonists can induce human cancer cell lines (Hep-2) apoptosis. So the correlation NK1-1 SP receptor expression and regulation of apoptosis mechanism may be regulated neuropeptide substance P during physiological fracture healing processes.Regulating biological effects of cell is inseparable with cell signaling pathways. Studies have confirmed that Wnt signaling pathway and MAPK signaling pathways involved in multiple cellular processes that SP could promote bone marrow stromal cell proliferation, differentiation and mineralization of osteoblasts, but the Wnt signaling pathway is involved in the anti-SP bone marrow stromal cells to serum-induced apoptosis has not been confirmed.Canonical Wnt signaling pathway plays an important role in bone metabolism physiological processes. Canonical Wnt pathway can be summarized as β-catenin (β-catenin) expression increases in the cytoplasm, then enters the nucleus when accumulated to a certain amount, causing the downstream Wnt-related genes such as c-myc transcript expression and so on. Studies have shown that, SP promotes bone marrow stromal stem cells and pre-osteoblast MC3T3-E1 cells the degree of differentiation associated with Wnt signaling pathway, whereas the Wnt signaling pathway and apoptosis pathway is also relevant. So whether SP anti-apoptotic effect can be regulated by canonical Wnt signaling pathway is unkown.Based on the above existing three issues, namely:whether substance P can regulate bone marrow stromal cells to serum-induced apoptosis? What would be the effect of its regulation of concentration and time characteristics? Its regulatory mechanisms involved in apoptosis signaling pathway in which the Wnt signaling pathway and change? Explain these issues will further deepen research on neural factors currently regulated by neuropeptides molecular mechanisms of fracture healing process, and the process of understanding the neuroprotective effect of fracture repair.This study is focused on regulation effects of serum-induced apoptosis by SP in rat bone marrow stromal cells and related classical Wnt/β-catenin pathway, also discussed NK-1 expression levels. This experiment experimental materials, methods, and conclusions include the following sections:Research purposes1. neuropeptide substance P affected apoptosis in rat bone marrow stromal cells.2. neuropeptide substance P regulate the serum-induced apoptosis pathway in rat bone marrow stromal cells.3. neuropeptide substance P and the regulation of apoptosis of bone marrow stromal cells relative to classical Wnt relations/β-catenin signal.Section 1 identified rat bone marrow stromal cells and NK-1 receptor detection Purpose:Get high purity in rat bone marrow stromal stem cells by using bone marrow culturing method and identified by flow purity, while studing the expression of NK-1 receptor.Experimental methods:1. Harvesting rat bone marrow stromal cells Select one month age or so animals weighting 80-100g SD rats, according to the experimental ethics requiring animals were killed off the neck in a clean condition, after cuting rat skin and subcutaneous muscle, femur and tibia are removed. Use a needle into the bone marrow cavity to washing, while adding DMEM medium (containing 10% fetal bovine serum) isolated and BMSCs are then cultured.2. cultured bone marrow stromal cellsPrimary cultures of rat bone marrow stromal cells after 48-72h are timely cleaning flasks after the first exchange of fluid removed after subculture non-adherent cells, according to 1:2 or 1:3 ratio after inoculation trypsin digestion and passage to culture flask, using containing fetal calf serum (10% volume fraction) and penicillin streptomycin solution (1% volume fraction) were cultured in DMEM medium. Bottle of liquid culture medium was changed every 2-3 days, cells were grown covered the bottom, after trypsin digestion by 1:2 passage through the light microscope and inverted microscope rat bone marrow stromal cell morphology characteristics.3. Identification of rat bone marrow stromal cellsTake three generations of bone marrow stromal cells, after washing with PBS, digestive cells to flow cytometry using a small stream to identify surface markers were detected by FL1-Height, CD90, IgG1, CD34, CD44, CD45, IgG2a, CD11b/C, HAMSTER and CD29 expression. By detecting CD34 and CD45 hematopoietic stem and progenitor cells to exclude the possibility of blood cells/fibroblasts, and to determine the bone marrow mesenchymal stem cells by detecting the purity of CD44, CD29.4. NK-1 receptor expression was detectedThe cells were plated in 96-well plates, after fixing using an antibody (anti-NK-1 receptor antibodies) were incubated, followed by FITC secondary antibody incubation, to be fully observed under fluorescence microscope after staining. After the cells were applied to normal and serum treatment, the NK-1 receptor protein is detected by western blot.Results:By using bone marrow culture method, the cells are successfully isolated and cultured in vitro which can be used for experiments. Flow cytometry identification showed FL1-Height (0.2%±1.7%), CD90 (99.7%±3.1%), IgG1(0.96%±1.3%), CD34 (1.06%±1.3%), CD44 (99.8%±4.1%), CD45 (1.7%±2.1%), IgG2a (0.35%± 2.9%), CDllb/C (0.6%±1.7%), HAMSTER (0.09%±0.7%), CD29 (99.88%± 3.1%) immune phenotypic characteristics consistent with bone marrow stromal cells. Meanwhile, NK-1 receptor expression in bone marrow stromal cells and cytoplasmic membrane, the protein expression level is relatively stable.Results:By using bone marrow culture method, we can get a lot of high purity rat bone marrow stromal cells, and its surface stably expressed NK-1 receptor, providing a theoretical basis for the cells for the next study.Section 2 substance P acts on activity of serum-induced bone marrow stem cellsPurpose:Preliminary observation is made on the cell activity changes under treatment of substance P during serum-induced process.Methods:1. Experimental groupsTake three generations of bone marrow stromal cells that divided into four groups, namely the control group (added the same amount of PBS), SP (10-8 mol/L) group, SP (10-10 mol/L) group, SP (10-12 mol/L) group were seeded in 96-well plates to 12 hours after induction of apoptosis and 24 hours respectively using MTT detecting cell viability assay. After the initial screening of suitable concentration and a suitable point in time, DAPI staining and Annexin V-FITC/PI staining are used.2. MTT cell viability assayEach hole by adding 15μl of MTT solution (1:10 dilution), at 37℃ environment within 4 h incubation, each well was added to 150μl of DMSO used to dissolve crystals produced, followed by the use of measured absorbance microplate reader (495 nm wavelength readings).3. DAPI fluorescence staining nucleiAfter taking third-generation rat bone marrow stromal cells, appropriately adjust the cell density, were seeded in 96-well plate experiments intervention added 24 h after 50μl of DAPI staining solution for 15 minutes, then discard the stain with UV light under a fluorescence microscope excitation observed and photographed.4. Detecting apoptosis rate by flow cytometry with Annexin V-FITC/PI staining.After taking the third generation cell experiments intervention after washing with DPBS trypsin EDTA-free, cells were collected in the streaming tubules then were added Annexin V-FITC and PI reagents thoroughly mixed, while the establishment of the negative control and positive control tube, machine measuring Annexin V-FITC and PI fluorescence signal.Results:MTT assay showed 24h to serum treatment,10-10 mol/L SP experimental treatment can improve cell activity, further DAPI staining showed that SP can protect nuclear morphology shrinkage deformation and other changes, and reduce the rate of apoptosis (Annexin V fluorescence notation). The experimental results:Substance P can increase the activity of bone marrow stromal cells after serum treatment to improve nuclear morphology changes and reduces the rate of apoptosis.Section 3 Preliminary study of P substance on bone marrow mesenchymal stem cells to serum-induced apoptosis and related endogenous/exogenous apoptotic pathway regulation mechanismPurpose: Explore apoptosis pathway signal changes of substance P regulation of bone marrow stromal cells during apoptosis.Methods:1. Experimental Packet ProcessingFirst, the third-generation rat bone marrow stromal cells were randomly divided into four groups, namely the control group, SP (10-8 mol/L) group, SP (10-10 mol/L) group, SP (10-12 mol/L) group,3-apoptotic protein expression was measured to explore the anti-substance P concentrations of specific apoptotic effects. Further settings in the control group, SP (10-10 mol/L) group, SP (10-10 mol/L)+NK-1 receptor antagonist (Spantide 10-8 mol/L) group to explore the anti-apoptotic effect SP mechanisms.2. immunofluorescence, Western blot, Q-PCR detection of apoptosis protein 3 (caspase-3) expressionCells taken after the experimental treatment, and paraformaldehyde, use 1% BSA for 30 minutes antigen, caspase-3 antibody was incubated overnight incubation at room temperature for 15 minutes the second antibody, photographed under a fluorescence microscope. After extraction of RNA and protein in the experimental group, the quantitative determination of caspase-3 mRNA and protein expression.3. Q-PCR to detect apoptotic gene changeRneasy kit utilizing the experimental group was extracted RNA, RNA concentration and purity determination. The use of synthesized cDNA reverse transcription reaction, fluorescence quantitative determination of Bcl-2, Bax, caspase-3, caspase-8, caspase-9 content, the results based on an amendment to the internal control GAPDH content.Results:Substance P acts on Serum after bone marrow stromal cells, the number of three-apoptotic protein staining positive cells decreased, reduce the associated RNA and protein expression to 10-10 mol/L most obvious. Apoptotic genes to change the display of substance P in 12 hours and 24 hours can reduce the apoptotic signal Bax/ Bcl-2 ratio regulation of caspase-8, caspase-9 and caspase-3 apoptotic genes changed.The experimental results:Substance P can regulate endogenous apoptosis pathway signal bone marrow stromal cells and reduce apoptosis protein 3 expression, thereby inhibiting cell apoptosis by serumSection 4 the regulation of serum-induced apoptosis by substance P in bone marrow stem cells related to Wnt/β-catenin pathway mechanismPurpose:Preliminary study of substance P canonical Wnt pathway regulation of bone marrow stromal cells signal-dependent mechanism of apoptosis.Methods:1. Experimental Packet ProcessingFirst set the control group, SP (10-10 mol/L) group, SP (10-10 mol/L)+Wnt pathway antagonists DKK (0.01g/L) group, SP (10-10 mol/L)+NK 1 receptor antagonist (Spantide 10-8 mol/L) group, the determination of the Wnt pathway and apoptosis pathway gene protein change, and set the controls again, SP (10-8 mol/L) group, SP (10-10 mol/L) group, SP (10-12 mol/L) group confirmed the Wnt pathway proteins related to changes in circumstances.2. Western blot, Q-PCR detection of gene and protein expression of Wnt pathway and apoptosis pathwayReverse transcription reaction in the experimental group after the extraction of total RNA, fluorescence quantitative determination of Bcl-2, Bax, caspase-3, caspase-8, caspase-9, β-catenin, GSK-3β, c-myc and cyclin Dl content. Correlation results at 12 hours and 24 hours was measured three times respectively.3. immunofluorescence assay β-catenin protein localizationCells were taken after three generations of the experimental treatment,4% paraformaldehyde for 15 minutes, using 0.4% Triton solution rupture 10 minutes, skim milk blocking antibodies for 30 minutes prior to 1:300 dilution of β-catenin antibody was incubated overnight PBST solution.After washing with PBS three times, the following FITC green fluorescent secondary antibodies were incubated in the dark at room temperature for 30 minutes, again after the liquid has been sucked out of DAPI staining 15 minutes, under a fluorescence microscope.Results:Substance P acts on the bone marrow stromal cells, promote the canonical Wnt pathway β-catenin, p-GSK-3β, c-myc and cyclin D1 gene and protein expression and increased β-catenin into the nucleus transfer, blocking Wnt pathway withered after anti-substance P death effect of weakening the Wnt pathway and reduce the level of performance.Experiments Conclusion:Bone marrow stromal cells can activate the canonical Wnt pathway, thereby regulating apoptosis by serum. |
PDF Full Text Request