Method for inducing ankylosing spondylitis synovial fibroblast osteogenesis in vitro

Abstract

The invention provides a method for inducing ankylosing spondylitis synovial fibroblast osteogenesis in vitro, and belongs to the technical field of cell culture, the ankylosing spondylitis hip joint synovial fibroblast is induced in vitro by using 100ng / mL of tumor necrosis factor-alpha (TNF-alpha) and 100ng / mL of bone morphogenetic protein 2 (BMP-2), and the ankylosing spondylitis hip joint synovial fibroblast can be converted into osteoblasts 12 days after induction. The ankylosing spondylitis synovial fibroblast osteogenesis is specifically induced, the interference of non-target cells is reduced, and the accuracy and reliability of the experiment are improved; the method disclosed by the invention is simple, strong in operability and good in controllability, lays a foundation for research on pathogenesis of new bone formation of ankylosing spondylitis, can greatly shorten the experimental period, and has better application prospects compared with traditional BMP-2 and TGF-beta induced fibroblasts. The osteogenesis model constructed by the method is closer to an osteogenesis mechanism of ankylosing spondylitis and is higher in conversion efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to a method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro. Background Art

[0002] Ankylosing spondylitis (AS) is a chronic inflammatory disease that primarily affects the sacroiliac joints, spinal apophysis, paraspinal soft tissues, and peripheral joints, and may also present with extra-articular manifestations. Unlike diseases such as rheumatoid arthritis, AS patients experience progressive inflammation and bone erosion accompanied by heterotopic ossification. The osteophytes formed by heterotopic ossification can cause joint stiffness, ankylosing, and deformity, significantly impacting normal spinal mobility and making it a major health hazard. Currently, the main goal of clinical treatment strategies is to control inflammation and halt disease progression with medications, including nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and TNF inhibitors. These medications are not only associated with significant side effects and high costs, but their ability to inhibit pathological new bone formation remains unproven. Therefore, new therapeutic strategies are urgently needed. A major challenge in treating AS lies in the lack of clarity regarding its pathogenesis, particularly the relationship between inflammation and pathological ossification.

[0003] To date, the pathogenesis of AS remains a mystery, a focus of ongoing exploration and attention by scientists. AS research has largely relied on drug-induced or transgenic rodent models. Without understanding the etiology, these models only partially retain clinical features, making it difficult to establish animal models that closely resemble clinical AS patients. Laboratory rodent models, including HLA-B27 humanized transgenic mouse models, ERAP1-deficient mouse models, and inflammation-induced mouse models, only partially recapitulate the genetic susceptibility to AS, often exhibit inconsistent pathology, and fail to simulate new bone formation in AS. According to literature reports, the most commonly used methods for inducing new bone formation in AS are to induce MSCs (mesenchymal stem cells) to transform into osteoblasts using BMP-2 and TGF-β (Transforming Growth Factor-β). However, this induction method has two limitations: 1. TGF-β is a cell-transforming factor that regulates cell growth and differentiation and generally has anti-inflammatory effects, which does not adequately mimic the inflammatory effects of AS. 2. Simulating new bone formation in AS is typically done by inducing MSCs into osteoblasts using BMP-2 and TGF-β. MSCs are primarily derived from bone marrow, whereas heterotopic ossification in AS typically occurs between bones. Whether MSCs participate in the mechanism of new bone formation in AS patients is currently unclear. Therefore, we proposed an in vitro method to induce osteogenesis in ankylosing spondylitis synovial fibroblasts. Summary of the Invention

[0004] The purpose of the present invention is to address the problems raised by the existing background technology. In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solution: a method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro, comprising the following steps:

[0005] Step 1. Extraction of ankylosing spondylitis synovial fibroblasts: Pretreated synovial tissue was minced thoroughly (3-5 mm) and added to a tissue digestion solution (20 times the volume of the tissue). Each ml of digestion solution contained: 1 mg of collagenase II, 1 mg of dispase II, 0.5 mg of DNase I, and DMEM medium containing a double-antibody (penicillin-streptomycin mixture). Digestion was performed at 37°C for 40 minutes. The digestion solution was passed through a 100-mesh sieve. The resulting cell suspension was centrifuged at 3000 g / min, the supernatant discarded, and the cells were resuspended in complete culture medium.

[0006] Step 2. Identification of synovial fibroblasts: After passage 3, adjust the cell concentration to 1 × 10 5 / mL inoculated in 25cm 2 Add 5 mL of DMEM culture medium to the cell culture flask and culture in a CO2 incubator until the cells cover the bottom of the flask and adhere to the wall. Purify the cells using natural purification and repeated adherence methods to obtain synovial fibroblasts. Observe the cells under a microscope, stain them, label them with Vimentin, and further identify the synovial fibroblasts using immunofluorescence.

[0007] Step 3. Inducing synovial fibroblast osteogenesis: First, prepare 10 μg / mL TNF-α and 2 μg / mL BMP-2 stock solutions, then dilute the stock solutions stepwise to prepare DMEM working solutions containing 200 ng / mL TNF-α + 200 ng / mL BMP, 100 ng / mL TNF-α + 100 ng / mL BMP, 50 ng / mL TNF-α + 50 ng / mL BMP, and 10 ng / mL TNF-α + 10 ng / mL BMP, respectively. Add these solutions to the third generation cells, and replace the DMEM working solution every 3 days for up to 12 days.

[0008] Step 4. Osteoblast identification: Osteoblast identification was performed using Alizarin red staining and alkaline phosphatase staining.

[0009] As a preferred technical solution of the present invention, step 1. Complete culture medium contains: 20% fetal bovine serum, DMEM culture medium containing double antibody (penicillin-streptomycin mixture); the resuspended liquid is seeded into a 25cm 2 Cell culture flasks were subcultured after 24 hours of adherence.

[0010] As a preferred technical solution of the present invention, step 4. Identification of osteoblasts: The specific steps of identifying osteoblasts using alizarin red staining are as follows: After the cell induction and differentiation are completed, the cells are digested with trypsin and seeded into a 6-well plate. After the cells are completely attached to the wall, the cell supernatant is aspirated and the cells are rinsed twice with PBS. 1 mL of formaldehyde fixative is added to each well, and the cells are fixed at room temperature for 15-30 minutes. The formaldehyde fixative is aspirated and the cells are rinsed twice with PBS. Alizarin red staining solution is added along the well wall and the cells are stained at room temperature for 30 minutes. The staining solution is aspirated and the cells are rinsed twice with PBS. After the floating color is completely removed, the alizarin red staining effect is observed under a microscope. Osteoblasts appear darker orange-red in the field of view due to calcium salt deposition.

[0011] As a preferred technical solution of the present invention, step 4. osteoblast identification: The specific operating steps of the alkaline phosphatase staining method for osteoblast identification are as follows: after the cell induction and differentiation are completed, the cells are digested with trypsin and seeded into a 6-well plate. After the cells are completely attached to the wall, the cell supernatant is aspirated, and the cells are rinsed twice with PBS. 1 mL of formaldehyde fixative is added to each well. After fixing at room temperature for 15-30 minutes, the formaldehyde fixative is aspirated, and the cells are rinsed twice with PBS. After adding alkaline phosphatase staining solution, the plate is protected from light and color is developed at room temperature for 30 minutes. The color is terminated by rinsing with PBS, and the floating color is removed. The staining effect is observed under a microscope. Osteoblasts appear blue in the field of view.

[0012] As a preferred technical solution of the present invention, in the step of inducing synovial fibroblasts to form osteoblasts, the third generation cells are induced to culture using a DMEM culture working solution containing 100 ng / mL TNF-α + 100 ng / mL BMP-2.

[0013] As a preferred technical solution of the present invention, the pretreated synovial tissue is fully broken; a digestion solution containing collagenase II, dispase II, and DNase I is added thereto and blown, cultured and digested, and then broken into single cells; after filtration, the filtrate is centrifuged to remove the lower layer of precipitate and inoculated into DMEM high-glucose culture medium for adherent culture; the cells are purified by natural purification method and repeated adherence method to obtain ankylosing spondylitis hip joint synovial fibroblasts.

[0014] As a preferred technical solution of the present invention, when the purified synovial fibroblasts reach the third generation, DMEM culture medium containing 100 ng / mL TNF-α + 100 ng / mL BMP-2 is added, and the culture medium is changed every 3 days for a total of 12 days.

[0015] As a preferred technical solution of the present invention, 100 ng / mL TNF-α and 100 ng / mL BMP-2 are used to induce ankylosing spondylitis hip joint synovial fibroblasts in vitro, thereby converting them into osteoblasts.

[0016] The invention discloses an application of a method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro, and an application of the method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro in constructing an ankylosing spondylitis new bone formation model.

[0017] As a preferred technical solution of the present invention, a method of inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro is used to study the pathogenesis of ankylosing spondylitis.

[0018] Compared with existing technologies, the present invention offers the following advantages: It provides an in vitro method for inducing osteogenesis in synovial fibroblasts from ankylosing spondylitis joints. Following 12 days of induction, the conversion rate of synovial fibroblasts into osteoblasts reached 25%. Furthermore, inducing synovial fibroblasts from hip joints of AS patients using BMP-2 and TNF-α more closely resembles the true pathogenesis of AS. Furthermore, this method offers a high conversion rate and a short cycle, meeting various experimental needs. Therefore, it is a rapid culture method for establishing an in vitro cell model of new bone formation in AS. This provides strong experimental support for studying the osteogenic pathogenesis of AS and has excellent practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Isolation and identification of AS synovial fibroblasts;

[0020] Figure 2 : Identification of osteoblasts from AS synovial fibroblasts induced by TNF-α and BMP-2 (Alizarin red staining);

[0021] Figure 3 : TNF-α and BMP-2 induced osteoblastic identification of AS synovial fibroblasts (alkaline phosphatase staining). DETAILED DESCRIPTION

[0022] To make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0023] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents some embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions in the embodiments can be combined with each other. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0024] Example 1: Please refer to Figure 1-Figure 3 A method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro comprises the following steps: Step 1. Extracting ankylosing spondylitis synovial fibroblasts: mincing the pretreated synovial tissue thoroughly (3-5 mm), adding tissue digestion solution (the volume is 20 times that of the tissue), wherein each milliliter of digestion solution contains: 1 mg of collagenase II, 1 mg of dispase II, 0.5 mg of DNase I, and DMEM medium containing a double antibody (penicillin-streptomycin mixture); digesting at 37°C for 40 minutes, filtering the digestion solution through a 100-mesh sieve, and centrifuging the resulting cell suspension at 3000 g / min, discarding the supernatant, centrifuging twice, and resuspending the cells in complete medium;

[0025] Step 2. Identification of synovial fibroblasts: After passage 3, adjust the cell concentration to 1 × 10 5 / mL inoculated in 25cm 2 Add 5 mL of DMEM culture medium to the cell culture flask and culture in a CO2 incubator until the cells cover the bottom of the flask and adhere to the wall. Purify the cells using natural purification and repeated adherence methods to obtain synovial fibroblasts. Observe the cells under a microscope, stain them, label them with Vimentin, and further identify the synovial fibroblasts using immunofluorescence.

[0026] Step 3. Inducing synovial fibroblast osteogenesis: First, prepare 10 μg / mL TNF-α and 2 μg / mL BMP-2 stock solutions, then dilute the stock solutions stepwise to prepare DMEM working solutions containing 200 ng / mL TNF-α + 200 ng / mL BMP, 100 ng / mL TNF-α + 100 ng / mL BMP, 50 ng / mL TNF-α + 50 ng / mL BMP, and 10 ng / mL TNF-α + 10 ng / mL BMP, respectively. Add these solutions to the third generation cells, and replace the DMEM working solution every 3 days for up to 12 days.

[0027] Step 4. Osteoblast identification: Osteoblast identification was performed using Alizarin red staining and alkaline phosphatase staining.

[0028] Step 1. Complete culture medium contains: 20% fetal bovine serum, DMEM culture medium containing double antibody (penicillin-streptomycin mixture); the resuspension is seeded into 25cm 2 Cell culture flasks were subcultured after 24 hours of adherence.

[0029] Step 4. Identification of osteoblasts: The specific steps for identifying osteoblasts using alizarin red staining are as follows: After the cell induction and differentiation are completed, the cells are digested with trypsin and seeded into 6-well plates. After the cells are completely attached to the wall, the cell supernatant is aspirated and the cells are rinsed twice with PBS. 1 mL of formaldehyde fixative is added to each well and the cells are fixed at room temperature for 15-30 minutes. The formaldehyde fixative is aspirated and the cells are rinsed twice with PBS. Alizarin red staining solution is added along the well wall and the cells are stained at room temperature for 30 minutes. The staining solution is aspirated and the cells are rinsed twice with PBS. After the floating color is completely removed, the alizarin red staining effect is observed under a microscope. Osteoblasts appear darker orange-red in the field of view due to the deposition of calcium salts.

[0030] Step 4. Identification of osteoblasts: The specific operating steps of the alkaline phosphatase staining method for identification of osteoblasts are as follows: After the cell induction and differentiation are completed, the cells are digested with trypsin and seeded into 6-well plates. After the cells are completely attached to the wall, the cell supernatant is aspirated and the plates are rinsed twice with PBS. 1 mL of formaldehyde fixative is added to each well. After fixing at room temperature for 15-30 minutes, the formaldehyde fixative is aspirated and the plates are rinsed twice with PBS. After adding alkaline phosphatase staining solution, the plates are protected from light and color developed at room temperature for 30 minutes. The color development is terminated by rinsing with PBS, and the floating color is removed. The staining effect is observed under a microscope. Osteoblasts appear blue in the field of view.

[0031] In the step of inducing synovial fibroblasts to form osteoblasts, the third generation cells were induced to culture using a DMEM culture working solution containing 100 ng / mL TNF-α+100 ng / mL BMP-2.

[0032] The pretreated synovial tissue was fully broken up; a digestion solution containing collagenase II, dispase II, and DNase I was added thereto and pipetted, cultured and digested, and then broken up into single cells; the filtrate was centrifuged and the lower layer of precipitate was removed and inoculated into DMEM high-glucose culture medium for adherent culture; the cells were purified by natural purification and repeated adherence methods to obtain ankylosing spondylitis hip joint synovial fibroblasts.

[0033] When the purified synovial fibroblasts reached the third passage, DMEM medium containing 100 ng / mL TNF-α and 100 ng / mL BMP-2 was added, and the medium was changed every 3 days for a total of 12 days.

[0034] In vitro, synovial fibroblasts of the hip joint of ankylosing spondylitis were induced to transform into osteoblasts by using 100 ng / mL TNF-α and 100 ng / mL BMP-2.

[0035] The invention discloses an application of a method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro, and an application of the method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro in constructing an ankylosing spondylitis new bone formation model.

[0036] Application of the method of inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro in studying the pathogenesis of ankylosing spondylitis.

[0037] The existing models cannot simulate the actual new bone formation of AS patients, which has created a huge obstacle for researchers to study AS. In view of this, in a typical embodiment of the present invention, a method for primary culture of ankylosing spondylitis synovial fibroblasts is provided, which comprises: fully crushing the pretreated synovial tissue (3-5 mm); adding a digestion solution containing 1 mg of collagenase II, 1 mg of dispase II, and 0.5 mg of DNase I (the volume is 20 times that of the tissue) and pipetting, culturing and digesting to break up into single cells; filtering the filtrate and centrifuging the precipitate to obtain 1×10 5 / mL was inoculated into DMEM high-glucose medium for adherent culture; the cells were purified by natural purification method and repeated adherent method to obtain ankylosing spondylitis hip joint synovial fibroblasts.

[0038] In another typical embodiment of the present invention, an in vitro method for inducing osteogenesis of ankylosing spondylitis cells is provided, the method comprising: waiting until the purified synovial fibroblasts reach the third generation, adding DMEM culture medium containing 100 ng / mL TNF-α + 100 ng / mL BMP-2, changing the culture medium every 3 days, and culturing for a total of 12 days, and identifying osteoblasts by alizarin red staining and alkaline phosphatase staining. The osteoblasts appear orange-red under a microscope after alizarin red staining and blue after alkaline phosphatase staining.

[0039] In another typical embodiment of the present invention, the synovial fibroblasts of ankylosing spondylitis obtained by the above-mentioned culture method can be successfully transformed into osteoblasts.

[0040] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] Experimental example:

[0042] 1. Main reagents, instruments and consumables

[0043] 1.1 Reagents

[0044]

[0045]

[0046] 1.2 Instrument consumables

[0047] 2 Methods

[0048] Instrument consumables name factory model centrifuge Beckman 20 Inverted microscope Zeiss Primovert Clean bench Jinan, Shandong VD / HD650 cell culture incubator ThemoFisherScientific Forma371 Fluorescence microscopy OLYMPUS BX53 cell counter Riverwood C100-Pro 25cm2 cell culture flask Seville Wuhan 6-well plate Seville Wuhan slides Seville Wuhan Cover glass Seville Wuhan 0.2 μm sterile filter Seville Wuhan 15mL centrifuge tube Seville Wuhan 50mL centrifuge tube Seville Wuhan 100 mesh sieve Seville Wuhan

[0049] 2.1 Isolation and culture of ankylosing spondylitis synovial fibroblasts

[0050] Synovial tissue from AS patients undergoing hip replacement surgery was obtained with patient consent and after passing the institutional ethics review. The removed synovial tissue was placed in sterile saline, sealed in sterile bags, and quickly transferred to the laboratory. In a clean bench, the synovial tissue was rinsed three times with 50 mL of PBS. Sterile ophthalmic scissors were used to carefully remove the cartilage and adipose tissue from the synovial tissue. The synovial tissue was rinsed twice with PBS and transferred to a 90 mm culture dish. The clean synovial tissue was minced thoroughly (<0.5 mm). Twenty times the volume of tissue was added to each dish in a digestion solution containing 1 mg of collagenase II, 1 mg of dispase II, 0.5 mg of DNase I, and a double-antibody (penicillin-streptomycin mixture) in DMEM per ml. After repeated pipetting with a sterile pipette, the cells were incubated at 37°C, 5% CO2, and digested for 40 min. The cells were then passed through a 100-mesh sieve and the resulting cell suspension was centrifuged at 3000 g / min, the supernatant discarded, and the cells were resuspended in complete culture medium. Complete culture medium contains: 20% fetal bovine serum, DMEM culture medium containing double antibody (penicillin-streptomycin mixture). 2 Cell culture flasks were subcultured after 24 hours of adherence. Medium was changed and subcultured according to the cell status. Cells were purified using natural purification and repeated adherence methods. Cells with high activity and purity at passages 3-6 were used for experiments.

[0051] 2.2 Identification of synovial fibroblasts

[0052] 2.2.1 Morphological observation

[0053] Use an inverted microscope to observe whether the first and second generation primary cells are fibroblasts. The morphology of fibroblasts is generally long spindle-shaped. Whether they grow adherently to the wall can be preliminarily judged to determine the purity and growth status of the primary fibroblasts.

[0054] 2.2.2 Cell counting

[0055] Primary cells were counted using a cell counter. Equal amounts of tissue were weighed and extracted using conventional and optimized methods, then seeded into 25 cm2 cell culture flasks. Cells were then purified by natural passage and finally digested with trypsin and counted using a cell counter.

[0056] 2.2.3 Cell identification

[0057] Adjust the cell concentration to 1×10 5 / mL inoculated in 25cm 2Add 5 mL of DMEM culture medium to the cell culture flask and culture in a CO2 incubator until the cells cover the bottom of the flask and adhere to the wall. Observe the cells under a microscope, stain them, and use Vimentin to label fibroblasts. Further identify synovial fibroblasts by immunofluorescence.

[0058] 2.3 Induction of synovial fibroblast osteogenesis

[0059] First, prepare 10 μg / mL TNF-α and 2 μg / mL BMP-2 stock solutions according to the reagent instructions. Then, serially dilute these stock solutions with DMEM complete medium to prepare the following DMEM working solutions: 200 ng / mL TNF-α + 200 ng / mL BMP, 100 ng / mL TNF-α + 100 ng / mL BMP, 50 ng / mL TNF-α + 50 ng / mL BMP, and 10 ng / mL TNF-α + 10 ng / mL BMP. Add these solutions to the third-generation cells, adding 5 mL per 25 cm2 cell culture flask. Change the DMEM working solution every 3 days for up to 12 days. Observe cell morphology and identify the cells.

[0060] 2.4 Osteoblast Identification

[0061] Osteoblasts were identified using Alizarin Red staining and alkaline phosphatase staining. ① Alizarin Red staining: After cell differentiation induction, trypsinize the cells and seed them into 6-well plates. Once the cells have fully adhered, remove the cell supernatant and rinse twice with PBS. Add 1 mL of formaldehyde fixative to each well and fix at room temperature for 15-30 minutes. Then, remove the formaldehyde fixative and rinse twice with PBS. Add Alizarin Red staining solution along the well walls and stain at room temperature for 30 minutes. Aspirate the staining solution and rinse twice with PBS. Once the floating color has completely disappeared, observe the Alizarin Red staining under a microscope. Osteoblasts appear darker orange-red in the field of view due to calcium salt deposition. ② Alkaline phosphatase staining: The specific steps are the same as for Alizarin Red staining. Note that after adding the alkaline phosphatase staining solution, protect from light and allow the color to develop at room temperature for 30 minutes. Rinse with PBS to terminate the color development and remove the floating color. Observe the staining under a microscope. Osteoblasts appear blue in the field of view.

[0062] 2.5 Calculation of osteoblast conversion rate

[0063] The osteoblast staining area after Alizarin Red and alkaline phosphatase staining was observed under a microscope, the staining area was estimated, the number of osteoblasts was converted according to the area, and the osteogenic conversion rate was calculated.

[0064] 3 Results

[0065] 3.1AS Identification of Synovial Fibroblasts

[0066] After the primary cells were cultured for 36 hours, the medium was changed to remove foreign cells. First, an inverted microscope was used to observe that all the cells were spindle-shaped synovial fibroblasts. In addition, Vimentin is a protein marker of fibroblasts. We used Vimentin to label fibroblasts and detected them by immunofluorescence to further confirm that the primary cells were synovial fibroblasts ( Figure 1 ).

[0067] 3.2 Osteoblast Identification

[0068] Osteoblasts were identified using Alizarin red staining and alkaline phosphatase staining. ① Alizarin red staining: Due to calcium salt deposition, osteoblasts appear darker orange-red in the microscope field ( Figure 2 ②Alkaline phosphatase staining: Due to calcium salt deposition, osteoblasts appear blue in the fiber microscope field of view ( Figure 3 ).

[0069] 3.3 Osteogenesis conversion rate

[0070] The osteogenic conversion rate was calculated by the staining area under a microscope, and the osteogenic conversion rate of AS synovial fibroblasts after 12 days of induction was 25%.

[0071] The present invention uses 100ng / mL TNF-α+100ng / mL BMP to induce synovial fibroblasts from the hip joint of AS patients to transform them into osteoblasts, and the osteoblasts are identified by alizarin red staining and alkaline phosphatase staining. The osteoblast conversion rate is above 25%, and the conversion efficiency is relatively high. Compared with the existing AS osteogenesis model, the synovial fibroblasts from the hip joint of AS patients used in the present invention are the initiating cells for the ossification of the AS ligament, and are induced by TNF-α and BMP. Studies have confirmed that TNF-α can trigger the generation of intracellular ROS, and the interaction of ROS and TNF-α promotes AS osteogenic differentiation and new bone formation. Therefore, compared with other methods, this method is closer to the osteogenic mechanism of AS and has a higher conversion efficiency. The method of the present invention is simple, highly operational, and has good controllability, greatly shortening the experimental cycle, laying the foundation for the study of the pathogenesis of new bone formation in ankylosing spondylitis.

[0072] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although this specification has described the present invention in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or equivalent replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. A method for inducing osteogenesis in ankylosing spondylitis synovial fibroblasts in vitro, characterized in that: The following steps are involved: Step 1. Extraction of ankylosing spondylitis synovial fibroblasts: Pretreated synovial tissue was minced thoroughly (3-5 mm) and added to a tissue digestion solution (20 times the volume of the tissue). Each ml of digestion solution contained: 1 mg of collagenase II, 1 mg of dispase II, 0.5 mg of DNase I, and DMEM medium containing a double-antibody (penicillin-streptomycin mixture). Digestion was performed at 37°C for 40 minutes. The digestion solution was passed through a 100-mesh sieve. The resulting cell suspension was centrifuged at 3000 g / min, the supernatant discarded, and the cells were resuspended in complete culture medium. Step 2. Identification of synovial fibroblasts: After passage 3, adjust the cell concentration to 1 × 10 5 / mL inoculated in 25cm 2 Add 5 mL of DMEM culture medium to the cell culture flask and culture in a CO2 chamber until the cells cover the bottom of the flask and adhere to the wall; The cells were purified by natural purification and repeated adherence methods to obtain synovial fibroblasts. The synovial fibroblasts were further identified by microscopic staining, Vimentin labeling, and immunofluorescence. Step 3. Inducing synovial fibroblast osteogenesis: First, prepare 10 μg / mL TNF-α and 2 μg / mL BMP-2 stock solutions, then dilute the stock solutions stepwise to prepare DMEM working solutions containing 200 ng / mL TNF-α + 200 ng / mL BMP, 100 ng / mL TNF-α + 100 ng / mL BMP, 50 ng / mL TNF-α + 50 ng / mL BMP, and 10 ng / mL TNF-α + 10 ng / mL BMP, respectively. Add these solutions to the third generation cells, and replace the DMEM working solution every 3 days for up to 12 days. Step 4. Osteoblast identification: Osteoblast identification was performed using Alizarin red staining and alkaline phosphatase staining.

2. The method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro according to claim 1, characterized in that: Step 1. Complete culture medium contains: 20% fetal bovine serum, DMEM culture medium containing double antibody (penicillin-streptomycin mixture); the resuspension is seeded into 25cm 2 Cell culture flasks were subcultured after 24 hours of adherence.

3. The method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro according to claim 2, characterized in that: Step 4. Identification of osteoblasts: The specific steps for identifying osteoblasts using alizarin red staining are as follows: After the cell induction and differentiation are completed, the cells are digested with trypsin and seeded into 6-well plates. After the cells are completely attached to the wall, the cell supernatant is aspirated and the cells are rinsed twice with PBS. 1 mL of formaldehyde fixative is added to each well and the cells are fixed at room temperature for 15-30 minutes. The formaldehyde fixative is aspirated and the cells are rinsed twice with PBS. Alizarin red staining solution is added along the well wall and the cells are stained at room temperature for 30 minutes. The staining solution is aspirated and the cells are rinsed twice with PBS. After the floating color is completely removed, the alizarin red staining effect is observed under a microscope. Osteoblasts appear darker orange-red in the field of view due to the deposition of calcium salts.

4. The method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro according to claim 3, characterized in that: Step 4. Identification of osteoblasts: The specific operating steps of the alkaline phosphatase staining method for identification of osteoblasts are as follows: After the cell induction and differentiation are completed, the cells are digested with trypsin and seeded into 6-well plates. After the cells are completely attached to the wall, the cell supernatant is aspirated and the plates are rinsed twice with PBS. 1 mL of formaldehyde fixative is added to each well. After fixing at room temperature for 15-30 minutes, the formaldehyde fixative is aspirated and the plates are rinsed twice with PBS. After adding alkaline phosphatase staining solution, the plates are protected from light and color developed at room temperature for 30 minutes. The color development is terminated by rinsing with PBS, and the floating color is removed. The staining effect is observed under a microscope. Osteoblasts appear blue in the field of view.

5. The method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro according to claim 4, characterized in that: In the step of inducing synovial fibroblasts to form osteoblasts, the third generation cells were induced to culture using a DMEM culture working solution containing 100 ng / mL TNF-α+100 ng / mL BMP-2.

6. The method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro according to claim 5, characterized in that: The pretreated synovial tissue was fully broken; a digestion solution containing collagenase II, dispase II, and DNase I was added thereto and pipetted, cultured and digested to break up into single cells; the filtrate was filtered and centrifuged to remove the lower precipitate, which was inoculated into DMEM high-glucose medium for adherent culture; The cells were purified by natural purification and repeated adherence methods to obtain ankylosing spondylitis hip joint synovial fibroblasts.

7. The method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro according to claim 6, characterized in that: When the purified synovial fibroblasts reached the third passage, DMEM medium containing 100 ng / mL TNF-α and 100 ng / mL BMP-2 was added, and the medium was changed every 3 days for a total of 12 days.

8. The method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro according to claim 7, characterized in that: In vitro, synovial fibroblasts of the hip joint of ankylosing spondylitis were induced to transform into osteoblasts by using 100 ng / mL TNF-α and 100 ng / mL BMP-2.

9. Application of a method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro, characterized in that: Application of the in vitro osteogenesis-inducing method of ankylosing spondylitis synovial fibroblasts in constructing an ankylosing spondylitis new bone formation model.

10. Use of the method for inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro according to claim 9, characterized in that: Application of the method of inducing osteogenesis of ankylosing spondylitis synovial fibroblasts in vitro in studying the pathogenesis of ankylosing spondylitis.

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