Method for inducing osteogenic differentiation of human amniotic mesenchymal stem cells through targeted cyclooxygenase 2 and culture medium used by method

Abstract

The invention relates to the technical field of biology, in particular to novel application of sanguinarine (SAN), a method for inducing human amniotic mesenchymal stem cells (hAMSCs) to be differentiated into osteoblasts through COX2 protein and an osteogenic induction culture medium for the inducing method, and the method can be applied to the fields of bone disease treatment drug research and development and clinical transformation. It is proved for the first time that SAN mediates hAMSCs osteogenic differentiation through COX2 protein, SAN can significantly improve expression of COX2 gene (PTGS2) in hAMSCs, the osteogenic effect of SAN can be reversed by adding a COX2 inhibitor (such as celecoxib), and direct experimental evidence is provided for'COX2 serving as a small molecule drug to promote osteogenic differentiation '.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biology, and particularly relates to a new application of sanguinarine and a method for inducing human amniotic mesenchymal stem cells (hAMSCs) to differentiate into osteoblasts by cyclooxygenase 2 (COX2) protein, and simultaneously relates to an osteogenic induction medium for the induction method, which can be applied to the fields of research and development and clinical transformation of bone disease treatment drugs. BACKGROUND

[0002] Human amniotic mesenchymal stem cells (hAMSCs) are derived from the amniotic membrane tissue of a pregnant woman's placenta and belong to adult tissue stem cells, which have the potential to differentiate into osteoblasts, adipocytes and chondroblasts. As a potential candidate cell for clinical application, hAMSCs have the advantages of low immunogenicity, strong differentiation ability and no ethical controversy, and thus become an ideal seed cell for tissue engineering, providing a new treatment idea for refractory bone diseases such as bone defects, fractures and osteoporosis. Bone remodeling is a core physiological process for maintaining skeletal homeostasis, which is dynamically regulated by the balance between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. The destruction of this balance will cause various bone diseases. New bone formation, as a key link of bone remodeling, depends on the synthesis, secretion and mineralization of bone matrix by osteoblasts, and drugs that regulate new bone formation play a role by regulating the proliferation of osteoblast lineage cells or inducing the differentiation of osteoblast progenitor cells. Current studies have shown that small molecule compounds such as homogentisic acid and bisdemethoxycurcumin can induce hAMSCs to differentiate into osteoblasts, suggesting that natural small molecule compounds and their derivatives are a potential way to induce mesenchymal stem cells to differentiate, and therefore it is necessary to develop natural or synthetic drugs with strong efficacy, high safety and low side effects. Sanguinarine (SAN) is an isoquinoline alkaloid extracted from plants of the Papaveraceae family (such as Chelidonium majus, Corydalis decumbens and Macleaya cordata), with a molecular formula of C 20 H 14 NO4 + and a molecular weight of 332.33 g / mol. It has been reported to have anti-inflammatory and anti-tumor pharmacological activities. In addition, studies have found that SAN can promote the osteogenic differentiation of mouse preosteoblasts MC3T3-E1 and alleviate osteoporosis in ovariectomized rat and mouse models, indicating that SAN is a natural drug with potential osteogenic ability, but its mechanism of promoting differentiation in hAMSCs has not been clearly defined. Cyclooxygenase 2 (COX2) plays a key role in bone metabolism: on the one hand, COX2 can be induced by inflammation and participate in the occurrence of diseases; on the other hand, epigallocatechin gallate regulates the differentiation and maturation of osteoblasts through COX2 to participate in bone formation, and celecoxib relieves bone loss caused by bone balance destruction in mice by inhibiting the expression of COX2, suggesting that COX2 may be an important bridge connecting small molecule compounds and osteogenic differentiation (which has not been verified).

[0003] Based on the above research status, the present application proposes an innovative scientific hypothesis: SAN may promote osteogenesis by specifically regulating COX2. This study first found that the isoquinoline carbocyclic skeleton compound SAN promotes the differentiation of hAMSCs into osteoblasts through COX2, providing a theoretical and experimental basis for the development of bone disease treatment drugs, targeted drug development and clinical application. SUMMARY

[0004] To solve the problems of insufficient hAMSCs osteogenic induction efficiency and unclear mechanism of natural small molecule drugs promoting osteogenesis in the prior art, the present application provides a method for inducing hAMSCs osteogenic differentiation through COX2 protein, and a sanguinarine osteogenic induction medium for the method, which clearly shows the mechanism of SAN regulating hAMSCs osteogenic differentiation through COX2, and provides a new strategy for bone disease treatment.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: In a first aspect, the present application provides the use of sanguinarine in the preparation of a reagent or medium for inducing human amniotic membrane mesenchymal stem cells to differentiate into osteoblasts, wherein the sanguinarine promotes the differentiation of hAMSCs into osteoblasts by regulating the expression or / and activity of cyclooxygenase 2. Further, the reagent or medium can also be used in combination with dexamethasone, ascorbic acid and beta-glycerophosphate sodium.

[0006] In a second aspect, the present application further provides a sanguinarine-containing human amniotic membrane mesenchymal stem cell osteogenic induction medium, which is based on a low-sugar DMEM medium supplemented with fetal bovine serum and penicillin, and further supplemented with dexamethasone, ascorbic acid, beta-glycerophosphate sodium, and in particular, sanguinarine solution.

[0007] Further, the concentration of fetal bovine serum in the base medium is 10%, the concentration of penicillin is 1%, the concentration of dexamethasone is 100nM, the concentration of ascorbic acid is 50μM, the concentration of beta-glycerophosphate sodium is 100nM, and the concentration of sanguinarine solution is 0.01-5μM.

[0008] Further, the sanguinarine solution is prepared by dissolving sanguinarine powder in sterile DMSO to form a 1mM stock solution, which is diluted to 0.01-5μM for use. The dilution medium is LG-DMEM / F12 medium containing 10% FBS, 1% penicillin-streptomycin, 100nM dexamethasone, 10mM β-glycerophosphate sodium and 50μM ascorbic acid.

[0009] In a third aspect, the present application further provides a method for inducing osteogenic differentiation of human amniotic mesenchymal stem cells by cyclooxygenase 2, comprising the following steps: S1, preparation and culture of hAMSCs S1-1, isolation: fresh placenta is taken from a healthy cesarean section pregnant woman, blood stains are removed, amnion is peeled off and cut into pieces, and epithelial cells are removed by oscillation digestion; S1-2, primary culture: after digestion, the cells are collected by centrifugation after filtration through a filter screen; the cells are inoculated in LG-DMEM / F12 complete medium containing FBS for primary culture, and are cultured in a constant temperature and CO2 environment, and are subcultured when the cell density reaches the standard; S1-3, subculture: the primary cells with a density reaching the standard are digested with trypsin, subcultured, and the specified passage hAMSCs are selected; S2, induction of osteogenic differentiation of hAMSCs S2-1, inoculation: the 3rd-5th passage hAMSCs subcultured are inoculated and cultured, and are cultured in a constant temperature and CO2 environment until the cell density reaches the standard; S2-2, induction: the supernatant is discarded, and the osteogenic induction medium of the second aspect of the present application is added; the osteogenic induction medium is replaced regularly, and the culture is continued for a specified period of time.

[0010] Further, the blood stains in S1-1 are rinsed with sterile PBS containing 1% penicillin-streptomycin.

[0011] Further, in S1-1, the epithelial cells are removed by oscillation digestion with 0.5% trypsin containing 0.02% EDTA-2Na at 37°C for 60-90min, and then with 0.5mg / ml collagenase type II containing 0.05mg / ml DNase I at 37°C for 60-90min.

[0012] Further, the cells are subcultured when the cell density reaches 80%, and are subcultured at a ratio of 1:2-1:3.

[0013] Further, in S2-1, the 3rd-5th passage hAMSCs are resuspended in LG-DMEM / F12 complete medium containing 10% FBS, and the cell concentration is adjusted to 1×10 5Cells were seeded in 6-well plates at a density of 1x104 cells / ml and cultured at 37℃ in a 5% CO2 incubator until the cell density reached 50%-60%.

[0014] The beneficial technical effects of the present application are: 1. High-efficiency promotion of osteogenic differentiation: Compared with ordinary hAMSCs osteogenic induction medium (without SAN), the addition of 0.01-5 μM SAN (optimal concentration 1 μM) can significantly improve the osteogenic differentiation efficiency of hAMSCs, which is specifically manifested in the up-regulation of early osteogenic-related proteins RUNX2, OSX, and ALP, the increase of late osteogenic-related proteins COL1α1, OPN, and OCN, and the significant increase of late mineralized nodule formation.

[0015] Verification method: During the culture, the cell morphological changes were observed, and the osteogenic differentiation effect was confirmed by alkaline phosphatase (ALP) staining, enzyme activity detection (early marker), alizarin red staining and quantification (late mineralized nodule, late marker), and Western blotting detection of osteogenic-related proteins (RUNX2, OSX, and ALP for early stage, and COL1α1, OPN, and OCN for late stage). Meanwhile, the mediation effect of COX2 was verified by adding a COX2 inhibitor (such as celecoxib), and the results showed that the addition of the inhibitor significantly reduced the osteogenic differentiation indicators (such as ALP activity and early osteogenic-related proteins RUNX2, OSX, and ALP), which proved that SAN regulates osteogenic differentiation through COX2.

[0016] 2. Clear mechanism: It is first confirmed that SAN mediates hAMSCs osteogenic differentiation through COX2 protein — SAN can significantly improve the expression of COX2 gene (PTGS2) in hAMSCs, and the addition of COX2 inhibitor (such as celecoxib) can reverse the osteogenic effect of SAN, which provides direct experimental evidence for "COX2 as a small molecule drug bridge for promoting osteogenic differentiation".

[0017] 3. Clinical transformation value: The induction method and medium provided by the present application provide a theoretical and experimental basis for the research and development of drugs for bone diseases (such as arthritis, fracture, osteoporosis, etc.) (such as the design of derivatives based on the structure of SAN), the development of COX2 targeted drugs, and the optimization scheme for the application of hAMSCs in tissue engineering (such as seed cell induction). BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Cell toxicity analysis chart of hAMSCs treated with different concentrations of sanguinarine (SAN) (*p<0.05 vs CON); Figure 2The effect of SAN on osteogenic differentiation of hAMSCs; (A): Alkaline phosphatase staining; (B): Alkaline phosphatase activity assay; (C): Alizarin red staining; (D): Alizarin red staining quantitative assay (*p<0.05 vs CON; # p<0.05 vs OIM); Figure 3 Effects of sanguinarine treatment on osteogenic-associated protein expression in hAMSCs; (A): Immunoblotting of early osteogenic-associated proteins; (B): Immunoblotting analysis of early osteogenic-associated proteins; (C): Immunoblotting of late osteogenic-associated proteins; (D): Immunoblotting analysis of late osteogenic-associated proteins (*p<0.05 vs CON; # p<0.05 vs OIM); Figure 4 The experiment was conducted to verify the function of COX2 in sanguinarine-induced osteogenic differentiation of hAMSCs; (A): COX2 gene (PTGS2) expression; (B): Effect of COX2 inhibitor on alkaline phosphatase staining, an early osteogenic marker; (C): Effect of COX2 inhibitor on alkaline phosphatase activity, an early osteogenic marker; (D): Changes in the expression of early osteogenic-related proteins and COX-2 after COX2 inhibition (*p<0.05 vs CON; # p<0.05 vs OIM). Detailed Implementation

[0019] (I) Preparation of experimental materials Cell source: Fresh placenta from healthy pregnant women undergoing cesarean section (ethical approval: Zunyi Lun Shen (2023) 1-130); Main reagents: 0.5% trypsin digestion solution containing 0.02% EDTA-2Na, type II collagenase (0.5 mg / ml), DNase I (0.05 mg / ml), LG-DMEM / F12 medium, fetal bovine serum (FBS), penicillin and streptomycin, dexamethasone, ascorbic acid, sodium β-glycerophosphate, sanguinarine (SAN, CAS No. 2447-54-3), celecoxib (CAS No. 169590-42-5), DMSO (sterile grade), CCK-8 kit, BCIP / NBT alkaline phosphatase staining kit, alkaline phosphatase activity assay kit, alizarin red S staining solution, 10% hexadecylpyridine chloride solution, RNA extraction kit, RT-qPCR kit, Western blotting reagents; Instrument and equipment: 37℃, 5% CO2 constant temperature incubator, clean bench, optical microscope, stereomicroscope, centrifuge (1200 rpm), microplate reader (405 nm, 450 nm, 562 nm wavelength), PCR instrument, Western blotting electrophoresis and development system. (II) Isolation and culture of hAMSCs Placenta processing: Place the fresh placenta on a high-pressure sterilized clean operating tray, gently rinse the surface bloodstains with sterile PBS containing 1% penicillin, and peel off the amnion on the surface of the placenta with sterilized scissors and forceps, and place it in a container containing sterile PBS; Cell digestion: In the clean bench, cut the amnion into 1cm~3cm, put it into a 50ml centrifuge tube, add 0.02% EDTA-2Na containing 0.5% trypsin digestion solution (2 times the volume of amnion), shake and digest at 37℃ constant temperature for 60-90min; filter with a 300 mesh stainless steel filter to remove epithelial cells, and collect the amnion residue, wash with D-PBS for 3 times; add 0.05mg / ml DNase I containing 0.5mg / ml type II collagenase (equal volume to amnion) to the residue, shake and digest at 37℃ constant temperature for 60-90min, shake manually every 15-20min; Primary culture: Filter again with a 300 mesh stainless steel filter, collect the filtered cell suspension, centrifuge at 1200rpm for 5min, discard the supernatant, and obtain hAMSCs precipitate; resuspend the cells with LG-DMEM / F12 complete medium containing 10% FBS, inoculate in T75 culture bottles, and place in a 37℃, 5% CO2 incubator; the next day, observe the cell state under a light microscope, discard the old culture medium and unattached cells, wash with sterile PBS for 3 times, and add fresh culture medium; observe the cell state every 2-3 days, and replace the culture medium as needed; Passage and cryopreservation: After 5-7 days of culture, the cells grow and fuse to a density of about 80%, and are digested with 0.25% trypsin, passaged at a ratio of 1:2 or 1:3, and passaged every 2~4 days; freeze the 3rd passage (P3) cells for subsequent experiments. (III) Preparation of sanguinarine (SAN) and celecoxib stock solution SAN stock solution: Weigh SAN powder, dissolve in sterile DMSO to prepare a stock solution with a concentration of 1mM; aliquot with EP tubes and seal, store in a -80℃ refrigerator, and dilute to 0.01μM, 0.1μM, 1μM, 5μM, 10μM, etc. working concentrations with osteogenic induction medium (positive control group (OIM) medium described below) when used; Celecoxib stock solution: 10 mg Celecoxib powder was weighed and dissolved in sterile DMSO to make a stock solution with a concentration of 1 mM; diluted to the required working concentration (25 mM) with the osteogenic induction medium containing SAN of the application and stored in a -20°C refrigerator. (IV) Experimental grouping design The following groups were set up for all experiments, with 3 replicate wells in each group and 3 repeated experiments: Control group (CON): LG-DMEM / F12 complete culture medium containing only 10% FBS and 1% penicillin-streptomycin; Positive control group (OIM): OIM osteogenic induction medium - LG-DMEM / F12 medium containing 10% FBS, 1% penicillin-streptomycin, 100 nM dexamethasone, 10 mM sodium beta-glycerophosphate, and 50 mM ascorbic acid; SAN combined induction group (SAN+OIM): SAN-containing osteogenic induction medium - OIM osteogenic induction medium + 1% penicillin-streptomycin + 0.01 mM to 5 mM SAN (select a specific concentration according to experimental requirements, with 1 mM being optimal); COX2 inhibitor verification group (SAN+OIM+Celecoxib): SAN+OIM medium + COX2 inhibitor (1 mM celecoxib was used in the application, which was diluted to 25 mM when used). (V) Cytotoxicity detection of SAN on hAMSCs (CCK-8 method) Cell inoculation: Take the second generation (P2) hAMSCs with good growth state and a density of 60%-80%, digest with 0.25% trypsin for 2 min, centrifuge at 1200 rpm for 5 min, resuspend with LG-DMEM / F12 medium containing 10% FBS, inoculate in 96-well plates at a density of 1x10 4 Cell inoculation: Take the second generation (P2) hAMSCs with good growth state and a density of 60%-80%, digest with 0.25% trypsin for 2 min, centrifuge at 1200 rpm for 5 min, resuspend with LG-DMEM / F12 medium containing 10% FBS, inoculate in 96-well plates at a density of 1x10 Drug treatment: Discard the supernatant in the 96-well plate, and add LG-DMEM / F12 medium containing 0.01 mM, 0.1 mM, 1 mM, 5 mM, and 10 mM SAN (containing 10% FBS), respectively, and CON group without SAN; Toxicity detection: After 24 h, 48 h, and 72 h of culture, respectively, add 10 μl of CCK-8 reagent to each well and continue to culture for 2 h; detect the absorbance value of each well at 450 nm wavelength with a microplate reader and calculate the cell survival rate; Results: As shown in Figure 1, compared with the CON group, SAN had no significant toxicity to hAMSCs at 0.01 μM-5 μM for 24 h, 48 h, and 72 h. (VI) Detection of the effect of SAN on the osteogenic differentiation of hAMSCs 1. Early osteogenic marker (alkaline phosphatase) detection Cell inoculation and induction: Take the third generation (P3) hAMSCs in good growth condition with a density of 60%-80%, resuspend them with LG-DMEM / F12 culture medium containing 10% FBS, inoculate them in a 12-well plate at a density of 1x10 5 cells / well, and place them in a 37°C, 5% CO2 incubator for culture to the logarithmic growth phase; discard the supernatant, wash it with D-PBS for 3 times, and add CON group, OIM group, and different concentrations of SAN+OIM group medium (0.01 μM, 0.1 μM, 1 μM, 5 μM) respectively, and change the medium every 2-3 days; Alkaline phosphatase staining (BCIP / NBT method): after 5 days of culture, the early marker alkaline phosphatase of osteoblasts was determined by BCIP / NBT staining method. Observe the staining under an optical microscope and take pictures; the results are determined as follows: Figure 2 (A) shows that the osteogenic differentiation cells are blue-violet, and the blue-violet staining depth of the SAN+OIM group is significantly stronger than that of the OIM group, and the 1 μM SAN group has the deepest staining.

[0020] Alkaline phosphatase enzyme activity detection: alkaline phosphatase enzyme activity detection was performed according to the kit instructions, the absorbance value of the sample was detected at 405 nm wavelength, and the activity was calculated according to the definition of alkaline phosphatase activity. As shown in (B), consistent with the alkaline phosphatase expression results, compared with the OIM group, the ALP activity of the 0.01 μM-5 μM SAN group was significantly increased, and the alkaline phosphatase showed the optimal enzyme activity when the SAN concentration was 1 μM. Figure 2

[0021] Late osteogenic marker (mineralized nodule and osteogenesis-related protein) detection Alizarin red staining and quantification (mineralized nodule detection) Staining steps: Take the third generation hAMSCs in good growth condition, divide the hAMSCs into CON group, OIM group, and SAN (1 μM)+OIM group, inoculate them in a 12-well plate at a density of 1x10 5 ​The number of cells in the wells was inoculated in a 12-well cell culture plate for osteogenic induction, and the liquid was changed every 2-3 days according to the cell state during the induction. After 21 days, the old culture medium supernatant was discarded, the cells were fixed, and then washed with pure water three times, incubated with alizarin red S staining solution at room temperature for 30 minutes, slowly washed with pure water three times, and observed under a stereomicroscope to observe the formation of calcium nodules.

[0022] Results: As shown in Figure 2 (C), compared with the OIM group, the addition of SAN promoted the formation of mineralized calcium nodules of hAMSCs osteogenic differentiation. The CON group had almost no red nodules, the OIM group had a small amount of red nodules, the SAN+OIM group had a significant increase in the number and size of nodules, and the 1 μM SAN group had the highest absorbance value. Further incubated with an equal amount of 10% cetylpyridinium chloride solution at room temperature for 30 minutes, and alizarin red S staining solution was fully compatible, and the OD value was detected at 562 nm using a microplate reader. As shown in Figure 2 (D), consistent with the results of mineralized calcium nodule formation, compared with the OIM group, the OD value was significantly improved after the addition of SAN.

[0023] (Seven) Western blotting detection of the effect of SAN on the expression of osteogenic proteins of hAMSCs hAMSCs were treated with SAN for 5 days and 21 days, and the expression of osteogenic proteins in the samples was detected by Western blotting. As shown in Figure 3 compared with the OIM group, the addition of SAN significantly promoted the expression of pre-osteogenic proteins RUNX2, OSX, ALP at the fifth day and post-osteogenic proteins COL1α1, OPN, OCN at the 21st day of hAMSCs differentiation.

[0024] (Eight) COX2 mediated action verification experiment 1. The effect of SAN on the expression of COX2 (PTGS2) gene of hAMSCs hAMSCs were divided into CON group, OIM group, SAN (1 μM)+OIM group, and cultured for 3 days after induction, RNA was extracted, and the expression of COX2 gene PTGS2 was detected by real-time fluorescent quantitative PCR (RT-qPCR), as shown in Figure 4 (A), when adding the solution of sanguinarine in the osteogenic induction medium, the expression of PTGS2 gene of hAMSCs was significantly improved.

[0025] 2. Evaluation of SAN regulating early markers of hAMSCs differentiation into osteoblasts after inhibiting the expression of COX2 protein According to the CON group, OIM group, SAN (1 μM) + OIM group, SAN (1 μM) + OIM + Celecoxib group, after hAMSCs adhered and were in the logarithmic growth phase, drug was added (corresponding to the above "(four) experimental grouping design" drug was added), and the early marker alkaline phosphatase of osteoblasts was determined by BCIP / NBT staining method on the fifth day.

[0026] Results, as shown in Figure 4 (B), compared with the OIM group, SAN treatment significantly increased the expression of alkaline phosphatase, but the addition of Celecoxib reversed this effect. Further alkaline phosphatase enzyme activity detection, the absorbance value of the sample was detected at 405 nm wavelength, and its activity was calculated according to the definition of alkaline phosphatase activity. Results, as shown in Figure 4 (C), compared with Figure 4 (B) results are consistent, the addition of Celecoxib reversed the SAN to increase the activity of alkaline phosphatase.

[0027] 3. After inhibiting the expression of COX2 protein, the expression of early proteins and COX-2 of SAN regulated hAMSCs differentiation into osteoblasts was changed According to the CON group, OIM group, SAN (1 μM) + OIM group, SAN (1 μM) + OIM + Celecoxib group, after hAMSCs adhered and were in the logarithmic growth phase, drug was added (corresponding to the above "(four) experimental grouping design" drug was added), and the early marker alkaline phosphatase of osteoblasts was determined by BCIP / NBT staining method on the fifth day. Figure 4 (D) shows, compared with the OIM group, the addition of SAN can significantly promote the expression of COX-2 protein of hAMSCs on the third day, and the expression of early osteogenic proteins RUNX2, OSX and ALP on the fifth day, but the addition of Celecoxib group reversed this effect.

[0028] The above only is the embodiment of the present application, and the specific technical solutions and / or characteristics of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be regarded as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in this application should be subject to the content of its claims, and the specific embodiments described in the specification can be used to explain the content of the claims.

Claims

1. Use of sanguinarine for the preparation of a reagent or a medium for inducing osteogenic differentiation of human amniotic mesenchymal stem cells, characterized in that: The sanguinarine promotes differentiation of the hAMSCs into osteoblasts by regulating expression or / and activity of cyclooxygenase 2.

2. Use according to claim 1, characterized in that: The reagent or medium can also be used in combination with dexamethasone, ascorbic acid, beta-glycerophosphate sodium.

3. A sanguinarine containing human amniotic mesenchymal stem cell osteogenic induction medium, the osteogenic induction medium is based on low sugar DMEM with fetal bovine serum, penicillin, and further adding dexamethasone, ascorbic acid, β-glycerophosphate sodium, characterized in that: The sanguinarine solution is added to the osteogenic induction medium.

4. The osteogenic induction medium according to claim 3, characterized in that: The concentration of fetal bovine serum in the basal medium is 10%, and the concentration of penicillin is 1%. The concentration of dexamethasone is 100 nM, the concentration of ascorbic acid is 50 μM, the concentration of beta-glycerophosphate sodium is 100 nM, and the concentration of the sanguinarine solution is 0.01-5 μM.

5. The osteogenic induction medium according to claim 4, characterized in that: The sanguinarine solution is prepared by dissolving sanguinarine powder in sterile DMSO to prepare a 1 mM stock solution, and diluted to 0.01 μM-5 μM for use.

6. A method of inducing osteogenic differentiation of human amniotic mesenchymal stem cells by cyclooxygenase 2, characterized by, The method comprises the following steps: S1, preparation and culture of hAMSCs S1-1, isolation: fresh placenta is taken from healthy cesarean section pregnant women, blood stains are removed, amnion is peeled off and cut into pieces, and epithelial cells are removed by oscillation digestion; S1-2, primary culture: after digestion, the cells are filtered through a filter screen and centrifuged to collect the cell precipitate; the cell precipitate is inoculated in LG-DMEM / F12 complete medium containing FBS for primary culture, and cultured in a constant temperature and constant CO2 environment; when the cell density reaches the standard, the cells are subcultured; S1-3, subculture: the primary cells with a density reaching the standard are digested with trypsin, subcultured, and the specified passage hAMSCs are selected; S2, osteogenic differentiation induction of hAMSCs S2-1, inoculation: the 3rd-5th passage hAMSCs obtained by subculture are inoculated and cultured, and cultured in a constant temperature and constant CO2 environment until the cell density reaches the standard; S2-2, induction: the supernatant is discarded, and the osteogenic induction medium of any one of claims 3-5 is added; the osteogenic induction medium is replaced regularly, and the culture is continued for a specified period of time.

7. The method of claim 6, wherein: In S1-1, the blood stains are washed with sterile PBS containing 1% penicillin.

8. The method of claim 6, wherein: In S1-1, the epithelial cells are removed by oscillation digestion with 0.5% trypsin solution containing 0.02% EDTA-2Na at 37°C for 60-90 min, and then with 0.5 mg / ml type II collagenase containing 0.05 mg / ml DNase I at 37°C for 60-90 min.

9. The method of claim 8, wherein: When the cell density reaches 80%, the cells are subcultured at a ratio of 1:2-1:

3.

10. The method of claim 9, wherein: In S2-1, the 3rd-5th generation hAMSCs were resuspended with LG-DMEM / F12 complete medium containing 10% FBS, and the cell concentration was adjusted to 1x10 5 6 / ml. The cells were inoculated in a six-well plate and cultured in a 37°C, 5% CO2 incubator until the cell density reached 50%-60%.

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