MiR-137 adipose-derived stem cell exosome as well as preparation method and application thereof
By preparing and transfecting miR-137 adipose stem cell exosomes, the limitations of adipose stem cell exosomes in the treatment of central stroke post-stroke pain were addressed, and an effective reduction in CPSP was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
- Filing Date
- 2025-10-21
- Publication Date
- 2026-04-21
AI Technical Summary
In the prior art, the therapeutic effect of exosomes derived from adipose stem cells on central post-stroke pain (CPSP) is unclear, and the application of miRNA in exosomes has not been fully studied.
A method for preparing adipose stem cell exosomes is provided, including collagenase digestion, incubation and multiple centrifugation steps, and further preparing miR-137 adipose stem cell exosomes by transfecting miR-137 adipose stem cells, which are then applied to the treatment of central post-stroke pain.
The release of exosomal miR-137 via ADSC significantly alleviated CPSP symptoms, providing insights into the molecular mechanism of ADSC-exosomes in CPSP treatment and demonstrating potential therapeutic value.
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Figure CN121896159A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and in particular relates to a miR-137 adipose stem cell exosome, its preparation method and application. Background Technology
[0002] Stroke is a group of sudden-onset cerebrovascular disorders, including ischemic and hemorrhagic strokes. Although only 8-18% of strokes are hemorrhagic, reports show that the mortality rate of hemorrhagic stroke is higher than that of ischemic stroke. Meanwhile, reports indicate that approximately 8-14% of hemorrhagic stroke survivors experience central post-stroke pain (CPSP), which is the most common and difficult symptom after a stroke. CPSP is a chronic neuropathic pain syndrome directly related to the focal point of the stroke; it is typically long-lasting, severe, or difficult to describe, significantly impacting patients' quality of life and social functioning. Treatment of CPSP is limited by insufficient basic diagnosis and the difficulty in controlling the condition.
[0003] Currently, stem cells are widely used in the treatment of neurological diseases such as spinal cord injury, Alzheimer's disease, and stroke. Adipose-derived stem cells (ADSCs) are a population of cells extracted from the matrix portion of adipose tissue, exhibiting multi-lineage differentiation potential and possessing immunomodulatory and anti-inflammatory functions. Exosomes are more commonly used than stem cells due to their advantages of ease of production and storage, and lack of immunogenicity. Exosomes contain various molecules and proteins that can regulate intercellular communication, and studies have shown that exosomes derived from stem cells have produced various beneficial effects in multiple disease models. However, the effects of ADSC-derived exosomes on CPSP remain unclear. Among the various inclusions of exosomes, microRNAs (miRNAs) have been the most extensively studied. Summary of the Invention
[0004] In view of this, the purpose of this invention is to provide a miR-137 adipose stem cell exosome, its preparation method and application.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing exosomes from adipose-derived stem cells, comprising the following steps: 1) After digesting adipose tissue, centrifuge to obtain cells, incubate the cells, and passage them to obtain adipose stem cells; 2) Adipose-derived stem cells were cultured in DMEM-F12 medium containing 5-15% serum-free EVs. The supernatant was collected by centrifugation, and the supernatant was centrifuged four times to obtain adipose-derived stem cell exosomes.
[0006] Preferably, in step 1), the digestion uses collagenase I, the concentration of collagenase I is 0.05~0.15% w / v of adipose tissue, the digestion temperature is 35~40℃, the digestion time is 0.5~1.5h, the incubation temperature is 35~40℃, the incubation time is 36~50h, the passage time is until the cell density reaches 85~95%, and the number of passages is 2~4 times.
[0007] Preferably, the density of adipose-derived stem cells in step 2) is 80-90%, and the conditions for the first centrifugation in the four centrifugations are 400-600g for 5-15min, the conditions for the second centrifugation in the four centrifugations are 1800-2200g for 15-25min, the conditions for the third centrifugation in the four centrifugations are 8000-12000g for 50-70min, and the conditions for the fourth centrifugation in the four centrifugations are 100000-140000g for 60-80min.
[0008] The present invention also provides adipose-derived stem cell exosomes prepared by the aforementioned preparation method.
[0009] This invention also provides a method for preparing exosomes from miR-137 adipose stem cells, comprising the following steps: 1) After digesting adipose tissue, centrifuge to obtain cells, incubate the cells, and passage them to obtain adipose stem cells; 2) Transfect miR-137 into adipose-derived stem cells, culture the transfected adipose-derived stem cells in DMEM-F12 medium with 5-15% serum-free EVs, collect the supernatant by centrifugation, and centrifuge the supernatant four times to obtain miR-137 adipose-derived stem cell exosomes. The nucleotide sequence of miR-137 is shown in SEQ ID No. 1.
[0010] Preferably, in step 1), the digestion uses collagenase I, the concentration of collagenase I is 0.05~0.15% w / v of adipose tissue, the digestion temperature is 35~40℃, the digestion time is 0.5~1.5h, the incubation temperature is 35~40℃, the incubation time is 36~50h, the passage time is until the cell density reaches 85~95%, and the number of passages is 2~4 times.
[0011] Preferably, in step 2), the transfection uses Lipofectamine® 2000 transfection reagent, the concentration of which is 10-30 nM, and the transfection time is 36-60 h.
[0012] Preferably, the density of adipose-derived stem cells in step 2) is 80-90%, and the conditions for the first centrifugation in the four centrifugations are 400-600g for 5-15min, the conditions for the second centrifugation in the four centrifugations are 1800-2200g for 15-25min, the conditions for the third centrifugation in the four centrifugations are 8000-12000g for 50-70min, and the conditions for the fourth centrifugation in the four centrifugations are 100000-140000g for 60-80min.
[0013] The present invention also provides miR-137 adipose stem cell exosomes prepared by the preparation method described above.
[0014] The present invention also provides the use of the adipose stem cell exosomes prepared by the preparation method, the adipose stem cell exosomes, the miR-137 adipose stem cell exosomes prepared by the preparation method, or the miR-137 adipose stem cell exosomes in the preparation of a drug for treating central post-stroke pain.
[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention uses a rat model of thalamic hemorrhage to discover that ADSC can alleviate CPSP by releasing exosomes miR-137, providing valuable insights into ADSC-exosomes as a treatment for CPSP and their molecular mechanisms. Future research can build on this to develop targeted therapies to improve the quality of life for CPSP patients. Attached Figure Description
[0016] Figure 1 The results of the isolation and identification of exosomes derived from ADSCs are as follows (where A is Oil Red O staining of isolated ADSCs, B is immunofluorescence staining of ADSCs biomarkers, C is the morphology of ADSCs-derived exosomes as shown by transmission electron microscopy, D is the size distribution of exosomes detected by NTA, E is the detection of exosome biomarkers by Western blot, and F is the verification of high expression of miR-137 in ADSCs-derived exosomes that overexpress miR-137). Figure 2 The effects of ADSC-Exo on mechanical response and heat tolerance in CPSP rats. Detailed Implementation
[0017] This invention provides a method for preparing exosomes from adipose-derived stem cells, comprising the following steps: 1) After digesting adipose tissue, centrifuge to obtain cells, incubate the cells, and passage them to obtain adipose stem cells; 2) Adipose-derived stem cells were cultured in DMEM-F12 medium containing 5-15% serum-free EVs. The supernatant was collected by centrifugation, and the supernatant was centrifuged four times to obtain adipose-derived stem cell exosomes.
[0018] In this invention, adipose tissue is digested and centrifuged to obtain cells, which are then incubated and passaged to obtain adipose-derived stem cells. The digestion uses collagenase I, and the concentration of collagenase I is preferably 0.05-0.15% w / v of the adipose tissue, more preferably 0.08-0.12% w / v, and even more preferably 0.01% w / v. The digestion temperature is preferably 35-40℃, more preferably 36-39℃, and even more preferably 37℃. The digestion time is preferably 0.5-1.5 h, more preferably 0.8-1.2 h, and even more preferably 1 h. After digestion, the tissue is washed using DMEM containing fetal bovine serum, and the concentration of the fetal bovine serum is preferably 5-15%, more preferably 6-13%, and even more preferably... The incubation process involves adding 5-15% bovine serum and 0.5-1.5% penicillin / streptomycin, wherein the concentration of bovine serum is preferably 6-13%, more preferably 10%, and the concentration of penicillin / streptomycin is preferably 0.7-1.3%, more preferably 1%; the incubation temperature is preferably 35-40℃, more preferably 36-39℃, more preferably 37℃; the incubation time is preferably 36-50h, more preferably 30-44h, more preferably 48h; the passage time is until the cell density reaches 85-95%, preferably 90%; and the number of passages is preferably 2-4 times, more preferably 3 times.
[0019] In this invention, adipose-derived stem cells are cultured in DMEM-F12 medium containing 5-15% serum-free EVs. The supernatant is collected by centrifugation, and the supernatant is subjected to four separate centrifugations to obtain adipose-derived stem cell exosomes. The density of the adipose-derived stem cells is 80-90%. The preferred conditions for the first centrifugation are 400-600g for 5-15 min, more preferably 450-550g for 8-12 min, and even more preferably 500g for 10 min. The preferred conditions for the second centrifugation are 1800-2200g for 15-25 min, more preferably 1900-2100g for 18-22 min, and even more preferably 2000g for 20 min. The preferred conditions for the third centrifugation of the four centrifugations are 8000-12000g and 50-70min, more preferably 9000-11000g and 55-65min, and even more preferably 10000g and 60min; the preferred conditions for the fourth centrifugation of the four centrifugations are 100000-140000g and 60-80min, more preferably 110000-130000g and 65-75min, and even more preferably 120000g and 70min.
[0020] The present invention also provides adipose-derived stem cell exosomes prepared by the aforementioned preparation method.
[0021] This invention also provides a method for preparing exosomes from miR-137 adipose stem cells, comprising the following steps: 1) After digesting adipose tissue, centrifuge to obtain cells, incubate the cells, and passage them to obtain adipose stem cells; 2) Transfect miR-137 into adipose-derived stem cells, culture the transfected adipose-derived stem cells in 10% serum-free DMEM-F12 medium, collect the supernatant by centrifugation, and obtain miR-137 adipose-derived stem cell exosomes by centrifugation four times in sequence. The nucleotide sequence of miR-137 is shown in SEQ ID No.1, specifically 5′-UAUUGCUUGAGAAUACACGUAG-3′.
[0022] In this invention, adipose tissue is digested and centrifuged to obtain cells, which are then incubated and passaged to obtain adipose-derived stem cells. The digestion uses collagenase I, and the concentration of collagenase I is preferably 0.05-0.15% w / v of the adipose tissue, more preferably 0.08-0.12% w / v, and even more preferably 0.01% w / v. The digestion temperature is preferably 35-40°C, more preferably 36-39°C, and even more preferably 37°C. The digestion time is preferably 0.5-1.5 h, more preferably 0.8-1.2 h, and even more preferably 1 h. After digestion, the tissue is washed using DMEM containing fetal bovine serum, and the concentration of the fetal bovine serum is preferably 5-15%. The concentration of bovine serum is preferably 6-13%, and even more preferably 10%; the incubation is performed with 5-15% bovine serum and 0.5-1.5% penicillin / streptomycin, wherein the concentration of bovine serum is preferably 6-13%, and even more preferably 10%, and the concentration of penicillin / streptomycin is preferably 0.7-1.3%, and even more preferably 1%; the incubation temperature is preferably 35-40℃, even more preferably 36-39℃, and even more preferably 37℃; the incubation time is preferably 36-50h, even more preferably 30-44h, and even more preferably 48h; the passage time is until the cell density reaches 85-95%, and the cell density is preferably 90%; the number of passages is preferably 2-4 times, and even more preferably 3 times.
[0023] In this invention, miR-137 is transfected into adipose-derived stem cells. The transfected adipose-derived stem cells are cultured in 10% serum-free DMEM-F12 medium, and the supernatant is collected by centrifugation. The supernatant is then centrifuged four times to obtain miR-137 adipose-derived stem cell exosomes. The transfection uses Lipofectamine® 2000 transfection reagent, and the concentration of the Lipofectamine® 2000 transfection reagent is preferably 10-30 nM, more preferably 15-25 nM, and even more preferably 20 nM. The transfection time is preferably 36-60 h, more preferably 42-54 h, and even more preferably 48 h. The density of the adipose-derived stem cells is 80-90%. The preferred conditions for the first centrifugation in the four centrifugations are 400-600g for 5-15 min, more preferably 450-550g for 8-12 min, and even more preferably 500g for 10 min. The preferred conditions for the second centrifugation in the four centrifugations are 1800-2200g for 15-25 min, more preferably 1900-2100g for 18-22 min, and even more preferably 2000g for 20 min. The preferred conditions for the third centrifugation of the four centrifugations are 8000-12000g and 50-70min, more preferably 9000-11000g and 55-65min, and even more preferably 10000g and 60min; the preferred conditions for the fourth centrifugation of the four centrifugations are 100000-140000g and 60-80min, more preferably 110000-130000g and 65-75min, and even more preferably 120000g and 70min.
[0024] The present invention also provides miR-137 adipose stem cell exosomes prepared by the preparation method described above.
[0025] The present invention also provides the use of the adipose stem cell exosomes prepared by the preparation method, the adipose stem cell exosomes, the miR-137 adipose stem cell exosomes prepared by the preparation method, or the miR-137 adipose stem cell exosomes in the preparation of a drug for treating central post-stroke pain.
[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0027] Example 1
[0028] A method for preparing exosomes from adipose-derived stem cells is as follows: Adipose tissue was collected from male rats and washed with PBS. The tissue was then mechanically minced and digested with 0.1% w / v collagenase I (Gibco, Darmstadt, Germany) at 37°C for 1 h with intermittent shaking. The digested tissue was washed with high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Merck, Darmstadt, Germany) and then centrifuged at 1000 rpm for 5 min to remove mature adipocytes. Cell pellets were resuspended in an incubator at 37°C with 5% CO2 supplemented with 10% bovine serum and 1% penicillin / streptomycin (PS; Gibco, Darmstadt, Germany). After 48 h of incubation, non-adherent cells were removed, and fresh culture medium was added to the flasks. Adherent cells were passaged with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) containing 0.02% EDTA. The culture medium was changed every 3 days. When the cells reached 90% confluence, the adherent cells were washed with phosphate-buffered saline (PBS), incubated with 1 ml of 0.25% trypsin and 1 mM EDTA at 37°C for 1 min, and the adherent cells were harvested to obtain adipose-derived stem cells.
[0029] To obtain third-generation ADSC cells with a density of 85%, we replaced the medium with DMEM-F12 medium containing 10% EVs-free serum and then collected the cell supernatant. To remove cells or apoptotic bodies, the supernatant was first centrifuged at 500g for 10 min, then filtered through a 0.22 μm filter, centrifuged at 2000g for 20 min, and further centrifuged at 10000g for 60 min to remove cell debris. The remaining supernatant was then ultracentrifuged at 120000g for 70 min to harvest adipose-derived stem cell exosomes.
[0030] Example 2
[0031] A method for preparing exosomes from adipose-derived stem cells is as follows: Adipose tissue was collected from male rats and washed with PBS. The tissue was then mechanically minced and digested with 0.05% w / v collagenase I (Gibco, Darmstadt, Germany) at 35°C for 0.5 h with intermittent shaking. The digested tissue was washed with high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Merck, Darmstadt, Germany) and then centrifuged at 1000 rpm for 5 min to remove mature adipocytes. Cell pellets were resuspended in an incubator at 35°C with 5% CO2 supplemented with 5% bovine serum and 0.5% penicillin / streptomycin (PS; Gibco, Darmstadt, Germany). After 36 h of incubation, non-adherent cells were removed, and fresh culture medium was added to the flask. Adherent cells were passaged with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) containing 0.02% EDTA. The culture medium was changed every 3 days. When the cells reached 85% confluence, the adherent cells were washed with phosphate-buffered saline (PBS), incubated with 1 ml of 0.25% trypsin and 1 mM EDTA at 35°C for 1 min, and the adherent cells were harvested to obtain adipose-derived stem cells.
[0032] To obtain second-generation ADSC cells with a density of 80%, we replaced the medium with DMEM-F12 medium containing 5% EVs-free serum and then collected the cell supernatant. To remove cells or apoptotic bodies, the supernatant was first centrifuged at 400g for 5 min, then filtered through a 0.22 μm filter and centrifuged at 1800g for 15 min, followed by further centrifugation at 8000g for 50 min to remove cell debris. The remaining supernatant was then ultracentrifuged at 100,000g for 60 min to harvest adipose-derived stem cell exosomes.
[0033] Example 3
[0034] A method for preparing exosomes from adipose-derived stem cells is as follows: Adipose tissue was collected from male rats and washed with PBS. The tissue was then mechanically minced and digested with 0.15% w / v collagenase I (Gibco, Darmstadt, Germany) at 40°C for 1.5 h with intermittent shaking. The digested tissue was washed with high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum (FBS; Merck, Darmstadt, Germany) and then centrifuged at 1000 rpm for 5 min to remove mature adipocytes. Cell pellets were resuspended in an incubator at 40°C with 5% CO2 supplemented with 15% bovine serum and 1.5% penicillin / streptomycin (PS; Gibco, Darmstadt, Germany). After 60 h of incubation, non-adherent cells were removed, and fresh culture medium was added to the flasks. Adherent cells were passaged with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) containing 0.02% EDTA. The culture medium was changed every 3 days. When the cells reached 95% confluence, the adherent cells were washed with phosphate-buffered saline (PBS), incubated with 1 ml of 0.25% trypsin and 1 mM EDTA at 40°C for 1 min, and the adherent cells were harvested to obtain adipose-derived stem cells.
[0035] To obtain ADSC cells at a density of 90% in the fourth generation, we replaced the medium with DMEM-F12 containing 15% EVs-free serum and collected the cell supernatant. To remove cells or apoptotic bodies, the supernatant was first centrifuged at 600g for 15 min, then filtered through a 0.22 μm filter, centrifuged at 2200g for 25 min, and further centrifuged at 12000g for 70 min to remove cell debris. The remaining supernatant was then ultracentrifuged at 140000g for 80 min to harvest adipose-derived stem cell exosomes.
[0036] Example 4
[0037] A method for preparing exosomes from miR-137 adipose stem cells is as follows: Adipose tissue was collected from male rats and washed with PBS. The tissue was then mechanically minced and digested with 0.1% w / v collagenase I (Gibco, Darmstadt, Germany) at 37°C for 1 h with intermittent shaking. The digested tissue was washed with high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Merck, Darmstadt, Germany) and then centrifuged at 1000 rpm for 5 min to remove mature adipocytes. Cell pellets were resuspended in an incubator at 37°C with 5% CO2 supplemented with 10% bovine serum and 1% penicillin / streptomycin (PS; Gibco, Darmstadt, Germany). After 48 h of incubation, non-adherent cells were removed, and fresh culture medium was added to the flasks. Adherent cells were passaged with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) containing 0.02% EDTA. The culture medium was changed every 3 days. When the cells reached 90% confluence, the adherent cells were washed with phosphate-buffered saline (PBS), incubated with 1 ml of 0.25% trypsin and 1 mM EDTA at 37°C for 1 min, and the adherent cells were harvested to obtain adipose-derived stem cells.
[0038] Third-generation ADS-C cells with a density of 85% were obtained. miR-137 (SEQ ID No. 1) was transfected into the cells using Lipofectamine® 2000 transfection reagent (Gibco Life Technologies) at a concentration of 20 nM, according to the manufacturer's instructions. Cells were harvested after 48 h, and the medium was replaced with DMEM-F12 medium containing 10% EVs-free serum. The cell supernatant was then collected. To remove cells or apoptotic bodies, the supernatant was centrifuged at 500 g for 10 min, filtered through a 0.22 μm filter, centrifuged at 2000 g for 20 min, and further centrifuged at 10000 g for 60 min to remove cell debris. The remaining supernatant was then ultracentrifuged at 120000 g for 70 min to harvest miR-137 ADS-C exosomes.
[0039] Example 5
[0040] A method for preparing exosomes from miR-137 adipose stem cells is as follows: Adipose tissue was collected from male rats and washed with PBS. The tissue was then mechanically minced and digested with 0.05% w / v collagenase I (Gibco, Darmstadt, Germany) at 35°C for 0.5 h with intermittent shaking. The digested tissue was washed with high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Merck, Darmstadt, Germany) and then centrifuged at 1000 rpm for 5 min to remove mature adipocytes. Cell pellets were resuspended in an incubator at 35°C with 5% CO2 supplemented with 5% bovine serum and 0.5% penicillin / streptomycin (PS; Gibco, Darmstadt, Germany). After 36 h of incubation, non-adherent cells were removed, and fresh culture medium was added to the flask. Adherent cells were passaged with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) containing 0.02% EDTA. The culture medium was changed every 3 days. When the cells reached 85% confluence, the adherent cells were washed with phosphate-buffered saline (PBS), incubated with 1 ml of 0.25% trypsin and 1 mM EDTA at 35°C for 1 min, and the adherent cells were harvested to obtain adipose-derived stem cells.
[0041] Second-generation ADS-137 cells with a density of 80% were obtained and transfected with miR-137 (SEQ ID No. 1) using Lipofectamine® 2000 transfection reagent (Gibco Life Technologies) at a concentration of 10 nM according to the manufacturer's instructions. Cells were harvested after 36 h, and the medium was replaced with DMEM-F12 medium containing 5% EVs-free serum. The cell supernatant was then collected. To remove cells or apoptotic bodies, the supernatant was first centrifuged at 400 g for 5 min, then filtered through a 0.22 μm filter, centrifuged at 1800 g for 15 min, and further centrifuged at 8000 g for 50 min to remove cell debris. The remaining supernatant was then ultracentrifuged at 100,000 g for 60 min to harvest miR-137 ADS-137 exosomes.
[0042] Example 6
[0043] A method for preparing exosomes from miR-137 adipose stem cells is as follows: Adipose tissue was collected from male rats and washed with PBS. The tissue was then mechanically minced and digested with 0.15% w / v collagenase I (Gibco, Darmstadt, Germany) at 40°C for 1.5 h with intermittent shaking. The digested tissue was washed with high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum (FBS; Merck, Darmstadt, Germany) and then centrifuged at 1000 rpm for 5 min to remove mature adipocytes. Cell pellets were resuspended in an incubator at 40°C with 5% CO2 supplemented with 15% bovine serum and 1.5% penicillin / streptomycin (PS; Gibco, Darmstadt, Germany). After 60 h of incubation, non-adherent cells were removed, and fresh culture medium was added to the flasks. Adherent cells were passaged with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) containing 0.02% EDTA. The culture medium was changed every 3 days. When the cells reached 95% confluence, the adherent cells were washed with phosphate-buffered saline (PBS), incubated with 1 ml of 0.25% trypsin and 1 mM EDTA at 40°C for 1 min, and the adherent cells were harvested to obtain adipose-derived stem cells.
[0044] Fourth-generation ADS-C cells with a density of 90% were obtained, and miR-137 (SEQ ID No. 1) was transfected into the cells using Lipofectamine® 2000 transfection reagent (Gibco Life Technologies) at a concentration of 30 nM according to the manufacturer's instructions. Cells were harvested after 60 h, and the medium was replaced with DMEM-F12 medium containing 15% EVs-free serum. The cell supernatant was then collected. To remove cells or apoptotic bodies, the supernatant was first centrifuged at 600 g for 15 min, then filtered through a 0.22 μm filter, centrifuged at 2200 g for 25 min, and further centrifuged at 12000 g for 70 min to remove cell debris. The remaining supernatant was then ultracentrifuged at 140000 g for 80 min to harvest miR-137 ADS-C exosomes.
[0045] Experimental Example 1
[0046] 1. Isolation, culture and identification of ADSCs
[0047] The ADSCs harvested in Example 1 were identified using relevant labeled antibodies by immunofluorescence assay, including CD29, CD44, CD105, CD90, ISO, and vWF (eBioscience, USA). Simultaneously, after culturing in lipid-inducing medium for 21 days, the lipid-inducing conditions were detected using Oil Red O staining (Sigma, USA).
[0048] To quantify and identify the ADSC-Exo prepared in Example 1, nanoparticle tracking analysis was used to detect its concentration and size distribution. Simultaneously, its characteristics were observed by transmission electron microscopy (TEM), and biomarkers were detected by Western blotting.
[0049] The ADSCs from Example 4 were transfected with either miR-137 mimic (nucleotide sequence SEQ ID No. 1: 5′-UAUUGCUUGAGAAUACACGUAG-3′) or NC (nucleotide sequence SEQ ID No. 2: 5′-UUC UCC GAA CGU GUCACG UTT-3′, GENCEFE, Wuxi, China) using Lipofectamine® 2000 transfection reagent (Gibco Life Technologies) at a concentration of 20 nM according to the manufacturer's instructions. Cells were harvested after 48 h, and exosomes were isolated following the method described in Example 4. ADSCs and ADSC-Exo were then validated by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR).
[0050] Experimental results: such as Figure 1 As shown. Obtained from major ADSCs, and by the appearance of lipid droplets after Oil Red O staining ( Figure 1 A) in the study preliminarily demonstrated its ability to differentiate and generate adipate.
[0051] Immunofluorescence results showed that third-generation ADSCs highly expressed CD29, CD44, CD90, and CD105, while showing negative expression for IOS and vWF. Figure 1 (B in the middle).
[0052] Next, the exosomes isolated from these ADSCs were identified. First, the exosomes exhibited a disc-like appearance under a transmission electron microscope. Figure 1 The C in the figure has a size distribution ranging from approximately 50 to 120 nanometers, mainly concentrated around 60 nanometers. Figure 1 (D in the middle).
[0053] Furthermore, ADSC-Exo showed positive expression of TSG101, CD9, and CD63 proteins, but no expression of β-actin. Figure 1(E in the text). These results all indicate that exosomes have been successfully isolated from ADSCs.
[0054] Finally, ADSCs expressing miR-137 (miR-137 mimics) were also cultured, and the high levels of miR-137 in ADSC-Exo were validated. Figure 1 (F in the text).
[0055] 2. Establishment and treatment of CPSP model
[0056] Male SD rats (300-400 g) were fed at room temperature with a 12-hour light-dark cycle and free access to food and water. A central post-stroke pain model was established based on the literature (Wasserman JK, Koeberle PD. Development and characterization of a hemorrhagic rat model of central post-stroke pain.[J]. Neuroscience, 2009.). In short, the pain model was obtained by injecting type IV collagenase (Sigma, USA) into the right posteromedial nucleus (VPM) / posterolateral nucleus (VPL) of the thalamus after anesthesia with 1% isoflurane. The control group (sham group) rats received only 0.5 μL of sterile saline.
[0057] Rats with successfully modeled CPSP were divided into a model group (CPSP), an Exo group, and an Exo-137 group. Exo group CPSP rats were treated 24 hours after injury with an injection of 20 micrograms of ADSC-Exo (Example 1) into the lateral ventricle. Exo group CPSP rats were treated 24 hours after injury with an injection of 20 micrograms of ADSC-Exo (Example 4) overexpressing miR-137 into the lateral ventricle. Healthy rats underwent surgery but received no modeling or treatment, serving as the sham group. To assess the behavior of these rats, two uninformed investigators analyzed mechanical force and heat tolerance on days 0, 7, 14, 21, and 35 post-treatment.
[0058] Mechanical and thermal pain response tests
[0059] (1) Mechanical stimulation pain (Von Frey test)
[0060] Place the animal in a ventilated, transparent plastic cage with a mesh bottom to facilitate stimulation of the animal's paws. Allow the animal to acclimatize to this environment for approximately 15 minutes. Use a set of Von Frey fibers that bend under pressure. Each fiber has a specific bending threshold, expressed in grams (g) or milligrams (mg). Start with the smallest fiber and gradually increase the pressure until the animal responds. Record the animal's responses to the stimulus, including lifting, licking, or shaking its paw. If the animal does not respond to a fiber, test with the next larger fiber. Repeat this process until the smallest fiber that elicits a response is found. The pain threshold can be calculated based on the animal's response to fibers at different pressures.
[0061] (2) Thermal pain response test
[0062] Transfer the animal from the feeding room to the laboratory, weigh and record its weight, and allow it to acclimatize to the laboratory environment for at least half an hour. Set the temperature of the hot plate to match the temperature of the testing chamber and clean the surface of the test plate. Place a single subject animal in the testing chamber and allow it to explore undisturbed for 15 minutes. Remove the animal and clean any urine or feces from the plate surface. Set the hot plate temperature to the test temperature, typically between 50°C and 56°C. Once the preset temperature is reached, place the acclimatized animal on the hot plate. Measure the latency period for the first occurrence of an injury response (e.g., licking paws, raising hind paws, or jumping).
[0063] Experimental results: such as Figure 2 As shown, ADSC-Exo treatment alleviated symptoms in a rat CPSP model. To analyze the function of ADSC-Exo in the CPSP rat model, exosomes were injected into the rats, revealing that the CPSP rats exhibited significant hyperalgesia under mechanical and thermal stimuli one week prior. On day 21, CPSP rats treated with ADSC-exo or ADSC-exo-miR-137 showed significantly improved responses compared to untreated CPSP rats, especially those treated with ADSC-exo-miR-137. These results indicate that ADSC-Exo containing high levels of miR-137 is effective in CPSP model rats.
[0064] As can be seen from the above examples and experimental cases, the present invention uses a rat model of thalamic hemorrhage to discover that ADSC can alleviate CPSP by releasing exosomes miR-137, providing valuable insights into ADSC-exosomes as a treatment for CPSP and its molecular mechanism. Future research can be based on this to develop targeted therapies to improve the quality of life of CPSP patients.
[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing exosomes from adipose-derived stem cells, characterized in that, Includes the following steps: 1) After digesting adipose tissue, centrifuge to obtain cells, incubate the cells, and passage them to obtain adipose stem cells; 2) Adipose-derived stem cells were cultured in DMEM-F12 medium containing 5-15% serum-free EVs. The supernatant was collected by centrifugation, and the supernatant was centrifuged four times to obtain adipose-derived stem cell exosomes.
2. The preparation method according to claim 1, characterized in that, Step 1) The digestion uses collagenase I, the concentration of collagenase I is 0.05~0.15% w / v of adipose tissue, the digestion temperature is 35~40℃, the digestion time is 0.5~1.5h, the incubation temperature is 35~40℃, the incubation time is 36~50h, the passage time is when the cell density reaches 85~95%, and the number of passages is 2~4 times.
3. The preparation method according to claim 1, characterized in that, Step 2) The density of the adipose stem cells is 80-90%. The conditions for the first centrifugation in the four centrifugations are 400-600g and 5-15min, the conditions for the second centrifugation in the four centrifugations are 1800-2200g and 15-25min, the conditions for the third centrifugation in the four centrifugations are 8000-12000g and 50-70min, and the conditions for the fourth centrifugation in the four centrifugations are 100000-140000g and 60-80min.
4. Adipose-derived stem cell exosomes prepared by the preparation method according to any one of claims 1 to 3.
5. A method for preparing exosomes from miR-137 adipose-derived stem cells, characterized in that, Includes the following steps: 1) After digesting adipose tissue, centrifuge to obtain cells, incubate the cells, and passage them to obtain adipose stem cells; 2) Transfect miR-137 into adipose-derived stem cells, culture the transfected adipose-derived stem cells in DMEM-F12 medium with 5-15% serum-free EVs, collect the supernatant by centrifugation, and centrifuge the supernatant four times to obtain miR-137 adipose-derived stem cell exosomes. The nucleotide sequence of miR-137 is shown in SEQ ID No.
1.
6. The preparation method according to claim 5, characterized in that, Step 1) The digestion uses collagenase I, the concentration of collagenase I is 0.05~0.15% w / v of adipose tissue, the digestion temperature is 35~40℃, the digestion time is 0.5~1.5h, the incubation temperature is 35~40℃, the incubation time is 36~50h, the passage time is when the cell density reaches 85~95%, and the number of passages is 2~4 times.
7. The preparation method according to claim 5, characterized in that, Step 2) The transfection uses Lipofectamine® 2000 transfection reagent, the concentration of which is 10~30 nM, and the transfection time is 36~60 h.
8. The preparation method according to claim 5, characterized in that, Step 2) The density of the adipose stem cells is 80-90%. The conditions for the first centrifugation in the four centrifugations are 400-600g and 5-15min, the conditions for the second centrifugation in the four centrifugations are 1800-2200g and 15-25min, the conditions for the third centrifugation in the four centrifugations are 8000-12000g and 50-70min, and the conditions for the fourth centrifugation in the four centrifugations are 100000-140000g and 60-80min.
9. miR-137 adipose stem cell exosomes prepared by the preparation method according to any one of claims 5 to 8.
10. The use of adipose-derived stem cell exosomes prepared by the preparation method according to any one of claims 1 to 3, the adipose-derived stem cell exosomes according to claim 4, the miR-137 adipose-derived stem cell exosomes prepared by the preparation method according to any one of claims 5 to 8, or the miR-137 adipose-derived stem cell exosomes according to claim 9 in the preparation of a drug for treating central post-stroke pain.