Method for inducing osteoblasts by using mesenchymal stem cells

Abstract

The invention discloses a method for inducing osteoblasts by using mesenchymal stem cells, and belongs to the technical field of biology. The method for inducing the osteoblasts comprises the step of adding brucella outer membrane protein into a conventional osteoblast induction culture medium to promote the dental pulp stem cells to be differentiated into the osteoblasts. According to osteoblast differentiation metaphase differentiation marker detection, the cell ALP activity of a group added with 800ng / mL of brucella outer membrane protein is 2.36 times that of a conventional osteoblast induction culture medium group; according to osteoblast differentiation end-stage differentiation marker detection, the calcium deposition amount of brucella outer membrane proteome cells added with 800ng / mL is 481.54 mu g / mg protein, and is 2.55 times of that of a conventional osteoblast induction culture medium group. By adding the brucella outer membrane protein into a conventional osteoblast induction culture medium, the osteogenic differentiation efficiency of the dental pulp stem cells can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biology, and in particular relates to a method for inducing osteoblasts from mesenchymal stem cells. Background Art

[0002] Osteoporosis is a systemic bone disease characterized by decreased bone mass and increased bone brittleness due to destruction of bone microstructure. Osteoporosis, in turn, leads to potential fracture risk, resulting in reduced quality of life and loss of autonomy in severe cases. It is a common health problem among middle-aged and elderly people, especially postmenopausal women, but it can also occur in other groups. With the trend of aging, the number of osteoporosis patients has increased year by year. The prevalence rate in women is significantly higher than that in men. The decline in estrogen in postmenopausal women accelerates bone loss, making them a high-risk group for osteoporosis.

[0003] Stem cells have become an important breakthrough in the medical field due to their unique self-renewal and multidirectional differentiation capabilities. Multipotent stem cells and dental pulp mesenchymal stem cells can play a promoting role in bone damage repair. At present, inducing stem cells into osteoblasts (osteoblastic differentiation) is a new research direction in regenerative medicine and bone tissue engineering, which is mainly achieved by simulating the microenvironment of bone formation in the body. However, the efficiency of inducing osteoblast differentiation of stem cells by existing technologies is low, and the degree of bone cell differentiation is insufficient, which cannot meet the needs of osteoporosis treatment.

[0004] Studies have shown that Brucella infection in animals can cause bone hyperplasia and bone bridge formation at the vertebral margins. The infection leads to the release of inflammatory mediators IL-1 and TNF-α, which stimulates osteoblast activation and forms reactive new bone. Using Brucella outer membrane protein to stimulate dental pulp mesenchymal stem cells to induce osteogenic differentiation and obtain highly differentiated osteoblasts is expected to supplement osteoblasts for osteoporosis patients, increase bone density, and improve osteoporosis symptoms. Summary of the invention

[0005] The purpose of the present invention is to provide a method for inducing mesenchymal stem cells into osteoblasts, thereby inducing the differentiation of dental pulp mesenchymal stem cells into osteoblasts.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Firstly, the present invention provides a method for inducing osteoblasts from mesenchymal stem cells. The method for inducing osteoblasts comprises adding Brucella outer membrane protein to a conventional osteoblast induction medium to promote the osteogenic differentiation of dental pulp stem cells.

[0008] Furthermore, the method for inducing osteoblasts comprises the following steps:

[0009] (1) Expression and purification of Brucella outer membrane protein;

[0010] (2) Prepare an osteoblast induction medium containing Brucella outer membrane protein;

[0011] (3) Isolate and culture dental pulp stem cells, with the passage number not exceeding 5 generations;

[0012] (4) The cell passage density is 5×103 / mL. Discard the original medium after culturing for 24 h, and replace it with the osteoblast induction medium. Change the medium once every 3 days and continuously culture for 21 days to harvest osteoblasts.

[0013] Further, the formulation of the osteoblast induction medium is as follows: Add 100 nM of dexamethasone, 10 mM of β-glycerophosphate, 50 μg / mL of vitamin C, 100 ng / mL of bone morphogenetic protein BMP-2 / 4 / 7, 100 ng / mL of TGF-β, and 800 ng / mL of Brucella outer membrane protein to the mesenchymal stem cell serum-free medium.

[0014] Further, the preparation method of the Brucella outer membrane protein includes the following steps:

[0015] (1) Inoculate E.coli BL21 / pET28a-OMP into LB culture medium containing 50 μg / mL of kanamycin and culture it with shaking at 37 °C until the OD600nm is 0.6 - 0.8;

[0016] (2) Add α-lactose to a final concentration of 0.5 mM / L and culture at 37 °C for 6 h;

[0017] (3) Centrifuge to collect the bacterial cells, add PBS with a volume 10 times the weight of the bacterial cells to suspend the bacterial cells, and disrupt them by ultrasonic wave; Centrifuge to collect the supernatant protein solution;

[0018] (4) Purify the recombinant Brucella outer membrane protein with Ni column;

[0019] (5) Dialyze with a dialysis bag with a cut-off molecular weight of 10 kD at 4 °C for 16 hours, filter and sterilize through a 0.22 μm filter, and store at 4 °C for later use.

[0020] Secondly, the present invention provides an osteoblast induction medium, and the formulation of the medium is as follows: Add 100 nM of dexamethasone, 10 mM of β-glycerophosphate, 50 μg / mL of vitamin C, 100 ng / mL of bone morphogenetic protein BMP-2 / 4 / 7, 100 ng / mL of TGF-β, and 800 ng / mL of Brucella outer membrane protein to the mesenchymal stem cell serum-free medium.

[0021] The present invention also provides a biological preparation for promoting osteoblast differentiation, and the biological preparation is Brucella outer membrane protein.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention expresses and purifies Brucella outer membrane protein by genetic engineering means as a biological agent for promoting osteoblast differentiation, and adds Brucella outer membrane protein to a conventional osteoblast induction medium to promote the differentiation of dental pulp stem cells into osteoblasts. For the detection of differentiation markers in the middle stage of osteoblast differentiation, the ALP activity of cells in the group added with 800 ng / mL Brucella outer membrane protein was 2.36 times that of the group with the conventional osteoblast induction medium; for the detection of differentiation markers in the final stage of osteoblast differentiation, the calcium deposition amount of cells in the group added with 800 ng / mL Brucella outer membrane protein was 481.54 μg / mg protein, which was 2.55 times that of the group with the conventional osteoblast induction medium. The Brucella outer membrane protein expressed and purified by the present invention can be used as a biological agent for promoting osteoblast differentiation, and adding Brucella outer membrane protein to a conventional osteoblast induction medium can effectively improve the osteogenic differentiation efficiency of dental pulp stem cells. Obtaining highly differentiated osteoblasts is expected to supplement osteoblasts for osteoporosis patients, increase bone density, and improve osteoporosis symptoms. Description of the Drawings

[0024] Figure 1 is the electrophoresis detection result of purified Brucella outer membrane protein;

[0025] Figure 2 is the detection result of the ALP activity of cells, which is a differentiation marker in the middle stage of osteoblast differentiation;

[0026] Figure 3 is the detection result of the calcium deposition amount of cells, which is a differentiation marker in the final stage of osteoblast differentiation. Detailed Embodiments

[0027] The following is a more detailed description of the present invention, which is elaborated through examples. It should be clear that these examples are only examples of the present invention, and their purpose is to illustrate the principles and functions of the present invention, rather than limiting the protection scope of the present invention.

[0028] Example 1 Expression and Purification of Brucella Outer Membrane Protein

[0029] (1) Construction of the expression plasmid According to the amino acid sequence AAB36693.1 (SEQ ID NO.1) of Brucella outer membrane protein OMP (outer membrane protein) in GenBank, the DNA sequence of the tandem multi-epitope protein was optimized for expression with E. coli-preferred codons, and SEQ ID NO.2 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned between the BamHI and XhoI sites of the pET28a plasmid, named pET28a-OMP.

[0030] (2) Construction of recombinant expression strain: The pET28a-OMP plasmid was transformed into competent cells of Escherichia coli BL21. Single colonies were picked and identified as positive transformants by PCR, named E. coli BL21 / pET28a-OMP. A single colony was cultured in 5 mL of LB medium containing 50 μg / mL kanamycin sulfate at 37 °C and 250 r / min on a shaker for 12 h as the seed culture.

[0031] (3) Expression of target protein: A single colony of E. coli BL21 / pET28a-OMP was picked and inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37 °C with shaking until the OD600nm reached 0.6 - 0.8. 100 mM / L of α-lactose was added to a final concentration of 0.5 mM / L, and the culture was continued at 37 °C for another 6 h. After that, it was centrifuged at 12000 r / min for 10 min to collect the bacterial cells. The bacterial cells were resuspended with 10 times the volume of PBS by weight, and disrupted by ultrasonic waves. Then it was centrifuged at 12000 r / min at 4 °C for 15 min, and the supernatant was collected.

[0032] (4) Purification of recombinant protein: Before loading the sample, the nickel column was equilibrated with 10 column volumes of Binding Buffer at a flow rate of 5 mL / min. After the protein solution was filtered through a 0.22 μm filter, it was loaded onto the equilibrated Ni column through a constant flow pump at a flow rate of 1 mL / min, and the flow-through was collected. The sample could be loaded repeatedly to improve the column binding efficiency of the sample. The Ni column bound with the target protein was equilibrated again with 10 column volumes of Binding Buffer at a flow rate of 5 mL / min to remove the unbound protein. The Ni column was eluted with an equal volume mixture of Elution Buffer and Binding Buffer at a flow rate of 5 mL / min, and the OD280 value was measured. The eluate corresponding to the protein absorption peak was collected. An appropriate amount of the eluate was subjected to SDS-PAGE electrophoresis detection. The size of the protein band was about 25 kD, and the electrophoresis result was as Figure 1 .

[0033] (5) Dialysis desalting of recombinant protein: The protein solution purified by the Ni column was loaded into a dialysis bag with a molecular weight cut-off of 10 kD. The dialysis bag was immersed in 10 times the volume of PBS solution and dialyzed at 4 °C for 16 h. The protein solution in the dialysis bag was collected, filtered through a 0.22 μm filter to sterilize, the protein concentration was measured, and it was stored at 4 °C for standby.

[0034] Example 2: Preparation of osteoblast induction medium

[0035] Osteoblast induction basal medium: Add 100 nM dexamethasone, 10 mM β-glycerophosphate, 50 μg / mL vitamin C, 100 ng / mL bone morphogenetic protein BMP-2 / 4 / 7, and 100 ng / mL TGF-β to the mesenchymal stem cell serum-free medium.

[0036] Modified osteoblast induction medium: Add Brucella outer membrane protein at different concentrations to the osteoblast induction basal medium.

[0037] Example 3 Isolation and culture of dental pulp stem cells

[0038] (1) Immerse the teeth with intact tooth surface and no caries collected from the hospital in PBS containing penicillin-streptomycin double antibody, wash repeatedly, and remove the soft tissues such as periodontal membrane attached to the tooth root and neck.

[0039] (2) Use a dental chisel to split the tooth longitudinally along the tooth axis, and use sterile forceps to take out the dental pulp tissue and place it in a sterile EP tube.

[0040] (3) After rinsing 3 times with PBS containing penicillin-streptomycin double antibody, add 500 μl PBS and cut the dental pulp tissue with scissors. Put the EP tube into a centrifuge and centrifuge at 1000 rpm / min for 5 minutes. Retain the precipitate and remove the supernatant.

[0041] (4) Add type I collagenase and place it in a carbon dioxide incubator at 37 °C for 30 min.

[0042] (5) Add an appropriate amount of mesenchymal stem cell culture medium to terminate digestion, centrifuge at 1000 rpm / min for 10 min, discard the supernatant, evenly spread the tissue mass on the bottom of the cell culture dish, add mesenchymal stem cell culture medium, and culture it in a carbon dioxide incubator with a CO2 concentration of 5%. Replace the medium every 2 - 3 days.

[0043] (6) Adjust the cell concentration of the first-generation human dental pulp stem cells to 10 - 20 cells / mL using the effective dilution method, and then inoculate the cells into a 96-well plate, 100 μl per well.

[0044] (7) After culturing until cell clones appear, use trypsin to digest the cells for passage, and then human dental pulp stem cells can be obtained. After detection and confirmation, they are used for subsequent experiments. The stem cells used in this study are the 3rd to 5th generations.

[0045] Example 4 Osteogenic induction culture of dental pulp stem cells

[0046] Passage the dental pulp stem cells into a 6-well cell culture plate, and adjust the cell density to 5×10 using the mesenchymal stem cell serum-free medium 3Cultured in a carbon dioxide incubator at 37 °C, 5% CO2 concentration for 24 h. For groups 1-5, discard the original medium and replace it with osteoblast induction basal medium, and add 0 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 800 ng / mL, and 1600 ng / mL Brucella outer membrane protein respectively; group 6 is the non-induced cell control, and the fresh mesenchymal stem cell serum-free medium is replaced. Change the medium every 3 days. Each group has 6 replicates.

[0047] Example 5 Detection of differentiation markers in the middle stage of osteoblast differentiation

[0048] Alkaline phosphatase (ALP) is a marker enzyme in the early stage of osteogenic differentiation, participates in the initial process of calcification, and its activity reaches a peak on the 7th - 14th day of differentiation.

[0049] (1) Cell treatment: For 3 wells of cells induced osteogenically on the 7th day in Example 4, aspirate the medium and gently rinse twice with PBS. Add lysis buffer (RIPA buffer + 1% protease inhibitor), and lyse on ice for 30 minutes. Scrape the cells with a cell scraper, centrifuge at 12000 rpm, 4 °C for 10 minutes, and take the supernatant of the cell lysate as the crude ALP enzyme solution.

[0050] (2) Protein concentration determination: Determine the protein concentration of the supernatant of the ALP cell lysate according to the Bradford method, which is used to standardize the ALP activity (the final result is expressed as ALP activity / total protein).

[0051] (3) Detection by pNPP colorimetric method

[0052] Prepare the substrate solution: Dissolve 1 mg / mL of p-nitrophenyl phosphate disodium (pNPP) in ALP buffer (0.1 M carbonate buffer, pH 9.8, containing 2 mM MgCl2).

[0053] Detection steps: Take 50 μL of the supernatant of the cell lysate + 50 μL of the pNPP substrate solution (96-well plate). Incubate at 37 °C in the dark for 30 minutes. Add 50 μL of 3 M NaOH to terminate the reaction. Detect the absorbance at 405 nm (OD value) with an enzyme-labeled instrument.

[0054] Standard curve: Gradient dilute p-nitrophenol (pNP) (0 - 100 μM) to establish a standard curve to calculate the ALP activity (unit: μM pNP / min / mg protein).

[0055] From Figure 2It can be seen that the ALP activity of the control group cells that were not induced into osteogenic differentiation was only 14.42 μM pNP / min / mg; the ALP activity of the cells in the conventional osteoblast induction medium group without adding Brucella outer membrane protein (0 ng / mL) was 113.61 μM pNP / min / mg; in the conventional osteoblast induction medium supplemented with 100 - 800 ng / mL Brucella outer membrane protein, the ALP activity of the induced group cells showed an obvious increasing trend with the increase of the concentration of Brucella outer membrane protein, reaching as high as 268.87 μM pNP / min / mg, which was 2.36 times that of the conventional osteoblast induction medium group. There was no significant difference in the ALP activity between the 800 ng / mL and 1600 ng / mL Brucella outer membrane protein groups. Therefore, adding 800 ng / mL Brucella outer membrane protein to the conventional osteoblast induction medium can achieve the best effect of inducing osteogenic differentiation.

[0056] Example 6 Detection of terminal differentiation markers of osteoblast differentiation

[0057] The alizarin red S staining experiment is used to detect calcium deposition (formation of calcium nodules) after the osteogenic differentiation of stem cells, and it is a classic method for evaluating the function at the terminal stage of osteogenic differentiation.

[0058] (1) Alizarin red S staining solution (2%, pH 4.1 - 4.3) Dissolve 2 g of alizarin red S powder in 100 mL of distilled water, and adjust the pH to 4.2 with NaOH / HCl (an acidic environment enhances staining specificity). Filter with a 0.45 nm filter and store in the dark (it can be stored at 4°C for 1 month).

[0059] (2) Cell fixation For 3 wells of the cells on the 21st day of osteogenic induction in Example 4, aspirate the culture medium, and gently rinse with PBS twice (avoid washing away the calcium nodules). Add 4% paraformaldehyde and fix at room temperature for 15 - 30 minutes. Rinse with PBS three times, 5 minutes each time.

[0060] (3) Alizarin red staining Add 2% alizarin red S staining solution (just cover the cell layer), and incubate at room temperature in the dark for 20 - 30 minutes (too long time is prone to background staining). Gently rinse with PBS until there is no red dye residue (avoid over - rinsing and dissolving the nodules). Observe under the microscope: The calcium nodules are orange - red to dark red.

[0061] (4) Quantitative analysis of calcium deposition by acetic acid dissolution method: Add 10% acetic acid (1 mL / well, 6-well plate), incubate at room temperature for 30 minutes, and gently shake to dissolve the calcium nodules. Scrape the cells with a cell scraper and transfer them to a 1.5 mL centrifuge tube, boil in a water bath for 10 minutes, and centrifuge (12,000 rpm, 10 minutes). Take the supernatant, neutralize it to pH 7.0 with NaOH (to avoid interference of acetic acid in detection). Measure the total protein concentration by BCA method. Detect the absorbance at 405 nm (OD value) with an enzyme-labeled instrument, and compare with the standard curve. The standard curve is drawn by gradient dilution of calcium chloride standard.

[0062] Under an inverted microscope, there was almost no red staining in the control group of cells without osteogenic differentiation induction, and orange-red nodules of varying degrees were observed in each group of cells induced for osteogenic differentiation. The group supplemented with Brucella outer membrane proteins had significantly denser nodule plaques and a redder color than the conventional osteoblast induction medium.

[0063] It can be seen from Figure 3 that in the quantitative analysis test of calcium deposition by acetic acid dissolution method, the calcium deposition amount of the control group of cells without osteogenic differentiation induction was only 3.46 μg / mg protein; the calcium deposition amount of the cells in the conventional osteoblast induction medium group without adding Brucella outer membrane protein (0 ng / mL) was 188.55 μg / mg protein; in the conventional osteoblast induction medium supplemented with 100 - 800 ng / mL Brucella outer membrane protein, the calcium deposition amount of the induced cells showed an obvious increasing trend with the increase of the concentration of Brucella outer membrane protein. The calcium deposition amount in the 1600 ng / mL group was 477.88 μg / mg protein, and the calcium deposition amount in the 800 ng / mL group was 481.54 μg / mg protein, which was 2.55 times that of the conventional osteoblast induction medium group. Therefore, adding 800 ng / mL Brucella outer membrane protein to the conventional osteoblast induction medium can achieve the best effect of inducing osteogenic differentiation.

[0064] In summary, adding Brucella outer membrane protein to the conventional osteoblast induction medium can effectively improve the osteogenic differentiation efficiency of dental pulp stem cells.

Claims

1. A method for inducing osteoblasts from mesenchymal stem cells, characterized in that: The method for inducing osteoblasts is to add Brucella outer membrane protein to a conventional osteoblast induction medium to promote the osteogenic differentiation of dental pulp stem cells.

2. The method according to claim 1, characterized in that The method for inducing osteoblasts comprises the following steps: (1) Expression and purification of Brucella outer membrane protein; (2) preparing an osteoblast induction medium containing Brucella outer membrane protein; (3) Isolation and culture of dental pulp stem cells, with the number of passages not exceeding 5; (4) The cell subculture density is 5×10 3 / mL, culture for 24 hours, discard the original culture medium, replace it with osteoblast induction medium, change the medium every 3 days, culture continuously for 21 days, and then harvest the osteoblasts.

3. The osteoblast induction medium according to claim 2, characterized in that The formula of the culture medium is: 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, 50 μg / mL vitamin C, 100 ng / mL bone morphogenetic protein BMP-2 / 4 / 7, 100 ng / mL TGF-β, and 800 ng / mL Brucella outer membrane protein are added to the mesenchymal stem cell serum-free culture medium.

4. Brucella outer membrane protein according to claim 3, characterized in that, The preparation method of the Brucella outer membrane protein comprises the following steps: (1) E. coli BL21 / pET28a-OMP was inoculated into LB medium supplemented with 50 μg / mL kanamycin and cultured at 37°C with shaking until the OD600nm reached 0.6-0.8; (2) Add α-lactose to a final concentration of 0.5 mM / L and culture at 37°C for 6 h; (3) Collect the cells by centrifugation, add PBS (10 times the cell weight) to suspend the cells, and disrupt them by ultrasonic wave; collect the supernatant protein solution by centrifugation; (4) Ni column purification of recombinant Brucella outer membrane protein; (5) Dialyze the solution using a dialysis bag with a molecular weight cutoff of 10 kD at 4°C for 16 h, filter and sterilize using a 0.22 μm filter, and store at 4°C until use.

5. An osteoblast induction medium, characterized in that The formula of the culture medium is: 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, 50 μg / mL vitamin C, 100 ng / mL bone morphogenetic protein BMP-2 / 4 / 7, 100 ng / mL TGF-β, and 800 ng / mL Brucella outer membrane protein are added to the mesenchymal stem cell serum-free culture medium.

6. A biological agent for promoting osteoblast differentiation, characterized in that: The biological agent is Brucella outer membrane protein.

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