Maxillary palatal side bone cortex remodeling mechanical response protein detection method

Abstract

The invention provides an upper jaw palatal side bone cortex remodeling mechanical response protein detection method, and belongs to the technical field of response protein detection.The method comprises the following steps of upper jaw anterior tooth adduction experiment animal model specimen construction, bone cortex structure and density change, bone morphological metrology analysis and osteogenesis and apoptosis related marker detection. Dyeing and detecting apoptotic cells, analyzing the change of a palate-side bone cortical fossa-bone tubule system in the maxillary anterior tooth adduction process and the mechanical response of the in-vitro cultured bone cells under a fluid shear force, and screening the in-vitro cultured bone cell mechanical response key protein A; and analyzing the action mechanism of the key protein A for regulating and controlling the functions of osteoblasts or osteoclasts in the bone cells cultured by the in-vitro fluid shear force. Flow of interstitial fluid in a bone fossa-bone tubule system caused by force load is in the form of bone cell recognition signals, and the biological regulation and control mechanism of bone cells under the condition that fluid in bone flows can promote people to deeply understand the bone cortex remodeling process caused by orthodontic force.

Description

Technical Field

[0001] The invention relates to the technical field of response protein detection, and in particular to a method for detecting mechanical response proteins of maxillary palatal cortical bone remodeling. Background Art

[0002] Skeletal Class II malocclusion Malocclusion is a common craniofacial deformity in orthodontic clinics, mainly manifested as obvious maxillary protrusion and (or) mandibular retrusion, which has a great damage to facial beauty and can affect the patient's mental health and even normal social interaction. Orthodontic-orthognathic combined treatment, or compensatory tooth movement for orthodontic camouflage treatment is a common means of correcting deformity. Due to certain surgical and anesthesia risks, as well as long treatment cycles and high costs, many patients are unable to undergo surgical treatment to improve their deformities. In recent years, with the widespread application of implant support in orthodontic diagnosis and treatment, the ability of orthodontists to move teeth has undergone a qualitative change, such as using absolute support to retract the anterior teeth, which has broadened the scope of orthodontic clinical application to a certain extent. However, at the same time, due to the long distance of tooth retraction or the congenital deficiency of the thickness of the alveolar bone in the anterior area, there is a risk of bone fenestration, bone cracking or root absorption of the palatal bone tissue after the anterior teeth are retracted. Therefore, whether the thickness of the palatal alveolar bone in the anterior area can be increased, or whether the remodeling of the palatal alveolar bone can be induced to expand the safe range of anterior tooth retraction is a key issue for skeletal Class II malocclusion. The key and difficulty of orthodontic compensation treatment of deformity. Therefore, it is necessary to design a detection method for the mechanical response protein of maxillary palatal cortical bone remodeling so that maxillary palatal cortical bone remodeling can become a reality. Summary of the invention

[0003] The purpose of the present invention is to provide a method for detecting mechanical response proteins of maxillary palatal cortical bone remodeling, so as to solve the technical problem that the existing maxillary palatal cortical bone remodeling cannot be achieved.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A method for detecting mechanical response proteins of maxillary palatal cortical bone remodeling, the method comprising the following steps:

[0006] Step 1: Construct the maxillary anterior teeth retraction experimental animal model specimen;

[0007] Step 2: Changes in cortical bone structure and density;

[0008] Step 3: bone histomorphometric analysis;

[0009] Step 4: Detection of osteogenesis and apoptosis-related markers;

[0010] Step 5: staining to detect apoptotic cells, dewax and hydrate the paraffin sections, add methyl green anti-staining solution, seal the sections with sealing solution containing anti-fluorescence quencher, observe under a laser scanning confocal microscope, and count the number of TUNEL-positive and TUNEL-negative cells in the bone cortex;

[0011] Step 6: Analyze the changes of the palatal cortical bone lacuna-canalicular system during the retraction of the maxillary anterior teeth;

[0012] Step 7: Mechanical response of in vitro cultured osteoblasts under fluid shear stress;

[0013] Step 8: Screening of key protein A in the mechanical response of bone cells cultured in vitro;

[0014] Step 9: Analyze the mechanism of action of key protein A in regulating osteoblast or osteoclast function in bone cells cultured under fluid shear force in vitro.

[0015] Furthermore, the specific process of step 1 is:

[0016] Eighteen healthy adult beagle dogs aged 24 months were selected. All animals met the international laboratory animal requirements and had a body mass of 10-15 kg. The experimental animals were randomly divided into two groups: a force group with a force value of 150 g to retract the maxillary incisors and a control group with no force applied to the maxillary anterior teeth;

[0017] After the two groups of experimental animals were adapted to caged conditions for one week, they were anesthetized by intramuscular injection of 3% pentobarbital solution at 30 mg / kg body weight, alginate was used to take molds and perfuse hard plaster models, individual bands were made and force-adding devices were welded, the third anterior teeth of the maxillary teeth on both sides were extracted, and force-adding devices were bonded. A nickel-titanium tension spring was used to apply traction force of 0g and 150g to the two groups of animals from the molars to the anterior teeth, respectively. The traction force was tested once a week with a dynamometer, and the oral cavity was cleaned with 2g / L chlorhexidine twice a week. The animals were fed a normal diet. The experimental animals were euthanized at the 4th, 7th, and 10th weeks of orthodontic force loading, and tissue specimens were collected.

[0018] Furthermore, the specific process of step 2 is:

[0019] The maxillary bones of the specimens were separated and cut to make experimental bone blocks and control bone blocks in the maxillary anterior area, which were preserved in 4% paraformaldehyde and fixed for 48 hours. Micro-computed tomography was used to measure the porosity, total cortical area, cortical area, cortical thickness, mineral content and mineral density of the cortical bone, and to analyze the changes in the cortical bone structure under the action of orthodontic force.

[0020] Furthermore, the specific process of step 3 is:

[0021] Tetracycline was injected intramuscularly 20 and 21 days before the animals were killed; Alizarin red was injected intramuscularly 13 and 14 days before the animals were killed, and calcein was injected intramuscularly 3 and 4 days before the animals were killed, with the dose of 15 mg / Kg.

[0022] Hard tissue specimens were removed from 4% paraformaldehyde, dehydrated with gradient ethanol, embedded in plexiglass, and removed after the tissue blocks were completely hardened. The tissue blocks were trimmed and sliced ​​using a Leitzl600 saw slicer. The specimens were mounted after ultrasonic cleaning, polished, and sealed with neutral gum after staining. Hematoxylin-eosin staining, Von Kossa staining, alkaline phosphatase staining, and tartrate-resistant acid phosphatase staining were used to detect the osteoid surface, osteoblast surface, relative number of osteoclasts, bone resorption surface and mineralization deposition rate. The bone cortex tissue morphometric analysis was performed to reflect the changes in bone turnover and bone remodeling at the cellular level.

[0023] Furthermore, the specific process of step 4 is:

[0024] Immunohistochemistry was used to detect the expression of osteogenic markers ALP, Runx2, and OCN, and apoptotic markers B-lymphocytoma-2 and caspase-3;

[0025] Hard tissue specimens were removed from 4% paraformaldehyde, and paraffin sections were prepared conventionally. After gradient ethanol dehydration, 0.3% methanol solution of hydrogen peroxide was added to eliminate the influence of endogenous peroxides. Primary antibodies diluted with serum diluent were added and incubated at 4°C overnight. Secondary antibodies, i.e., biotinylated goat anti-rabbit IgG, were added and incubated at room temperature for 2h. Color developing solution was added for immunohistochemical color development. After gradient alcohol dehydration, the sections were sealed and photographed. The force application time of the group with the maximum osteogenic activity was selected as the parameter for the later experiments.

[0026] Furthermore, the specific process of step 6 is:

[0027] (1) Observation of the morphological changes of bone lacuna and canalicular system using laser scanning confocal microscopy

[0028] The hard tissue specimens were immersed in gradient ethanol containing 1% basic fuchsin for dehydration and staining, and then embedded in polymethyl methacrylate and sliced ​​using the EXAKT cutting and grinding system to make 10 μm slices. The slice surface was polished with sandpaper. The morphology, area, and direction of the bone lacuna and canaliculus in the bone cortex were observed using a laser scanning confocal microscope. The fluorescence excitation wavelength of the filter was 580 nm. 21 layers of optical slices were cut under a 100x oil immersion lens, with an interval of 0.5 μm between each layer and a total thickness of 10 μm.

[0029] (2) Scanning electron microscopy observation of the structural changes of the lacunae-canaliculi system

[0030] After PMMA embedding, the hard tissue sections were etched with 9% orthophosphoric acid for 20 seconds with the polished surface facing up, then washed with deionized water and immersed in 5% sodium hypochlorite for 5 minutes, washed with deionized water, air-dried and sprayed with gold. The canalicular diameter and bone lacuna density were observed with a Quanta 250 scanning electron microscope.

[0031] Furthermore, the specific process of step 7 is:

[0032] (1) Construction of a fluid shear force loading model for in vitro cultured bone cells

[0033] ① Isolation, culture and identification of bone cells

[0034] Take the tissue block under the femoral neck, place it in α-MEM containing 10% penicillin-streptomycin, remove the excess tissue and repeatedly rinse the bone marrow surface, rinse with HBSS 3 times, cut the bone tissue into 1 cm2 small pieces, add 2 mg / ml type I collagenase, digest for 15 minutes, wash 3 times with HBSS, repeat 2 times, add 0.02% EDTA containing 1% BSA, culture in the incubator for 25 minutes, discard the supernatant, wash 3 times with HBSS, repeat adding type I collagenase, digest for 25 minutes, wash 3 times with HBSS, collect the washing liquid and supernatant, centrifuge at 1200r / min for 5 minutes, discard the supernatant, resuspend in the culture medium and implant it in a 6-well plate coated with type I rat tail collagen with the digested bone tissue, culture in the incubator, and identify bone cells by morphological observation, E11 / GP38 fluorescence staining, and ALP staining after 2-3 generations of subculture;

[0035] ② Construction of fluid shear force loading model

[0036] A parallel plate flow chamber was used to apply fluid shear force to the bone cells. The force-adding device was made of two layers of organic glass, with a 1cm×1cm×0.02cm square groove in the center of the lower layer, which could hold a coverslip covered with bone cells. There was a 6cm×1.35cm×0.04cm rectangular groove on the upper layer, which formed a channel for liquid flow with the upper organic glass. An opening was opened at each end of the channel for injecting and discharging liquid. The liquid was a liquid flow with a fixed shear force provided by the perfusion system. The third generation of bone cells were inoculated on the coverslip, which was placed in the square groove in the center of the flow chamber system and divided into four groups. The fluid shear forces loaded in each group were 0.8Pa, 1.2Pa, 1.4Pa and 1.6Pa, respectively, and the loading lasted for 10min, 15min, 30min and 60min. The experiment was repeated 6 times in each time period, and no force was applied to the control group.

[0037] (2) Detection of bone cell functional status, proliferation activity and secretion factors

[0038] MTT colorimetry and flow cytometry were used to identify the functional status and proliferation activity of osteocytes under different force values ​​and time. Enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction-protein blotting were used to detect the changes in the gene and protein expression of OPG, PGE2, and Sost in osteocytes after fluid shear force.

[0039] Furthermore, the specific process of step 8 is:

[0040] (1) Experimental groups

[0041] The osteocytes cultured by fluid shear force in vitro were divided into two groups: a control group and an experimental group. The osteocytes in the control group were not subjected to force, while the osteocytes in the experimental group were subjected to the optimal fluid shear force according to the above experimental results.

[0042] (2) Transcriptomics screening of differentially expressed genes in response to osteocyte mechanochemistry

[0043] The total RNA of bone cells in the force group and the control group was extracted by Trizol method, and the extracted total RNA was reverse transcribed to synthesize cDNA and add adapter sequence; the cDNA was divided according to the fragment size; the cDNA was cyclized and sequenced on a machine. After quantitative analysis of sample transcription, differentially expressed genes were screened, and the common differential genes were imported into relevant software for interaction analysis to obtain clusters of potential targets of differentially expressed genes. The identified genes were subjected to gene function cluster analysis and Kyoto Encyclopedia of Genes and Genomes analysis to screen out key genes for mechanical response of bone cells;

[0044] (3) iTRAQ technology combined with LC-ESI-MSMS to screen differentially expressed proteins in response to mechanical changes in bone cells

[0045] Total protein samples were extracted from the force group and the control group. The samples were subjected to SDS-PAGE electrophoresis, and the protease peptides were labeled with different isotope relative and absolute quantitative labels. The samples were separated by liquid phase using UltremexSCX column and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry based on Triple TOF 5600. The same bioinformatics method was used to screen out the key proteins of bone cell mechanical response.

[0046] (4) Combined transcriptomic and proteomic analysis reveals the mechanism by which orthodontic force regulates maxillary palatal cortical bone remodeling

[0047] The identified proteins were associated with the transcriptome results; the common targets were projected to KEGG, and the KEGG enrichment results were plotted into a KEGG pathway enrichment clustering heat map. Clustering was performed at the protein level according to the differential expression multiples to obtain the key gene A, and RT-PCR and Western Blot techniques were used to perform biological identification of A.

[0048] Furthermore, the specific process of step 9 is as follows:

[0049] (1) Isolation and culture of osteoblasts and osteoclasts

[0050] ① Isolation, culture and identification of osteoblasts

[0051] After the dog was euthanized, part of the femur was cut off under sterile conditions, the medullary cavity was washed with sterile injection water, the bone blocks were broken into a diameter of 1-2 mm, and placed in a 6-well plate. An appropriate amount of type I collagenase solution was added to each well at a concentration of 1 mg / mL, and incubated in a 37°C constant temperature shaker for 15 minutes. The culture medium was replaced and incubated on ice for 5 minutes, and repeated 3 times. Then, 0.5 mM EDTA solution was used for decalcification for 5 minutes, and the enzymatic solution was alternated for 15 minutes. The above steps were repeated 3 times, and the enzymatic solution and EDTA solution collected in the last 3 times were centrifuged, and the obtained lower layer cells were cultured in a culture incubator. After 2-3 generations of subculture, osteoblasts were identified by morphological observation and ALP staining;

[0052] ② Isolation, culture and identification of osteoclasts

[0053] Peripheral blood was collected from dogs, and peripheral blood mononuclear cells were isolated by density gradient centrifugation. The isolated PBMCs were cultured and inoculated in six-well plates. Macrophage colony stimulating factor was added, and the supernatant was discarded after overnight incubation. 50ng / ml RANKL reagent was added for culture. After 2-3 generations of subculture, osteoclasts were identified by morphological observation and TRAP staining;

[0054] (2) The effect of silencing / overexpressing key protein A on the regulation of osteoblastic or osteoclast function of osteocytes cultured by fluid shear force in vitro. Protein A is gene A

[0055] ① Experimental grouping

[0056] According to different treatment methods, the osteoblasts / osteoclasts co-cultured osteocytes were divided into three groups: blank control group, A gene transfection group, i.e. A siRNA interference group or A overexpression group, and negative control group. siRNA interference plasmid or overexpression plasmid was constructed for key gene A, and the extracted canine osteocytes were transfected after lentivirus packaging to inhibit / upregulate the expression of key gene A.

[0057] ②Construction of osteocyte-osteoblast / osteoclast conditioned medium co-culture model

[0058] According to the above experiments, the best force value and action time were selected, and fluid shear force was applied to the three groups of bone cells. After stopping the force, the cells were washed twice with PBS solution, and 4 mL of serum-free culture medium was added; the culture was continued for 24 hours, the supernatant was collected, centrifuged at 4°C, 1200 rpm, 5 minutes, and stored in a -80°C ultra-low temperature refrigerator. 1×105 osteoblasts or osteoclasts / well were cultured at a volume ratio of conditioned medium: total culture medium of 1:2, and the culture plate was placed in a packing box for 3-5 days;

[0059] ③The effect of silencing / overexpressing key gene A on the function of osteoblasts or osteoclasts regulated by osteocytes cultured by fluid shear force in vitro

[0060] A gene and osteocyte secretory factors: OPG, PGE-2, Sost gene and protein expression were detected; RT-PCR, Western Blot, and ELISA were used to detect osteoblast markers ALP, Runx2, and OCN gene and protein expression, and ALP staining was used to observe changes in osteoblast function; R was used to detect osteoclast markers nuclear factor of activated T cells 1, cathepsin K, and tartrate-resistant acid phosphatase gene and protein expression, and TRAP staining was used to observe changes in osteoclast function.

[0061] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0062] (1) The force load of the present invention causes the flow of tissue fluid in the bone lacuna-canalicular system, which is a form of bone cell recognition signal. The biological regulation mechanism of bone cells under the flow of fluid in the bone will promote people's in-depth understanding of the process of orthodontic force-induced cortical bone remodeling. One of the main research directions in the field of bone cell mechanics is the in vitro experiment of cell fluid mechanics. On the basis of constructing an in vitro cultured bone cell fluid shear force loading model, transcriptomics and proteomics were further combined for analysis to try to obtain the differentially expressed genes and proteins in bone cells in response to fluid flow. By analyzing the changes in the transcription and translation levels of the differentially expressed genes, the gene expression regulation mechanism of bone cell mechanical response was fully explained, providing a technical basis for the subsequent role of gene A in cortical bone remodeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a flow chart of analyzing the changes of the maxillary palatal cortical bone and the bone lacuna-bone canalicular system in the present invention by orthodontic force regulation;

[0064] Figure 2 is a flow chart of the mechanism of analyzing the response of in vitro cultured bone cells to fluid shear force of the present invention;

[0065] Figure 3 The present invention analyzes the effect of key gene (protein) A on the regulation of osteoblast / osteoclast function of bone cells cultured by fluid shear force in vitro. DETAILED DESCRIPTION

[0066] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0067] like Figure 1-3 As shown, a method for detecting mechanical response proteins of maxillary palatal cortical bone remodeling comprises the following steps:

[0068] Step 1: Changes in the palatal cortical bone during maxillary anterior tooth retraction

[0069] (1) Experimental animals

[0070] Eighteen healthy adult beagle dogs aged 24 months were selected. All animals met the international laboratory animal requirements and had a body weight of (10-15) kg. The experimental animals were randomly divided into two groups: a force-added group (150 g force to retract the maxillary incisors) and a control group (no force added to the maxillary anterior teeth).

[0071] (2) Construction of an animal model for maxillary anterior teeth retraction experiment

[0072] After one week of adaptation of the two groups of experimental animals to captivity, they were anesthetized by intramuscular injection of 3% pentobarbital solution at 30 mg / kg body weight, and alginate was used to take molds and perfuse hard plaster models to make individual bands and weld force devices. The third anterior teeth of the maxillary teeth on both sides were extracted and the force device was bonded. A nickel-titanium tension spring was used to apply traction force of 0g and 150g to the two groups of animals from the molars to the anterior teeth, and the traction force was tested once a week with a dynamometer. The oral cavity was cleaned with 2g / L chlorhexidine twice a week. A normal diet was maintained, and the experimental animals were euthanized at the 4th, 7th, and 10th week of orthodontic force loading, and tissue specimens were collected.

[0073] (3) Changes in cortical bone structure and density

[0074] After the force was applied, the experimental animals were killed, and the maxilla was separated and cut to make the experimental bone blocks and control bone blocks of the maxillary anterior area, which were stored in 4% paraformaldehyde and fixed for 48 hours. Micro computed tomography (Micro-CT) was used to measure the cortical porosity (Ct.Po), total tissue areas (Tt.Ar), cortical area (Ct.Ar), cortical width (Ct.Th), mineral content (Bone mineral content, BMC), mineral density (Bone mineral density, BMD), etc., to analyze the changes in the microstructure of the cortical bone under the action of orthodontic force.

[0075] (4) Bone morphometric analysis

[0076] Tetracycline was injected intramuscularly 20 and 21 days before the animals were killed; Alizarin red was injected intramuscularly 13 and 14 days before the animals were killed; calcein was injected intramuscularly 3 and 4 days before the animals were killed, and the dose was 15 mg / Kg.

[0077] Remove the hard tissue specimen from 4% paraformaldehyde, dehydrate with gradient ethanol, and embed in organic glass. After the tissue block is completely hardened, remove the specimen and trim the tissue block. Use Leitzl600 saw slicer for slicing. After ultrasonic cleaning, paste the slice, grind and polish, and seal the slice with neutral gum after staining. Hematoxylin-eosin staining (HE), Von Kossa staining, alkaline phosphatase (ALP) staining, and tartrate resistant acid phosphatase (TRAP) staining were used to detect the osteoid surface (OS / BS), osteoblast surface (Ob.S / BS), relative number of osteoclasts (osteoclast number, No.Oc. / B.Pm), bone resorption surface (ES / BS), mineral apposition rate (MAR), etc., and cortical bone tissue morphometric analysis was performed to reflect the changes in bone turnover and bone remodeling at the cellular level.

[0078] (5) Detection of osteogenesis and apoptosis-related markers

[0079] Immunohistochemistry was used to detect the expression of osteogenic markers ALP, Runx2, OCN and apoptosis markers B-cell lymphoma-2 (Bcl-2) and cysteine ​​protease-3 (caspase-3). Hard tissue specimens were removed from 4% paraformaldehyde and paraffin sections were prepared routinely. After gradient ethanol dehydration, a 0.3% methanol solution of hydrogen peroxide was added to eliminate the effects of endogenous peroxides. Primary antibodies (ALP, Runx2, OCN, Bcl-2, caspase-3) diluted with serum diluent were added and incubated at 4°C overnight. Secondary antibodies (biotinylated goat anti-rabbit IgG) were added and incubated at room temperature for 2 hours. Color development solution was added for immunohistochemical color development. After gradient alcohol dehydration, the sections were sealed and photographed. The force application time of the group with the highest osteogenic activity was selected as the parameter for the later experiments.

[0080] (6) TUNEL staining to detect apoptotic cells

[0081] Paraffin sections were dewaxed and hydrated, and methyl green anti-staining solution was added according to the reagent manufacturer's instructions, and the sections were sealed with sealing solution containing anti-fluorescence quencher. The sections were observed under a laser scanning confocal microscope, and the number of TUNEL-positive and TUNEL-negative cells in the bone cortex was counted.

[0082] Step 2: Changes in the palatal cortical bone lacuna-canalicular system during maxillary anterior tooth retraction

[0083] (1) Observation of the morphological changes of the lacunae-canaliculi system using laser scanning confocal microscopy

[0084] The hard tissue specimens were immersed in gradient ethanol containing 1% basic fuchsin for dehydration and staining; after embedding in polymethylmethacrylate (PMMA), 10 μm sections were made using the EXAKT cutting and grinding system. The surface of the sections was polished with sandpaper. The morphology, area size, and direction of the bone lacunas and canaliculi in the bone cortex were observed under a laser scanning confocal microscope, and the fluorescence excitation wavelength of the filter was 580 nm. 21 layers of optical sections were cut under a 100x (NA 1.4) oil immersion lens, with an interval of 0.5 μm between each layer and a total thickness of 10 μm.

[0085] (2) Scanning electron microscopy observation of the structural changes of the lacunae-canaliculi system

[0086] After PMMA embedding, the hard tissue sections were etched with 9% orthophosphoric acid for 20 seconds with the polished surface facing up. Then they were washed with deionized water and immersed in 5% sodium hypochlorite for 5 minutes. After being washed with deionized water and air-dried, they were sprayed with gold and the canalicular diameter and bone lacuna density were observed with a Quanta 250 scanning electron microscope.

[0087] Step 3: Analyze the mechanical response of osteoblasts cultured in vitro under fluid shear stress

[0088] (1) Construction of a fluid shear force loading model for in vitro cultured bone cells

[0089] ① Isolation, culture and identification of bone cells

[0090] After the beagle dogs were euthanized, the tissue blocks below the femoral neck were taken and placed in α-MEM containing 10% penicillin-streptomycin. The excess tissue was removed and the bone marrow surface was repeatedly rinsed. After rinsing with HBSS three times, the bone tissue was cut into 1 cm2 small pieces. 2 mg / ml type I collagenase was added, digested for 15 minutes, and then washed with HBSS three times. Repeated twice. 0.02% EDTA containing 1% BSA was added, and the supernatant was discarded after incubation in an incubator for 25 minutes, and washed with HBSS three times. Repeatedly add type I collagenase, digested for 25 minutes, washed with HBSS three times, and then the washing liquid and supernatant were collected, centrifuged at 1200r / min for 5 minutes, and the supernatant was discarded. After resuspending in the culture medium, the digested bone tissue was implanted in a 6-well plate coated with type I rat tail collagen and cultured in an incubator. After 2-3 generations of subculture, bone cells were identified by morphological observation, E11 / GP38 fluorescence staining, and ALP staining.

[0091] ② Construction of fluid shear force loading model

[0092] A parallel plate flow chamber was used to apply fluid shear force to osteocytes. The force-adding device was made of two layers of plexiglass, with a 1cm×1cm×0.02cm square groove in the center of the lower layer, which could hold a coverslip covered with osteocytes. Above this groove was a 6cm×1.35cm×0.04cm rectangular groove, which formed a channel for liquid flow with the upper plexiglass. An opening was opened at each end of the channel for injecting and outflowing liquid. This liquid was a liquid flow with a certain shear force provided by the perfusion system. The third generation osteocytes were inoculated on the coverslip, which was placed in the square groove in the center of the flow chamber system and divided into 4 groups. The fluid shear forces loaded in each group were 0.8Pa, 1.2Pa, 1.4Pa and 1.6Pa, respectively, and the loading was 10min, 15min, 30min, and 60min. The experiment was repeated 6 times in each time period. No force was applied to the control group.

[0093] (2) Detection of bone cell functional status, proliferation activity and secretion factors

[0094] MTT colorimetry and flow cytometry were used to identify the functional state and proliferation activity of osteocytes under different force values ​​and time, and the optimal force value and action time were selected to lay the foundation for subsequent experiments. Enzyme-Linked ImmunoSorbent Assay (ELISA), Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and Western Blot were used to detect the changes in the gene and protein expression of OPG, PGE2, and Sost in osteocytes after fluid shear force.

[0095] Step 3: Screening of key genes (proteins) for mechanical response of osteoblasts cultured in vitro

[0096] (1) Experimental groups

[0097] The osteocytes cultured with fluid shear force in vitro were divided into two groups: a control group and an experimental group. The osteocytes in the control group were not subjected to force, while the osteocytes in the experimental group were subjected to the optimal fluid shear force according to the above experimental results.

[0098] (2) Transcriptomics screening of differentially expressed genes in response to osteocyte mechanochemistry

[0099] The total RNA of bone cells in the force group and the control group was extracted by Trizol method, and the extracted total RNA was reverse transcribed, and cDNA was synthesized and the adapter sequence was added; the cDNA was segmented according to the fragment size; the cDNA was cyclized and sequenced. After quantitative analysis of sample transcription, differentially expressed genes were screened, and the common differential genes were imported into relevant software for interaction analysis to obtain clusters of potential targets of differentially expressed genes. The identified genes were subjected to gene function clustering (gene ontology, GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to screen out key genes for mechanical response of bone cells.

[0100] (3) iTRAQ technology combined with LC-ESI-MSMS to screen differentially expressed proteins in response to mechanical changes in bone cells

[0101] Total protein samples were extracted from the force group and the control group. The samples were subjected to SDS-PAGE electrophoresis, and the protease peptides were labeled with different isotope relative and absolute quantitative labels. The samples were separated by liquid phase using an UltremexSCX column. High-performance liquid chromatography-electrospray tandem mass spectrometry analysis based on Triple TOF 5600 was used to screen out key proteins of osteocyte mechanical response using the same bioinformatics method.

[0102] (4) Combined transcriptomic and proteomic analysis reveals the mechanism by which orthodontic force regulates maxillary palatal cortical bone remodeling

[0103] The identified proteins were associated with the transcriptome results; the common targets were projected to KEGG, and the KEGG enrichment results were plotted into a KEGG pathway enrichment clustering heat map, and clustered at the protein (gene) level according to their differential expression folds to obtain the key gene (protein) A. RT-PCR and Western Blot techniques were used to identify A biologically.

[0104] Step 4: Analyze the mechanism of action of key gene A in regulating osteoblast (or osteoclast) function in osteocytes cultured by fluid shear force in vitro

[0105] (1) Isolation and culture of osteoblasts and osteoclasts

[0106] ① Isolation, culture and identification of osteoblasts

[0107] After the dog was euthanized, part of the femur was cut off under sterile conditions, the medullary cavity was washed with sterile injection water, the bone blocks were broken into a diameter of 1-2 mm, and placed in a 6-well plate. An appropriate amount of type I collagenase solution was added to each well at a concentration of 1 mg / mL. The plate was incubated in a constant temperature shaker at 37°C for 15 minutes, and the culture medium was replaced and incubated on ice for 5 minutes, and repeated 3 times. Then, decalcification was performed with 0.5mM EDTA solution for 5 minutes, and the enzymatic solution was alternated for 15 minutes. The above steps were repeated 3 times, and the enzymatic solution and EDTA solution collected in the last 3 times were centrifuged, and the lower layer of cells was cultured in a culture incubator. After 2-3 generations of subculture, osteoblasts were identified by morphological observation and ALP staining.

[0108] ② Isolation, culture and identification of osteoclasts

[0109] Peripheral blood was collected from dogs, and peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. The isolated PBMC were cultured and inoculated in a six-well plate, and macrophage colony stimulating factor (M-CSF) was added. After overnight incubation, the supernatant was discarded and 50ng / ml RANKL reagent was added for culture. After 2-3 generations of subculture, osteoclasts were identified by morphological observation and TRAP staining.

[0110] (2) Effects of silencing / overexpressing key gene A on the regulation of osteoblast (or osteoclast) function by osteocytes cultured by fluid shear force in vitro

[0111] ① Experimental grouping

[0112] According to different treatment methods, the osteoblasts / osteoclasts co-cultured osteocytes were divided into three groups: blank control group, A gene transfection group (A siRNA interference group or A overexpression group), and negative control group (empty vector group). siRNA interference plasmids or overexpression plasmids were constructed for the key gene A, and the extracted canine osteocytes were transfected after lentivirus packaging to inhibit / upregulate the expression of the key gene A.

[0113] ②Construction of osteocyte-osteoblast / osteoclast conditioned medium co-culture model

[0114] According to the above experiment, the optimal force value and action time were selected to apply fluid shear force to the three groups of bone cells. After stopping the force, the cells were washed twice with PBS solution, and 4 mL of serum-free culture medium was added; the culture was continued for 24 hours, the supernatant was collected, centrifuged at 4°C, 1200 rpm, 5 minutes, and stored in a -80°C ultra-low temperature refrigerator; 1×105 osteoblasts or osteoclasts were cultured at a volume ratio of conditioned medium: total culture medium of 1:2. The culture plate was placed in a packing box and cultured for 3-5 days.

[0115] ③The effect of silencing / overexpressing key gene A on the regulation of osteoblast (or osteoclast) function by osteocytes cultured by fluid shear force in vitro

[0116] RT-PCR, Western Blot, and ELISA were used to detect the gene and protein expression of A and osteocyte secretory factors: OPG, PGE-2, and Sost. RT-PCR, Western Blot, and ELISA were used to detect the gene and protein expression of osteoblast markers ALP, Runx2, and OCN, and ALP staining was used to observe the functional changes of osteoblasts. RT-PCR, Western Blot, and ELISA were used to detect the gene and protein expression of osteoclast markers: Nuclear factor of active T cells 1 (NFATc1), Recombinant cathepsin K (CTSK), and Acidphosphatase 5 (ACP5), and TRAP staining was used to observe the functional changes of osteoclasts.

[0117] Then the key gene A of this application can be used to regulate the maxillary palatal cortical bone remodeling under orthodontic force and then check the effect.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting mechanoresponsive proteins in maxillary palatal cortical bone remodeling, characterized in that: The method comprises the following steps: Step 1: Construct the maxillary anterior teeth retraction experimental animal model specimen; Step 2: Changes in cortical bone structure and density; Step 3: bone histomorphometric analysis; Step 4: Detection of osteogenesis and apoptosis-related markers; Step 5: staining to detect apoptotic cells, dewax and hydrate the paraffin sections, add methyl green anti-staining solution, seal the sections with sealing solution containing anti-fluorescence quencher, observe under a laser scanning confocal microscope, and count the number of TUNEL-positive and TUNEL-negative cells in the bone cortex; Step 6: Analyze the changes of the palatal cortical bone lacuna-canalicular system during the retraction of the maxillary anterior teeth; Step 7: Mechanical response of in vitro cultured osteoblasts under fluid shear stress; Step 8: Screening of key protein A in the mechanical response of bone cells cultured in vitro; Step 9: Analyze the mechanism of action of key protein A in regulating osteoblast or osteoclast function in bone cells cultured under fluid shear force in vitro.

2. The method for detecting mechanoresponsive proteins of maxillary palatal cortical bone remodeling according to claim 1, characterized in that: The specific process of step 1 is: Eighteen healthy adult beagle dogs aged 24 months were selected. All animals met the international laboratory animal requirements and had a body mass of 10-15 kg. The experimental animals were randomly divided into two groups: a force group with a force value of 150 g to retract the maxillary incisors and a control group with no force applied to the maxillary anterior teeth; After the two groups of experimental animals were adapted to caged conditions for one week, they were anesthetized by intramuscular injection of 3% pentobarbital solution at 30 mg / kg body weight, alginate was used to take molds and perfuse hard plaster models, individual bands were made and force-adding devices were welded, the third anterior teeth of the maxillary teeth on both sides were extracted, and force-adding devices were bonded. A nickel-titanium tension spring was used to apply traction force of 0g and 150g to the two groups of animals from the molars to the anterior teeth, respectively. The traction force was tested once a week with a dynamometer, and the oral cavity was cleaned with 2g / L chlorhexidine twice a week. The animals were fed a normal diet. The experimental animals were euthanized at the 4th, 7th, and 10th weeks of orthodontic force loading, and tissue specimens were collected.

3. The method for detecting mechanoresponsive proteins of maxillary palatal cortical bone remodeling according to claim 1, characterized in that: The specific process of step 2 is: The maxillary bones of the specimens were separated and cut to make experimental bone blocks and control bone blocks in the maxillary anterior area, which were preserved in 4% paraformaldehyde and fixed for 48 hours. Micro-computed tomography was used to measure the porosity, total cortical area, cortical area, cortical thickness, mineral content and mineral density of the cortical bone, and to analyze the changes in the cortical bone structure under the action of orthodontic force.

4. The method for detecting mechanoresponsive proteins of maxillary palatal cortical bone remodeling according to claim 1, characterized in that: The specific process of step 3 is: Tetracycline was injected intramuscularly 20 and 21 days before the animals were killed; Alizarin red was injected intramuscularly 13 and 14 days before the animals were killed, and calcein was injected intramuscularly 3 and 4 days before the animals were killed, with the dose of 15 mg / Kg. Hard tissue specimens were removed from 4% paraformaldehyde, dehydrated with gradient ethanol, embedded in plexiglass, and removed after the tissue blocks were completely hardened. The tissue blocks were trimmed and sliced ​​using a Leitzl600 saw slicer. The specimens were mounted after ultrasonic cleaning, polished, and sealed with neutral gum after staining. Hematoxylin-eosin staining, Von Kossa staining, alkaline phosphatase staining, and tartrate-resistant acid phosphatase staining were used to detect the osteoid surface, osteoblast surface, relative number of osteoclasts, bone resorption surface and mineralization deposition rate. The bone cortex tissue morphometric analysis was performed to reflect the changes in bone turnover and bone remodeling at the cellular level.

5. The method for detecting mechanoresponsive proteins of maxillary palatal cortical bone remodeling according to claim 1, characterized in that: The specific process of step 4 is: Immunohistochemistry was used to detect the expression of osteogenic markers ALP, Runx2, and OCN, and apoptotic markers B-lymphocytoma-2 and caspase-3; Hard tissue specimens were removed from 4% paraformaldehyde, and paraffin sections were prepared conventionally. After gradient ethanol dehydration, 0.3% methanol solution of hydrogen peroxide was added to eliminate the influence of endogenous peroxides. Primary antibodies diluted with serum diluent were added and incubated at 4°C overnight. Secondary antibodies, i.e., biotinylated goat anti-rabbit IgG, were added and incubated at room temperature for 2h. Color developing solution was added for immunohistochemical color development. After gradient alcohol dehydration, the sections were sealed and photographed. The force application time of the group with the maximum osteogenic activity was selected as the parameter for the later experiments.

6. The method for detecting mechanoresponsive proteins of maxillary palatal cortical bone remodeling according to claim 1, characterized in that: The specific process of step 6 is: (1) Observation of the morphological changes of bone lacuna and canalicular system using laser scanning confocal microscopy The hard tissue specimens were immersed in gradient ethanol containing 1% basic fuchsin for dehydration and staining, and then embedded in polymethyl methacrylate and sliced ​​using the EXAKT cutting and grinding system to make 10 μm slices. The slice surface was polished with sandpaper. The morphology, area, and direction of the bone lacuna and canaliculus in the bone cortex were observed using a laser scanning confocal microscope. The fluorescence excitation wavelength of the filter was 580 nm. 21 layers of optical slices were cut under a 100x oil immersion lens, with an interval of 0.5 μm between each layer and a total thickness of 10 μm. (2) Scanning electron microscopy observation of the structural changes of the lacunae-canaliculi system After PMMA embedding, the hard tissue sections were etched with 9% orthophosphoric acid for 20 seconds with the polished surface facing up, then washed with deionized water and immersed in 5% sodium hypochlorite for 5 minutes, washed with deionized water, air-dried and sprayed with gold. The canalicular diameter and bone lacuna density were observed with a Quanta 250 scanning electron microscope.

7. The method for detecting mechanoresponsive proteins of maxillary palatal cortical bone remodeling according to claim 1, characterized in that: The specific process of step 7 is: (1) Construction of a fluid shear force loading model for in vitro cultured bone cells ① Isolation, culture and identification of bone cells Take the tissue block under the femoral neck, place it in α-MEM containing 10% penicillin-streptomycin, remove the excess tissue and repeatedly rinse the bone marrow surface, rinse with HBSS 3 times, cut the bone tissue into 1 cm2 small pieces, add 2 mg / ml type I collagenase, digest for 15 minutes, wash 3 times with HBSS, repeat 2 times, add 0.02% EDTA containing 1% BSA, culture in the incubator for 25 minutes, discard the supernatant, wash 3 times with HBSS, repeat adding type I collagenase, digest for 25 minutes, wash 3 times with HBSS, collect the washing liquid and supernatant, centrifuge at 1200r / min for 5 minutes, discard the supernatant, resuspend in the culture medium and implant it in a 6-well plate coated with type I rat tail collagen with the digested bone tissue, culture in the incubator, and identify bone cells by morphological observation, E11 / GP38 fluorescence staining, and ALP staining after 2-3 generations of subculture; ② Construction of fluid shear force loading model A parallel plate flow chamber was used to apply fluid shear force to the bone cells. The force-adding device was made of two layers of organic glass, with a 1cm×1cm×0.02cm square groove in the center of the lower layer, which could hold a coverslip covered with bone cells. There was a 6cm×1.35cm×0.04cm rectangular groove on the upper layer, which formed a channel for liquid flow with the upper organic glass. An opening was opened at each end of the channel for injecting and discharging liquid. The liquid was a liquid flow with a fixed shear force provided by the perfusion system. The third generation of bone cells were inoculated on the coverslip, which was placed in the square groove in the center of the flow chamber system and divided into four groups. The fluid shear forces loaded in each group were 0.8Pa, 1.2Pa, 1.4Pa and 1.6Pa, respectively, and the loading lasted for 10min, 15min, 30min and 60min. The experiment was repeated 6 times in each time period, and no force was applied to the control group. (2) Detection of bone cell functional status, proliferation activity and secretion factors MTT colorimetry and flow cytometry were used to identify the functional status and proliferation activity of osteocytes under different force values ​​and time. Enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction-protein blotting were used to detect the changes in the gene and protein expression of OPG, PGE2, and Sost in osteocytes after fluid shear force.

8. The method for detecting mechanoresponsive proteins of maxillary palatal cortical bone remodeling according to claim 1, characterized in that: The specific process of step 8 is: (1) Experimental groups The osteocytes cultured by fluid shear force in vitro were divided into two groups: a control group and an experimental group. The osteocytes in the control group were not subjected to force, while the osteocytes in the experimental group were subjected to the optimal fluid shear force according to the above experimental results. (2) Transcriptomics screening of differentially expressed genes in response to osteocyte mechanochemistry The total RNA of bone cells in the force group and the control group was extracted by Trizol method, and the extracted total RNA was reverse transcribed to synthesize cDNA and add adapter sequence; the cDNA was divided according to the fragment size; the cDNA was cyclized and sequenced on a machine. After quantitative analysis of sample transcription, differentially expressed genes were screened, and the common differential genes were imported into relevant software for interaction analysis to obtain clusters of potential targets of differentially expressed genes. The identified genes were subjected to gene function cluster analysis and Kyoto Encyclopedia of Genes and Genomes analysis to screen out key genes for mechanical response of bone cells; (3) iTRAQ technology combined with LC-ESI-MSMS to screen differentially expressed proteins in response to mechanical changes in bone cells Total protein samples were extracted from the force group and the control group. The samples were subjected to SDS-PAGE electrophoresis, and the proteolytic peptides were labeled with different isotope relative and absolute quantitative labels. The samples were separated by liquid phase using UltremexSCX column and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry based on TripleTOF 5600. The same bioinformatics method was used to screen out the key proteins of bone cell mechanical response. (4) Combined transcriptomic and proteomic analysis reveals the mechanism by which orthodontic force regulates maxillary palatal cortical bone remodeling Correlate the identified proteins with the transcriptome results; The common targets were projected to KEGG, and the KEGG enrichment results were plotted into a KEGG pathway enrichment cluster heat map. Clustering was performed at the protein level according to the differential expression fold to obtain the key gene A, which was biologically identified using RT-PCR and Western Blot techniques.

9. The method for detecting mechanoresponsive proteins of maxillary palatal cortical bone remodeling according to claim 1, characterized in that: The specific process of step 9 is: (1) Isolation and culture of osteoblasts and osteoclasts ① Isolation, culture and identification of osteoblasts After the dog was euthanized, part of the femur was cut off under sterile conditions, the medullary cavity was washed with sterile injection water, the bone blocks were broken into a diameter of 1-2 mm, and placed in a 6-well plate. An appropriate amount of type I collagenase solution was added to each well at a concentration of 1 mg / mL, and incubated in a 37°C constant temperature shaker for 15 minutes. The culture medium was replaced and incubated on ice for 5 minutes, and repeated 3 times. Then, 0.5 mM EDTA solution was used for decalcification for 5 minutes, and the enzymatic solution was alternated for 15 minutes. The above steps were repeated 3 times, and the enzymatic solution and EDTA solution collected in the last 3 times were centrifuged, and the obtained lower layer cells were cultured in a culture incubator. After 2-3 generations of subculture, osteoblasts were identified by morphological observation and ALP staining; ② Isolation, culture and identification of osteoclasts Peripheral blood was collected from dogs, and peripheral blood mononuclear cells were separated by density gradient centrifugation. The separated PBMCs were cultured and inoculated in six-well plates. Macrophage colony stimulating factor was added, and the supernatant was discarded after overnight incubation. 50ng / ml RANKL reagent was added for culture. After 2-3 generations of subculture, osteoclasts were identified by morphological observation and TRAP staining; (2) The effect of silencing / overexpressing key protein A on the regulation of osteoblastic or osteoclast function of osteocytes cultured by fluid shear force in vitro. Protein A is gene A ① Experimental grouping According to different treatment methods, the osteoblasts / osteoclasts co-cultured osteocytes were divided into three groups: blank control group, A gene transfection group, i.e. A siRNA interference group or A overexpression group, and negative control group. siRNA interference plasmid or overexpression plasmid was constructed for key gene A, and the extracted canine osteocytes were transfected after lentivirus packaging to inhibit / upregulate the expression of key gene A. ②Construction of osteocyte-osteoblast / osteoclast conditioned medium co-culture model According to the above experiments, the best force value and action time were selected, and fluid shear force was applied to the three groups of bone cells. After stopping the force, the cells were washed twice with PBS solution, and 4 mL of serum-free culture medium was added; the culture was continued for 24 hours, the supernatant was collected, centrifuged at 4°C, 1200 rpm, 5 minutes, and stored in a -80°C ultra-low temperature refrigerator. 1×105 osteoblasts or osteoclasts / well were cultured at a volume ratio of conditioned medium: total culture medium of 1:2, and the culture plate was placed in a packing box for 3-5 days; ③The effect of silencing / overexpressing key gene A on the function of osteoblasts or osteoclasts regulated by osteocytes cultured by fluid shear force in vitro A gene and osteocyte secretory factors: OPG, PGE-2, Sost gene and protein expression were detected; RT-PCR, Western Blot, and ELISA were used to detect osteoblast markers ALP, Runx2, and OCN gene and protein expression, and ALP staining was used to observe changes in osteoblast function; R was used to detect osteoclast markers nuclear factor of activated T cells 1, cathepsin K, and tartrate-resistant acid phosphatase gene and protein expression, and TRAP staining was used to observe changes in osteoclast function.

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