CN115364120A - MSC (mesenchymal stem cell) source exosome cultured by hydrogel 3D and application thereof in cerebral ischemia repair - Google Patents

Abstract

The invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to an MSC (mesenchymal stem cell) source exosome cultured by hydrogel 3D and application thereof in cerebral ischemia repair. The invention is researched and proved for the first time that the application of 3D-Exo has the effects of relieving MCAO complications, improving prognosis and the like. Specifically, the 3D-Exo can effectively improve symptoms such as neurologic impairment after MCAO, so that the 3D-Exo is a potential treatment method, can relieve local microenvironment in a brain injury area after MCAO and promote recovery of neurologic function, and has good practical application value.

Description

MSC (mesenchymal stem cell) source exosome cultured by hydrogel 3D and application thereof in cerebral ischemia repair

Technical Field

The invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to an MSC (mesenchymal stem cell) source exosome cultured by hydrogel 3D and application thereof in cerebral ischemia repair.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The stroke is a disease seriously threatening the health of human beings, accounts for the third cause of death worldwide and accounts for 75-80% of the total stroke. After cerebral ischemia, a strong neuroinflammatory response is a major cause of secondary brain injury, and plays an indispensable role in cerebral ischemia-reperfusion injury (I/R). Microglia, which is the only innate immune cell in the Central Nervous System (CNS), responds earliest after brain injury and plays an essential role in regulating the inflammatory environment induced by brain injury.

The mesenchymal stem cell-derived exosome (MSC-Exo) can promote the nerve vessel reconstruction and the recovery of nerve function after brain injury, and has great application prospect. In addition, MSC-Exo has good circulation stability, biocompatibility, biological barrier permeability, low toxicity and low immunogenicity, and these properties mainly depend on various bioactive substances (proteins, miRNAs) encapsulated in exosomes to exert the regulation function among cells. However, the inventor finds that how to greatly improve the generation efficiency of the MSC-Exo is still a difficult problem for researchers.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an MSC source exosome cultured by hydrogel 3D and application thereof in cerebral ischemia repair. According to the invention, the 3D-Exo is extracted by culturing the MSC with GelMA photo-curing hydrogel 3D and is used for treating the mouse I/R, and researches prove that the 3D-Exo can relieve I/R-induced neuroinflammatory reaction, apoptosis and glial scar after MCAO (middle cerebral artery occlusion), and is helpful for relieving MCAO complications and improving prognosis. The present invention has been completed based on the above results.

The technical scheme of the invention is as follows:

the first aspect of the invention provides application of exosome in preparing a product for repairing cerebral ischemia injury.

Wherein the cerebral ischemic injury is specifically cerebral ischemic reperfusion injury, more specifically, in one embodiment of the invention, the cerebral ischemic injury is cerebral ischemic reperfusion injury mediated by Middle Cerebral Artery Occlusion (MCAO).

Specifically, the cerebral ischemic injury repair is characterized in that:

(a) Alleviating MCAO-induced neurological deficit;

(b) Reducing brain tissue damage caused by MCAO;

(c) Reducing MCAO-induced loss of the nissl bodies;

(d) Inhibiting MCAO-induced apoptosis;

(e) Promoting MCAO results in a restorative increase in cerebral blood flow locally to the brain;

(f) Promoting the expression of CD31 and TGF beta 1 in MCAO damaged brain tissues;

(g) Inhibiting the expression of TNF-alpha and IL-6 after MCAO and promoting the expression of IL-10;

(h) Promoting the up-regulated expression of the nerve repair protein;

(i) Promoting the up-regulation expression of the nerve repair related miRNAs;

(j) Promoting angiogenesis of ischemic tissue after MCAO.

The exosome is derived from mesenchymal stem cells; more particularly, the mesenchymal stem cell is a bone marrow mesenchymal stem cell.

The diameter of the exosome is 30-150nm.

Wherein the exosome is derived from mesenchymal stem cells cultured in 2D and/or GelMA hydrogel 3D; further preferably, the exosome is GelMA hydrogel 3D-cultured bone marrow mesenchymal stem cell-derived exosome (3D-MSC-Exo). The GelMA hydrogel 3D is adopted to culture the mesenchymal stem cells, and soluble secretion can generate Exo with enhanced treatment efficacy to regulate paracrine signals of the mesenchymal stem cells, so that angiogenesis, neurogenesis and immunoregulation are enhanced, and nerve function recovery is mediated. In addition, secretion of soluble factors of MSCs is affected by mechanical stretching, thereby remarkably promoting secretion of cytokines, whereas 3D-cultured MSCs secrete more Exo than 2D-cultured or mono-cultured cells.

In a second aspect of the invention, there is provided a product comprising an exosome as described above. More specifically, the exosome is an exosome derived from mesenchymal stem cells cultured in 2D and/or GelMA hydrogel 3D; further preferably, the exosome is a GelMA hydrogel 3D-cultured bone marrow mesenchymal stem cell-derived exosome.

The product has the following effects:

(a) Alleviating MCAO-induced neurological deficit;

(b) Reducing brain tissue damage caused by MCAO;

(c) Reducing MCAO-induced loss of the nissl bodies;

(d) Inhibiting MCAO-induced apoptosis;

(e) Promoting MCAO results in a restorative increase in regional cerebral blood flow in the brain;

(f) Promoting the expression of CD31 and TGF beta 1 in MCAO damaged brain tissues;

(g) Inhibiting the expression of TNF-alpha and IL-6 after MCAO and promoting the expression of IL-10;

(h) Promoting the up-regulated expression of the nerve repair protein;

(i) Promoting the up-regulated expression of the neurorepair-related miRNAs;

(j) Promoting angiogenesis of ischemic tissue after MCAO;

(k) Is used for treating MCAO mediated cerebral ischemia reperfusion injury.

In a third aspect of the present invention, there is provided a method for repairing cerebral ischemic injury, the method comprising: administering to the subject the above-described inhibitory exosomes or the above-described product.

The beneficial effects of one or more of the above technical solutions are as follows:

the technical scheme is researched and confirmed for the first time, 3D-Exo is extracted after MSC is cultured by GelMA hydrogel 3D, the 3D-Exo is injected into MCAO mice intravenously, the neuroinflammation reaction is relieved, the number of new blood vessels is increased, and the neuroprotective effect is good. Therefore, the 3D-Exo is a potential strategy for treating MCAO, can relieve local microenvironment after MCAO and promote nerve function recovery, and has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows MSC culture and MSC-Exo identification in examples of the present invention. (A) 2D-and 3D-MSC morphological images under light microscopy. (B) Live/dead cell staining was performed by calcein-AM/PI. Dead cells were stained with PI (red fluorescence) and live cells were stained with calcein-AM (green fluorescence). (C) SOX2 immunofluorescent staining in 2D-or 3D-MSCs. (D) immunofluorescence staining of Nanog in 2D-or 3D-MSCs. (E) SOX2 representative western blotting strip. (F) quantitative analysis of SOX2 protein expression. (G) Nanog representative western blotting strip. (H) quantitative analysis of Nanog protein expression level. Data are expressed as mean ± Standard Deviation (SD) (n = 3). * P <0.01, p <0.001, post hoc comparisons according to ANOVA and Bonferroni tests.

FIG. 2 shows the extraction and characterization of MSC-Exo in an embodiment of the present invention. (A) 2D-and 3D-Exo extraction scheme. (B) Transmission Electron microscopy morphology of MSC-Exo. (C) MSC-Exo particle size analysis by NanoSight. (D) MSC-Exo positive marker (TSG 101, CD63 and CD 9) and negative marker (Calnexin) protein expression.

FIG. 3 is a graph showing the effect of BV2 on the endocytosis of MSC-Exo and on LPS-induced inflammation in vitro in the present example. (A) BV2 cells take PKH67 labeled MSC-Exo confocal microscopy images. (B) Representative western blotting bands for TNF-alpha, IL-10, beta-actin. (C) quantitative analysis of TNF-alpha protein expression. (D) quantitative analysis of IL-6 protein expression. Data are presented as mean ± standard deviation (n = 3). * p <0.05, p <0.01, p <0.001, post-hoc comparisons were performed according to ANOVA and Bonferroni test.

FIG. 4 shows the in vivo BBB crossing efficiency of MSC-Exo in the example of the present invention. (A) MSC-Exo crossing BBB efficiency representative immunofluorescence images. DAPI: blue, exo: and (4) green. (B) 2D-or 3D-Exo distribution in mice.

FIG. 5 is a graph showing the effect of MSC-Exo on MCAO-induced neuronal pathological changes and neurological function recovery in an example of the present invention. (A) Representative mouse brain tissue TTC stained coronal sections per group. And (B) quantitatively analyzing the cerebral infarction volume of each group of mice. (C) post MCAO neurological deficit score. (D) representative HE and Nissl stained images after MCAO. Data are presented as mean ± standard deviation (n = 3). * p <0.05, p <0.001, p <0.0001, post-hoc comparisons were performed according to ANOVA and Bonferroni assays.

FIG. 6 is a graph of the effect of MSC-Exo on the local blood flow (CBF) in brain tissue after MCAO in an example of the present invention. (a) CBF representative images in each group based on LSFI. (B) 0s, 10s and 60s postcerebral local CBF change statistical analysis.

FIG. 7 shows the effect of MSC-Exo on angiogenesis after MCAO in an example of the present invention. Representative immunofluorescent staining images for each group of TGF β 1, CD 31. DAPI: blue, TGF β 1: green, CD31: red.

FIG. 8 shows the neuroinflammatory response and angiogenesis effects of MSC-Exo on MCAO in brain tissue in an example of the present invention. (A) Representative western blotting bands for TNF-alpha, IL-10, beta-actin. (B) quantitative analysis of TNF-alpha protein expression. (C) quantitative analysis of IL-10 protein expression. (D) Typical western blotting bands for TGF-beta 1, CD31, beta-actin. (E) quantitative analysis of TGF beta 1 protein expression. (F) quantitative analysis of CD31 protein expression. Data are presented as mean ± standard deviation (n = 3). * p <0.05, p <0.01, p <0.001, p <0.0001, post hoc comparisons were performed according to ANOVA and Bonferroni assays.

FIG. 9 shows the mass spectrometric and bioinformatic analysis of MSC-Exo proteins in examples of the present invention. (A) GO enrichment analysis histogram of differentially expressed proteins. (B) KEGG enrichment analysis the first 20 differentially expressed protein bubble profiles.

FIG. 10 shows the sequencing and bioinformatic analysis of MSC-Exo internal miRNAs in examples of the present invention. (A) differential analysis of the GO target genes of the miRNAs. (B) enrichment analysis of differentially expressed genes of the first 20 KEGG pathways.

FIG. 11 is a Pearson correlation heatmap of the molecular differences of proteins between 2D-Exo and 3D-Exo in examples of the present invention.

FIG. 12 is a graph showing the cluster analysis of Differentially Expressed Proteins (DEPs) in 2D-Exo and 3D-Exo in examples of the present invention.

FIG. 13 is a graph showing the clustering analysis of differentially expressed miRNAs in 2D-Exo and 3D-Exo in the present example.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the following detailed description is given with reference to specific embodiments.

As shown above, how to greatly improve the generation efficiency of MSC-Exo is still a difficult problem for researchers. And whether exosome extracted from mesenchymal stem cells plays a role in physiological pathological change caused by MCAO and a related action mechanism are not researched yet.

The inventor researches and discovers that the three-dimensional (3D) culture of the MSC can not only simulate the in-vivo growth environment of cells to the maximum extent, but also obviously improve the production efficiency of exosomes (3D-Exo) compared with the traditional two-dimensional (2D) culture. Here, we cultured MSCs in 3D using methacrylyl gelatin (GelMA) as a carrier, and 3D cell culture allowed cells to interact with extracellular matrix (ECM) and activate a stronger cell signaling network than 2D cell culture. The hydrogel can simulate the water environment of human tissues, has excellent biocompatibility and higher biodegradability, and is an ideal biomaterial for tissue engineering and 3D cell culture at present. After MSC is cultured in GelMA hydrogel under the 3D environment, 3D-Exo is extracted to study the treatment effect of MSC on cerebral ischemia. In vitro experiments prove that MSC-Exo can be endocytosed by microglia to relieve neuroinflammatory reaction. In vivo experiments show that MSC-Exo injected into MCAO mice through tail veins can efficiently permeate BBB and be enriched in cerebral ischemia areas to play a role. After being endocytosed by target cells in vivo, the MSC-Exo plays a powerful neuroprotective role by alleviating neuroinflammatory reactions and promoting angiogenesis. Furthermore, 3D-Exo showed higher neuroprotective efficiency compared to 2D-Exo.

In a typical embodiment of the invention, the application of the exosome in preparing a product for repairing the cerebral ischemia injury is provided.

In a further embodiment of the invention, the cerebral ischemic injury is in particular a cerebral ischemic reperfusion injury, more in particular in one embodiment of the invention, the cerebral ischemic injury is a cerebral ischemic reperfusion injury mediated by Middle Cerebral Artery Occlusion (MCAO).

In another embodiment of the present invention, the cerebral ischemic injury repair is characterized by:

(a) Reducing MCAO-induced neurological deficit;

(b) Reducing brain tissue damage caused by MCAO;

(c) Reducing MCAO-induced loss of the nissl bodies;

(d) Inhibiting MCAO-induced apoptosis;

(e) Promoting MCAO results in a restorative increase in regional cerebral blood flow in the brain;

(f) Promoting the expression of CD31 and TGF beta 1 in MCAO damaged brain tissues;

(g) Inhibiting the expression of TNF-alpha and IL-6 after MCAO and promoting the expression of IL-10;

(h) Promoting the up-regulated expression of the nerve repair protein;

(i) Promoting the up-regulated expression of the neurorepair-related miRNAs;

(j) Promoting angiogenesis of ischemic tissue after MCAO.

The exosome is derived from mesenchymal stem cells; more particularly, the mesenchymal stem cell is a bone marrow mesenchymal stem cell.

The diameter of the exosome is 30-150nm.

In yet another embodiment of the present invention, the exosome is a bone marrow mesenchymal stem cell-derived exosome cultured in 2D and/or GelMA hydrogel 3D; further preferably, the exosome is GelMA hydrogel 3D-cultured bone marrow mesenchymal stem cell-derived exosome (3D-MSC-Exo). The bone marrow mesenchymal stem cells cultured by GelMA hydrogel 3D can generate Exo with enhanced treatment efficacy through soluble secretion to regulate paracrine signals of the bone marrow mesenchymal stem cells, enhance angiogenesis, neurogenesis and immunoregulation, and mediate nerve function recovery. In addition, secretion of soluble factors of MSCs is affected by mechanical stretching, thereby remarkably promoting secretion of cytokines, whereas 3D-cultured MSCs secrete more Exo than 2D-cultured or mono-cultured cells.

In a further embodiment of the invention, there is provided a product comprising an exosome as described above. More specifically, the exosome is an exosome derived from mesenchymal stem cells cultured in 2D and/or GelMA hydrogel 3D; further preferably, the exosome is a GelMA hydrogel 3D-cultured bone marrow mesenchymal stem cell-derived exosome.

The product has the following effects:

(a) Alleviating MCAO-induced neurological deficit;

(b) Reducing brain tissue damage caused by MCAO;

(c) Reducing MCAO-induced loss of the nissl bodies;

(d) Inhibiting MCAO-induced apoptosis;

(e) Promoting MCAO results in a restorative increase in regional cerebral blood flow in the brain;

(f) Promoting the expression of CD31 and TGF beta 1 in MCAO damaged brain tissues;

(g) Inhibiting the expression of TNF-alpha and IL-6 after MCAO and promoting the expression of IL-10;

(h) Promoting the up-regulated expression of the nerve repair protein;

(i) Promoting the up-regulated expression of the neurorepair-related miRNAs;

(j) Promoting angiogenesis of ischemic tissue after MCAO;

(k) Is used for treating MCAO mediated cerebral ischemia reperfusion injury.

The product may be a pharmaceutical or a test agent for use in basic research and thus may be used to construct relevant cellular or animal models.

When the product is a pharmaceutical, the pharmaceutical may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, emulsifier, suspending agent, stabilizer, preservative, excipient, filler, coagulant and blender, surfactant, dispersing agent or antifoaming agent.

The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a virus, a microcapsule, a liposome, a nanoparticle, or a polymer, and any combination thereof. The delivery vehicle for the pharmaceutically acceptable carrier can be a liposome, biocompatible polymer (including natural and synthetic polymers), lipoprotein, polypeptide, polysaccharide, lipopolysaccharide, artificial viral envelope, inorganic (including metal) particle, and bacterial or viral (e.g., baculovirus, adenovirus and retrovirus), phage, cosmid, or plasmid vector.

The medicament can also be combined with other medicaments for preventing and/or treating cerebral ischemic injury, and other preventive and/or therapeutic compounds can be simultaneously administered with the main active ingredients, even in the same composition.

The medicament may also be administered separately to other prophylactic and/or therapeutic compounds, in a separate composition or in a different dosage form than the main active ingredient. Some of the doses of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the agents of the invention may be adjusted during the course of treatment depending on the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

The medicament of the present invention can be administered into the body by a known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In another embodiment of the present invention, there is provided a method for repairing cerebral ischemic injury, the method comprising: administering to the subject the above-described inhibitory exosomes or the above-described product.

The subject of the present invention refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. All the starting materials used in the examples are, unless otherwise specified, commercially available.

Examples

1. Experimental animals: adult (6-8 weeks) male wild-type C57/BL6J mice were selected as experimental subjects purchased from Jinnanpo animal Co., ltd (accession number: SYXK-LU2020 0022), and were kept in SPF-grade animal rooms of the first subsidiary hospital of Shandong first medical university 7 days before the experiment in natural light/dark (. Apprxeq.12 h-12 h) cycles. The animal protocol was approved by the institutional animal care and use committee of the first medical university in shandong, according to the guidelines outlined in the national institutes of health, animal care and use guidelines. The personnel studying the animal models received systematic training as specified by the institutional animal Care and use Commission guidelines (IACUC).

2.MSC 2D culture: 1 newborn SD suckling mouse is sacrificed randomly and soaked in 75% ethanol by volume fraction for 12min for disinfection. Separating femur and tibia from each other under aseptic condition, cutting bone ends, washing marrow cavity with sterile PBS, filtering with 70 μm cell filter screen, and centrifuging. Taking the sediment, using a DMEM medium containing l0% fetal calf serum and 1% penicillin-streptomycin for resuspension, then inoculating the sediment into a cell culture bottle, and replacing the solution after 72 hours to remove the non-adherent cells. Passage was performed when the cells grew to 90% confluence. MSCs after passage 3 (P3) were taken for this study.

3, culturing MSC 3D: the lyophilized GelMA-60 was dissolved in Phosphate Buffered Saline (PBS) containing the photoinitiator phenyllithium-2,4,6-trimethylbenzoylphosphonate (LAP) to make a 5% GelMA solution. The above MSC cells were collected and resuspended in 5% GelMA solution to form bio-ink with cell density of about 1.5X 10 ⁶ mL, add cell solution to the immobilization ring. Curing for 10s by a timing blue light source, adding a complete culture medium, culturing for 5min in a cell culture box at 37 ℃, pouring the culture medium, and adding a brand-new culture medium for continuous culture.

4, collection of MSC-Exo: and (3) taking 3-5 generation MSC, washing the cells for 2 times by using sterile PBS when the cells are fused to 80%, and then continuously culturing the cells for 48 hours by using a serum-free DMEM medium. When the cell density reached 80%, the medium was collected. Centrifuging at 2000g for 5min at 4 deg.C; centrifuging, taking the supernatant, placing the supernatant in another centrifuge tube, centrifuging at 4 ℃ and 3500g for 15min, continuously taking the supernatant, placing the supernatant in a new centrifuge tube, centrifuging at 4 ℃ and 10000g for 30min, taking the supernatant, sucking the supernatant, transferring the supernatant into an ultra-high speed centrifuge tube, using a pipette gun to balance with 1 XPBS buffer solution on a balance capable of accurately achieving 0.001g, placing the balance into an ultra-high speed centrifuge, centrifuging at 4 ℃ and 120000g for 140min at ultra-high speed, discarding the supernatant after resuspending the precipitate with 1 XPBS buffer solution, filtering the precipitate with a 0.22um sterile filter, sucking the precipitate into a centrifuge tube matched with the ultra-high speed centrifuge again, centrifuging at 4 ℃ and 120000g for 70min at ultra-high speed, discarding the supernatant to obtain a centrifuge tube bottom exosome, carefully blowing the bottom of the centrifuge tube with 100uL of 1 XPBS solution, sucking the supernatant into a 0.5mL centrifuge tube, using a pipette gun to blow the precipitate gently to dissolve the exosome completely, and determining the exosome concentration by a bisinching method, wherein the bisinchoninic acid (BCA) protein method is used for storage at-80 ℃.3D-MSC-Exo was collected as above, and the hydrogel was removed by filtration when the supernatant was collected.

5, identifying MSC-Exo electron microscope: identifying morphology by using a transmission electron microscope, dripping 8 mu L of MSC-Exo suspension onto a carrier copper mesh (220 meshes), standing for 2min, dripping 8 mu L of 1% phosphotungstic acid negative dye solution onto the copper mesh after the filter paper sucks the periphery, re-dyeing at room temperature for 2min, and baking the copper mesh under an incandescent lamp for about 10min. The 2D-MSC-Exo and the 3D-MSC-Exo were observed under a transmission electron microscope and photographed.

MSC-Exo surface marker identification: detecting the surface specificity positive marker protein of the MSC-Exo by using Western blotting, adding fresh MSC-Exo into RIPA for lysis, then, quantifying the protein concentration by using a BCA method, and detecting the expression of CD63, CD9 and TSG101 by using conventional Western blotting. Negative markers were quantified with Calnexin.

MSC-Exo particle size analysis: and (3) identifying the particle size of the MSC-Exo by using a qNano instrument, diluting the MSC-Exo by using PBS according to a ratio of 1. qNano instrument parameters are as follows: the voltage of 0.76V, the current of 125nA, and the distance directly between the nanopore arms were set to 47.12mm. The calibration process is performed using the software ics.3.3.2.2000.

8. Live/dead cell staining: the MSC viability was determined using a live/dead cytotoxicity kit (KeyGEN, china). The reagent stock solutions of calcein AM and PI were equilibrated at room temperature for 30min to prepare working solutions of the desired concentrations. The 2D-or 3D-MSCs were washed with PBS, working solution was added in sufficient quantity to ensure that the cells were covered, and then incubated at room temperature for 45min. Cell survival and proliferation were observed under a confocal laser microscope (TCS SP8, germany, come).

MSC-Exo fluorescent labeling: MSC-Exo was labeled using the PKH-67 fluorescent cell linker kit. Briefly, MSC-Exo was added to dilute solution C (CC), then PKH-67 dye was added to CC and incubated for 4min. After termination of the reaction by addition of BSA, MSC-Exo was suspended in PBS and free dye was removed at 200,000 Xg for 2h for subsequent experiments.

MSC-Exo and BV2 cells Co-incubation: BV2 cells were seeded in DMEM medium containing 5% inactivated fetal calf serum at 37 ℃ with 5% CO ₂ Culturing in an incubator. Cells were cultured in the whole medium containing 100ng/mL lipopolysaccharide for 24h and replaced with new medium. Then, 10ug of PKH-67 labeled MSC-Exo (2D or 3D) was added to each well and cultured for 24h and photographed under a fluorescence confocal microscope to observe the endocytosis of MSC-Exo by BV2 cells.

MSC-Exo protein marker-free quantification: proteins in MSC-Exo were extracted and quantified by BCA method, and 20. Mu.g of the sample was subjected to 12-vol SDS-PAGE. After electrophoresis, the gel was removed and stained with Coomassie Brilliant blue (R-250). The peptide fragments were collected after 4h of trypsin degradation and quantified (OD 280). The samples were then separated using a nano-elution system with a nano-liter flow rate and subjected to mass spectrometry analysis for 120 min. The mass-to-charge ratios of the polypeptides and polypeptide fragments were collected as follows: 10 fragment modes after each full scan (MS 2 scan), isolation window 2m/z, secondary mass spectral resolution 17, 500, MS1, secondary maximum IT 45MS, normalized collision energy 27eV.

Sequencing and generating information analysis of miRNAs in MSC-Exo: total RNA was extracted from MSC-Exo and RNA of different fragment sizes was separated using PAGE. The 18-30nt band was excised and the small RNA was recovered. The 3 'and 5' ligation systems were prepared by homogeneous mixing, centrifugation, and then ligated at the appropriate time and temperature. RNA preparation, reverse transcription and PCR were performed. The PCR product was separated by PAGE and dissolved in ethidium bromide to complete the library construction. The 49 nt sequences obtained were sequenced using the Hiseq of Illumina or the MGI platform of BGI and authentic target sequences were obtained by filtration. And obtaining the information of each component in the sample and the expression level by classifying and labeling the target sequence. Prediction of new miRNAs on small RNA fragments was not annotated. According to GO annotation results and official classification, the target genes of the differential miRNAs are subjected to functional classification, and meanwhile, a phyper function in R software is used for enrichment analysis. And (3) carrying out biological pathway classification on the differential miRNAs target genes, and simultaneously carrying out enrichment analysis by using a phyper function in R software.

13. Establishing a mouse MCAO model: the MCAO model was created by a wire-embolisation method, and after anaesthesia with isoflurane, a longitudinal incision was made in the neck, exposing the neck artery under a microscope. After ligation of the external carotid artery, the internal carotid artery and the common carotid artery were temporarily occluded with an artery clamp, and the external carotid artery was inserted with a suture (Guangzhou Jialing biotechnology Co., ltd.; model: L1800) and gently inserted into the middle cerebral artery. After 1.5h of ischemia, the reperfusion process was initiated by removing the wire plug in the external carotid artery after re-anaesthetising the mice. In the sham group, the procedure was as before, but the wire plug was not inserted into the internal carotid artery, but was left in the external carotid artery for 1.5h. After surgery, mice were raised in individual cages and water and food were available ad libitum.

14. Experiment design: in this study, mice were randomized into 4 groups: (1) sham group, (2) MCAO group, (3) MCAO +2D-Exo group, and (4) MCAO +3D-Exo group. MSC-Exo (100. Mu.g per mouse, volume 100. Mu.L) was administered by tail vein injection for 3 consecutive days.

15. Scoring for nerve injury: at the corresponding time points, mice were scored for loss of neural function according to Longa method. The Longa score was made from 0 to 4 points, with 0 being the lightest and 4 being the heaviest.

16. Extracting a mouse brain tissue specimen: after a mouse is anesthetized by isoflurane, the chest cavity of the mouse is opened and the heart is exposed, a 20-gauge puncture needle is inserted into the left ventricle and points to the direction of the ascending aortic arch, 250mL of normal saline is firstly and quickly filled, and a small opening is cut in the right auricle, so that diluted blood flows out. And slowly infusing 4% paraformaldehyde, gradually whitening the sternum and liver of the mouse after about 20min, opening the whole skull after the limbs are stiff, completely taking out the brain, soaking the brain in a fresh formaldehyde solution, fixing for 48h, and carrying out paraffin embedding and slicing in a pathology department, wherein the slice thickness is 5 mu m. Sections were subjected to HE staining and under-mirror MCAO.

17. Hematoxylin-eosin (HE) staining: after the spinal cord tissue is fixed for 24 hours by 40g/L paraformaldehyde solution, the spinal cord tissue is subjected to gradient dehydration, paraffin embedding and fixing, and is sliced by a slicer, the thickness of the spinal cord tissue is 5-10 mu m, and the slice is kept to be vertical to the long axis of the spinal cord. The slices are dewaxed and rehydrated conventionally, stained with hematoxylin for 5min, subjected to color separation by 0.5% hydrochloric acid ethanol and water washing and bluing, stained with eosin for 30s, transparent with xylene, sealed with neutral gum, and observed under a microscope for spinal cord tissue morphology and photographed.

18. And (3) Nie dyeing: paraffin sections are dewaxed, and then are degreased by alcohol step by step and washed by distilled water. Dyeing in 1% tar violet solution, dyeing in 37 deg.C incubator for 20min, washing with distilled water, separating color with 95% alcohol, and examining under microscope until the neuron Neisseria is clear. Dehydrated by absolute alcohol, transparent xylene and sealed by neutral gum.

19.2,3,5-Triphenyltetrazolium chloride (TTC) staining and cerebral infarction volume measurement rats were sacrificed after intraperitoneal anesthesia with lethal doses of chloral hydrate at the respective time points. Completely taking out rat brain, freezing in a refrigerator at-20 deg.C for 20min, and slicing brain tissue layer by layer from front to back with blade to obtain slices with thickness of about 2mm, and cutting into 6 slices. Then, the brain tissue slice group was immersed in a 2.0% TTC solution preheated to 37 ℃ in advance, incubated in a water bath at 37 ℃ in the dark for 30min, observed and photographed. The normal brain tissue is stained bright red, and the brain tissue in the area of ischemic infarction is wax white. After fixation in 4% formalin for 24h, the percent by volume of cerebral infarction was analyzed by Image J software.

Tunel staining: dewaxing paraffin section, dropping 20 ug/mL protease K without DNase, standing at 37 deg.C for 30min, washing with PBS for 3 min/times × 3 times, 3%H ₂ O ₂ Incubating at room temperature for 20min, washing with PBS for 3 min/times multiplied by 3 times, dripping 50 mu L of TUNEL detection solution, incubating at 37 ℃ in a dark place for 90min, washing with PBS for 3min, dripping a labeling reaction termination solution, incubating at room temperature in a wet place for 10min, washing with PBS for 3 min/times multiplied by 3 times, dripping DAB developing solution for incubating at room temperature for 10min, washing with PBS for 3 min/times multiplied by 3 times, counterstaining with hematoxylin, washing with PBS for 3 min/times multiplied by 3 times, and sealing with gum cover. Analyzing results and taking pictures under a fluorescence microscope and a photographic system, taking brownish yellow particles in cell nuclei as TUNEL positive marked cells, taking 5 visual fields for each section, respectively counting apoptosis positive cells and total number of cells in an outer nuclear layer, and reflecting the apoptosis condition by an apoptosis index.

21. And (3) immunofluorescence staining: after antigen retrieval, the tissue sections were incubated with dropwise BSA for 30min. TGF beta 1 and CD31 antibodies were incubated overnight at 4 ℃ and then the corresponding secondary antibodies were added and incubated for 50min at room temperature in the dark. DAPI was added dropwise to the sections, and the sections were dried and incubated at room temperature for 10min. Finally, the sections were sealed with an anti-fluorescence quencher.

Western blotting: proteins were extracted from cells or tissue homogenates and assayed using the BCA protein assay kit. Protein samples were then separated on SDS-PAGE gels and then transferred to PVDF membranes. Incubate with primary antibody on a shaker overnight at 4 ℃. The fluorescently bound secondary antibody was incubated for 1h at room temperature and then washed with TBST solution. The PVDF membrane was washed 3 times with TBST solution for 5min each time. Observing an immunoreaction zone for 3-5min by using an Enhanced Chemiluminescence (ECL) reagent, developing by using the ECL luminescence reagent, carrying out development detection by using an Image J gel Image system, analyzing gray values of a target strip and an internal reference, calculating a ratio of a target protein to the internal reference beta-actin, and analyzing the relative protein amount.

23. Statistical analysis: data analysis was performed using MATLAB/GraphPad Prism software (version 8.3). Data are expressed as means ± standard deviation, differences between groups were assessed using one-way anova, post hoc comparisons were performed using Bonferroni test. Statistical significance criteria were set at p <0.05 (. Sup.p <0.05,. Sup.p <0.01,. Sup.p <0.001, and. Sup.p < 0.0001).

The experimental results are as follows:

2D and 3D culture of MSC and identification of MSC-Exo

In this study, we cultured MSCs in conventional culture flasks (2D) and photo-cured GelMA hydrogel (3D), respectively. As shown in fig. 1A, MSCs in 2D culture environment grew flat at the bottom of the flask, whereas MSCs in hydrogel 3D culture were smaller and tightly connected. The cytotoxicity of the hydrogels on MSCs was detected by live/dead cell staining. The results show that: the hydrogel cultured MSCs grew well with few dead cells seen (fig. 1B). In addition, immunofluorescence results show: the cell dryness indicators (SOX 2 and Nanog) fluorescence intensity were increased in 3D-MSC compared to 2D-MSC (FIGS. 1C, 1D). Western blotting results show that: SOX2 and Nanog protein expression were increased 3.46-fold and 3.3-fold in the 3D-MSC group, respectively, compared to the 2D-MSC group (FIGS. 1E-1H).

MSC-Exo extraction and characterization

2D-or 3D-MSC supernatants were collected and MSC-Exo was extracted by differential centrifugation (FIG. 2A). TEM results show that: MSC-Exo is a disk-like bilayer membrane structure with diameters between 30-150nm (FIG. 2B). NanoSight analysis showed: the MSC-Exo mean particle size was about 120nm (FIG. 2C). Western blotting results show that: TSG101 and CD9 were highly enriched in MSC-Exo, while Calnexin (endoplasmic reticulum stress protein) was barely detectable (FIG. 2D).

BV2 on MSC-Exo endocytosis and its effects on inflammation

MSC-Exo can exert a regulatory effect only after being endocytosed by target cells. The endocytosis efficiency of the target cells was verified by incubating 2D-or 3D-Exo (PKH-67 labeled) with BV2 cells. The immunofluorescence results show that: PKH-67 labeled MSC-Exo was endocytosed by BV2 cells and 3D-Exo was endocytosed more efficiently (FIG. 3A). MSC-Exo can play a role in neuroprotection by regulating and controlling a brain injury induced immune inflammation microenvironment. TNF- α expression was reduced 0.57-fold and 0.3-fold in 2D-and 3D-Exo groups, respectively, compared to sham group in LPS-induced BV2 inflammatory model. While IL-10 expression was increased by 1.96-fold and 1.46-fold, respectively (FIGS. 3B-3D).

4.3D-Exo ability to target ischemic areas of the brain

To evaluate the efficiency of MSC-Exo in targeting cerebral ischemic area after penetrating BBB, 100 μ g of PKH-67 labeled 2D-or 3D-Exo was injected into MCAO mice via tail vein, and brain tissue was taken out 12h after immunofluorescence assay, and the results showed: 3D-Exo aggregated more efficiently in the ischemic areas of the brain than 2D-Exo (FIG. 4A). In order to observe the distribution of the MSC-Exo in the body, after 12 hours of MSC-Exo injection, the brain, heart, liver, lung, kidney and spleen of the animal are taken out, and the fluorescence intensity of PKH-67 is detected by a fluorescence imaging system. The results show that: after 3D-Exo injection, aggregation in liver and kidney was significantly reduced and enrichment in ischemic brain tissue was significantly increased, compared to the 2D-Exo group, confirming that 3D-Exo could significantly enhance its targeting ability to ischemic brain regions (fig. 4B).

Effect of MSC-Exo on alleviation of neurological deficit and neuropathological injury

MSC-Exo can reduce the cerebral infarction volume of mice after MCAO, and the TTC result shows that: compared with the MCAO group, the cerebral infarction volume of mice in the 2D-and 3D-Exo groups was reduced by 0.53-fold and 0.23-fold respectively (FIGS. 5A and 5B), and the neurological score was improved by 0.64-fold and 0.41-fold respectively (FIG. 5C). Compared to the MCAO group, 3D-Exo treatment significantly reduced pathological damage, increased Nissl body numbers (fig. 5D).

6, the influence of MSC-Exo on the local cerebral blood flow of MCAO hindbrain

Mouse bilateral cerebral cortex local Cerebral Blood Flow (CBF) was detected using laser speckle blood flow imaging (LSF). The results show that: after MCAO, CBF was dramatically reduced in local brain tissue in the injured side (right) compared to the healthy side (left). Compared to the left, the local brain tissue CBF was significantly increased in the right side of the 2D-and 3D-Exo groups, and the CBF was more significantly increased in the 3D-Exo group than in the contralateral side (FIG. 6A). FIG. 6B shows the statistics of CBF in mice at 0s, 30s, 60 s. The immunofluorescence results show that: compared with the MCAO group, the expression of CD31 and TGF beta 1 is obviously increased in the 2D-and 3D-Exo groups, and the increase in the 3D-Exo group is more obvious (figure 7).

Effect of MSC-Exo on inflammatory response after cerebral ischemia reperfusion injury

In order to further verify the neuroinflammatory response and the angiogenesis influence of 3D-Exo on the ischemic tissues after MCAO of mice, the expression of TNF-alpha, IL-10, CD31 and TGF beta 1 in brain tissues is detected by using western blotting. The results show that: TNF- α expression levels were reduced by 0.5-fold and IL-10 levels were increased by 2.53-fold in the 3D-Exo group compared to the MCAO group (FIGS. 8A-8C). In the 3D-Exo group, the levels of CD31 and TGF-beta 1 were increased 4.32-fold and 2.09-fold, respectively, as compared with the MCAO group (FIGS. 8D-8F). 3D-Exo is shown to be more efficient in relieving neuroinflammatory reaction of ischemic brain tissues and promoting angiogenesis.

MSC-Exo protein mass spectrometry sequencing and bioinformatics analysis

3D-Exo has stronger neuroprotective effect and is related to the fact that the 3D-Exo is rich in biological protective factors related to nerve repair. In this study, we used protein mass spectrometry sequencing to detect protein molecular differences within 2D-and 3D-Exo. 3 samples were taken for testing and the Pearson correlation heatmap between samples is shown in FIG. 11. The quantitative proteomics results show that: compared with 2D-Exo, 19 proteins in 3D-Exo are up-regulated, and 33 proteins are down-regulated. Clustering analysis of Differentially Expressed Proteins (DEPs) in 2D-and 3D-Exo is shown in FIG. 12. GO analysis of DEPs showed: DEPs were significantly enriched in cell adhesion regulation and protein heterodimer activity (fig. 9A). KEGG analysis results show: DEPs are mainly concentrated in necrosis, apoptosis, and other related signaling pathways (fig. 9B).

9 sequencing and bioinformatics analysis of mRNAs in MSC-Exo

Changes in the microenvironment of the secreting cells can lead to changes in proteins and miRNAs in MSC-Exo. We used the limma R software package for transcriptome sequencing and differential expression analysis for 2D and 3D-Exo, respectively. Clustering analysis of differentially expressed miRNAs is shown in fig. 13. The RNAhybrid, miRBase and TargetScan were used to predict target genes that differentially express mRNAs. The accumulation of GO function in predicted target genes was shown to be mainly enriched in neuron-synaptic connections and dependence on ATP activity (fig. 10A). The related differential regulatory genes were analyzed by KEGG enrichment analysis to identify the top 20 pathways, and the top 3 KEGG pathways were significantly enriched in PI3K-Akt signaling pathway, calcium regulatory signaling pathway, and MAPK signaling pathway (fig. 10B).

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The exosome is applied to preparing a product for repairing cerebral ischemia injury; wherein the exosome is a mesenchymal stem cell-derived exosome; the cerebral ischemia injury is cerebral ischemia reperfusion injury.

2. The use of claim 1, wherein the cerebral ischemic injury is cerebral ischemic reperfusion injury mediated by Middle Cerebral Artery Occlusion (MCAO).

3. The use of claim 1, wherein the repair of cerebral ischemic injury is characterized by:

(a) Alleviating MCAO-induced neurological deficit;

(b) Reducing brain tissue damage caused by MCAO;

(c) Reducing MCAO-induced loss of nissl bodies;

(d) Inhibiting MCAO-induced apoptosis;

(e) Promoting MCAO results in a restorative increase in cerebral blood flow locally to the brain;

(f) Promoting the expression of CD31 and TGF beta 1 in MCAO damaged brain tissues;

(g) Inhibiting the expression of TNF-alpha and IL-6 after MCAO and promoting the expression of IL-10;

(h) Promoting the up-regulated expression of the nerve repair protein;

(i) Promoting the up-regulated expression of the neurorepair-related miRNAs;

(j) Promoting angiogenesis of ischemic tissue after MCAO.

4. The use of claim 1, wherein the mesenchymal stem cell is a bone marrow mesenchymal stem cell.

5. The use of claim 1, wherein the exosomes are 2D cultured and/or GelMA hydrogel 3D cultured bone marrow mesenchymal stem cell-derived exosomes.

6. The use of claim 1, wherein the exosomes are GelMA hydrogel 3D cultured mesenchymal stem cell-derived exosomes.

7. A product, wherein said product comprises exosomes; the exosome is derived from bone marrow mesenchymal stem cells cultured in a 2D mode and/or a GelMA hydrogel 3D mode.

8. The product of claim 7, wherein the exosomes are GelMA hydrogel 3D-cultured bone marrow mesenchymal stem cell-derived exosomes.

9. The product according to claim 7 or 8, characterized in that it has the following effects:

(a) Alleviating MCAO-induced neurological deficit;

(b) Reducing brain tissue damage caused by MCAO;

(c) Reducing MCAO-induced loss of the nissl bodies;

(d) Inhibiting MCAO-induced apoptosis;

(e) Promoting MCAO results in a restorative increase in regional cerebral blood flow in the brain;

(f) Promoting the expression of CD31 and TGF beta 1 in MCAO damaged brain tissue;

(g) Inhibiting the expression of TNF-alpha and IL-6 after MCAO and promoting the expression of IL-10;

(h) Promoting the up-regulated expression of the nerve repair protein;

(i) Promoting the up-regulated expression of the neurorepair-related miRNAs;

(j) Promoting angiogenesis of ischemic tissue after MCAO;

(k) Is used for treating MCAO mediated cerebral ischemia reperfusion injury.

10. The product of claim 9, wherein the product is a pharmaceutical or a test agent for use in basic research.

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