CN115501252A - Application of dental pulp stem cell small cell outer vesicle in preparation of medicine for treating inflammatory bone resorption - Google Patents
Application of dental pulp stem cell small cell outer vesicle in preparation of medicine for treating inflammatory bone resorption Download PDFInfo
- Publication number
- CN115501252A CN115501252A CN202211242011.1A CN202211242011A CN115501252A CN 115501252 A CN115501252 A CN 115501252A CN 202211242011 A CN202211242011 A CN 202211242011A CN 115501252 A CN115501252 A CN 115501252A
- Authority
- CN
- China
- Prior art keywords
- sev
- dental pulp
- pulp stem
- bone resorption
- stem cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Physical Education & Sports Medicine (AREA)
- Veterinary Medicine (AREA)
- Rheumatology (AREA)
- Public Health (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses application of a dental pulp stem cell small extracellular vesicle in preparation of a medicine for treating inflammatory bone resorption, and relates to the technical field of biological medicines. The inventor of the application finds that the DPSC-sEV has a regulation effect on macrophage inflammatory reaction and osteoclast generation, and can promote inflammatory bone resorption repair and regeneration. Not only provides a new source for preparing the medicine for treating the inflammatory bone resorption, but also develops new medicinal value of the pulp stem cell small extracellular vesicle.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of dental pulp stem cell small cell outer vesicles in preparation of medicines for treating inflammatory bone resorption.
Background
In inflammatory bone resorption diseases, such as osteomyelitis, periodontitis, peri-implantitis, rheumatoid Arthritis (RA), and suppurative arthritis, etc., mononuclear/macrophages are recruited and polarized towards the pro-inflammatory M1 type. Persistently activated M1 macrophages produce large amounts of proinflammatory cytokines such as IL-1 β, IL-6, and TNF- α, among others, which induce inflammatory cascades and osteoclast over-formation and activation. Given the critical role of pro-inflammatory M1 macrophages and excessive osteoclastogenesis in bone destruction, inducing the conversion of M1 macrophages to anti-inflammatory M2 types, inhibiting osteoclastogenesis, have been considered as effective strategies for the treatment of inflammatory bone resorption. Currently, commonly used anti-bone resorption drugs, including bisphosphonates, cathepsin K inhibitors and RANKL inhibitors (e.g., denosumab), are effective in inhibiting bone resorption, but these drugs have a number of side effects, including nephrotoxicity, induction of allergic reactions, jaw necrosis, etc. In addition, these drugs do not induce M2 macrophage formation to improve the inflammatory microenvironment. Therefore, new therapeutic regimens for the treatment and prevention of inflammatory bone resorption are urgently under investigation.
Mesenchymal Stem Cells (MSCs) source small cell outer vesicles (MSC-sEV) are proved to have obvious immunoregulation function, can effectively induce macrophage M2 to form, promote repair and regeneration of defective bone tissues, and are expected to become a new scheme for treating inflammatory bone resorption diseases. Dental Pulp Stem Cells (DPSCs) are mesenchymal stem cells isolated from adult dental pulp, widely available, and can be obtained from extracted teeth without ethical problems. Compared with classical bone marrow mesenchymal stem cells (BMSCs), DPSCs have a higher proliferation rate. In addition, it was reported that DPSC-sEV showed stronger immunomodulatory function than BMSC-sEV. DPSC-sEV has also been demonstrated to have therapeutic effects on skin wounds, spinal cord injuries, and mandibular and cranial defects. However, it is not clear whether DPSC-sEV can promote inflammatory bone resorption repair and regeneration by directly inhibiting both macrophage inflammatory response and osteoclastogenesis.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of the small extracellular vesicles of the dental pulp stem cells in preparing a medicament for treating inflammatory bone resorption.
In order to achieve the purpose, the invention adopts the technical scheme that: use of a dental pulp stem cell small extracellular vesicle in the manufacture of a medicament for the treatment of inflammatory bone resorption.
Dental Pulp Stem Cells (DPSCs) are mesenchymal stem cells isolated from adult dental pulp, and have a higher proliferation rate than classical bone marrow mesenchymal stem cells (BMSCs). Research shows that the DPSCs small extracellular vesicles (sEV) have stronger immunomodulatory effects than BMSC-sEV, and have therapeutic effects on skin wounds, spinal cord injuries and mandibular and cranial defects. However, no report is available on the treatment of inflammatory bone resorption by DPSC-sEV. The inventor of the application finds that the DPSC-sEV has a regulation effect on macrophage inflammatory reaction and osteoclast generation, and can promote inflammatory bone resorption repair and regeneration.
In a preferred embodiment of the application of the present invention, the dental pulp stem cell small extracellular vesicles are treated with hypoxia. MSCs cultured or expanded in vitro are typically exposed to normoxia (21% O) 2 ) This is different from the in vivo oxygen concentration under natural physiological conditions. The inventor of the present application researches and discovers hypoxia (1%O) 2 ) Pretreatment may facilitate the release of DPSC-sEV. In addition, with normoxia (21% O) 2 ) Compared with DPSC-sEV obtained under the condition, DPSC-sEV pretreated by hypoxia can enhance and induce macrophage M2 to polarize, inhibit osteoclast formation and reduce inflammatory bone resorption induced by Lipopolysaccharide (LPS).
As a preferred embodiment of the use according to the invention, the hypoxia is an oxygen concentration of 1%.
As a preferred embodiment of the application of the present invention, the preparation method of the small extracellular vesicles of dental pulp stem cells comprises the following steps:
(1) Obtaining pulp tissue from a tooth ex vivo;
(2) Culturing dental pulp tissues to obtain dental pulp stem cells;
(3) Culturing dental pulp stem cells growing to the density of 80-90% in a serum-free culture medium under a low-oxygen condition, and collecting a culture solution;
(4) And (4) centrifuging the culture solution obtained in the step (3) to obtain the dental pulp stem cell small extracellular vesicles.
As a preferred embodiment of the use according to the invention, the medicament induces a switch from M1 macrophages to M2 macrophages. The inventor researches and discovers that the DPSC-sEV can reduce the number of M1 macrophages in inflammatory bone tissues and increase the number of M2 macrophages, and the effect of the hypoxia-treated DPSC-sEV on inducing the conversion of the M1 macrophages to the M2 macrophages is stronger.
As a preferred embodiment of the use according to the invention, the medicament inhibits the expression of proinflammatory cytokines from macrophages and promotes the expression of anti-inflammatory cytokines. The inventor researches and discovers that the DPSC-sEV can inhibit the expression of proinflammatory cytokines IL-6 and TNF-alpha, and the low-oxygen-treated DPSC-sEV can not only inhibit the expression of the proinflammatory cytokines IL-1 beta, IL-6 and TNF-alpha, but also efficiently induce the expression of Arg1, CD163 and CD 206.
As a preferred embodiment of the use according to the invention, the medicament inhibits osteoclast formation.
As a preferred embodiment of the use according to the invention, said inflammatory bone resorption comprises osteomyelitis, periodontitis, peri-implantitis, rheumatoid arthritis or suppurative arthritis.
As a preferred embodiment of the application of the invention, the concentration of the small extracellular vesicles of the dental pulp stem cells in the medicament is 30 μ g/mL.
The invention also provides a pharmaceutical composition comprising the hypoxia-treated dental pulp stem cell small extracellular vesicles and a pharmaceutically acceptable carrier.
The invention has the beneficial effects that: the invention provides application of a small extracellular vesicle of a dental pulp stem cell in preparing a medicament for treating inflammatory bone resorption. The invention discloses an action mechanism of promoting inflammatory bone resorption repair and regeneration by simultaneously enhancing the regulation and control effects of the DPSC-sEV on macrophage inflammatory reaction and osteoclast generation through hypoxia treatment for the first time. The hypoxia-treated dental pulp stem cell extracellular vesicles are applied to the treatment of inflammatory bone resorption, so that a new source is provided for preparing medicines for treating inflammatory bone resorption, and a new medicinal value of the dental pulp stem cell extracellular vesicles is developed.
Drawings
FIG. 1 is a graph showing the results of phenotypic identification of rat DPSCs; wherein A is a DPSCs surface marker flow cytometry analysis chart; b is an immunofluorescence staining pattern of vimentin and cytokeratin; c is alizarin red staining and oil red O staining pattern of DPSCs.
FIG. 2: a is a transmission electron micrograph of DPSC-sEV; b is a comparison graph of the NTA particle size analysis of DPSC-sEV; c is a particle number comparison graph of DPSC-sEV; d is a protein concentration comparison graph of DPSC-sEV; and E is a protein blotting picture of the vesicle-related marker of DPSC-sEV.
FIG. 3: a is the experimental flow chart of example 2; b is a Micro-CT scanning and skull 3D reconstruction map; c is a bone volume/tissue volume contrast map; d is skull HE staining diagram; e is a skull erosion area comparison graph.
FIG. 4: a is CD68 of LPS, LPS + Nor-sEV, LPS + Hypo-sEV group observed by laser scanning confocal microscope + iNOS + Macrophage (M1) profile; b is LPS, LPS + Nor-sEV, LPS + Hypo-sEV group CD68 + iNOS + Macrophage (M1) number map; c is CD68 observed by laser scanning confocal microscope in LPS, LPS + Nor-sEV, LPS + Hypo-sEV groups + Arg1 + Macrophage (M2) profile; d is LPS, LPS + Nor-sEV, LPS + Hypo-sEV group CD68 + Arg1 + Macrophage (M2) number map; e is an mRNA expression comparison chart of LPS, LPS + Nor-sEV, LPS + Hypo-sEV group IL-1 beta, IL-6, TNF-alpha, IL-10 and Arg 1; f is a Western blot diagram of macrophage-related markers in a group of LPS, LPS + Nor-sEV and LPS + Hypo-sEV; g is a CD86 flow cytometry analysis chart of LPS, LPS + Nor-sEV and LPS + Hypo-sEV group; h is the analysis chart of LPS, LPS + Nor-sEV, LPS + Hypo-sEV group CD163 flow cytometry; i is LPS, LPS + Nor-sEV, LPS + Hypo-sEV group CD206 flow cytometry analysis chart.
FIG. 5: a and B are LPS, LPS + Nor-sEV, LPS + Hypo-sEV group TRAP + Osteoclast number comparison graph; c and D are RANKL, RANKL + Nor-sEV, RANKL + Hypo-sEV group TRAP + Multinucleated osteoclast number contrast plots; e is an expression level diagram of RANKL, RANKL + Nor-sEV, RANKL + Hypo-sEV group Acp5, CTSK, c-FOS, DC-STAMP and Atp v0d 2.
Detailed Description
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
EXAMPLE 1 isolation culture of rat DPSCs
Removing maxillary incisors from 5-week-old SD rats under local anesthesia and removing part of enamel and dentin with forceps, separating dental pulp tissue and cutting with ophthalmic scissors in alpha-MEM, culturing the cut dental pulp tissue in 10 cm-diameter culture dish containing DPSCs culture solution, replacing the culture medium once every three days at 37 deg.C and 5% CO 2 After culturing for 7-10 days, passages of the DPSCs grown from adherent clones were established, and the 3 rd to 5 th generation DPSCs were used for the subsequent experiments.
Example 2 phenotypic characterization of rat DPSCs
This example identifies the phenotype of the DPSCs prepared in example 1 as follows:
2.1 flow cytometry analysis of surface markers
Digesting 3 rd generation DPSCs trypsin with good growth state, centrifuging at 500 Xg for 5 minutes, and washing with FACS solution containing 3% fetal bovine serum PBS for 2 times; FITC-anti-rat CD34, PE-anti-rat CD45, CD44, CD90 and APC-anti-rat CD29 antibodies are respectively added, isotype control and blank control without the antibodies are set up, incubation is carried out for 30 minutes at 4 ℃ in a dark place, and FACS liquid is used for washing cells for 2 times and then analyzing the cells by an up-flow cytometer.
2.2 immunofluorescence staining of vimentin and cytokeratin
DPSCs at 5X 10 4 The density of the cells is/mL, the cells are inoculated in a 24-hole plate in which a cell slide is placed, 1mL of each hole, and the cells are cultured for 24 hours; 4% PFA-fixed cells for 15min, permeabilized and blocked with PBS containing 0.1% Triton X-100 and 5% BSA; primary antibody (vimentin or cytokine-14 antibody) was added at 4 ℃ and incubated overnight, followed by Alexa Fluor488/568 secondary antibody incubation at room temperature for 1h, and post-DAPI staining for nuclear mounting.
2.3 Osteogenic and adipogenic differentiation potential of DPSCs
Will be 3X 10 5 One DPSCs/well was seeded in 6-well plates. To induce osteogenic or adipogenic differentiation of DPSCs, the culture medium was changed to an osteogenic or adipogenic inducing solution, the solution was changed every 3 days, after induction for 14 days, the cells were washed with PBS, 4% PFA was fixed at room temperature for 30 minutes, followed by alizarinRed or oil red O staining, and microscopic observation of mineralized nodules and lipid droplet formation.
As shown in fig. 1, it is understood from the flow cytogram of fig. 1 that the positive expression rate of MSCs markers CD29, CD44, and CD90 in DPSCs exceeds 96%, and the expression rate of hematopoietic stem cell surface marker CD34 and leukocyte surface marker CD45 is only less than 3.2%. Immunofluorescence analysis showed that DPSCs positively expressed MSC marker vimentin, and did not express epithelial marker cytokeratin. Furthermore, alizarin red staining results show that DPSCs can form mineralized matrices, and lipid droplets, which are shown by oil red O staining, can form in DPSCs.
Example 3 osteogenic and adipogenic differentiation potential of DPSCs
After DPSCs have grown to a density of about 80-90%, the culture medium is replaced with serum-free medium and incubated in 21% 2 (normoxia) or 1%O 2 (hypoxia) 48 hours. Conditioned medium was collected and centrifuged at 800 Xg for 10 minutes, 3000 Xg for 10 minutes in sequence, cell debris and large vesicles were removed, then ultracentrifuged at 100,000 Xg for 60 minutes at 4 ℃ and after discarding the supernatant, DPSC-sEV obtained under either normoxic (Nor-sEV) or hypoxic (Hypo-sEV) conditions was resuspended in sterile PBS and stored at-80 ℃. Detecting the total protein concentration of sEV according to the BCA protein concentration determination kit; detecting the shape and size distribution of sEV by Transmission Electron Microscopy (TEM) and Nanosight; and detecting whether the extracted sEV expresses the specific markers TSG101, CD63 and CD9 or not by using Westernblot.
As shown in FIG. 2, the results of transmission electron microscopy showed that both Nor-sEV and Hypo-sEV were "cup-disk" structures. NTA showed Nor-sEV and Hypo-sEV peak diameters of 133nm and 124nm, respectively; furthermore, hypo-sEV concentration or particle count is higher compared to Nor-sEV; the protein concentration of Hypo-sEV is also significantly higher than Nor-sEV; western blot results show that Nor-sEV and Hypo-sEV both express sEV related protein markers CD63, TSG101 and CD9; the expression level of TSG101 and CD9 in Hypo-sEV is higher than Nor-sEV. However, hypoxia significantly reduced the expression of CD63 and CD9 in DPSCs, indicating that hypoxia induced sEV release from DPSCs, rather than affecting sEV production in DPSCs.
Example 4 building of LPS-induced skull inflammatory bone resorption model
The specific experimental method comprises the following steps: after anesthesia of C57BL6 mice, subcutaneous injections of 25mg/kg LPS (Escherichia coli O111: B4) were received at the sagittal suture of the skull. To evaluate the therapeutic effect of Nor-sEV and Hypo-sEV on LPS-induced cranial inflammatory bone resorption, each mouse was injected with either Nor-sEV or Hypo-sEV simultaneously with LPS injection. After 7 days, all mice were sacrificed, the cranium was collected and fixed in 4-pfa for 24 hours for further micro-CT and histological analysis.
And (3) a microscopic CT analysis method: calvarial bone was scanned using a high resolution micro-CT (μ CT-50, SCANCO Medical AG) with the following scanning parameters: 70kV, 114. Mu.A and 7 μm. Three-dimensional (3D) image reconstruction and analysis of bone volume/tissue volume (BV/TV) were performed using μ CT Evaluation Program V6.6 software.
Pathological tissues of the skull and immunofluorescence staining: the skull was decalcified in 10% EDTA (pH = 7.4) for 2 weeks, routinely dehydrated, paraffin embedded and sectioned for routine HE staining. Inflammatory cell infiltration and bone resorption were observed and analyzed under an optical microscope.
The experimental results are shown in figure 3, and Micro-CT scanning and skull 3D reconstruction show that compared with a control group, the bone absorption of mice in an LPS group is obviously increased, and BV/TV is reduced. HE staining analysis shows that inflammatory cell infiltration and bone resorption area are obviously increased after LPS injection. Nor-sEV failed to significantly reduce LPS-induced inflammatory bone resorption. While Hypo-sEV obviously reduces inflammatory cell infiltration, increases BV/TV, reduces bone erosion area, and effectively inhibits LPS-induced skull inflammatory bone absorption
Example 5
In this example, the role of the hypoxia-treated DPSC-sEV in inducing macrophage M2 polarization in vivo and in vitro was studied, and the specific experimental method was as follows:
immunofluorescence staining observed macrophage polarization in the skull: after the skull was embedded by OCT, frozen sections were frozen, and then blocked with blocking buffer containing 0.1% Triton X-100 and 5% serum for 60 minutes, incubated overnight at 4 ℃ with primary antibody (CD 68, iNOS, arg-1 antibody), then incubated at room temperature with Alexa Fluor488/568 secondary antibody for 1h, and then sealed after DAPI staining. Use ofCD68 observation by laser scanning confocal microscope + iNOS + Macrophages (M1) and CD68 + Arg1 + Macrophage (M2) distribution and proportion.
Real-time fluorescent quantitative PCR (RT-PCR): extracting RNA from RAW264.7 cells stimulated by LPS or RANKL by using an RNA-Quick Purification Kit (ESscience, beijing, china); the RNA was then reverse transcribed into cDNA using the PrimeScriptTM RT kit (TaKaRa co., kyoto, japan); finally, fast SYBR Green Master Mix (Thermo Fisher, waltham, MA, USA) is used for implementing RT-PCR, and beta-actin is used as an internal reference; the primer sequence table 1 of the detected mRNA is shown in the specification.
Flow cytometry analysis of macrophage surface markers: RAW264.7 macrophages were stimulated with 500ng/mL LPS and 30. Mu.g/mL Nor-sEV or Hypo-sEV for 12 hours. Then TrypLE (Gibco, USA) and after centrifugation at 500 × g for 5 minutes, washed 2 times with FACS liquid containing 3% fetal bovine serum PBS; the samples were incubated with CD86-PerCP, CD163-APC and CD206-FITC monoclonal antibodies (eBioscience, san Diego, calif., at 4 ℃ for 30 minutes in the absence of light, and the cells were washed 2 times with FACS solution and analyzed by flow cytometry.
TABLE 1
The results of the experiment are shown in FIG. 4, and the immunofluorescence results show that LPS increases CD68 in bone tissue + iNOS + The number of macrophages (M1 macrophages); nor-sEV and Hypo-sEV can reduce CD68 in inflammatory bone tissue + iNOS + Macrophage number and increase CD68 + Arg1 + Macrophage (M2 macrophage) number. Furthermore, with LPS + Nor-sEV had less CD68 in LPS + Hypo-sEV group than in Nor-sEV group + iNOS + Macrophages and more CD68 + Arg1 + Macrophages are provided.
In vitro PCR results show that LPS stimulation significantly induces mRNA expression of proinflammatory cytokines IL-1 beta, IL-6 and TNF-alpha in RAW264.7 cells, and inhibits mRNA expression of anti-inflammatory cytokine IL-10. Nor-sEV inhibits the expression of IL-6 and TNF- α, with no significant effect on the expression of IL-1 β or IL-10. Hypo-sEV not only inhibits the expression of IL-1 β, IL-6 and TNF- α, but also upregulates the expression of Arg1, a key effector and marker of IL-10 and M2 macrophages. In addition, hypo-sEV significantly reduced the expression of IL-1 β and TNF- α and promoted the expression of IL-10 and Arg1 compared to Nor-sEV. Western blot and flow cytometry analysis results showed that Nor-sEV upregulated the expression of M2 macrophage markers Arg1, CD163, and CD206 in RAW264.7 cells after LPS stimulation. However, it had no significant effect on the expression of the M1 macrophage markers iNOS and CD 86. Hypo-sEV induces expression of Arg1, CD163 and CD206 more efficiently than Nor-sEV. In addition, hypo-sEV significantly inhibited the expression of iNOS and CD 86. These results indicate that Hypo-sEV inhibits LPS-induced macrophage inflammatory responses and induces macrophage M2 polarization.
Example 6
This example explores the effect of hypoxia-treated DPSC-sEV on osteoclast formation in vitro and in vivo, and the specific experimental method is as follows:
in vivo osteoclast formation: the skull is conventionally embedded in paraffin and sectioned for TRAP staining, and the formation of TRAP-positive osteoclasts is observed under an optical microscope and the number of the TRAP-positive osteoclasts is counted.
In vitro osteoclast formation: RAW264.7 cells were plated at 2.5X 10 4 The density of the seed/mL was changed to 50ng/mL sRANKL (R) after 24 hours for each well in a six-well plate at 2mL&D Systems, minneapolis, MN, USA) were incubated continuously with 30. Mu.g/mL Nor-sEV or Hypo-sEV medium for 4 days, with fluid changes every other day. After 4 days, the cells were rinsed twice with PBS, stained with a TRAP staining kit (387-A), washed 3 times with double distilled water after DAPI staining, and observed for TRAP staining under an inverted microscope. TRAP + And the cells with the cell nucleus number more than or equal to 3 are counted as osteoclasts.
The results of the experiment are shown in FIG. 5, and TRAP staining indicates that LPS injection results in TRAP in the skull + Osteoclast number is significantly increased, which is in contrast to enhanced bone resorptionThus, the method can be used for the treatment of the tumor. Nor-sEV and Hypo-sEV both reduced LPS-induced TRAP + Osteoclast number. Furthermore, TRAP in the LPS + Hypo-sEV group + The number of osteoclasts was lower than in the LPS + Nor-sEV group. In vitro results show that RANKL can induce RAW264.7 cells to form a large amount of TRAP + Multinucleated osteoclasts. Nor-sEV and Hypo-sEV inhibited RANKL-induced osteoclast differentiation and significantly reduced osteoclast number. In addition, hypo-sEV inhibits osteoclastogenesis more strongly than Nor-sEV. PCR results show that RANKL significantly induces anti-tartrate acid phosphatase 5 (Acp 5), cathepsin K (CTSK), c-FOS, dendritic cell-specific transmembrane protein (DC-STAMP) and ATPaseH in RAW264.7 cells + mRNA expression for the trafficking V0 subunit D2 (Atp V0D 2). Nor-sEV only significantly inhibited Acp5 expression, although expression of CTSK, c-FOS and DC-STAMP declined after Nor-sEV treatment. Notably, hypo-sEV significantly inhibited RANKL-induced expression levels of Acp5, CTSK, c-FOS, DC-STAMP, and Atp v0d 2. The RANKL + Nor-sEV group Acp5, CTSK, DC-STAMP and Atp v0d2 were expressed at lower levels than the RANKL + Nor-sEV group. The above results indicate that Hypo-sEV directly inhibits osteoclastogenesis in vitro and in vivo.
The experimental data of the embodiment of the invention adopts the mean value plus or minus standard deviationAll experiments were repeated 3-4 times and analyzed using the SPSS17.0 software. The one-way ANOVA (one-way ANOVA) was used for the comparison between groups, and if there was a significant difference, dunnett's test (Dunnett test) was used for the comparison between experimental groups and a control group, bonferronit test (Bonferronittest) was used for the comparison between groups, and P was used for the comparison between groups<A significant difference was found at 0.05.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. Use of a dental pulp stem cell small extracellular vesicle in the manufacture of a medicament for the treatment of inflammatory bone resorption.
2. The use according to claim 1, wherein the dental pulp stem cell small extracellular vesicles are treated with hypoxia.
3. Use according to claim 2, wherein the hypoxia is an oxygen concentration of 1%.
4. The use according to claim 2, wherein the method of preparing the small extracellular vesicles of dental pulp stem cells comprises the steps of:
(1) Obtaining pulp tissue from a detached tooth;
(2) Culturing dental pulp tissues to obtain dental pulp stem cells;
(3) Culturing dental pulp stem cells growing to the density of 80-90% in a serum-free culture medium under a low-oxygen condition, and collecting a culture solution;
(4) And (4) centrifuging the culture solution in the step (3) to obtain the small extracellular vesicles of the dental pulp stem cells.
5. The use of claim 1, wherein the medicament induces a switch from M1 macrophages to M2 macrophages.
6. The use according to claim 1, wherein the medicament inhibits the expression of pro-inflammatory cytokines by macrophages and promotes the expression of anti-inflammatory cytokines.
7. The use of claim 1, wherein the medicament inhibits osteoclast formation.
8. The use of claim 1, wherein the inflammatory bone resorption comprises osteomyelitis, periodontitis, peri-implantitis, rheumatoid arthritis, or suppurative arthritis.
9. The use of claim 1, wherein the concentration of dental pulp stem cell small extracellular vesicles in the medicament is 30 μ g/mL.
10. A pharmaceutical composition comprising a hypoxic treated dental pulp stem cell small extracellular vesicle and a pharmaceutically acceptable carrier.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211242011.1A CN115501252B (en) | 2022-10-11 | 2022-10-11 | Application of dental pulp stem cell small extracellular vesicles in preparation of medicines for treating inflammatory bone resorption |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211242011.1A CN115501252B (en) | 2022-10-11 | 2022-10-11 | Application of dental pulp stem cell small extracellular vesicles in preparation of medicines for treating inflammatory bone resorption |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115501252A true CN115501252A (en) | 2022-12-23 |
CN115501252B CN115501252B (en) | 2023-07-11 |
Family
ID=84511018
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211242011.1A Active CN115501252B (en) | 2022-10-11 | 2022-10-11 | Application of dental pulp stem cell small extracellular vesicles in preparation of medicines for treating inflammatory bone resorption |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115501252B (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116688101A (en) * | 2023-06-26 | 2023-09-05 | 南通大学附属医院 | Application of high-expression IL-10 neutrophil extracellular vesicles in treatment of temporomandibular arthritis |
CN117064766A (en) * | 2023-07-17 | 2023-11-17 | 中山大学附属口腔医院 | Composite ROS (reactive oxygen species) responsive hydrogel as well as preparation method and application thereof |
WO2023248845A1 (en) * | 2022-06-20 | 2023-12-28 | Dexonファーマシューティカルズ株式会社 | Ptx3 expression control agent, prophylactic drug or therapeutic drug for vasculitis or hardening of skin associated with rheumatoid arthritis, autoimmune disease, method for improving vasculitis or hardening of skin associated with rheumatoid arthritis, autoimmune disease |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150366917A1 (en) * | 2013-02-13 | 2015-12-24 | National University Corporation Nagoya University | Composition for preventing or treating inflammatory disease |
WO2019241462A1 (en) * | 2018-06-13 | 2019-12-19 | Texas Tech University System | Stem cells for the treatment of conditions and diseases |
CN111467373A (en) * | 2020-04-24 | 2020-07-31 | 西安交通大学 | Dental pulp stem cell exosome preparation, preparation method and application thereof |
CN112755052A (en) * | 2021-01-29 | 2021-05-07 | 北京大学口腔医学院 | Application of human deciduous tooth pulp stem cell exosome |
-
2022
- 2022-10-11 CN CN202211242011.1A patent/CN115501252B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150366917A1 (en) * | 2013-02-13 | 2015-12-24 | National University Corporation Nagoya University | Composition for preventing or treating inflammatory disease |
WO2019241462A1 (en) * | 2018-06-13 | 2019-12-19 | Texas Tech University System | Stem cells for the treatment of conditions and diseases |
CN111467373A (en) * | 2020-04-24 | 2020-07-31 | 西安交通大学 | Dental pulp stem cell exosome preparation, preparation method and application thereof |
CN112755052A (en) * | 2021-01-29 | 2021-05-07 | 北京大学口腔医学院 | Application of human deciduous tooth pulp stem cell exosome |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023248845A1 (en) * | 2022-06-20 | 2023-12-28 | Dexonファーマシューティカルズ株式会社 | Ptx3 expression control agent, prophylactic drug or therapeutic drug for vasculitis or hardening of skin associated with rheumatoid arthritis, autoimmune disease, method for improving vasculitis or hardening of skin associated with rheumatoid arthritis, autoimmune disease |
CN116688101A (en) * | 2023-06-26 | 2023-09-05 | 南通大学附属医院 | Application of high-expression IL-10 neutrophil extracellular vesicles in treatment of temporomandibular arthritis |
CN116688101B (en) * | 2023-06-26 | 2024-05-10 | 南通大学附属医院 | Application of high-expression IL-10 neutrophil extracellular vesicles in treatment of temporomandibular arthritis |
CN117064766A (en) * | 2023-07-17 | 2023-11-17 | 中山大学附属口腔医院 | Composite ROS (reactive oxygen species) responsive hydrogel as well as preparation method and application thereof |
CN117064766B (en) * | 2023-07-17 | 2024-04-05 | 中山大学附属口腔医院 | Composite ROS (reactive oxygen species) responsive hydrogel as well as preparation method and application thereof |
Also Published As
Publication number | Publication date |
---|---|
CN115501252B (en) | 2023-07-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN115501252A (en) | Application of dental pulp stem cell small cell outer vesicle in preparation of medicine for treating inflammatory bone resorption | |
Zhu et al. | Dental pulp stem cells overexpressing stromal-derived factor-1α and vascular endothelial growth factor in dental pulp regeneration | |
Greenberger | Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro | |
Huang et al. | An efficient protocol to generate placental chorionic plate-derived mesenchymal stem cells with superior proliferative and immunomodulatory properties | |
US20220184136A1 (en) | Highly functional manufactured abcb5+ mesenchymal stem cells | |
Liang et al. | Macrophage M1/M2 polarization dynamically adapts to changes in microenvironment and modulates alveolar bone remodeling after dental implantation | |
CN103585177A (en) | Applications of mesenchymal stem cell and genetically modified mesenchymal stem cell | |
Wang et al. | Porcine tooth germ cell conditioned medium can induce odontogenic differentiation of human dental pulp stem cells | |
KR20120046430A (en) | Composition comprising hydrogel for transplant to cartilage | |
CN110616190B (en) | Method for regulating and controlling periodontal ligament stem cell osteogenic differentiation based on extracellular matrix | |
WO2010083730A1 (en) | New uses of tooth related stem cells | |
Chen et al. | Exosomal Lnc NEAT1 from endothelial cells promote bone regeneration by regulating macrophage polarization via DDX3X/NLRP3 axis | |
CN113995766A (en) | Application of digoxin in preparation of medicine for treating and/or preventing macrophage M1 type polarization related diseases | |
Zhang et al. | Enamel matrix derivative enhances the odontoblastic differentiation of dental pulp stem cells via activating MAPK signaling pathways | |
Sun et al. | Wireless electric cues mediate autologous DPSC‐loaded conductive hydrogel microspheres to engineer the immuno‐angiogenic niche for homologous maxillofacial bone regeneration | |
CN114652845B (en) | Alendronate coupled polyvinyl alcohol polymer, preparation method and application thereof | |
Trubiani et al. | Dental pulp stem cells bioadhesivity: evaluation on mineral-trioxide-aggregate | |
CN117157389A (en) | Preparation method and application of mesenchymal stem cells of hair follicle | |
CN110664993B (en) | New application of fibrinogen gamma chain in tooth regeneration field and kit thereof | |
CN111454892A (en) | Tooth mesenchymal stem cell culture medium and activity verification method in dental pulp stem cells | |
Weiss et al. | Isolation and characterization of stem cells derived by human dental pulp from harvest based in rotary and manual techniques used in endodontic therapy | |
CN112126621A (en) | Application of AMPK activator in preparation of product for improving AMPK signal pathway abnormity in mesenchymal stem cells | |
KR101452286B1 (en) | Method for culturing cell expressing gdf5 as single clone in serum free medium with mtx and zeocin | |
Coles et al. | Effect of mutagen on cultured Schistosoma mansoni | |
WO2021227573A1 (en) | Xeno-free culture medium and method for expansion of mesenchymal stem cells by means of using same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |