CN112755052A - Application of human deciduous tooth pulp stem cell exosome - Google Patents
Application of human deciduous tooth pulp stem cell exosome Download PDFInfo
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- CN112755052A CN112755052A CN202110125452.2A CN202110125452A CN112755052A CN 112755052 A CN112755052 A CN 112755052A CN 202110125452 A CN202110125452 A CN 202110125452A CN 112755052 A CN112755052 A CN 112755052A
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Images
Classifications
-
- 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
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Abstract
The invention relates to an application of human deciduous tooth pulp stem cell exosome in preparing a medicament for promoting bone tissue repair or treating bone loss diseases related to inflammation. The exosome of the human deciduous tooth pulp stem cells has wide sources, is simple to prepare and easy to store, has the advantages of source cells, has no side effect or small side effect, can be applied to the preparation of medicines for promoting bone tissue repair and medicines for treating bone loss diseases related to inflammation, and can be effectively and long-term used for treating or preventing clinical aged bone loss related diseases.
Description
Technical Field
The invention relates to the field of tissue engineering repair and regeneration, in particular to application of a human deciduous tooth pulp stem cell exosome in preparation of a medicine for promoting bone tissue repair and a medicine for treating bone-related diseases.
Background
Bone defect diseases are common diseases in clinic and affect physical and mental health of patients. As a gold standard for the treatment of bone defects, autologous bone graft and allogeneic bone graft are often used in clinical practice to avoid immune rejection. The body promotes healing by stimulating a bone regeneration mechanism after bone transplantation, but if the body lacks a certain bone regeneration capability, even ordinary fractures may cause difficulty in healing. Clinically, obesity, bone cancer, osteoporosis, and aging population may all contribute to a decrease in bone regeneration capacity. Population aging is an inevitable trend in the development of today's society, and aging is accompanied by a reduction in bone density leading to high risk of bone fracture and bone loss, and therefore repair of bone defects in the aging population becomes a major challenge for the treatment of clinicians.
For bone defects of middle-aged and elderly people, the gold-standard bone transplantation still has great defects, firstly, the bone density of the old people is low, the regeneration capability of the body bone is reduced, the bone quantity is insufficient, and secondly, complications such as fracture, nonunion, infection and the like still occur after the transplantation. In addition, the high surgical costs and long healing time impose severe psychological stress on the patient. The combined treatment of biological materials needs to consider the safety of the materials to the body. Stem cell therapy addresses the problem of immune rejection, but does not allow for accurate quantification and efficient use of cells, and may lead to tumors and emboli. The extracellular vesicles are nanoscale vesicles secreted by stem cells, have biological functions similar to those of donor cells, and can participate in information exchange among cells. At present, extracellular vesicles gradually become a novel treatment strategy for treating bone defects instead of stem cells due to high stability of the extracellular vesicles.
The extracellular vesicle is a new carrier for intercellular genetic material transfer, and plays a key role in various pathological and physiological processes such as tumor markers, cancer treatment, tissue regeneration and the like. The exosome is a vesicle with the diameter of 30-150nm in an extracellular vesicle, previous researches show that the exosome derived from the mesenchymal stem cell has higher capability of multidirectional differentiation than exosome derived from mature and aged stem cells, and exosomes secreted by different types of mesenchymal stem cells have different biological functions because stem cells are different in source and have different substances such as lipid, protein, nucleic acid and the like. Human exfoliated deciduous tooth pulp stem cells are stem cells which have high proliferation and pluripotent differentiation capacity and are easy to obtain, and the effective contents of extracellular vesicles such as exosomes derived from the stem cells and the modification application of the extracellular vesicles are not researched.
The deciduous tooth pulp stem cells of children are odontogenic mesenchymal stem cells, have strong self-renewal and multidirectional differentiation potentials, have no report on osteogenic differentiation research of exosomes on bone marrow mesenchymal stem cells, and have less research on osteogenic capacity of age-related organism-derived BMSCs.
Disclosure of Invention
The invention aims to provide a novel application of human exfoliated deciduous tooth pulp stem cell exosomes.
The technical scheme for solving the technical problems is as follows: the invention provides application of human deciduous tooth pulp stem cell exosome in preparing a medicament for promoting bone tissue repair or treating bone loss diseases related to inflammation.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the bone tissue repair comprises osteogenic differentiation of mesenchymal stem cells.
Further, the mesenchymal stem cells are bone marrow mesenchymal stem cells.
Further, the bone tissue repair includes alveolar bone repair and regeneration.
Further, the inflammation-related bone loss disease is periodontitis, osteoarthritis, or osteomyelitis.
The invention has the beneficial effects that: the inventor firstly discovers that the exosome of the human exfoliated deciduous tooth dental pulp stem cell can directionally promote the osteogenic differentiation capacity of the bone marrow mesenchymal stem cell and simultaneously promote the proliferation capacity of the cell. In addition, exosomes may inhibit LPS-induced up-regulation of inflammatory factors. In animal models, it was again verified that human exfoliated deciduous tooth pulp stem cell exosomes can promote alveolar bone repair and regeneration using periodontitis models. The exosome of the human deciduous tooth pulp stem cells has wide sources, is simple to prepare and easy to store, has the advantages of source cells, has no side effect or small side effect, can be applied to the preparation of medicines for promoting bone tissue repair and medicines for treating bone loss diseases related to inflammation, and can be effectively and long-term used for treating or preventing clinical aged bone loss related diseases.
Drawings
FIG. 1 is a morphological photomicrograph of deciduous tooth pulp stem cells exfoliated by the present inventors;
FIG. 2 is a transmission electron micrograph of exosomes of deciduous tooth pulp stem cells exfoliated by the present inventors;
FIG. 3 shows the identification of exosome-specific surface markers expressed by human exfoliated deciduous tooth dental pulp stem cells (SHED), human exfoliated deciduous tooth dental pulp stem cell exosomes (SHED-Exo) and conditioned medium enrichment (SHED-CM) of SHED by Western Blot according to the present invention;
FIG. 4 is a photograph of alizarin red staining for osteogenesis induction for 14 days;
fig. 5 shows the quantitative results of 14-day alizarin red staining after osteogenic induction (n-6-8). P <0.05, P <0.01 compared to Ctrl group; compared to the PBS group, # P <0.05, # P < 0.01. Error line: mean ± standard deviation (Means ± SD);
FIG. 6 shows the gene expression assay 14 days after osteogenic induction. P <0.05, P < 0.01; error line: mean ± standard deviation (Means ± SD);
FIG. 7 shows the expression of pro-inflammatory genes after SHED-Exo and SHED-CM treatment of bone marrow mesenchymal cells, P <0.05 and P < 0.01; error line: mean ± standard deviation (Means ± SD);
FIG. 8 is a graph of the effect of human exfoliated deciduous tooth dental pulp stem cell exosome (SHED-Exo) formulations on periodontal bone loss treatment;
FIG. 9 shows the analysis of major active ingredients in human exfoliated deciduous tooth dental pulp stem cell exosomes (SHED-Exo).
Detailed Description
The principles and features of this invention are described in connection with the drawings and the detailed description of the invention, which are set forth below as examples to illustrate the invention and not to limit the scope of the invention.
Example 1 culture of human exfoliated deciduous tooth pulp Stem cells (SHED)
(1) SHED extraction:
1) taking an in-vitro tooth which meets the clinical deciduous tooth extraction indication, exposing a pulp cavity, taking out pulp, putting D-Hank's solution containing 2 multiplied by antibiotics, and soaking for 0.5-1 hour at 4 ℃;
2) transferring the dental pulp tissue into a new culture dish, trimming, transferring into a 1.5mL centrifuge tube, and cutting into 1-2 mm pieces with small bending scissors3And (5) small blocks. Adding 0.5mL of enzyme digestion solution (4mg/mL type I collagenase and 3mg/mL type II lyase) and oscillating and digesting in a constant-temperature water bath at 37 ℃ for 30-60 minutes;
3) the cell suspension was collected, filtered through a 100 mesh cell screen, and centrifuged at 1000rpm for 5 min. Washing with D-Hank's twice, rinsing to remove supernatant, adding DMEM complete medium (containing 10% fetal calf serum and 1% penicillin/streptomycin), placing at 37 deg.C and 5% CO2And (5) incubating in the incubator. The liquid is changed every other day or every two days, and the cells can be passaged after 80% confluence.
(2) SHED culture
DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin was prepared and SHED to passage 4-7 cells were cultured in 100mm dishes. Cell culture medium was collected for subsequent exosome formulation extraction: when the cell fusion degree reaches 70%, the old culture medium is sucked away, washed by PBS and repeated for 2 times, and after the DMEM culture medium containing 1% penicillin/streptomycin but no serum is added for 24 hours, cell culture solution is collected and the DMEM culture medium containing serum is replaced. When the culture is carried out until the SHED of the 4 th to 7 th generations, the cells grow on the bottom of the culture dish in an adherent manner, the growth rate is stable, and the microscopical shape is shown in figure 1 and is in a spindle or spindle shape with uniform size.
Example 2 extraction and preparation of exosomes of stem cells of deciduous teeth of human
(1) Sucrose cushion combined ultracentrifugation method centrifugation
The cell culture medium was collected into 50ml centrifuge tubes (supernatant volume not exceeding 2/3 of the centrifuge tube volume) and subjected to the following centrifugation steps using a Beckman bench top refrigerated centrifuge: centrifuging at 300 Xg 4 deg.C for 10min to remove dead cells, centrifuging at 2000 Xg 4 deg.C for 10min to remove cell debris, and centrifuging at 5000 Xg 4 deg.C for 30min to remove macromolecular vesicles. Transferring the supernatant to 10KD and 100KD ultrafiltering tubes respectively for concentrating after centrifuging, and centrifuging at 5000 Xg 4 deg.C for 30min with 10KD ultrafiltering tube to obtain SHEDConditioned Medium Concentrate (CM), named SHED-CM; centrifuging at 5000 × 4 deg.C for 10min with 100KD ultrafiltering tube to obtain culture medium concentrated solution. Placing 100KD ultrafiltration concentrate into a centrifuge tube, then extracting 5ml of 30% sucrose cushion (30% sucrose cushion is prepared by sucrose and heavy water according to the mass/volume ratio of 30%) with a sterile syringe, pushing a needle against the bottom of the centrifuge tube, slowly injecting 30% sucrose cushion solution, allowing the culture medium and sucrose cushion to separate, slowly adding the rest concentrated culture medium along the wall of 1/3 tube on the centrifuge tube to make the liquid level reach the position 3mm away from the tube opening, and using Beckman OptimaTMCentrifuging SW 32Ti horizontal rotor of XPN-100 ultracentrifuge at 100000 Xg 4 deg.C for 70min, carefully sucking off the culture medium on the sucrose cushion after centrifugation is finished, and collecting the sucrose cushion containing exosome below.
(2) Washing and collecting exosome
Diluting a sucrose cushion containing exosomes by PBS, placing the sucrose cushion in an ultrafiltration tube with the temperature of 100KD, centrifuging for 10min at the temperature of 5000 Xg 4 ℃, repeatedly centrifuging for 3 times to remove sucrose heavy water solution, collecting concentrated solution which is exosomes (Exo) from human exfoliated deciduous tooth pulp stem cells (SHED), namely the human exfoliated deciduous tooth pulp stem cell exosomes (SHED-Exo), filtering and sterilizing by a 0.22 mu m needle head type filter, subpackaging and storing in a refrigerator with the temperature of-80 ℃.
(3) Identification of human exfoliated deciduous tooth pulp stem cell exosome (SHED-Exo)
1) Transmission Electron Microscope (TEM) observation
Fixing a small amount of SHED-Exo concentrated solution in 2% paraformaldehyde, washing with PBS, loading onto a polyethylene formaldehyde-carbon coating net, washing with PBS again, fixing with 2% glutaraldehyde for 2min, fixing with 2% phosphotungstic acid for 5min, washing, drying, and observing the exosome form with a transmission electron microscope. The result is shown in fig. 2, the human exfoliated deciduous tooth pulp stem cell exosome (SHED-Exo) is a spherical membrane-encapsulated structure with the diameter range smaller than 100nm, and conforms to the morphological characteristics of the vesicle with the exosome diameter range of 30-150 nm.
2)Western blot
Adding appropriate amount of SHED-Exo solution into PIPA lysate containing protease inhibitor, performing ice lysis for 30min, crushing with ultrasonic crusher, packaging, storing at-20 deg.C, and storing at-80 deg.C for a long time. Western blot was used to detect CD63, GM130 and GAPDH in SHED-Exo, SHED-CM and SHED, respectively, and the results are shown in FIG. 3. the collected exosomes (SHED-Exo) and conditioned medium concentrate (SHED-CM) of human exfoliated deciduous tooth pulp stem cells and CD63 of human exfoliated deciduous tooth pulp stem cells (SHED) were positive, CD63 is a member of a four-time transmembrane protein, is localized near the membrane structure, and is involved in the formation and secretion process of exosome vesicles, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and GM130 (Golgi matrix protein) are cell-specific protein components, GM130 and Gapdh expression are negative in SHED-Exo and SHED-CM and positive in the source cells (SHED).
Example 3 biological Regulation of bone marrow mesenchymal Stem cells (BMSCs) by human exfoliated deciduous tooth pulp Stem cell exosomes (SHED-Exo)
(1) Human deciduous tooth pulp stem cell exosome (SHED-Exo) for promoting osteogenic differentiation of bone marrow mesenchymal stem cells
Bone marrow-derived mesenchymal stem cells were isolated and cultured intramedully from tibia and femur of 10-month-old mice. Cells cultured to passage P1-P2 were spindle-shaped for subsequent experiments.
Preparing a bone culture medium: bone medium (OM) was prepared by adding 10nM dexamethasone, 10mM sodium beta-glycerophosphate and 100. mu. M L-ascorbic acid 2-phosphate to alpha-MEM medium containing 10% fetal bovine serum and 1% penicillin streptomycin antibiotic.
Osteogenic induction of BMSCs: BMSCs at 1.0X 105And/well inoculating on a 24-well culture plate, and dividing into four groups when the cell fusion degree reaches 90-95 percent: a ctrl group, a PBS group, an Exo group, and a CM group, wherein the ctrl group was supplemented with a general medium containing no osteogenic inducing component as a blank control, the PBS group was supplemented with an Osteogenic Medium (OM) and PBS, the exosoma of human exfoliated deciduous dental pulp stem cells (SHED-Exo) and an Osteogenic Medium (OM) were supplemented with an exosoma of human exfoliated deciduous dental tooth dental pulp stem cells, and the CM group was supplemented with a conditioned medium concentrate of SHED (SHED-CM) and an Osteogenic Medium (OM), and cultured for 14 days, respectively.
Alizarin red staining: after induction, washing with PBS, fixing with 4% stationary liquid for 30min, washing with distilled water, dyeing with 1% alizarin red for 10min, washing with distilled water for 4-5 times to remove redundant dye liquid, scanning the pore plate, and observing the formation of mineralized nodules by using an inverted phase contrast microscope. And after alizarin red staining, removing liquid in the pore plate, adding 100mM cetyl pyridinium chloride solution, measuring the absorbance value at the wavelength of 570nm, and quantitatively analyzing the mineralized nodule content of each treatment group. Microscopic results are shown in fig. 4, BMSCs differentiated into osteoblasts 14 days after osteogenic induction, and formed extracellular mineralized matrix, quantitative results are shown in fig. 5, alizarin red staining and quantitative results show that, compared with the control group, PBS group, Exo group, and CM group significantly promoted mineralized nodule formation (× P <0.01), and staining is orange red; compared with the osteogenesis inducing capacity of PBS, the SHED-Exo and SHED-CM have stronger capacity of promoting the formation of mineralized nodules of BMSCs than that of the PBS group, but the SHED-Exo can obviously promote the formation of mineralized nodules (# # P <0.01) of the BMSCs, and the formed mineralized nodules are connected in a net shape and are uniform in size. Therefore, after 14 days of osteogenic induction, the SHED-Exo and SHED-CM can promote osteogenic differentiation of BMSCs, and the SHED-Exo has stronger capability of promoting the BMSCs to form bones than the SHED-CM.
Detecting the expression level of the osteogenesis related gene: after 3 days, 7 days and 14 days of osteogenic induction, the gene expression levels of osteogenic genes Osx and Runx2 are respectively detected, and the specific steps are as follows: washing with sterile PBS, adding 400 μ l Trizol solution into each hole, cracking at room temperature for 5min, repeatedly blowing and beating a tip without RNase, adding 1.5ml RNase-free centrifuge tube, adding chloroform (1/5 volume Trizol), manually shaking for 15s, and standing at room temperature; centrifuging at 12000 Xg at 4 deg.C for 15min, separating into colorless layer, white middle layer and red lower layer, carefully sucking the upper colorless layer, and transferring into a new 1.5ml RNase-free centrifuge tube; adding isovolumetric isopropanol solution, reversing and mixing uniformly, and standing at room temperature for 10 min; centrifuging at 12000 Xg at 4 deg.C for 10min, carefully pouring out supernatant, adding 1ml 75% ethanol solution, suspending and precipitating, centrifuging at 7500 Xg at 4 deg.C for 5min, gently pouring out supernatant, air drying at room temperature for 5-10min, and adding 15-20 μ l DEPC water when precipitate becomes semitransparent; RNA concentration was measured using Nanodrop (RNA purity was based on 260/280 absorbance values ranging from 1.8 to 2.0). After RNA is reversely transcribed into cDNA, a real-time fluorescence quantitative system is used for amplification and analyzing the gene expression levels of osteogenic genes Alp and Runx2 (taking Gapdh as a reference standard), and after osteogenesis is induced for 3 days, SHED-Exo has no obvious induction effect on the osteogenic genes Alp and Runx 2; after 7 days of osteogenesis induction, SHED-Exo enhanced the activity of alkaline phosphatase (Alp) and gradually exerted osteogenesis, and after 14 days of induction, the results are shown in FIG. 6, and compared with the blank control group (ctrl) and PBS osteogenesis induction, SHED-Exo can both obviously promote the enhancement of the expression level of transcription related factors Runx2 and Osx genes.
Alizarin red staining results and osteogenesis related gene expression level detection both prove that the human exfoliated deciduous tooth dental pulp stem cell exosome can specifically promote osteogenic differentiation of bone marrow mesenchymal stem cells.
(2) Down-regulation of Lipopolysaccharide (LPS) -induced BMSCs inflammation-related factor expression by human exfoliated deciduous tooth pulp stem cell exosome (SHED-Exo)
BMSCs at 1.0X 105After being subcultured in a 12-well plate to 80% cell concentration, the cells were divided into 5 groups, control group, LPS + Exo/Exo1.0 group, Exo10.0 group and LPS + CM group, wherein the control group was not treated, the LPS group was added with 1. mu.g/ml LPS, the LPS + Exo/Exo1.0 group was added with 1. mu.g/ml LPS and 1. mu.g/ml SHED-Exo, the Exo10.0 group was added with 1. mu.g/ml LPS and 10. mu.g/ml SHED-Exo, the LPS + CM group was added with 1. mu.g/ml LPS and 1. mu.g/ml SHED-CM, and after 24 hours of culture, total RNA of cells were extracted by Trizol, reverse transcription cDNA was performed, and RT-PCR was performed to analyze the expression of proinflammatory primers Il-6 and Tnf-alpha, using Gapdh as a reference standard.
BMSCs were induced with LPS (1. mu.g/ml) for 24 hours, and qRT-PCR was performed to measure the gene expression associated with inflammation in each of the control group, LPS + Exo/Exo1.0 group, Exo10.0 group and LPS + CM group. The results are shown in FIG. 7, and qRT-PCR showed that both SHED-Exo (1. mu.g/ml) and SHED-CM decreased the expression of Il-6 and Tnf- α (# # P <0.01, # P <0.05) (FIG. 7A), indicating that 1. mu.g/ml SHED-Exo decreased the inflammatory response; however, high concentrations of exosomes (10. mu.g/ml SHED-Exo) instead promoted Il-6 and Tnf- α expression (FIG. 7B).
Example 4 periodontitis model establishment and application of human exfoliated deciduous tooth pulp stem cell exosomes
(1) Periodontitis mouse model establishment
1) Selecting a male CD-1 mouse (10-month-old) to establish a mouse periodontitis model, carrying out intraperitoneal injection anesthesia on the 10-month-old mouse by using 4% chloral hydrate according to the weight of each 10g of mouse and the dosage range of 105 mul, after the mouse enters a deep anesthesia state, fixing the mouse in a supine position, opening an opening angle, carrying out first molar ligation on the mouse by using a 5-0 silk thread, placing a ligation thread head in the center of the buccal side of the first molar, recording the retention condition of a ligature of the mouse after ligation for 14 days, and removing the ligature stimulation;
2) grouping: taking more than 3 mice to euthanasia, taking maxilla tissues and naming the maxilla tissues as a period group, and dividing the rest periodontitis model mice into three groups: PBS group, SHED-Exo group, each group containing more than 4 samples, injecting PBS, SHED and SHED-Exo respectively, and taking healthy mice of the same month as Control group;
3) preparing an exosome injection of the deciduous tooth pulp stem cells of the human being: selecting frozen SHED-Exo, rewarming at room temperature, diluting to 1 μ g/μ l with normal saline, and temporarily storing on ice to obtain SHED-Exo injection;
4) PBS or SHED-Exo injection therapy: the experimental mice were injected with 10 μ l of each buccal and palatine side local injection using a 10 μ l microsyringe (hamilton) at the first molar in the upper jaw of the mice, 1 time per week, for 2 weeks, while removing the ligature. After 2 weeks the mice were euthanized and the maxilla of the mice was fixed in 4% paraformaldehyde for 24 hours for micro CT scanning.
(2) Scanning the upper jaw of a mouse by using a microcomputer tomography (Micro CT), fixing a sample of the upper jaw of the mouse in 4% paraformaldehyde for 24 hours, placing and scanning the sample by using the Micro CT according to the set parameters such as 8.99 mu m pixel size, 80kv voltage, 500 mu A current, 1500ms exposure time and the like, reconstructing a three-dimensional image of the upper jaw of the mouse by using Inveon Research workbench 3.0 software (Siemens), adjusting the sagittal plane measurement of a first molar and a second molar, and calculating the distance from a first molar near-enamel cementum boundary (CEJ) to an alveolar crest (ABC).
(3)3D reconstruction results As shown in FIG. 8, bone resorption was evident in buccal, palatal and root bifurcation areas of the first molar of the upper jaw (M1) in the Perio group compared to the Control group; compared with the Control group, the bone absorption of M1 in buccal, palatal and root bifurcation areas is obvious in the PBS injection group, and compared with the periodo group, no obvious bone regeneration is observed in bone on the buccal and lingual sides; compared with the Perio group, the SHED group and the SHED-Exo group have obvious regeneration of bone on the cheek-tongue side, and new bone is generated in the root bifurcation area.
Example 5 assay of exosome activity of human exfoliated deciduous tooth dental pulp stem cells
(1) Extracting exosome protein: total protein quantification was performed on the extracted SHED-Exo: uniformly mixing the reagent A and the reagent B according to the volume ratio of 50:1 by using a BCA method to obtain a BCA working solution; a BSA standard was then prepared. After each SHED-Exo sample is diluted by 10 times by using PBS in a proper amount, BSA standard solution (20 mu l/hole) with different concentrations and a protein sample to be detected (20 mu l/hole, each sample is repeated for 3 times) are respectively added into a 96-well plate, then 160 mu l/hole BCA standard solution is added, incubation is carried out for 30min at 37 ℃, absorbance of each hole is detected at the wavelength of 570nm on a microplate reader, a standard curve is drawn according to the absorbance of the BCA standard solution, and finally the protein concentration of the sample is calculated according to the standard curve, the absorbance of the sample and the dilution times.
(2) Exosome protein mass spectrometry
And (3) purifying, reducing and enzyme-cutting the extracted exosome protein, separating a peptide segment by HPLC, and detecting and analyzing a proteome in a sample by adopting a Data Independent Acquisition (DIA) mode.
As shown in fig. 9, the human exfoliated deciduous dental pulp stem cell exosomes are rich in proteins related to the PI3K-Akt pathway, which is involved in regulating various cell functions such as cell proliferation, differentiation, apoptosis and energy metabolism, and is one of important active protein components of exosomes derived from deciduous dental pulp stem cells.
The above examples demonstrate that human exfoliated deciduous tooth dental pulp stem cell exosomes can directionally promote osteogenic differentiation capacity of bone marrow mesenchymal stem cells; and simultaneously promote the proliferation capacity of cells. In addition, exosomes may inhibit LPS-induced up-regulation of inflammatory factors. In animal models, we again demonstrated, using the periodontitis model, that human shed deciduous tooth pulp stem cell-derived exosomes promote alveolar bone repair and regeneration. Therefore, exosomes derived from human exfoliated deciduous tooth dental pulp stem cells are expected to be a novel therapeutic strategy for promoting bone regeneration.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. Application of the human deciduous tooth pulp stem cell exosome in preparing a medicament for promoting bone tissue repair or treating bone loss diseases related to inflammation.
2. Use according to claim 1, wherein the bone tissue repair comprises osteogenic differentiation of mesenchymal stem cells.
3. The use of claim 2, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells.
4. Use according to claim 1, wherein the bone tissue repair comprises alveolar bone repair and regeneration.
5. Use according to claim 1, wherein the inflammation-related bone loss disorder is periodontitis, osteoarthritis or osteomyelitis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113322230A (en) * | 2021-05-31 | 2021-08-31 | 重庆医科大学附属口腔医院 | Dental pulp stem cell exosome and preparation method and application thereof |
| CN115501252A (en) * | 2022-10-11 | 2022-12-23 | 中山大学附属口腔医院 | Application of dental pulp stem cell small cell outer vesicle in preparation of medicine for treating inflammatory bone resorption |
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Non-Patent Citations (2)
| Title |
|---|
| JINYAN WU等: "Exosomes Secreted by Stem Cells from Human Exfoliated Deciduous Teeth Promote Alveolar Bone Defect Repair through the Regulation of Angiogenesis and Osteogenesis", 《ACS BIOMATER. SCI. ENG.》 * |
| JIZHEN WEI等: "Exosomes derived from human exfoliated deciduous teeth ameliorate adult bone loss in mice through promoting osteogenesis", 《JOURNAL OF MOLECULAR HISTOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322230A (en) * | 2021-05-31 | 2021-08-31 | 重庆医科大学附属口腔医院 | Dental pulp stem cell exosome and preparation method and application thereof |
| CN113322230B (en) * | 2021-05-31 | 2024-03-12 | 重庆医科大学附属口腔医院 | Dental pulp stem cell exosome and preparation method and application thereof |
| CN115501252A (en) * | 2022-10-11 | 2022-12-23 | 中山大学附属口腔医院 | Application of dental pulp stem cell small cell outer vesicle in preparation of medicine for treating inflammatory bone resorption |
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