Application of pilose antler bud cell secretions in preparation of anti-osteoporosis drugs

Abstract

The invention belongs to the technical field of biotechnology and tissue engineering, and particularly relates to application of pilose antler bud cell secretions in preparation of anti-osteoporosis drugs. The pilose antler bud cell secreta is obtained by adopting gradient centrifugation, and cell experiments show that the pilose antler bud cell secreta provided by the invention has the effect of promoting osteogenic differentiation of cells; animal experiment results show that the cornu cervi pantotrichum bud cell secreta provided by the invention can significantly improve the bone mineral density, bone volume fraction, bone trabecula number, bone trabecula thickness, bone trabecula separation degree and cortical bone mineral density of mice, increase the bone trabecula area of primary osteogenesis and secondary osteogenesis centers of the mice, improve osteoporosis symptoms of the mice, and improve osteoporosis of the mice. The effect is obviously better than that of bone marrow mesenchymal stem cell secretions, part of indexes are equivalent to the treatment effect of diphosphates, and the antler bud cell secretions provided by the invention can be used for preparing medicines for preventing, relieving or treating osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biotechnology and tissue engineering, and specifically relates to the application of antler blastema progenitor cell secretions in the preparation of anti-osteoporosis drugs. Background Art

[0002] Osteoporosis is a bone disease mainly characterized by thinning of bones, reduced bone density, and susceptibility to fractures. Currently, the treatment methods for osteoporosis mainly include drug therapy (such as bisphosphonates, estrogen replacement therapy), physical therapy, and surgical treatment. However, these methods have problems such as strong side effects, unstable efficacy, and decreased tolerance after long-term use. Therefore, there is an urgent need for a safer and more efficient treatment strategy.

[0003] In recent years, natural drugs and bioactive substances have shown great potential in the treatment of osteoporosis. For example, plant extracts such as soy isoflavones and icariin can promote osteogenesis due to their estrogen-like effects. Bone Marrow Stem Cells (BMSCs) can promote bone regeneration by differentiating into osteoblasts or through paracrine effects. As a traditional Chinese medicine, antler has significant anti-inflammatory and tissue repair-promoting effects. The inventors' previous research found that transplanting Antler Blastema Progenitor Cells (ABPCs) into the femoral condyle defect site of rabbits can successfully achieve bone defect regeneration, and the effect is much stronger than that of Bone Marrow Stem Cells (BMSCs). Therefore, developing a product prepared from antler blastema progenitor cells is of great significance for the development of drugs for treating bone diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of antler blastema progenitor cell secretions in the preparation of anti-osteoporosis drugs to solve the problems existing in the above-mentioned prior art. The antler blastema progenitor cell secretions prepared by the present invention can effectively increase the bone density, bone volume fraction, trabecular bone number, trabecular bone thickness, trabecular bone separation, and cortical bone density of mice, improve the symptoms of osteoporosis, and have a more significant improvement effect on osteoporosis than the secretions of bone marrow mesenchymal stem cells. The antler blastema progenitor cell secretions provided by the present invention can be used to prepare drugs for preventing, alleviating, or treating osteoporosis.

[0005] The first aspect of the present invention provides an application of antler blastema progenitor cell secretions in the preparation of anti-osteoporosis drugs. The preparation method of the antler blastema progenitor cell secretions is as follows: After inoculating antler bud cells in a serum-containing medium for culture, the medium is replaced with a serum replacement medium for incubation. The incubated culture solution is collected. After gradient centrifugation of the incubated culture solution, the centrifugal supernatant obtained is the secretion of antler bud cells; The conditions for the gradient centrifugation are as follows: The cell supernatant is centrifuged at 150 g to 500 g for 10 min to 30 min, and then the centrifugal supernatant is collected and centrifuged at 1500 g to 3000 g for 10 min to 30 min.

[0006] Further, when the antler bud cells grow to a confluence of 80% to 85% in the serum-containing medium, the serum replacement medium is replaced; the incubated culture solution is the culture solution incubated for 45 h to 50 h.

[0007] Further, the serum-containing medium is DMEM high-glucose medium; the serum replacement medium is LONZA Ultra CULTURE™ serum-free medium.

[0008] Further, after the centrifugation, nuclease is added, and the addition amount of nuclease is 45 U to 50 U per milliliter of centrifugal supernatant.

[0009] Further, the addition amount of nuclease is 50 U per milliliter of centrifugal supernatant.

[0010] Further, the osteoporosis is postmenopausal osteoporosis, senile osteoporosis or idiopathic osteoporosis.

[0011] Further, the antler bud cell secretion is used to prepare a drug for promoting cell osteogenic differentiation, increasing bone density, increasing bone volume fraction, increasing trabecular bone number, increasing trabecular bone thickness, increasing trabecular bone separation or increasing cortical bone density.

[0012] The second aspect of the present invention provides an application of a pharmaceutical composition in the preparation of a drug for preventing, alleviating or treating osteoporosis. The pharmaceutical composition is composed of the antler bud cell secretion and pharmaceutically acceptable excipients.

[0013] Further, the only active ingredient of the pharmaceutical composition is the antler bud cell secretion.

[0014] Further, the excipients include one or several of antioxidants, pH regulators, bactericides, isotonic agents, metal ion chelators, buffers and surfactants.

[0015] In summary, compared with the prior art, the present invention has the following beneficial advantages and effects: (1) After gradient centrifugation of the cultured antler bud cells in the present invention, a secretion of antler bud cells is obtained. The present invention discovers that this secretion of antler bud cells has a significant therapeutic effect on osteoporosis. The results of both cell experiments and animal experiments show that the secretion of antler bud cells has extremely strong potential to promote osteogenic differentiation of cells, can improve the symptoms of osteoporosis in mice, significantly increase bone mineral density, bone volume fraction, trabecular bone number, trabecular bone thickness, trabecular bone separation degree and cortical bone density, increase the trabecular bone area of the primary and secondary osteogenic centers of the femur in mice, and the effect is significantly stronger than that of the secretion of bone marrow mesenchymal stem cells ( p < 0.05), and some indicators are comparable to the therapeutic effect of bisphosphonates. It can be used to prepare anti-osteoporosis drugs, providing new methods and ideas for the clinical treatment and development of osteoporosis drugs.

[0016] (2) The present invention discloses for the first time that the secretion of antler bud cells can be used to prepare drugs for treating osteoporosis. Traditional drugs for treating osteoporosis, such as hormonal drugs, may cause a series of adverse reactions when used for a long time, such as aggravated osteoporosis, increased risk of infection, etc. The secretion of antler bud cells provided by the present invention, as a natural drug, has active ingredients similar to natural substances in the human body, has better biocompatibility and safety, and the preparation method of the secretion of antler bud cells is simple and easy to operate, and the obtained secretion of antler bud cells has high activity, and has potential application value in the treatment or adjuvant treatment of orthopedic diseases. Description of the Drawings

[0017] Figure 1 To detect the effect of ABPC-P at different concentrations on the proliferation of BMSC by CCK8 method, the abscissa is the concentration of the untreated control group, ABPC-P and the secretion of bone marrow mesenchymal stem cells (BMSC-P), and the ordinate is the OD 450 value of BMSC; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

[0018] Figure 2 To detect the effect of ABPC-P and BMSC-P at different concentrations on the migration of BMSC by cell scratch assay. The upper row is before treatment (0 h), the middle row is treated with BMSC-P for 24 h, and the lower row is treated with ABPC-P for 24 h. The two black lines in the figure represent the original scratch boundary.

[0019] Figure 3 For Figure 2 statistical analysis of the results, the abscissa is the concentration of the untreated control group, ABPC-P and BMSC-P, and the ordinate is the number of invading cells; ** indicates p < 0.01, **** indicatesp <0.0001.

[0020] Figure 4 For the detection of the osteogenic differentiation function of antler bud cell secretions (ABPC-P) in inducing bone marrow mesenchymal stem cells (BMSCs), 100 μg / mL of ABPC-P and 100 μg / mL of bone marrow mesenchymal stem cell secretions (BMSC-P) were respectively added during the osteogenic induction of BMSCs. After 14 days, alkaline phosphatase staining (ALP) was used to detect the osteogenic differentiation, and after 21 days, alizarin red staining (ARS) was used to detect the osteogenic differentiation; Figure 4 Figure A shows the schematic diagram of the experimental procedure; Figure 4 Figure B shows the ALP and ARS staining results. The upper row shows the ALP staining results at 14 d, and the lower row shows the ARS staining results at 21 d; Figure 4 Figures C and D respectively show the statistical analysis results of ALP and ARS; *** indicates p <0.001, **** indicates p <0.0001.

[0021] Figure 5 Figure shows the micro-CT images of the distal femur of mice for the detection of the anti-osteoporosis function of ABPC-P. The upper row shows the overall longitudinal section, and the lower row shows the cross-section of the primary osteogenic center; CTRL represents the normal control group, PBS represents the negative control group (PBS treatment after ovariectomy), BPs represents the positive control group (bisphosphonate treatment after ovariectomy), BMSC-P represents the BMSC secretion group (BMSC-P treatment after ovariectomy), and ABPC-P represents the ABPC secretion group (ABPC-P treatment after ovariectomy).

[0022] Figure 6 Figure shows the statistical chart for the detection of the anti-osteoporosis function of ABPC-P. Among them, Figures A, B, C, and D respectively show the detection results of the bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp) of the primary osteogenic center; ns indicates that there is no significant difference between other groups and the ABPC-P group, * indicates that there is a difference between other groups and the ABPC-P group, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0023] Figure 7 Figure shows the HE staining results of the sections of the distal femur of mice; Figure 7 Figure A shows the HE staining map of the sections of the distal femur of mice. The lower row of pictures is the enlarged view of the red box area in the upper row of pictures;Figure 7 Figure B in p <0.001, **** indicates p <0.0001. Detailed implementation manners

[0024] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

[0025] In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0026] In some embodiments of the present invention, the pilose antler is derived from Shuangyang Sika Deer Farm in Jilin Province, and all sika deer are artificially farmed; the culture reagents used in the present invention are all purchased from Gibco.

[0027] Existing treatment methods for osteoporosis include drug treatment, physical treatment, surgical treatment, etc., but there are problems such as strong side effects and unstable curative effects. In recent years, natural drugs and bioactive substances have shown great potential in the treatment of osteoporosis. The inventors' previous research found that transplantation of antler blastema progenitor cells (ABPC) into the defect site of the rabbit femoral condyle can successfully achieve defect regeneration, and the effect is far stronger than that of bone marrow mesenchymal stem cells (BMSC). Therefore, the development of a product prepared based on antler blastema cells is of great significance for the development of drugs for treating bone diseases.

[0028] The present invention provides the application of antler blastema cell secretions in the preparation of anti-osteoporosis drugs. By obtaining antler blastema cells and performing cell culture, gradient centrifugation of the culture medium, adding nuclease and filtering, and freeze-drying, an antler blastema cell secretion is obtained; through cell experiments, it is verified that the prepared antler blastema cell secretion has the effect of promoting cell osteogenic differentiation. Through mouse experiments, it is found that the prepared antler blastema cell secretion can significantly increase bone density, bone volume fraction, trabecular bone number, trabecular bone thickness, trabecular bone separation degree, and cortical bone density, increase the trabecular bone area of the primary and secondary osteogenic centers of the mouse femur, improve the osteoporosis symptoms of mice, and the effect is significantly stronger than that of bone marrow mesenchymal stem cell secretions ( p <0.05), and some indicators are comparable to the treatment effect of bisphosphonates.

[0029] Example 1: Obtaining of ABPC secretions (ABPC-P) 1. Primary culture of ABPC: Select male sika deer aged 3 - 4 years. After the antler pedicles fall off for 2 - 10 days, professional veterinarians anesthetize the deer. Use a scalpel to scrape off the hair on the antler pedicles, and then disinfect them step - by - step with iodophor and alcohol. Circumcise the skin of the antler pedicles (at the distal 1 / 2), then saw off the distal antler pedicle tissue, disinfect the tissue step - by - step, open the skin in a biosafety cabinet to expose the antler regeneration bud tissue, then cut out the bud tissue and cut it into pieces about 0.2 mm. After washing 3 times with PBS, digest it with type I collagenase mixture (200 U / mL) at 37°C for about 30 minutes. Then, transfer and disperse the tissue blocks to the bottom of the culture flask for primary cell culture (with a small amount of medium). After the cells crawl out, add sufficient medium. When the cells grow to 80% density, harvest the cells.

[0030] 2. Preparation of ABPC secretions: Inoculate ABPC at a density of 6000 cells / cm 2 into a cell culture flask, and culture it using high - glucose DMEM medium. When the cell density reaches 80%, replace it with a medium containing serum replacement (LONZA Ultra CULTURE™ serum - free medium). After 48 h, collect the cell culture medium. Then, perform gradient centrifugation on the collected medium: centrifuge at 300 g for 10 min and 2000 g for 10 min to remove cells and dead cells respectively. Then, use nuclease (DNase and RNase, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to remove residual nucleic acids. The addition amount of nuclease is 50 U per milliliter of centrifuged supernatant. Then, inactivate the nuclease, and filter the medium through a 0.22 - μm filter membrane and freeze - dry it to obtain ABPC secretions.

[0031] Example 2: Function verification of ABPC secretions 1. Verification of the promoting effects of ABPC secretions on cell proliferation, migration and osteogenic differentiation To explore the promoting effects of the ABPC secretions obtained in Example 1 on cell proliferation and migration. In this invention, BMSC cells are inoculated into 96 - well plates. After the cells adhere, different concentrations of ABPC secretions and BMSC secretions are added for treatment. When the cells grow to 80% confluence, the effects of ABPC secretions and BMSC secretions on cell proliferation are detected by the CCK8 method. BMSC cells are inoculated into 24 - well plates. After the cells adhere, different concentrations of ABPC secretions and BMSC secretions are added for treatment. When the cells grow to 80% confluence, the effects of ABPC secretions and BMSC secretions on cell migration are detected by the cell scratch assay.

[0032] The results are as Figures 1 to 3As shown, ABPC secretions can significantly promote the proliferation and migration of BMSCs, and the effect is stronger than that of BMSC secretions; when the added concentration of ABPC secretions is 100 μg / mL, the effect is the best; it is proved that ABPC secretions have extremely strong potential for promoting cell proliferation and cell migration.

[0033] In order to explore the function of ABPC secretions obtained in Example 1 in promoting cell osteogenic differentiation. In the present invention, BMSC cells were seeded in a 12-well plate. When the cells grew to 80% confluence, the medium was replaced with osteogenic induction medium (purchased from Cyagen Biosciences), and 100 μg / mL of ABPC secretions was added to the medium. After 14 days, the formation of calcium nodules in the cells was detected by alizarin red staining.

[0034] The results are as Figure 4 shown, ABPC secretions can significantly promote the formation of calcium nodules in BMSCs, proving that ABPC secretions have extremely strong potential for promoting cell osteogenic differentiation.

[0035] 2. Verification of the anti-osteoporosis effect of ABPC secretions First, female KM mice aged 6 - 8 weeks (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were selected for ovariectomy. Four weeks later, they were respectively given PBS treatment (PBS was given after ovariectomy as a negative control), BPs treatment (5 mg / kg bisphosphonate was given after ovariectomy as a positive control), BMSC-P treatment (5 mg / kg BMSC cell secretions were given after ovariectomy), and ABPC-P treatment (5 mg / kg ABPC secretions prepared in Example 2 were given after ovariectomy), twice a week for 8 weeks. The mice were euthanized, and the femur tissues were harvested for detection of osteoporosis indicators.

[0036] The micro-CT detection results are as Figure 5 and Figure 6 shown, ABPC secretions can significantly increase bone mineral density, bone volume fraction, trabecular number, trabecular thickness, trabecular separation, and cortical bone density, and the effect is significantly stronger than that of BMSC secretions ( p < 0.05), and the effect of some indicators is comparable to that of bisphosphonate treatment.

[0037] The HE staining results are as Figure 7 shown, ABPC secretions significantly increased the trabecular area of the primary and secondary osteogenic centers of the femurs of mice, and the effect was significantly stronger than that of BMSC secretions, even reaching the level of bisphosphonate treatment.

[0038] In summary, the experiments of the present invention have confirmed that the secretions of pilose antler bud cells provided by the present invention can effectively increase the bone density, bone volume fraction, trabecular bone number, trabecular bone thickness, trabecular bone separation degree and cortical bone density of mice, improve the symptoms of osteoporosis, and the improvement effect of the secretions of pilose antler bud cells is more significant than that of the secretions of bone marrow mesenchymal stem cells.

[0039] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0040] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0041] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of the secretion of pilose antler blastema cells in the preparation of anti-osteoporosis drugs, characterized in that, The preparation method of the antler bud cell secretion is as follows: After inoculating antler bud cells in a serum-containing medium for culture, it is then replaced with a serum replacement medium for incubation. The incubated culture solution is collected, and after gradient centrifugation of the incubated culture solution, the centrifuged supernatant is the antler bud cell secretion; The conditions for the gradient centrifugation are: after centrifuging the cell supernatant at 150 g to 500 g for 10 min to 30 min, the centrifuged supernatant is collected and centrifuged at 1500 g to 3000 g for 10 min to 30 min.

2. Use of the antler blastema cell secretion according to claim 1 in the preparation of an anti-osteoporosis drug, characterized in that, When the antler bud cells grow to a confluence of 80% to 85% in the serum-containing medium, the serum replacement medium is replaced; the incubated culture solution is the culture solution incubated for 45 h to 50 h.

3. Use of the antler blastema cell secretion according to claim 2 in the preparation of an anti-osteoporosis drug, characterized in that, The serum-containing medium is DMEM high-glucose medium; the serum replacement medium is LONZA UltraCULTURE™ serum-free medium.

4. Use of the antler blastema cell secretion according to claim 1 in the preparation of an anti-osteoporosis drug, characterized in that, After gradient centrifugation, a nuclease needs to be added, and the addition amount of the nuclease is 45 U to 55 U per milliliter of the centrifuged supernatant.

5. Use of the antler bud cell secretion according to claim 1 in the preparation of an anti-osteoporosis drug, characterized in that, The osteoporosis is postmenopausal osteoporosis, senile osteoporosis or idiopathic osteoporosis.

6. Use of the antler bud cell secretion according to claim 1 in the preparation of an anti-osteoporosis drug, characterized in that, The antler bud cell secretion is used to prepare a drug for promoting cell osteogenic differentiation, increasing bone density, increasing bone volume fraction, increasing trabecular bone number, increasing trabecular bone thickness, increasing trabecular bone separation or increasing cortical bone density.

7. Use of a pharmaceutical composition in the preparation of a medicament for preventing, alleviating or treating osteoporosis, characterized in that, The pharmaceutical composition is composed of the antler bud cell secretion described in claim 1 and pharmaceutically acceptable excipients.

8. The application according to claim 7, wherein The only active ingredient of the pharmaceutical composition is the antler bud cell secretion described in claim 1.

9. The application according to claim 7, wherein The excipients include any one or several of antioxidants, pH regulators, bactericides, isotonic agents, metal ion chelators, buffers and surfactants.

Images