Method for inducing osteogenic differentiation of mesenchymal stem cells

Abstract

The invention relates to the technical field of bone marrow mesenchymal stem cell osteogenic differentiation, and particularly discloses a method for inducing bone marrow mesenchymal stem cell osteogenic differentiation, which is technically characterized by comprising the following steps: S1, preparing rat bone marrow mesenchymal stem cells; s2, performing osteogenesis induction; s3, cell differentiation induction: replacing the osteogenic induction culture medium every 2-3 days, continuously culturing for 14-21 days in the culture environment of 37 DEG C and 5% CO2, paying attention to observation of cell morphological change, and detecting the osteogenic differentiation condition of the rat bone marrow mesenchymal stem cells; wherein a mulberry leaf extract is added into the osteogenesis induction culture medium. According to the invention, the mulberry leaf extract is added into the osteogenic induced differentiation culture medium, so that osteogenic differentiation of bone marrow mesenchymal stem cells can be promoted, tissue regeneration is promoted, and the mulberry leaf extract is applied to preparation of the medicine for treating periodontitis; a new experimental basis and a new theoretical basis are provided for research, development and clinical application of the folium mori extract periodontitis treatment medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of osteogenic differentiation of bone marrow mesenchymal stem cells, and in particular to a method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells. Background Art

[0002] One of the main pathological changes of periodontitis is alveolar bone resorption, which is the most active process in human bone reconstruction. The development of periodontitis can lead to loose teeth and tooth displacement. What's more serious is that teeth affected by periodontitis may fall out in severe cases, which has a negative impact on the patient's chewing, pronunciation function and appearance. Currently, periodontitis has become a global burden, so periodontal tissue regeneration has attracted widespread attention worldwide. Current clinical treatments for periodontitis, such as periodontal scaling and root planing, can control the progression of periodontal inflammation and prevent further loss of periodontal supporting tissues, but cannot achieve the ideal regeneration effect of lost tissues. Guided tissue regeneration and bone transplantation provide new ideas for the treatment of periodontitis.

[0003] In the field of periodontal tissue engineering, how to select suitable seed cells has always been a hot topic of scholars. Because stem cells have good multidirectional differentiation potential, they are considered to be ideal seed cells that can be used in the field of periodontal tissue engineering. Bone marrow mesenchymal stem cells (BMMSCs) were first proposed by Friedenstein in the late 1960s. They are a type of bone marrow-derived stem cells with multidirectional differentiation potential. They are widely used in regenerative therapy for their superior immunoregulatory and proliferation properties. BMMSCs are a type of multipotent mesenchymal stem cells that exist in the bone marrow stroma. They can differentiate into mesoderm cells (osteoblasts, adipocytes, and chondrocytes), and have the characteristics of hematopoietic support, immune regulation, and self-replication. In recent years, many scholars have used BMMSCs to achieve the regeneration of periodontal tissues (alveolar bone, cementum, and periodontal ligament). Studies have found that BMMSCs can successfully repair Class III bone defects in the jaws of beagle dogs.

[0004] Bone formation involves a series of complex processes, including the proliferation and differentiation of osteoprogenitor cells, which ultimately lead to the formation of mineralized extracellular matrix. At the cellular level, the smallest functional unit is mainly composed of the communication and coupling between bone cell types, osteoblasts that form bone, and osteoclasts that degrade bone. Since new bone formation is mainly the function of osteoblasts, drugs that regulate bone formation mainly play a drug function by increasing the proliferation of osteoblast lineage cells or inducing the differentiation of osteoblast progenitor cells. Based on the above theory, with the help of specific and targeted drugs that promote bone regeneration, early prevention and treatment may reduce the incidence and harm of periodontitis. Therefore, the development of drugs that stimulate osteoblast differentiation is an effective therapeutic strategy for the prevention and treatment of periodontitis. In recent years, natural medicines have been widely used due to their advantages such as easy access and low toxicity. Therefore, more and more natural medicines are used as medicines and foods to prevent and treat common oral and systemic diseases. Therefore, it is very necessary to find and screen natural medicines or other compounds synthesized from natural ingredients for the prevention and treatment of periodontitis with high safety, strong tolerance and low side effects. During bone growth and development, the expression of Runx2 is the beginning of osteoblast differentiation. Runx2 can initiate and activate the differentiation of bone marrow stromal stem cells into osteoblasts. At the same time, it can upregulate the expression of many osteogenic genes, which are key markers of bone formation. In addition to its biological role in osteoblasts, Runx2 is highly expressed in hypertrophic chondrocytes and promotes chondrocyte hypertrophy and endochondral ossification. Runx2 also promotes osteoclast differentiation by inducing RANKL and Opn to play a corresponding role. In addition, Runx2 can also promote the synthesis of related extracellular matrix proteins, including Col-1, Ocn and bone sialoprotein (BSP). Runx2 is also a transcription factor required for the activation of osteoblast differentiation and the expression of bone formation-related genes ALP, Ocn and Opn. Bmp is an important member of the transforming growth factor-β (Protransforming growth factor alpha, TGF-β) superfamily, and it has been shown that targeting Runx2 can induce bone formation by stimulating osteoblast differentiation in mesenchymal stem cells. In addition, Bmp2 can bind to Bmp receptors to induce heteromeric complexes and phosphorylation of Smad1 / 5 / 9, subsequently inducing osteoblast differentiation.

[0005] Studies have found that quercetin has anti-diabetic and anti-apoptotic properties, and can also stimulate angiogenesis in skin defects. In addition, studies have found that hyperoside can enhance the proliferation and tube formation ability of endothelial progenitor cells, and isoquercetin can promote the osteogenic differentiation of human BMSCs and alleviate osteoporosis in mice induced by ovariectomy. These are the main components of mulberry leaf extract, so mulberry leaf extract is a natural medicine with potential osteogenic ability. However, whether mulberry leaf extract can promote the osteogenic differentiation of bone marrow mesenchymal stem cells, as well as the safety range and mechanism of action of the drug are not fully understood. In this study, representative rat bone marrow mesenchymal stem cells were used to study the biological performance of mulberry leaf extract after acting on bone marrow stem cells, and to evaluate whether it can promote stem cell differentiation and osteogenesis, and whether it can promote tissue regeneration. Summary of the invention

[0006] The purpose of the present invention is to solve the technical problems raised in the above background technology and provide a method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells.

[0007] The above-mentioned purpose of the present invention is achieved like this:

[0008] The present invention provides a method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells, comprising the following steps:

[0009] S1. Preparation of rat bone marrow mesenchymal stem cells:

[0010] The rat bone marrow mesenchymal stem cells were isolated and cultured by the whole bone marrow cell suspension adherence method. The isolated bone marrow cell suspension was subjected to primary culture, and the culture flask was moved to a 37°C, 5% CO2 constant temperature incubator. When the cell growth density reached 80%, the cells were subcultured, and trypsin (0.25%) was used for digestion. The cells were cultured in 1:2 iterative manner, and one iteration was started every 2-4 days.

[0011] S2. Osteogenesis Induction:

[0012] Select the 3rd-4th generation rat bone marrow mesenchymal stem cells iteratively cultured in step S1 and adjust the cell concentration to 2×10 4 / mL, inoculate and culture, culture at 37°C, 5% CO2 environment until the cell density reaches 60-70%, discard the supernatant, and add osteogenic differentiation medium;

[0013] S3. Cell differentiation induction: Replace osteogenic induction medium every 2-3 days, continue culturing at 37°C, 5% CO2 for 14-21 days, and observe cell morphology changes and detect osteogenic differentiation of rat bone marrow mesenchymal stem cells;

[0014] Among them, mulberry leaf extract is added to the osteogenic induction medium.

[0015] Furthermore, the added amount of the mulberry leaf extract is 10 mg / L-100 mg / L.

[0016] Furthermore, the added amount of the mulberry leaf extract is 10 mg / L.

[0017] Furthermore, the added amount of the mulberry leaf extract is 100 mg / L.

[0018] Another aspect of the present invention provides the use of mulberry leaf extract in the osteogenic differentiation of bone marrow mesenchymal stem cells.

[0019] Another aspect of the present invention provides a method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells and its application in preparing a drug for treating periodontitis.

[0020] The difficulty and significance of the technical problem solved by the present invention are:

[0021] The main feature of periodontitis is the progressive destruction of periodontal supporting tissues, especially the loss of alveolar bone. As periodontitis progresses, the height of the alveolar bone will gradually decrease, the tooth roots will be exposed, and the teeth will become loose. If not treated in time, it will eventually lead to tooth loss. Extensive tooth loss will seriously damage chewing efficiency, nutritional intake and mental health, and have an important impact on people's lives. Bone marrow mesenchymal stem cells are a type of stem cells with multiple potentials. They are derived from the bone marrow and play a key role in the development of bones. Bone marrow mesenchymal stem cells can be directed to differentiate according to specific molecular signals present in the bone marrow microenvironment. In addition to being able to differentiate into osteoblasts, they can also differentiate into several other cell types, such as adipocytes and chondrocytes, thus playing a vital role in maintaining bone health and repairing damaged bones. At the same time, bone marrow mesenchymal stem cells have the advantages of strong proliferation ability and low immunogenicity. They can be used for cell therapy and tissue engineering research, and have been proven to be used to repair bone damage and treat bone and joint diseases. In-depth study of the molecular mechanism of bone marrow mesenchymal stem cell differentiation regulation and exploration of new strategies to induce their differentiation into osteoblasts are current hot topics in bone biology research. By promoting the proliferation of bone marrow mesenchymal stem cells and accelerating new bone formation, it helps to enhance the bone metabolism function of osteoporosis patients, thereby playing a role in treating the disease. The mulberry leaf extract in the scheme of the present invention can promote the osteogenic differentiation of rBMMSCs, and experiments have confirmed that the expression of osteogenic related genes and proteins are upregulated, and has potential tissue repair ability.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] In the method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells provided by the present invention, mulberry leaf extract is added to the osteogenic induction differentiation medium, which can promote the osteogenic differentiation of bone marrow mesenchymal stem cells and promote tissue regeneration, so that the mulberry leaf extract is applied to the preparation of drugs for treating periodontitis, providing a new experimental basis and a new theoretical basis for the research, development and clinical application of periodontitis treatment drugs based on the development of mulberry leaf extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 rBMMSCs identification in the embodiment of the present invention ((A) cell morphology under microscope; (B) alizarin red staining; (C) alcian blue staining; (D) oil red O staining; (E) flow cytometry identification);

[0025] Figure 2 is the inhibition rate of rBMMSCs proliferation by mulberry leaf extracts of different concentrations in the present invention (data are expressed as mean ± standard deviation (n = 3). Compared with the control group, ***P < 0.001, **P < 0.01, *P < 0.05);

[0026] Figure 3 The effects of different concentrations of mulberry leaf extracts on apoptosis and reactive oxygen species in rBMMSCs in the examples of the present invention ((A) apoptosis flow cytometry image; (B) apoptosis flow analysis; (C) flow cytometry image; (D) reactive oxygen species analysis. FICT, fluorescein isothiocyanate; PI, propidium iodide. Data are expressed as mean ± standard deviation (n = 3), and the upper letters represent differences between different groups (P < 0.05));

[0027] Figure 4 The effects of different concentrations of mulberry leaf extracts on osteogenic differentiation of rBMMSCs in the examples of the present invention ((A) alkaline phosphatase staining and alizarin red staining; (B) quantitative results of alkaline phosphatase activity; (C) quantitative results of alizarin red. Data are expressed as mean ± standard deviation (n = 3), and the upper letters represent differences between different groups (P < 0.05));

[0028] Figure 5 The effect of different concentrations of mulberry leaf extract on the expression of osteogenic-related genes in rBMMSCs in the embodiment of the present invention ((A) grayscale image of protein expression; (A) ALP; (B) Runx2; (C) Col-1; (D) Ocn; (E) BMP2; (F) Osterix. The data are expressed as mean ± standard deviation (n = 3), and the upper letters represent the differences between different groups (P < 0.05));

[0029] Figure 6The effects of different concentrations of mulberry leaf extracts on the expression of osteogenic-related proteins in rBMMSCs in the examples of the present invention ((A) grayscale image of protein expression; (B) ALP; (C) Runx2; (D) Col-1; (E) Ocn; (F) BMP2; (G) Osterix. The data are expressed as mean ± standard deviation (n = 3), and the letters above represent the differences between different groups (P < 0.05)). DETAILED DESCRIPTION

[0030] 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 in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0032] As shown in the drawings, a preferred embodiment of the present invention is provided.

[0033] Embodiment: The embodiment of the present invention provides a method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells, comprising the following steps:

[0034] Step 1: Preparation of rat bone marrow mesenchymal stem cells: isolation and culture of rat bone marrow mesenchymal stem cells were completed by whole bone marrow cell suspension adherence method, primary culture was performed on the isolated bone marrow cell suspension, and the culture flask was moved to a 37°C, 5% CO2 constant temperature incubator; when the cell growth density reached 80%, subculture was started, during which trypsin (0.25%) was used for digestion, and 1:2 cell iterative culture was performed, and one iteration was started every 2-4 days;

[0035] Step 2: Osteogenesis induction: Select the 3rd-4th generation rat bone marrow mesenchymal stem cells iteratively cultured in step S1 and adjust the cell concentration to 2×10 4 / mL, inoculate and culture, culture at 37°C, 5% CO2 environment until the cell density reaches 60-70%, discard the supernatant, and add osteogenic differentiation medium;

[0036] Step 3: Cell differentiation induction: Replace osteogenic induction medium every 2-3 days, continue culturing at 37°C, 5% CO2 for 14-21 days, and observe cell morphology changes and detect osteogenic differentiation of rat bone marrow mesenchymal stem cells;

[0037] Wherein, mulberry leaf extract is added to the osteogenic induction medium, and the added amount of mulberry leaf extract is 100 mg / L.

[0038] In the scheme of this embodiment, the mulberry leaf extract is applied to the osteogenic differentiation of bone marrow mesenchymal stem cells and to the preparation of a drug for treating periodontitis.

[0039] The following is a specific experiment of the implementation scheme of the present invention:

[0040] 1. Experimental content and methods

[0041] 1.1 Experimental subjects

[0042] 1.1.1 Cell culture reagents and consumables

[0043] αMEM (αMinimum Essential Medium) culture medium, trypsin-EDTA containing phenol red (0.25%) were purchased from Gibco TM Company (USA); Fetal bovine serum (FBS, catalog number: 04-001-1ACS), phosphate buffered saline (PBS), and 1% penicillin-streptomycin (10000 units / mL) were purchased from BI Company (Israel); Cell culture grade dimethyl sulfoxide (DMSO, catalog number: D2650) was purchased from Sigma Company (USA); Rat bone marrow mesenchymal stem cell osteogenic / adipogenic / chondrogenic differentiation kit was purchased from China T25 cell culture flasks, cell cryopreservation tubes, laser confocal microscope culture dishes, 15mL / 50mL centrifuge tubes and 6-well, 12-well and 96-well culture plates were purchased from Wuxi Nice Life Sciences Co., Ltd. (China) (Cat. No.: RAXMX-90021 / RAXMX-90031 / RAXMX-90041); sealing films (Cat. No.: PM-996) were purchased from Parafilm (USA); disposable filters (0.22μm) were purchased from Biosharp Co., Ltd. (China).

[0044] 1.1.2. Isolation of rat BMMSCs

[0045] BMSCs were isolated and cultured by the whole bone marrow cell suspension adherence method, and SD rats were killed by cervical dislocation. Rats were immediately soaked in 75% ethanol (ET). After the rats were taken out, the limbs were wiped with iodine and alcohol cotton balls for disinfection. The femur and tibia of the rat were separated with sterilized forceps and scissors, the tendons and muscles were removed, and they were repeatedly rinsed with PBS; then the femur and tibia were soaked in DMEMF-12 culture medium, the epiphyses of the tibia and femur were cut off, and a No. 5 empty needle was inserted into the bone marrow cavity, and the epiphyses and the other end of the bone marrow were repeatedly rinsed with αMEM culture medium. The bone marrow washing liquid in the culture dish was repeatedly blown with a pipette to eliminate cell clusters and obtain a single cell suspension; the bone marrow cell suspension was centrifuged at room temperature, 1000rpm, centrifuged for 5 minutes, the supernatant was discarded, and the precipitate was diluted in αMEM culture medium containing 15% fetal bovine serum (FBS).

[0046] 1.1.3. Culture of rat BMMSCs

[0047] The isolated bone marrow cell suspension was subjected to primary culture, and the culture flask was moved to a constant temperature incubator at 37°C and 5% CO2. On the second day, the status of BMSCs was checked under a microscope, the old cell culture medium in the culture flask was cleaned out, and the flask was washed 2 or 3 times with PBS solution, and then fresh culture medium was added for continued culture, so that the non-adherent cells in the culture flask could be cleaned out. Thereafter, the cell status was observed every 2-3 days, and the culture medium was replaced according to the actual situation. BMSCs generally take about 7-10 days for cell growth and fusion. When the cell growth density reaches 80%, it enters the subculture, during which it is digested with trypsin (0.25%), and a 1:2 cell subculture is performed, with the first subculture starting at 2-4 days. The 3rd-4th generation cells were selected to complete subsequent experiments.

[0048] 1.1.4. Identification of rat BMMSCs

[0049] Osteogenesis induction: Inoculation of BMSCs: Prepare logarithmically growing cells to a cell concentration of 2 × 10 4 / mL, inoculate and culture, culture at 37℃, 5% CO2 culture environment until the cell density reaches 60-70%, discard the supernatant, add osteogenic differentiation medium; cell differentiation induction: replace osteogenic induction medium every 2-3 days, continue to culture at 37℃, 5% CO2 culture environment for 14-21 days, and pay attention to observe the changes in cell morphology. According to the crystallization of calcium salts and the formation of calcium nodules in cells, the time to terminate cell induction is determined, and staining identification is performed. Cell fixation: remove the culture medium, wash once with an appropriate amount of 1×PBS, discard it, take an appropriate amount of 4% neutral formaldehyde solution to cover the bottom of the culture vessel, fix at room temperature for 30-60min, recover the fixative, and wash the residual fixative with PBS; ARS staining: cover the bottom of the culture vessel with ARS dye, after 3-5min, recover the dye, wash the residual dye with PBS, and add an appropriate amount of 1×PBS to prevent the cells from drying; induction evaluation: microscopic observation, image acquisition and induction evaluation. When the induction is successful, the calcified nodules will appear red or orange after combining with the Alizarin Red dye.

[0050] Adipogenic induction: Inoculation of BMSCs: Take cells in the logarithmic growth phase and adjust the cell concentration to 2×10 4 / mL, inoculate, culture the cells at 37°C, 5% CO2 environment to a density of 90-100%, discard the supernatant, and add adipogenic differentiation medium (divided into induction medium A and maintenance medium B).

[0051] Cell differentiation induction: Add solution A and culture at 37°C, 5% CO2 for 3 days, then replace with solution B and culture for 1 day, repeat the cycle. Induce for 14-21 days according to the above solution change frequency, and pay attention to observe the changes in cell morphology. Determine the time to terminate cell induction based on the number and size of lipid droplets formed by cell induction, and perform staining identification.

[0052] Cell fixation: remove the culture medium, wash once with an appropriate amount of 1×PBS, discard it and take an appropriate amount of 4% neutral formaldehyde solution to cover the bottom of the culture vessel, fix at room temperature for 30-60 minutes, discard the fixative and wash twice with 1×PBS.

[0053] Chondrogenic induction: Digest the cells and count the cells. 4 × 10 5Transfer the cells to a 15mL centrifuge tube and centrifuge at 1200rpm for 4min. Aspirate the supernatant, add 0.5mL induction solution, resuspend the precipitate obtained from the previous centrifugation to wash the mesenchymal stem cells, and centrifuge at 150g for 5min at room temperature. Repeat the steps 3 times and wash the cells again. Resuspend the precipitate obtained in the previous step with 0.5mL mesenchymal stem cell chondrogenic induction complete medium. Centrifuge at 1000rpm for 5min at room temperature. Loosen the cap of the centrifuge tube to facilitate gas exchange, place it in an incubator at 37℃ and 5% CO2, and keep the centrifuge tube still for at least 24h. When the cell clusters appear to gather (usually after 24h or 48h, depending on the actual cell growth), flick the bottom of the centrifuge tube to detach the chondrocytes from the bottom of the tube and suspend them in the liquid. Starting from the start of inoculation, replace the cells with fresh chondrogenic induction complete medium every 2-3d, with about 0.5mL chondrogenic induction differentiation complete medium per tube. After changing the medium, flick the bottom of the centrifuge tube to detach the chondrocytes from the bottom of the tube and suspend them in the liquid. Slightly loosen the cap of the centrifuge tube and place it in an incubator at 37°C and 5% CO2 to continue induction culture. Generally, after 21-28 days of induction, the chondrocytes can be fixed with formalin, embedded in paraffin, and sectioned, and finally stained with Alecin blue.

[0054] Flow cytometry detection and identification of stem cell surface markers:

[0055] (1) Cell preparation: After digestion, centrifuge the cultured cells (1000 rpm, 5 min), discard the supernatant, and resuspend them in PBS solution. Pay attention to the degree of cell dispersion and whether there are clumps. If there are clumps, let it stand for 3-5 min, discard the clumps, and let it stand for another 3-5 min. Continue to observe the degree of cell dispersion. If there are still clumps, repeat the above steps, add a certain amount of PBS to wash once, and discard the supernatant;

[0056] (2) Mix the cells, calculate the amount of cells to be measured, and use a cell counter to count the amount of cells in the cell suspension, dilute to the required number of cells, and resuspend the cells to between 5 × 106 and 6 × 106 / mL;

[0057] (3) Take 100 μL of cells and place them in an EP tube or flow cytometry tube. Add the antibody according to the amount. When adding the antibody, it must be fully mixed with the cells. You can mix it slightly on a shaker. Incubate in a 4°C refrigerator for 30 min.

[0058] (4) Pipette 1.5-2 mL of buffer solution, add it to the flow tube, resuspend the cells, centrifuge at 1000 rpm for 4 min, and discard the supernatant;

[0059] (5) Resuspend the cells again and centrifuge to retain the cell pellet. Pipette 100 μL of buffer solution, add it to the flow cytometry tube, and resuspend the cells;

[0060] (6) Pipette 1.5-2 mL of buffer solution into the flow tube, carefully blow to mix thoroughly, and centrifuge at 1000 rpm for 4 minutes to remove the supernatant.

[0061] (7) Pipette 400 μL of buffer solution, add it into the flow cytometry tube, gently pipette to mix, filter the cells with a filter, and detect on the flow cytometry machine.

[0062] 1.1.5 Preparation of mulberry leaf extracts at different concentrations

[0063] Weigh 4 mg of mulberry leaf extract powder, dissolve it in 1 mL of DMSO, put it into an EP tube, seal it, and store it in a refrigerator at minus twenty degrees. According to the required drug concentration, take the mother solution and dilute it with α-MEM to the specified concentration. After dissolution, filter it with a 0.22μm disposable filter membrane for use. Finally, mulberry leaf extract solutions with concentrations of 0.01μg / mL, 0.1μg / mL, 1μg / mL, 10μg / mL, 100μg / mL, 200μg / mL, and 400μg / mL were obtained. 1.1.6CCK8 detection of cell viabilityCCK8 (Cell Counting Kit-8) is a kit based on WST-8 and widely used in rapid and highly sensitive detection of cell proliferation and cytotoxicity. It also shows the advantage of being non-cytotoxic. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the color depth is proportional to the number of cells. We use the CCK8 method to determine the concentration of mulberry leaf extract that is most suitable for cell proliferation. The specific steps are as follows:

[0064] (1) Select rBMMSCs cells that have been cultured for four generations to the logarithmic phase, remove the culture medium, digest the cells with 0.25% trypsin, and then count the cells. Inoculate 1500 cells per well in a 96-well plate with a culture medium volume of 100 μL per well;

[0065] (2) To avoid errors caused by volatilization, add PBS buffer equal to the volume of culture medium to the outermost wells of the 96-well culture plate;

[0066] (3) Place the inoculated 96-well plate in a cell culture incubator at 37°C and 5% CO2;

[0067] (4) After about 24 hours, when the cells in the 96-well plate are fully attached and the degree of confluence reaches 40-60%, the drug intervention group is replaced with mulberry leaf extract culture medium of different concentrations, and the control group is replaced with α-MEM complete culture medium; the blank control group is α-MEM complete culture medium without cells, and six parallel controls are set up for each group;

[0068] (5) The treated 96-well culture plate was placed in a cell culture incubator for 1, 3, 5, and 7 days; the culture medium was replaced every two days; after the intervention, 10 μL of CCK8 solution was added to each well, and the 96-well culture plate was gently shaken to avoid the generation of bubbles and to ensure that the CCK8 solution and cells were fully mixed and in contact;

[0069] (6) The 96-well culture plate was placed in a cell culture incubator at 37°C and 5% CO2 and incubated for 1.5 h away from light;

[0070] (7) After incubation, the cells were gently mixed on an orbital shaker for 1 min to evenly distribute the color. The absorbance of the 96-well culture plate was measured at a wavelength of 450 nm using a microplate reader, and the proliferation activity of each group of cells was calculated based on the absorbance.

[0071] 1.1.7 Reactive oxygen species

[0072] (1) rBMMSCs cultured to the logarithmic phase of P3 were selected, the culture medium was removed, and the cells were digested with 0.25% trypsin. Then, the cells were counted and diluted to a cell number of 1×10 5 Each bottle contains 4×10 5 The cells were inoculated into T25 cell culture flasks, with a cell inoculation volume of 4 mL per flask, and cultured for 24 hours until the cells were completely attached to the wall;

[0073] (2) After complete attachment, remove the old culture medium, wash the culture dish with 3 mL of PBS buffer, and then aspirate the PBS buffer. Add α-MEM complete culture medium to the control group, add α-MEM complete culture medium containing different concentrations of mulberry leaf extract (1 μg / mL, 10 μg / mL, 100 μg / mL) to the mulberry leaf extract experimental group, and add a blank group. Each group has 3 replicates. The cells that have been intervened are continued to be cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours;

[0074] (3) Collect the cell supernatant, add 2 mL of 0.25% trypsin, gently shake the culture dish to allow the trypsin to fully and evenly cover the bottom of the bottle, and digest for 1-2 minutes. After it is fully removed, immediately add the collected cell supernatant to stop digestion, and collect the cells into a 15 mL centrifuge tube;

[0075] (4) Centrifuge at 1000g for 5 min at room temperature, then discard the supernatant, collect the cells, add about 1 mL of ice-cold PBS, resuspend the cells, and transfer to a 1.5 mL centrifuge tube. Centrifuge again to precipitate the cells, carefully remove the supernatant, and gently tap the bottom of the centrifuge tube to disperse the cells to avoid cell clumping;

[0076] (5) Add 1 mL of 70% ethanol in an ice bath, gently pipette to mix, and fix at 4°C for 12-24 h;

[0077] (6) Centrifuge at 1000 g for 5 min, discard the supernatant, collect the cells, add about 1 mL of ice-cold PBS, resuspend the cells, and repeat the centrifugation twice;

[0078] (7) Prepare reactive oxygen species probe (1:1000) (8) Add 0.5 mL of probe solution to each sample and incubate at 37°C in the dark for 30 min. Mix the solution every 10 minutes to ensure sufficient reaction.

[0079] (8) After completion, centrifuge and discard the supernatant. Wash twice with PBS, add 500 μL PBS to each group for re-suspending, filter, and measure green fluorescence using a flow cytometer (BD). Plate the intervention cells as described above. Place 1×10 4 The cells were inoculated into a 30 mm cell culture dish, with a medium volume of 1 mL per well. The dye was then added as described above, and the cells were observed and photographed under a fluorescence microscope after washing.

[0080] 1.1.8. Apoptosis

[0081] Annexin is a class of calcium-dependent phospholipid-binding proteins widely distributed in the cytoplasm of eukaryotic cells, which can selectively bind to phosphatidylserine (PS). PS is mainly distributed on the inner side of the cell membrane, that is, the side adjacent to the cytoplasm. In the early stages of cell apoptosis, different types of cells will externalize PS to the cell surface. Annexin V can bind to PS on the cell surface and block the procoagulant and proinflammatory reactions of PS. Annexin V-FITC, which is an important feature of apoptosis, can be detected by labeling Annexin V with the green fluorescent probe FITC. PI can stain necrotic cells or cells that have lost their cell membrane integrity in the late stage of apoptosis, showing red fluorescence. For necrotic cells, since the integrity of the cell membrane has been lost, Annexin V-FITC enters the cytoplasm and binds to PS on the inner side of the cell membrane, thereby also making the necrotic cells show green fluorescence. Flow cytometry detection can obtain a histogram with four quadrants: the lower left, upper left, lower right, and upper right represent normal living cells, damaged cells, early apoptotic cells, late apoptotic cells, and necrotic cells, respectively.

[0082] (1) Select rBMMSCs cultured to the P3 logarithmic phase, remove the culture medium, digest the cells with 0.25% trypsin, count the cells, and dilute them to a cell count of 1×105 / mL. Inoculate 4×105 cells per dish into a 60 mm cell culture dish with a cell inoculation volume of 3 mL per dish, and culture for 24 h until the cells are completely attached to the wall;

[0083] (2) After complete attachment, remove the old culture medium and gently wash the culture dish with 3 mL of PBS buffer. Then aspirate the PBS buffer and add α-MEM complete culture medium to the control group (Control) and α-MEM complete culture medium containing mulberry leaf extract (1 μg / mL, 10 μg / mL, 100 μg / mL) to the experimental group. Set up a blank group, FITC adjustment compensation group, PI adjustment compensation group and FITC-PI control group, and repeat three times for each group. Place the cells after intervention in a cell culture incubator at 37°C and 5% CO2 for 24 hours;

[0084] (3) Collect the cell supernatant (which contains some apoptotic cells), add 2 mL of 0.25% trypsin without EDTA, gently shake the culture dish to allow the trypsin to fully and evenly cover the bottom of the bottle, and digest for 1-2 minutes. After the cells are fully detached, immediately add the collected cell supernatant to terminate the digestion, and collect the cells into a 15 mL centrifuge tube;

[0085] (4) Centrifuge at 2000 rpm for 5-10 min at room temperature, discard the supernatant, collect the cells, resuspend the cells once with pre-cooled PBS (4°C), and centrifuge at 2000 rpm for 5-10 min to wash the cells;

[0086] (5) Dilute 10× Binding Buffer to 1× Binding Buffer with deionized water, and then add 300 μL of 1× Binding Buffer to suspend the cells;

[0087] (6) Experimental group: Annexin V-FITC labeling: add 5 μL of Annexin V-FITC; no dye was added to the blank zeroing group, and Annexin V-FITC and PI were added to the Annexin V-FITC zeroing group and PI zeroing group for single staining zeroing. After mixing Annexin V-FITC in the dark, incubate at room temperature for 15 minutes; add 5 μL of PI staining 5 minutes before the machine; before the machine, add 200 μL of 1×BindingBuffer. Detect within 10 minutes;

[0088] (7) The flow cytometer was set to an excitation wavelength of 488 nm, Annexin V-FITC (green fluorescence) was detected in the FL1 channel, and PI (red fluorescence) was detected in the FL2 channel. After debugging, the staining was detected. The intervention cells were plated as described above. 1×104 cells were inoculated into a 30 mm cell culture dish per well, and the culture medium volume per well was 1 mL. Then, the dye was added and incubated as described above, and the cells were observed and photographed under a fluorescence microscope.

[0089] 1.2.9 Alkaline phosphatase staining and activity assay

[0090] 1.2.9.1. Alkaline phosphatase staining

[0091] ALP is alkaline phosphatase, a type of hydrolase, widely used in protein and nucleic acid detection. It is also commonly used for dephosphorylation (demonophosphorylation) of the 5' and 3' ends of DNA or RNA, especially dephosphorylation of the 5' end of plasmids to avoid plasmid self-ligation. BCIP / NBT is a common substrate for ALP. NBTformazan, which is catalyzed by ALP, turns dark blue to blue-purple. ALP in cells can be stained with the BCIP / NBT alkaline phosphatase colorimetric kit. (1) Select rBMMSCs cells cultured to the P3 logarithmic phase, remove the culture medium, digest the cells with 0.25% trypsin, and then count the cells and dilute them to a cell number of 1×10 4 / mL. 1×104 cells were inoculated into each well of a 12-well plate, with a cell inoculation volume of 1.5mL per well, and the cells were cultured for 24 hours until the cells were completely attached to the wall; (2) After complete attachment, the old culture medium was removed, and the wells were gently washed with 2mL PBS buffer, and the PBS buffer was aspirated. According to the negative control group (Control), α-MEM complete culture medium was added, and the OD (osteoblastic induction) group was added with osteoblastic induction medium, and the drug experimental group was added with osteoblastic induction medium containing mulberry leaf extract (1μg / mL, 10μg / mL, 100μg / mL), and 3 replicates were set up for each group; (3) The 12-well plate after intervention was placed in a cell culture incubator at 37°C and 5% CO2 for 7 and 14 days, and the corresponding culture medium was replaced every 2-3 days; (4) After the culture period is reached, remove the old culture medium, gently wash each well with PBS 2-3 times, add 1mL 4% paraformaldehyde to fix for 10-15min, and then wash with PBS 3 times after fixation, 5min each time; (5) Prepare ALP staining solution: prepare the staining working solution according to the proportion, 3mL alkaline phosphatase colorimetric buffer, 66μL / 10mL BCIP solution, and 33μL / 10mL NBT solution to prepare 10mL BCIP / NBT staining working solution; (6) After removing the PBS solution, add 1mL BCIP / NBT staining working solution to each well to cover the cell surface; (7) Incubate at room temperature and avoid light for 10min. After staining, remove the staining solution, wash with distilled water 1-2 times to terminate the color development reaction, and observe and take pictures under an optical microscope.

[0092] 1.2.9.2 Alkaline phosphatase activity assay

[0093] The CIAP / CIP method can be used to detect ALP activity in cells or tissues. Para-nitrophenylphosphate (pNPP) is a commonly used chromogenic substrate for phosphatase. Under alkaline conditions, it can generate para-nitrophenol under the action of ALP. Under alkaline conditions, it is yellow and the absorbance can be detected at 400-415nm. The darker the yellow, the higher the ALP activity, and vice versa.

[0094] 1.2.9.2.1. Sample collection

[0095] (1) Select rBMMSCs cells in the P3 logarithmic phase and inoculate them in a 12-well plate according to the method in 1.2.7.1; (2) After complete adhesion, remove the old culture medium and perform group induction intervention according to the method in 1.2.7.1, with 3 replicates in each group; (3) On days 7 and 14, replace the corresponding culture medium every 2-3 days; After reaching the culture period, remove the old culture medium, place on ice, and gently wash each well 3 times with pre-cooled PBS, each time for 5 minutes, then aspirate and place on ice; (4) Add an appropriate amount of pre-cooled RIPA (5) Use a small cell scraper to scrape the lysate thoroughly and lyse it on ice for 15 min to avoid protein loss; (6) Collect the lysate into 1 mL EP tubes and centrifuge at 4°C and 12,000 rpm for 15 min; (7) Pipette the supernatant and transfer it to another set of new EP tubes, take a small amount for BCA method to measure the protein content of the sample (try to keep the operation on ice to avoid protein decomposition), and set aside.

[0096] 1.2.9.2.2. Determination of cell protein content by BCA method

[0097] The BCA protein concentration assay kit is one of the two most commonly used protein concentration detection methods. It has the advantages of high sensitivity, a detection concentration limit of 25μg / mL, a minimum detection protein amount of 0.5μg, a minimum sample volume of 1-20μL, and a good linear relationship in the range of 50-2000μg / mL. (1) Preparation of protein standard: Take 0.8mL of protein standard preparation solution and add it to a tube of protein standard (20mgBSA). After it is fully dissolved, prepare a 25mg / mL protein standard solution. Use immediately after preparation, and the remaining can be stored at -20℃ for a long time. Take an appropriate amount of 25mg / mL protein standard and dilute it to a final concentration of 0.5mg / mL. For example, take 20μL of 25mg / mL protein standard and add 980μL of diluent to prepare a 0.5mg / mL protein standard. The diluted 0.5 mg / mL protein standard can be stored at -20°C for a long time; (2) Preparation of BCA working solution: According to the number of samples, prepare an appropriate amount of BCA working solution with BCA reagent A and BCA reagent B at a ratio of 50:1 and mix thoroughly. BCA working solution is stable within 24 hours at room temperature; (3) Determination of protein concentration: Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μL, which is equivalent to a standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Add an appropriate volume of sample to the sample wells of the 96-well plate. Add 200 μL of BCA working solution to each well and place at 37°C for 20-30 minutes. Use an enzyme reader to measure the optical density at 562 nm. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0098] 1.2.9.2.3. Determination of ALP activity

[0099] (1) Preparation of standard working solution: Take 10 μL p-nitrophenol solution (10 mM) and dilute it to 0.2 mL with detection buffer, with a final concentration of 0.5 mM; (2) Preparation of chromogenic substrate solution: Take a tube of chromogenic substrate and dissolve it in 2.5 mL of detection buffer (you can first dissolve it in 1 mL of detection buffer, and after sufficient dissolution and mixing, transfer it to a 15 mL centrifuge tube and then add 1.5 mL of detection buffer), fully dissolve and mix, and place it on ice for later use; (3) Experimental group: Add 50 μL of the sample to be tested and 50 μL of chromogenic substrate to each well; Blank control group: Add 50 μL of detection buffer and 50 μL of chromogenic substrate to each well; Standard group: Add 4, 8, 16, 24, 32 and 40 μL of standard solution to each well, and add 96, 92, 84, 76, 68 and 60 μL of detection buffer to each well, respectively. Mix thoroughly on a shaker, incubate at 37°C for 10 min, (4) add 100 μL of reaction stop solution to each well to terminate the reaction. At this point, the standard and sample wells will show different shades of yellow depending on the concentration of ALP; (5) measure the absorbance at 405 nm and calculate the ALP activity in the sample. 1.2.10 Alizarin Red Staining and Quantitative Determination Alizarin Red S (ARS) staining is a commonly used staining method. It is an anthraquinone derivative that can chelate with a variety of metal ions to form stable orange-red or dark red complexes. It is also a good metal indicator. In histology and pathology, ARS is often used to stain or locate calcium deposits in tissues because it can chelate with calcium salts in calcium carbonate or calcium phosphate to form orange-red complexes.

[0100] 1.2.10.1. Alizarin red staining

[0101] (1) Cell selection was the same as before. The culture medium was removed and the cells were digested with 0.25% trypsin. The cells were then counted and diluted to a cell number of 4 × 10 4 1×10 5The cells were inoculated into 6-well plates with a cell inoculation volume of 2.5 mL per well and cultured for 24 h to adhere to the wall; (2) after complete adhesion, the old culture medium was removed and the wells were gently washed with 2.5 mL of PBS buffer. The PBS buffer was then aspirated and α-MEM complete culture medium was added to the control group (Control), osteogenic induction culture medium was added to the ODM group, and osteogenic induction culture medium containing mulberry leaf extract (1 μg / mL, 10 μg / mL, 100 μg / mL) was added to the drug experimental group. Each group had 3 replicate wells (10, 1, and 0.1 μM). Each group had 3 replicate wells. ; (3) Place the 6-well plate after intervention in a cell culture incubator at 37°C and 5% CO2 for continued culture for 14 and 21 days, and replace the corresponding culture medium every 2-3 days; (4) After the culture period, remove the old culture medium, gently wash each well once with PBS, add 2mL 70% ethanol to fix for 20 minutes, and wash three times with PBS after fixation, each time for 5 minutes; (5) Remove PBS, add 2mL ARS staining solution to each well, evenly cover the cells, and stain at room temperature for 30 minutes; (6) Wash thoroughly with distilled water, and then observe and photograph under an optical microscope.

[0102] 1.2.10.2 Quantitative analysis of Alizarin red staining

[0103] (1) After the above-mentioned Alizarin Red staining observation and photography are completed, add 10% (w / v) cetylpyridinium chloride (CPC) solution to each well; (2) Incubate at room temperature for 30 minutes to allow the stained mineral deposits to fully dissolve; (3) Then, the mineralized matrix solution in each well is respectively pipetted into a 96-well plate and the absorbance is measured at a wavelength of 562 nm using an ELISA reader.

[0104] 1.2.11 RT-qPCR detection

[0105] Sangon Biotechnology Co., Ltd. was responsible for the design and synthesis of primers. The primer sequences are shown in Table 1 below.

[0106] Table 1 Primer sequences

[0107]

[0108] 1.2.11.1. Preparation of primer solution

[0109] 1.2.11.2. Cell sample preparation and processing

[0110] (1) Select cells that have been cultured to the logarithmic phase, remove the culture medium, digest the cells with 0.25% trypsin, and then count the cells at 5×10 per dish. 5The cells were inoculated into 100 mm cell culture dishes with a cell inoculation volume of 5 mL per dish and allowed to adhere to the wall. (2) After they were fully adhered to the wall and the degree of confluence reached 50%, the old culture medium was removed and the cells were gently washed with 3 mL of PBS buffer. Three parallel cells were set up in each group and placed in a cell culture incubator at 37°C and 5% CO2 for a further 72 h. The groups were divided into the same groups as before.

[0111] 1.2.11.3. RNA extraction, quantification, cDNA template synthesis and reverse transcription

[0112] Same as Part 2.

[0113] 1.2.12 Western blot detection

[0114] Western blot is a commonly used experimental method in molecular biology, biochemistry, etc. The basic principle is to stain samples treated with gel electrophoresis using specific antibodies. The level of protein expression can be evaluated by analyzing the location of staining.

[0115] 1.2.12.1. Cell preparation and protein sample preparation

[0116] (1) Select cells cultured to the P3 logarithmic phase, remove the culture medium, digest the cells with 0.25% trypsin, and then count the cells at 1×10 per dish. 6The cells were inoculated into 100 mm cell culture dishes with a cell inoculation volume of 5 mL per dish and cultured statically to allow them to fully adhere to the wall; (2) After they were fully adhered to the wall and the degree of fusion reached 60-70%, the old culture medium was removed and they were gently washed with 4 mL of PBS buffer. The PBS buffer was aspirated and osteogenic induction was started. Three parallel cells were set up in each group and cultured in a cell culture incubator at 37°C and 5% CO2 for 7 days. The groups were grouped the same as before; (3) The PBS buffer was pre-cooled on ice and the entire protein extraction process needed to be carried out on ice; after the cell intervention was completed, the culture dish was gently washed with the cooled PBS buffer. This step was repeated twice and finally 4 mL of pre-cooled PBS was added; (4) The cells were gently scraped off with a cell scraper and the above The cell suspension is collected in a 15 mL centrifuge tube, and 2 mL of the culture dish is added to rinse the dish. This portion of the cell suspension is also added to the 15 mL centrifuge tube. The entire process is carried out on ice. The cell suspension is centrifuged at 1000 rpm for 15 min, the supernatant is discarded, 120 μL of cell lysis buffer (RIPA: protease inhibitor: phosphatase inhibitor = 100:1:1) is added to each tube, and the mixture is fully mixed on ice. The mixture is placed on ice for lysis for 30 min, and vortexed every 10 min to ensure that the mixture is fully lysed. (5) After the lysis is completed, the cell suspension is centrifuged at 4°C and 12000 rpm for 15 min using a low-temperature high-speed centrifuge. The supernatant obtained is the cell protein extract. It is marked and stored at -80°C, or the next step is performed directly.

[0117] 1.2.12.2. Protein concentration determination by BCA method

[0118] Determine the cell protein content using the BCA method as described in 1.2.7.2.2 above.

[0119] 1.2.12.3 Protein denaturation

[0120] According to the standard curve drawn by the BCA standard, the concentration of the protein sample was calculated, and the protein sample was diluted with PBS buffer to make the concentration of the protein after denaturation uniform to 1μg / μL; the protein loading buffer was added according to the volume ratio of 4×ProteinSDS PAGE Loading Buffer (4×ProteinSDS PAGE Loading Buffer, 9173, Takara) to the volume of the protein sample at 1:3, and the two were mixed repeatedly, and then placed in an instant centrifuge for centrifugation; a dry constant temperature metal bath was used for high temperature denaturation, and the conditions were set to heat the protein sample at 100℃ for 8min. After the protein sample was labeled with information after high temperature denaturation, it can be directly loaded with electrophoresis, or stored in a -80℃ refrigerator, but care should be taken to avoid repeated freezing and thawing.

[0121] 1.2.12.4. Glue making

[0122] (1) Calculate the concentration of the gel according to the molecular weight of the antigen corresponding to the antibody to be detected, and then calculate the amount of each component of the separation gel; use distilled water to clean the glass plate, then rinse it with ddH2O, and place it on a drying rack with the side in contact with the gel tilted downward to dry; (2) Prepare the separation gel and concentrated gel in advance according to the amount (except AP and TEMED), filter the above gel solution and store it at 4°C as a storage solution away from light for 1 month; take it out in advance to balance at room temperature when it is used, otherwise the gelation process will be delayed. The heat generated will cause the gas dissolved in the storage solution at low temperature to precipitate and cause bubbles. Then add 10% APS (0.7-0.8:100, the higher the separation gel concentration, the lower the APS concentration. 15% separation gel can be used up to 0.5:100) and TEMED (0.4:1000 for separation gel, 0.3:1000 for 15%, and 0.8:1000 for concentrated gel); (3) Sealing: After filling 2 / 3 of the separation gel, seal the gel immediately. Use anhydrous ethanol for sealing. Remember not to move after sealing. Let it stand for 30 minutes. When the gel interface is obvious and completely solidified, pour out the sealing liquid, rinse it with ddH2O, and absorb the remaining water droplets with absorbent paper; (4) After the concentrated gel is completely solidified, remove the comb before loading the sample to prevent bubbles from entering the comb holes and deforming the comb holes. After pulling out the comb, rinse the gel holes with ddH2O to remove the residual glue, and then load the sample.

[0123] 1.2.12.5. Protein loading

[0124] Place the two prepared gels in the electrophoresis tank, slowly fill the tank between the two glass plates with 1×RunningBuffer, and observe for 5-10 minutes to see if there is any leakage at the edge of the glass plate. If there is leakage, reinstall the glass plate and repeat the leak test. If there is no leakage, carefully pull out the comb, clean the loading well and start loading. Use a 20μL gel electrophoresis special loading pipette to add protein samples to the loading well. In order to eliminate the influence of edge effects, concentrate the protein samples in the middle lane; at the same time, add 3μL pre-stained protein marker to the left lane of the protein sample, and add the same volume of 1×Loadingbuffer as the protein sample to the remaining lanes except the protein sample and marker. After the above operations are completed, add 1×RunningBuffer to the electrophoresis tank until the indicator scale line.

[0125] 1.2.12.6. Electrophoresis separation

[0126] Connect the electrophoresis tank, tank cover and power supply in the correct direction of the tank electrodes, and set the power supply parameters at the same time; the electrophoresis parameters selected for the concentrated gel are: 80V, 30-50min, and the specific time should be based on the time when the protein sample runs out of the concentrated gel; after the protein sample runs out of the concentrated gel, set the separation gel electrophoresis parameters: the voltage is set to 120V, and the time is temporarily set to 120min, but the specific time should be adjusted according to the molecular weight of the target protein and the corresponding position of the pre-stained protein marker.

[0127] 1.2.12.7. Rotational mold (wet method)

[0128] Put on gloves and start preparation about 20 minutes before the end of electrophoresis. (1) Soaking PVDF membrane: Spread the PVDF membrane flat on the methanol surface, absorb water naturally by capillary action, and then completely immerse it in water for 1 minute to remove bubbles, then rinse it with ddH2O for 2 minutes, and then immerse it in pre-cooled 1×Transferbuffer for 5 minutes before starting subsequent operations; at the same time, immerse the filter paper in pre-cooled 1×Transferbuffer; (2) Take out the gel: Remove the gel after electrophoresis, gently rinse the glass plate with ddH2O, gently pry up the shorter side of the glass with a gel lifter, and cut off the excess gel above and below the target protein and on both sides of the marker. You can also keep 30-100kD or a wider molecular weight range of gel (select the gel retention range according to the molecular weight of the target protein), then cut from the upper left corner, soak it slightly in pre-cooled 1×Transferbuffer, place it on a clean glass plate, and lay the membrane and two filter papers on each side in order. Be careful to use a glass rod to expel bubbles and cut off the excess parts of the filter paper and membrane; (3) Wet transfer: rinse the electrophoresis tank with deionized water and dry it, then add 1000mL of electrotransfer solution. Place the sponge, filter paper, glue, and PVDF membrane flat on the transfer rack from bottom to top, and use a glass rod to expel the bubbles between the PVDF membrane and the glue; after ensuring that there are no bubbles, place the filter paper and sponge on the PVDF membrane again, clamp the transfer clamp, and then place them in the transfer tank in the order of black to black and red to white. Place the electrophoresis tank in an ice-water mixture to ensure operation at low temperature. Set the transfer parameters: constant current 100mA overnight or 300mA for 60-90min. The transfer conditions for different proteins are different, and specific transfer parameters should be set according to the specific situation.

[0129] 1.2.12.8, Closure

[0130] Remove the PVDF membrane from the electroporation tank, rinse it with TBST with the front side facing up, and then immerse it in 5% skim milk powder blocking solution and shake it slowly for 1-2 hours. If necessary, stain it with Ponceau red to observe the protein bands, then wash off the Ponceau red with deionized water and TBST and block it.

[0131] 1.2.12.9. Membrane cutting and primary antibody incubation

[0132] After blocking, the PVDF membrane was transferred to another incubation box, and an appropriate amount of TBST working solution was added and washed on a shaker for 5 minutes, and the washing was repeated 3 times. After washing, the cutting position was determined according to the molecular weight of the target protein and the molecular weight position of the PVDF membrane marker, and the target protein was cut out in sequence. The cut protein bands were marked and transferred to an incubation box containing the primary antibody diluted with blocking solution, placed on a shaker and shaken slowly, and incubated overnight at 4°C.

[0133] 1.2.12.10. Secondary Antibody Incubation

[0134] After overnight incubation with the primary antibody, the protein bands were removed from the antibody incubation box, the primary antibody was recovered, and the protein bands were placed in another antibody incubation box. An appropriate amount of TBST working solution was added and the cells were washed on a shaker for 10 minutes. This process was repeated three times. After the washing was completed, the secondary antibody diluted with the blocking solution was added to the antibody incubation box, and the cells were placed on a horizontal shaker at room temperature and incubated for 1 hour with slow shaking.

[0135] 1.2.12.11. Protein band development

[0136] When the secondary antibody incubation is completed, remove the secondary antibody, add TBST working solution to the incubation box, and wash on a shaker for 10 minutes. This process can be repeated 3 times; the Azure500 multifunctional fluorescence imaging system needs to be opened in advance, precooled for 30 minutes, so that the CCD temperature of the imaging system reaches -20°C; prepare ECL chemiluminescence detection reagent, mix equal volumes of A solution and B solution, and prepare a luminescence detection substrate working solution (the usage amount is about 1mL per 10cm2 membrane); drip the luminescence substrate working solution onto the protein blot membrane, ensure that the working solution is evenly covered on the membrane, and place it for 1-2 minutes; place the membrane in the darkroom of the multifunctional fluorescence imaging system. This process should be completed carefully to avoid bubbles between the membranes, and develop and fix immediately. According to the results, adjust the exposure time. You can expose according to 0.5, 1, 3 and 5 minutes, and then develop and fix together to observe the results. Save and copy the above results, and then use ImageJ software for grayscale analysis.

[0137] 1.3 Statistical analysis

[0138] The data obtained from the experiment are expressed as mean ± standard deviation (mean ± SD), and the mean is the average of multiple independent repetitions. SPSS26.0 was used for statistical analysis. The data were tested for normality, and the data in this study were all in accordance with normal distribution. Then the variance homogeneity test was performed. If the variance was homogeneous, the LSD of one-way ANOVA was used for pairwise comparison; if the variance was unequal, the Kruskal-Wallis rank sum test in the nonparametric test was used for pairwise comparison of the differences between groups. GraphPad Prism9.0 software was used for graphics drawing in this study.

[0139] 2. Experimental results (such as Figure 1-Figure 6 (shown)

[0140] 2.1 Identification of rBMMSCs

[0141] In the P3 generation, the cells were elongated, and the number of spindle cells was more than that in the P1 generation. The nuclei were oval, the nucleoli were clear, and the protrusions of different lengths were extended. They adhered well to the wall and appeared in piles. After 21 days of osteogenic differentiation, the formation of calcium nodules was observed by using Alizarin red staining. Figure 1 A in Figure 1. After 21 days of adipogenic differentiation, a large number of lipid droplets were observed in the cells using Oil Red O staining. After 21 days of chondrogenic differentiation, chondrocytes were formed. After embedding and staining with Alcian blue, the cartilage in the chondrocytes was blue. Flow cytometry was used to detect the surface antigens of P3 bone marrow mesenchymal stem cells. The results are shown in Figure 1. Figure 2 As shown in the figure, the positive rates of CD90, CD44, CD34, and CD11 are 98.2%, 93.3%, 2.6%, and 4.4%, respectively. It can be seen that CD44 and CD90 are positive, and CD34 and CD11 are negative, thus distinguishing them from hematopoietic stem cells. Figure 1 .

[0142] 2.2. CCK8 detection of cell viability

[0143] rBMMSCs cells at various drug concentrations were cultured in 96-well culture medium for 1, 3, 5 and 7 days. The results show that there was no difference in cell activity between the groups of cells on the first day, but starting from the third day. The cell activity of the three concentrations of 1μg / mL, 10μg / mL and 100μg / mL increased, and the difference was statistically significant compared with the blank control group. In the drug concentration range of 1μg / mL-100μg / mL, the cell activity increased with the drug concentration. After the drug concentration exceeded 100μg / mL, the cell viability decreased, but there was no difference from the blank control group, and no obvious cytotoxic effect was shown, which can prove that mulberry leaf extract has good biosafety and a wide range of drug concentration selection. Figure 2 Since the concentration range of 1μg / mL-100μg / mL promoted cell proliferation, these three concentrations were selected for further screening in subsequent experiments.

[0144] 2.3 Effects of different concentrations of mulberry leaf extract on apoptosis and reactive oxygen species production in rBMMSCs

[0145] In conventional culture without osteogenic induction, we can see that mulberry leaf extract has the strongest effect on promoting cell proliferation at a concentration of 100μg / mL. However, the osteogenic induction solution contains dexamethasone, which may cause cell apoptosis and increased reactive oxygen species, and some substances in the extract may interact with the components in the osteogenic induction solution to cause cell apoptosis. Therefore, it is necessary to verify whether mulberry leaf extract can produce cytotoxicity to cells during osteogenic induction. Flow cytometry showed that compared with the blank group and the osteogenic induction group, the mulberry leaf water extract (1μg / mL, 10μg / mL, 100μg / mL) group had no significant effect on the apoptosis of rBMMSCs cells. The first quadrant is composed of late apoptotic cells. The second quadrant refers to PI-negative cells, and the third quadrant refers to normal living cells that have not been stained. The fourth quadrant refers to early apoptotic cells. Most of the cells in all groups are located in the third quadrant, and there is no significant difference between the groups. The apoptosis rate is (15.15±2.96)%, which is within the normal range. Figure 3 A in the figure proves that mulberry leaf water extract has no obvious cytotoxicity to cells. The flow cytometry results show that compared with the control group without drug addition and osteogenic induction, neither osteogenic induction nor different concentrations of drugs will cause rBMMSCs to produce more ROS, and there is no significant difference between the intervention group and the control group (P>0.05). Figure 3 B and C in the figure. Fluorescence staining also shows that the cells in each group have normal morphology, are spindle-shaped, and are in close contact with each other. No obvious green fluorescence is observed under the fluorescence microscope, which proves that the cell activity is normal. The osteogenic induction solution and drugs of different concentrations did not cause obvious oxidative stress damage to the cells. Figure 3 D. It was proved that different concentrations of mulberry leaf extract did not produce obvious cytotoxicity to cells during osteogenic induction. Further experiments on subsequent osteogenesis can be carried out.

[0146] 2.4 Effects of different concentrations of mulberry leaf extract on osteogenic differentiation of rBMMSCs

[0147] Bone marrow mesenchymal stem cells are a type of stem cell with multiple potentials. They originate from the bone marrow and play a key role in the development of bones. They can be used for cell therapy and tissue engineering research. This experiment verified that mulberry leaf extract can promote the osteogenesis of rBMMSCs. ALP is one of the early markers of osteogenic differentiation. Compared with the blank control group, after osteogenic induction, the color of alkaline phosphatase staining in each group was darker than that in the control group, and the ALP activity was significantly increased. Compared with the OD group, the alkaline phosphatase staining of 10μg / mL and 100μg / mL mulberry leaf extracts was darker, and the ALP activity was significantly increased. Among them, the 100μg / mL mulberry leaf extract group had the most significant effect, proving that mulberry leaf extract can promote the osteogenic differentiation of rBMMSCs. The ARS results showed that compared with the Blank group, a certain number of calcium nodules were observed in the rBMMSCs of the other groups after 14 and 21 days of osteogenic induction in the osteogenic induction medium. The number of calcium nodules increased with the increase of time and drug concentration. The same trend was also observed in the ARS quantitative experiments at 14 and 21 days, which fully proved that mulberry leaf extract can promote the differentiation and mineralization ability of rBMMSCs, among which the 100 μg / mL mulberry leaf extract group had the best effect (P<0.001).

[0148] 2.5 Effects of different concentrations of mulberry leaf extract on the expression of osteogenic-related genes in rBMMSCs

[0149] We then used RT-qPCR to detect the gene expression of osteoblast differentiation markers ALP, Ocn, Runx2, Osterix, Bmp2 and Col-1, and the results were consistent with the conclusions of alkaline phosphatase and alizarin red staining. Compared with the Blank group, the expression of osteoblast-related genes in the other groups was significantly increased, among which the expression of osteoblast genes in the 10μg / mL and 100μg / mL groups increased significantly compared with the OD group, and the expression of histones in the 100μg / mL group increased most significantly.

[0150] 2.6 Effects of different concentrations of mulberry leaf extract on the expression of osteogenic-related proteins in rBMMSCs

[0151] Western blot also further confirmed the effect of different concentrations of mulberry leaf extract on the expression of osteoblast-related proteins in rBMMSCs. After cell intervention, total protein was separated and the expression of osteoblast differentiation markers ALP, Ocn, Runx2, Osterix, Bmp2 and Col-1 was analyzed. Compared with the Blank group, the osteoblast-related proteins in the other groups increased significantly. Among them, the expression of osteoblast proteins in the 10μg / mL and 100μg / mL groups increased significantly compared with the OD group, and the protein expression in the 100μg / mL group increased most significantly. In the ALP and Alizarin Red staining experiments, mulberry leaf extract can promote the osteogenic differentiation and mineral deposition of rBMMSCs, and the subsequent expression of osteoblast-related genes and proteins also demonstrated this point. The main principle is that flavonoids can activate the Wnt / β-catenin signaling pathway, which leads to the phosphorylation of glycogen synthase kinase 3β (GSK-3β), which then induces the nuclear accumulation of β-catenin, leading to the transcriptional activation of Wnt target genes, thereby promoting osteogenesis. Isoflavones, as isomers of flavonoids, are aromatic oxygen-containing heterocyclic compounds with a similar structure to estrogen receptor (ER). In vitro experiments have found that chickpea sprout isoflavones can increase alkaline ALP activity and promote calcium deposition, upregulate the expression of Runx2, Ocn and Col-1, and stimulate osteoblast differentiation. This differentiation is inhibited after the addition of estrogen receptor inhibitors, indicating that chickpea sprout isoflavones can promote differentiation through ER-related pathways. In addition, isoflavone compounds can also promote osteogenic differentiation through other signaling pathways. Studies have found that biochanin A can promote the phosphorylation level of p38 and activate the p38 / mitogen-activated protein kinase (MAPK) signaling pathway, which is believed to be related to the regulation of osteoblast differentiation, matrix deposition and mineralization. Hyperoside can activate transforming growth factor-β (TGF-β) and BMP-2, and the expression and phosphorylation levels of TGF-β substrates Smad2 and Smad3 are also increased, stimulating osteogenic differentiation through the TGF-β pathway; isocarpone glycosides can promote the proliferation of bone marrow mesenchymal stem cells in vitro, increase the expression of ALP, regulate the osteogenic differentiation of cells, and increase bone formation and increase the expression level of BMP-2 in vivo; icariin can downregulate the expression of connective tissue growth factor (CTGF), thereby enhancing BMP-2-induced osteoblast differentiation. BMP-2 may become a key protein in studying the effects of flavonols on osteogenic differentiation.Other flavonoids, such as the dihydroflavonoid compound psoralen, can increase the expression of ALP in bone marrow mesenchymal stem cells and promote osteogenic differentiation by regulating the cyclic adenosine monophosphate (cAMP) / protein kinase A (PKA) / cAMP response element binding protein (CREB) signaling pathway. All of the above types of flavonoids can be found in mulberry leaf extract, so mulberry leaf extract has the effect of promoting osteogenesis and is a potential drug for the treatment of periodontitis.

[0152] In summary, the above experiments confirmed that mulberry leaf extract can promote rBMMSCs proliferation and osteogenic differentiation, and the expression of osteogenic-related genes and proteins are upregulated, which has potential tissue repair ability. The above experiments of the present invention separated and identified rat BMMSCs cells, and screened out the optimal concentration of mulberry leaf extract to promote rBMMSCs proliferation and osteogenic differentiation. The present invention provides further theoretical basis for the treatment of periodontitis with mulberry leaf extract.

[0153] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that: The following steps are involved: S1. Preparation of rat bone marrow mesenchymal stem cells: The rat bone marrow mesenchymal stem cells were isolated and cultured by the whole bone marrow cell suspension adherence method. The isolated bone marrow cell suspension was subjected to primary culture, and the culture flask was moved to a 37°C, 5% CO2 constant temperature incubator. When the cell growth density reached 80%, the cells were subcultured, and trypsin (0.25%) was used for digestion. The cells were cultured in 1:2 iterative manner, and one iteration was started every 2-4 days. S2. Osteogenesis Induction: Select the 3rd-4th generation rat bone marrow mesenchymal stem cells iteratively cultured in step S1 and adjust the cell concentration to 2×10 4 / mL, inoculate and culture, culture at 37°C, 5% CO2 environment until the cell density reaches 60-70%, discard the supernatant, and add osteogenic differentiation medium; S3. Cell differentiation induction: Replace osteogenic induction medium every 2-3 days, continue culturing at 37°C, 5% CO2 for 14-21 days, and observe cell morphology changes and detect osteogenic differentiation of rat bone marrow mesenchymal stem cells; Among them, mulberry leaf extract is added to the osteogenic induction medium.

2. The method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 1, characterized in that: The added amount of the mulberry leaf extract is 10 mg / L-100 mg / L.

3. The method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 2, characterized in that: The added amount of the mulberry leaf extract is 10 mg / L.

4. The method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 2, characterized in that: The added amount of the mulberry leaf extract is 100 mg / L.

5. Application of mulberry leaf extract in osteogenic differentiation of bone marrow mesenchymal stem cells.

6. The method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 5, characterized in that: The method is used in preparing medicine for treating periodontitis.

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