CN112915105B - Application of small extracellular vesicles secreted by mesenchymal stem cells in preparation of drugs for treating CP/CPPS - Google Patents
Application of small extracellular vesicles secreted by mesenchymal stem cells in preparation of drugs for treating CP/CPPS Download PDFInfo
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
- A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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Abstract
The invention relates to application of small extracellular vesicles secreted by mesenchymal stem cells in preparation of drugs for treating CP/CPPS. The invention extracts small extracellular vesicles secreted by mesenchymal stem cells derived from human pluripotent stem cells, researches the relieving effect of the small extracellular vesicles on CP/CPPS, and constructs small extracellular vesicles loaded with celecoxib for treating CP/CPPS. The invention not only can utilize the immune regulation function of the small extracellular vesicles derived from mesenchymal stem cells, but also can solve the problems of low bioavailability and weak penetrability of celecoxib in the body. The small extracellular vesicles secreted by the mesenchymal stem cells wrapped with the CP/CPPS therapeutic drug can reduce infiltration of inflammatory cells to inhibit inflammatory reaction and promote repair of prostate tissues; has analgesic and immunoregulatory effects and is effective for promoting prostate tissue repair.
Description
Technical Field
The invention belongs to the technical fields of cell biology, molecular biology and drug research and development, and particularly relates to application of small extracellular vesicles secreted by mesenchymal stem cells in preparation of drugs for treating CP/CPPS.
Background
Chronic prostatitis type III/chronic pelvic pain syndrome, CP/CPPS, is the most common prostatitis, accounting for about 90% of all types; the clinical manifestations are various, and mainly include pain and distension of pubic area, groin, perineum area, scrotum and penis root, dysfunction of lower urinary tract (frequent urination, urgent urination and incomplete urination), sexual hypofunction, depression, anxiety, fatigue and other neuropsychiatric symptoms. CP/CPPS is not directly life threatening, but severely affects the physical and mental health and quality of life of the patient. CP/CPPS has high heterogeneity, and the etiology and pathophysiology mechanisms of the occurrence and development are still not very definite, and the duration of the disease is easy to repeat, so that the treatment is one of the problems facing the clinic all the time.
The clinical treatment aiming at the CP/CPPS is mainly symptomatic treatment, one of the most commonly used treatment medicines is selacib, which is a selective COX-2 inhibitor, and is a nonsteroidal anti-inflammatory analgesic, and the medicine can relieve clinical symptoms of patients, but needs to be taken for a long time and is easy to relapse after stopping taking; in addition, celecoxib is extremely insoluble in water (3-7 ug/ml), has low oral bioavailability, is difficult to penetrate the prostate capsule to reach the target organ of the prostate, and has poor effect on partial patients. Therefore, it is clinically significant to develop a novel therapeutic approach for the treatment of CP/CPPS. Recent clinical studies have shown that the development and progression of CP/CPPS is mainly related to abnormal activation of immune responses and disturbance of immune function in the body. Thus, reestablishing immune balance in vivo is critical for the treatment of CP/CPPS.
In addition, many drug delivery vehicles are currently under study, including microspheres, liposomes, and nanoparticles. These exogenous carriers are stable in physicochemical properties outside the body, but are easily recognized and cleared by reticuloendothelial system of the body after entering the body, and are easily broken due to the actions of antibodies, opsonin and the like, so that the encapsulated drugs cannot leak to the destination, and the defects greatly limit the clinical application.
The existing research proves that the mesenchymal stem cells have strong anti-inflammatory and immunoregulatory functions, and the transplanted mesenchymal stem cells have good clinical application prospects in the aspects of treating autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, graft versus host diseases and the like, but the application of the mesenchymal stem cells also has safety concerns including vascular embolism after transplantation, abnormal differentiation and genetic stability. In recent studies, in vivo tracking of mesenchymal stem cells after transplantation revealed that the time for which most cells survive in vivo is short after mesenchymal stem cell transplantation does not rely mainly on self-renewal, proliferation and directed differentiation into effector cells after homing themselves in local tissues, but rather by secretion of small extracellular vesicles capable of carrying various signaling molecules. The small extracellular vesicles derived from the mesenchymal stem cells refer to nanoscale vesicles with lipid bilayer membrane structures secreted by the mesenchymal stem cells in a resting or activated state, and the diameters are between 30 and 150 nm. The cell-specific bioactive molecules are rich in the cell-specific bioactive molecules, including lipids, proteins, miRNAs, mRNAs and the like, and the bioactive molecules enter a lipid bilayer after being specially selected and packaged by parent cells, so that the cell-specific bioactive molecules are key for exciting a signal transduction mechanism in an organism and play a key role in the processes of immunoregulation and inhibition of inflammatory reactions. Compared with parent cells, the nano-scale small extracellular vesicles derived from the self tissue cells of the organism are stable in the organism and can successfully reach target organs through biological barriers in the organism. The small extracellular vesicles derived from mesenchymal stem cells have been proved to have good curative effects in immune abnormality diseases such as rheumatoid arthritis and inflammatory bowel diseases, but whether the small extracellular vesicles derived from mesenchymal stem cells can treat chronic prostatitis type III/chronic pelvic pain syndrome has not been reported yet.
Disclosure of Invention
Based on the defect of the prior art that celecoxib is used for treating CP/CPPS, the invention provides application of small extracellular vesicles secreted by mesenchymal stem cells in preparation of medicines for treating CP/CPPS.
The aim of the invention can be achieved by the following technical scheme:
in a first aspect of the invention, there is provided a small extracellular vesicle secreted by a mesenchymal stem cell.
In one embodiment of the invention, the small extracellular vesicles secreted by the mesenchymal stem cells are small extracellular vesicles secreted by mesenchymal stem cells derived from human pluripotent stem cell induction.
In the practice of the present invention, the mesenchymal stem cells may be derived from commercially available mesenchymal stem cells.
The small extracellular vesicles derived from the mesenchymal stem cells refer to nanoscale vesicles with lipid bilayer membrane structures secreted by the mesenchymal stem cells in a resting or activated state, and the diameters of the small extracellular vesicles are between 50 and 150 nm. The cell-specific bioactive molecules are rich in the cell-specific bioactive molecules, including lipids, proteins, miRNAs, mRNAs and the like, and the bioactive molecules enter a lipid bilayer after being specially selected and packaged by parent cells, so that the cell-specific bioactive molecules are key for exciting a signal transduction mechanism in an organism and play a key role in the processes of immunoregulation and inhibition of inflammatory reactions.
In a second aspect of the present invention, there is provided a method for preparing small extracellular vesicles secreted by mesenchymal stem cells, comprising the steps of: and taking the mesenchymal stem cells cultured to 5-10 generations to extract small extracellular vesicles secreted by the mesenchymal stem cells.
A mesenchymal stem cell marker expressing specificity, comprising: CD44, CD73, CD90, CD105; without expressing CD34, CD45, CD11b, CD19 and HLR-DR.
In one embodiment of the present invention, there is provided a method of culturing mesenchymal stem cells, comprising the steps of:
1. thawing the pluripotent stem cells;
2. uniformly mixing a cell suspension of the pluripotent stem cells with a cell complete culture medium NuwacellTM Nova;
3. Centrifuging to remove the supernatant, collecting cells, adding cell complete medium NuwacellTM Nova to resuspend cells, inoculating cells, and adding fresh cell complete medium NuwacellTM Nova;
4. the inoculated cells were cultured.
And 5, when the confluency of the cells reaches 85%, replacing a mesenchymal stem cell culture medium (Nuwacell Biotechnology, RP 020101) by passage, replacing the culture medium periodically, and transferring to the 4 th generation to obtain the cells which are in a typical long spindle shape, wherein the 5th generation is used for identifying the mesenchymal stem cells, and the cell supernatant of the 5 th-10 th generation culture is used for extracting small extracellular vesicles.
In one embodiment of the invention, the method of extracting small extracellular vesicles secreted by mesenchymal stem cells is to extract small extracellular vesicles using ultracentrifugation.
In one embodiment of the invention, the extraction of small extracellular vesicles secreted by mesenchymal stem cells using ultracentrifugation comprises the steps of:
1. collecting serum-free culture medium for culturing human pluripotent stem cell induced source mesenchymal stem cells;
2. centrifuging to remove cell debris, and collecting supernatant;
3. filtering and centrifuging the collected supernatant to remove impurities in the supernatant;
4. and (5) centrifuging for multiple times, wherein the obtained precipitate is the small extracellular vesicles.
In a third aspect of the invention, there is provided a small extracellular vesicle secreted by mesenchymal stem cells encapsulated with a CP/CPPS therapeutic agent. The small extracellular vesicles secreted by the mesenchymal stem cells are wrapped with a CP/CPPS therapeutic drug.
In one embodiment of the invention, small extracellular vesicles secreted by mesenchymal stem cells encapsulated with celecoxib are provided.
In one embodiment of the invention, celecoxib is associated with small extracellular vesicles in an amount of 80-800ug celecoxib per 10 10 small extracellular vesicles.
Celecoxib chemical name 4- [5- (4-tolyl) -3- (trifluoromethyl) -1 hydrogen-1-pyrazol-1-yl ] benzenesulfonamide has a molecular formula of C 17H14F3N3O2 S and a molecular weight of 381.38. Celecoxib is white or white-like crystalline powder, is easily dissolved in methanol, absolute ethyl alcohol, acetone and dimethyl sulfoxide, is dissolved in dichloromethane, and is almost insoluble in water; the melting point is 157-159 ℃; the dissociation constant pKa was 11.1.
In a fourth aspect of the present invention, a method for preparing small extracellular vesicles secreted by mesenchymal stem cells coated with a CP/CPPS therapeutic agent is provided, wherein a dose of the CP/CPPS therapeutic agent can be coated into the small extracellular vesicles secreted by mesenchymal stem cells by incubation, electrotransformation, extrusion, ultrasound, freeze thawing, or saponin treatment.
In a fifth aspect of the invention, there is provided the use of small extracellular vesicles secreted by mesenchymal stem cells in the manufacture of a medicament for the treatment of CP/CPPS.
In one embodiment of the present invention, there is provided the use of small extracellular vesicles secreted by mesenchymal stem cells to induce directional differentiation of pluripotent stem cells in the preparation of a medicament for treating CP/CPPS.
In a sixth aspect of the invention, there is provided the use of small extracellular vesicles secreted by mesenchymal stem cells encapsulated with a CP/CPPS therapeutic agent for the manufacture of a medicament for the treatment of CP/CPPS.
In one embodiment of the present invention, the small extracellular vesicles secreted by the mesenchymal stem cells coated with the CP/CPPS therapeutic agent are selected from small extracellular vesicles secreted by the mesenchymal stem cells coated with celecoxib.
In a seventh aspect of the invention, there is provided a formulation of small extracellular vesicles secreted by mesenchymal stem cells encapsulated with a CP/CPPS therapeutic drug, selected from any one of the following forms:
A. Suspending agent: dissolving small extracellular vesicles secreted by mesenchymal stem cells coated with a CP/CPPS therapeutic drug in a solvent, and allowing the small extracellular vesicles to exist in the form of a suspending agent;
B. Complex of slow release exosomes: the small extracellular vesicles secreted by the mesenchymal stem cells coated with the CP/CPPS therapeutic drug are combined with a carrier to form a compound of the slow-release small extracellular vesicles, wherein the carrier comprises various hydrogels, biological membranes, biological ceramic materials and biological materials with nano structures;
C. the additive takes small extracellular vesicles secreted by mesenchymal stem cells wrapped with CP/CPPS therapeutic drugs as the effective components;
D. The medicine liquid takes small extracellular vesicles secreted by mesenchymal stem cells wrapped with CP/CPPS therapeutic medicine as an effective component.
In one embodiment of the invention, in the preparation of small extracellular vesicles secreted by mesenchymal stem cells coated with a CP/CPPS therapeutic agent, celecoxib is associated with small extracellular vesicles in an amount of 80-800ug per 10 10 small extracellular vesicles.
In one embodiment of the present invention, a preparation of small extracellular vesicles secreted by mesenchymal stem cells coated with celecoxib is provided, further comprising instructions for: when the preparation coated with small extracellular vesicles secreted by the mesenchymal stem cells of celecoxib is used, the effective dosage of celecoxib is 0.1-5mg/kg, preferably 1mg/kg.
Although small extracellular vesicles derived from mesenchymal stem cells have been demonstrated to have good therapeutic effects in immune-related diseases such as rheumatoid arthritis, inflammatory bowel disease, since different conditions are applicable to a large variety of drugs, and the pathogenesis of chronic prostatitis type III/chronic pelvic pain syndrome (CP/CPPS) of which the present application is concerned and the therapeutic manner are greatly different, conventional drugs for other immune-related diseases such as rheumatoid arthritis, inflammatory bowel disease, etc. are not applicable to the treatment of CP/CPPS, there has been no report before the present application as to whether small extracellular vesicles derived from mesenchymal stem cells can treat chronic prostatitis type III/chronic pelvic pain syndrome. Whether small extracellular vesicles derived from mesenchymal stem cells can treat chronic prostatitis type III/chronic pelvic pain syndrome or not was not known to those skilled in the art before the present application, and the present application has been confirmed by a great deal of theoretical studies and experimental verification that small extracellular vesicles derived from mesenchymal stem cells can treat chronic prostatitis type III/chronic pelvic pain syndrome. And the small extracellular vesicles secreted by the mesenchymal stem cells coated with the CP/CPPS therapeutic drug have better effect of treating chronic prostatitis type III/chronic pelvic pain syndrome.
The invention extracts small extracellular vesicles secreted by mesenchymal stem cells from human pluripotent stem cells, researches the relieving effect of the small extracellular vesicles on CP/CPPS, constructs small extracellular vesicles loaded with celecoxib for treating the CP/CPPS, and can not only utilize the immunoregulation function of the small extracellular vesicles from mesenchymal stem cells, but also solve the problems of low bioavailability and weak penetrability of celecoxib in organisms.
Compared with the prior art, the technical scheme of the invention has the advantages that:
1. The small extracellular vesicles derived from the mesenchymal stem cells and the small extracellular vesicles secreted by the mesenchymal stem cells coated with the CP/CPPS therapeutic drug can reconstruct the balance of Th1/Th2 and Th17/Treg immune cells in vivo by reducing Th1, th17 cells and increasing Treg cells, and the small extracellular vesicles derived from the mesenchymal stem cells and the small extracellular vesicles secreted by the mesenchymal stem cells coated with the CP/CPPS therapeutic drug can reduce infiltration of inflammatory cells to inhibit inflammatory response and promote repair of prostate tissues; has analgesic and immunoregulatory effects and is effective for promoting prostate tissue repair.
2. The small extracellular vesicles derived from the mesenchymal stem cells not only can play the role of the maternal cells for treating related diseases, but also are ideal carriers for drug delivery. Therefore, the small extracellular vesicles from the mesenchymal stem cells are wrapped with the CP/CPPS therapeutic drug, so that the problem that the traditional CP/CPPS therapeutic drug carrier is difficult to simultaneously consider the transfer efficiency and the biocompatibility can be solved.
Drawings
Fig. 1 is a photomicrograph of the mesenchymal stem cells provided in example 1;
FIG. 2 is a representation of the mesenchymal stem cell expressing cell markers provided in example 1;
FIG. 3 is an electron micrograph of mesenchymal stem cell-derived small extracellular vesicles provided in example 2;
FIG. 4 is a graph showing the particle size distribution of small extracellular vesicles derived from mesenchymal stem cells provided in example 2;
FIG. 5 is a graph showing the results of the test of the pain response of mice in each group by von Frey test in example 4;
FIG. 6 shows the results of the in vivo immune cell change by flow assay in example 4;
FIG. 6 includes FIGS. 6-1, 6-2, 6-3, and 6-4;
FIG. 7 shows the histological results of the different experimental groups of example 4.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
Example 1
The embodiment provides a specific preparation method of small extracellular vesicles secreted by mesenchymal stem cells, comprising the following steps:
1. The water bath is preheated to 37 ℃, and the pluripotent stem cells stored in liquid nitrogen are taken out and placed on dry ice to be transported to a cell room.
2. And (3) placing the frozen tube into a preheated water bath kettle, thawing while shaking uniformly, thawing within 1min, and taking out when ice crystals in the cell suspension disappear quickly and completely by naked eyes.
3. Immediately sucking the cell suspension into a 15mL centrifuge tube, dropwise adding 9mL of preheated cell complete culture medium NuwacellTM Nova, and uniformly mixing; meanwhile, 1mL of preheated cell culture fluid is taken to wash the freezing tube.
The supernatant was removed by centrifugation at 4.200 g.times.5 min, cells were collected, resuspended in 1-2mL complete medium, and the cells were seeded in six well plates with appropriate amount of pre-warmed fresh complete medium.
5. After shaking horizontally for 3 times, the cells were placed in an incubator at 37℃with a concentration of 5% CO2 and a saturation humidity of 95%.
6. When the confluency of the cells reaches 85%, the mesenchymal stem cell culture medium (Nuwacell Biotechnology, RP 020101) is replaced by passage, and the mesenchymal stem cell culture medium is replaced by liquid every 2 days in general, and the mesenchymal stem cell culture medium is transferred to the 4 th generation to form the typical long spindle shape, wherein the 5 th generation is used for identifying the mesenchymal stem cells, and the cell supernatant of the 5-10 th generation culture is used for extracting small extracellular vesicles.
In this embodiment, the mesenchymal stem cells are derived from human pluripotent stem cells, and various ways of obtaining human pluripotent stem cells are available from commercial sources, for example, from the Ministry of biosciences, inc., whereas in this embodiment, the mesenchymal stem cells derived from human pluripotent stem cell line (iPS-01) are adopted, and are provided by the research of biochemistry and cell biology of the national academy of sciences.
The mesenchymal stem cell diagrams provided in this embodiment are shown in fig. 1 and fig. 2, fig. 1 shows that the cells exhibit typical mesenchymal stem cell morphology (long fusiform), and fig. 2 shows that the cells express specific mesenchymal stem cell markers, including: CD44, CD73, CD90, CD105; without expressing CD34, CD45, CD11b, CD19 and HLR-DR.
Example 2
The present embodiment provides a method for extracting small extracellular vesicles secreted by mesenchymal stem cells using an ultracentrifugation method, comprising the steps of:
1. collecting serum-free culture medium for culturing human pluripotent stem cell induced source mesenchymal stem cells under aseptic condition, about 200-250ml, and packaging in 50ml centrifuge tube.
2. All subsequent centrifugation is carried out at 4 ℃, an ice box is needed in the transportation process, and the operation is carried out on ice;
cell debris was removed by centrifugation at 3.300g for 10min and the supernatant was taken to a new 50ml centrifuge tube.
4.2000G was centrifuged for 10min, and the cell debris was further removed and the supernatant was taken to a new 50ml centrifuge tube.
5. The collected supernatant was filtered through a 0.22 μm needle filter to remove impurities.
6.10000G was centrifuged for 30min to further remove impurities from the supernatant and the liquid was transferred to a Beckman ultracentrifuge tube.
7. After trimming, centrifuging in an ultracentrifuge for 70min at 100000g, and obtaining a precipitate as a mixture containing small extracellular vesicles.
8. Taking the centrifuged sediment to a new ultra-high centrifuge tube, adding PBS at 4 ℃ for balancing, centrifuging for 70min with 100000g, and repeating the process for three times to obtain the small extracellular vesicles.
The diagrams of the small extracellular vesicles derived from the mesenchymal stem cells provided in this example are shown in fig. 3 and 4. The small extracellular vesicles are seen in the typical cup shape in FIG. 3, and the diameter of the small extracellular vesicles is between 50 and 150nm in FIG. 4.
Example 3
This example provides small extracellular vesicles secreted by mesenchymal stem cells encapsulated with celecoxib, and small extracellular vesicles secreted by mesenchymal stem cells not encapsulated with celecoxib as a comparison
Celecoxib was entrapped in small extracellular vesicles by incubation, 55mg celecoxib was dissolved in 10ml dimethyl sulfoxide, the dissolved celecoxib was then mixed with the small extracellular vesicles and incubated on a shaker at 37 ℃ for 1 hour, after which it was added to a purification column (MW 3000 Exosome Spin columns), 750g, and centrifuged for 10 minutes to remove non-entrapped celecoxib. Ultracentrifugation 100000g,70 min, 3 washes with phosphate buffer.
Measuring the drug-loading rate of the small extracellular vesicles by using a high performance liquid chromatography, adding an equal volume of acetonitrile into the small extracellular vesicles, fully and uniformly mixing, performing ultrasonic membrane rupture, centrifuging for 13000g and 10 minutes to remove impurities, filtering by using a 0.22um sterile filter, diluting by using a mobile phase, transferring into a sample bottle, sampling, and detecting the content of celecoxib according to standard curve chromatographic conditions.
Example 4
This example provides small extracellular vesicles secreted by mesenchymal stem cells coated with celecoxib, and the effect of small extracellular vesicles secreted by mesenchymal stem cells not coated with celecoxib on chronic prostatitis type III
First, preparation of model III chronic prostatitis is carried out
1. 4% Chloral hydrate is prepared, 4g chloral hydrate is added into 100ml sterilized water injection water, and the mixture is uniformly mixed and stored in a sealing way at 4 ℃.
2.1Ml/100g chloral hydrate was intraperitoneally injected into anesthetized SD rats, which were fixed on a rat fixing plate.
3. Mixing an equal volume of prostate antigen solution with Complete Freund's Adjuvant (CFA), and emulsifying by syringe method, namely: one syringe is used for sucking antigen solution, the other syringe is used for sucking CFA with equal volume, the two syringes are repeatedly sucked back and forth in a three-way pipe connection for about 1-2 hours until the liquid drops to the water surface to form a sphere without diffusion, and the periphery of the syringes is wrapped by a four-degree ice bag during emulsification, and the temperature is kept low.
4.4% Chloral hydrate anesthetized mice, after local skin preparation and disinfection, injecting 1ml of emulsifier into the neck, two sides of tail root, sole, groin and other points of the mice in a subcutaneous mode, and injecting 0.5ml of Bai-broken vaccine in an intraperitoneal mode; in addition, this operation was repeated after 15 days, 30 days, and modeling was completed after one month.
The small extracellular vesicles secreted by the mesenchymal stem cells coated with celecoxib and the small extracellular vesicles secreted by the mesenchymal stem cells not coated with celecoxib in example 3 were used for treating chronic prostatitis of type III by tail intravenous injection, wherein a normal group and a model group were set in addition to the small extracellular vesicles secreted by the mesenchymal stem cells coated with celecoxib and the small extracellular vesicles secreted by the mesenchymal stem cells not coated with celecoxib. That is, the experiments were divided into four groups: normal group, model group, small extracellular vesicle group, drug-loaded small extracellular vesicle group.
Wherein 200ul of the small extracellular vesicles containing 1X 10 10/small extracellular vesicles are administered to the small extracellular vesicles group, 200ul of the small extracellular vesicles containing 1X 10 10/small extracellular vesicles coated with celecoxib are administered to the small extracellular vesicles group, and the normal group and the model group are administered with equal amounts of PBS (wherein the model group is not given small extracellular vesicles after molding, only equal amounts of PBS are given to the small extracellular vesicles, and the normal group is an animal with normal prostate without intervention). Four groups of pain changes were examined at time points of 2 weeks, 4 weeks and 6 weeks, respectively, and changes in immune cells in vivo, and after 6 weeks, the material was HE stained to observe the inflammatory conditions in the prostate tissue.
The results of the von Frey experiments to detect the response of each group of mice to pain show that the 2, 4 and 6 weeks (shown in figure 5) of small extracellular vesicles and the drug-loaded small extracellular vesicles can relieve the response of the rat prostate perineal region to the stimulus, but the drug-loaded small extracellular vesicles have more remarkable effect.
The results of the flow-through detection of the change of immune cells in vivo are shown in FIG. 6, wherein FIG. 6 comprises FIG. 6-1, FIG. 6-2, FIG. 6-3 and FIG. 6-4, and the bar graphs corresponding to each time point in FIG. 6-1, FIG. 6-2, FIG. 6-3 and FIG. 6-4 respectively show a normal group, a model group, a small extracellular vesicle group and a drug-loaded small extracellular vesicle group from left to right.
FIGS. 6-1, 6-2, 6-3, and 6-4 show that small extracellular vesicles and drug-loaded small extracellular vesicles have similar functions in immunomodulation, both by decreasing Th1, th17 cells and increasing Treg cells to reconstitute the balance of Th1/Th2 and Th17/Treg immune cells in vivo.
The histological results are shown in fig. 7, and fig. 7 shows that the small extracellular vesicles and the drug-loaded small extracellular vesicles can reduce infiltration of inflammatory cells to inhibit inflammatory reaction and promote repair of prostate tissues.
Comparative example
In the case of small extracellular vesicles not involved in mesenchymal stem cell secretion, the effective amount of celecoxib is 4mg/kg in the case of treatment of CP/CPPS with celecoxib alone, i.e. the effective amount of celecoxib required per kilogram of body weight is 4mg.
When the small extracellular vesicles secreted by the mesenchymal stem cells coated with celecoxib are used, the effective dosage of celecoxib is 0.1-5mg/kg, preferably 1mg/kg. That is, in the case of using the small extracellular vesicles secreted by the mesenchymal stem cells coated with celecoxib of the present invention for the treatment of CP/CPPS, the optimal effective amount of the small extracellular vesicles secreted by the mesenchymal stem cells coated with celecoxib per kg body weight is 1mg. Much lower than celecoxib alone.
The results show that the celecoxib-loaded small extracellular vesicles combine the advantages of the celecoxib-loaded small extracellular vesicles and have the effects of easing pain and regulating immunity to promote prostate tissue repair.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (2)
1. The application of small extracellular vesicles secreted by mesenchymal stem cells in preparing a medicament for treating CP/CPPS is characterized in that the small extracellular vesicles secreted by the mesenchymal stem cells are wrapped with celecoxib, and the relationship between the celecoxib and the small extracellular vesicles is that 80-800ug of celecoxib is contained in every 10 10 small extracellular vesicles;
The mesenchymal stem cells are derived from human pluripotent stem cells;
the small extracellular vesicles secreted by the mesenchymal stem cells refer to nanoscale vesicles with lipid bilayer membrane structures secreted by the mesenchymal stem cells in a resting or activated state, the diameters of the vesicles are between 50 and 150nm, and cell-specific bioactive molecules including lipids, proteins, miRNAs and mRNAs are carried in the vesicles.
2. The use according to claim 1, wherein small extracellular vesicles secreted by mesenchymal stem cells encapsulated with celecoxib are formulated, said formulation further comprising instructions for: when the preparation of small extracellular vesicles secreted by the mesenchymal stem cells coated with celecoxib is used, the effective dosage of the celecoxib is 1 mg/kg.
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