Use of rvg-exo-mir-3059-5p in treating stroke

Abstract

The application discloses application of RVG-Exo-miR-3059-5 in treating cerebral stroke. The RVG-Exo-miR-3059-5 is prepared by using gene engineering technology to perform brain targeting modification on EPCs-Exo and load miR-3059-5p drugs. The experiment proves that the RVG-Exo-miR-3059-5 can effectively target brain tissues, can prevent or treat cerebral stroke, and can improve the prognosis of cerebral stroke patients.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to the use of RVG-Exo-miR-3059-5p in treating cerebral stroke. BACKGROUND

[0002] Gene drug therapy represented by miRNA is a new disease treatment strategy, which can achieve precise and personalized treatment of diseases by specifically regulating the expression of target genes. However, due to the negative charge of miRNA, it is difficult to enter cells, and the half-life in vivo is short, the stability is poor, and it is easy to be degraded by RNA enzymes in circulation. How to effectively deliver therapeutic miRNA to the brain lesion site is a great challenge. In order to solve this problem, researchers have made many attempts, such as introducing chemical modification into miRNA or using synthetic nanoparticles as carriers for transportation, but most of them cannot be clinically transformed due to the problems of complex preparation or purification process, self-toxicity and immunogenicity. Exosomes have unique advantages in delivering miRNA as biological nanocarriers. First, exosomes are isolated and extracted from biological fluids or cells, which have lower toxicity and immunogenicity; second, the nanometer characteristics endow them with the ability to penetrate deep tissues and longer circulation time; in addition, the lipid bilayer membrane structure can protect the therapeutic molecules loaded inside it, achieving stable transportation in circulation. Therefore, exosomes can be an ideal carrier for miRNA drug delivery. However, after intravenous injection, exosomes are often mainly distributed in the liver, lungs, spleen and other organs, and the effective concentration in the brain is very low. To improve the effective concentration of exosomes in brain tissue, the problem of targeting must be solved.

[0003] Genetic engineering is one of the most commonly used techniques for targeted modification of exosomes. In this process, the gene sequence of a targeting ligand or polypeptide is fused with the gene sequence encoding the transmembrane protein on the surface of exosomes. The parent cells are transfected with the fusion plasmid to secrete engineered exosomes with targeting ligands on the surface. Rabies virus glycoprotein (RVG) is a widely recognized brain tissue targeting peptide that can specifically target nerve cells and brain vascular endothelial cells by binding to nicotinic acetylcholine receptors. Lysosome-associated membrane protein 2b (Lamp2b) is a transmembrane protein highly expressed on the membrane of exosomes. Alvarez-Erviti et al. pioneered the use of genetic engineering technology to transfect RVG-Lamp2b fusion plasmid into dendritic cells, resulting in engineered exosomes carrying RVG on the surface. After injection into the tail vein of mice, these exosomes can specifically deliver siRNA to mouse brain neurons, microglia and oligodendrocytes, resulting in target gene knockdown. Since then, the RVG-Lamp2b fusion plasmid has been widely used to mediate brain tissue targeted delivery of exosomes. SUMMARY

[0004] To solve the technical problems existing in the prior art, the purpose of the present application is to use genetic engineering technology to modify EPCs-Exo for brain targeting and load miR-3059-5p drugs, to prepare gene-modified exosomes that can specifically target brain tissue to treat stroke.

[0005] The present application provides a genetically modified cell, which expresses miR-3059-5p.

[0006] Preferably, the genetically modified cell expresses a tissue targeting protein and miR-3059-5p.

[0007] Preferably, the cell secretes a membrane structure.

[0008] The cells that can be used in the present application can be prokaryotes or eukaryotes. Preferably, the cells are eukaryotic cells, such as yeast cells, plant cells, insect cells or mammalian cells.

[0009] The cells that can be used in the present application to generate the membrane structures can be referred to as source cells, meaning any type of cells that can be able to produce membrane structures under suitable conditions, for example in suspension culture or in adherent culture or any in other type of culture system. The source cells according to the present application can also include cells that produce exosomes in vivo. The source cells according to the present application can be selected from a wide range of cells and cell lines, which can be grown in suspension or adherent culture or adapted to grow in suspension. The source cells of the present application can be selected from the group comprising mesenchymal stem cells or stromal cells (obtainable from e.g. bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, placenta, dental bud, umbilical cord blood, skin tissue, etc.), fibroblasts, amniotic cells (and more particularly amniotic epithelial cells, optionally expressing various early markers), myeloid suppressor cells, M2 polarized macrophages, adipocytes, endothelial cells, fibroblasts, etc. Cell lines of particular interest include human umbilical cord endothelial cells (HUVEC), human embryonic kidney (HEK) cells, endothelial cell lines (such as microvascular or lymphatic endothelial cells), red blood cells, erythroid progenitor cells, chondrocytes, MSC of different origin, amniotic cells, amniotic epithelial (AE) cells, any cells obtained by amniocentesis or from placenta, epithelial cells from airways or alveoli, fibroblasts, endothelial cells, etc. Furthermore, immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DC) are also within the scope of the present application, and essentially any type of cells that are able to produce membrane structures are encompassed herein. In general, the membrane structures can essentially be derived from any cell source, whether it is a primary cell source or an immortalized cell line. The source cells from which the membrane structures are derived can be any embryonic, fetal and adult somatic stem cell type, including induced pluripotent stem cells (iPSC) and other stem cells obtained by any method. When treating diseases of the nervous system, one can consider utilizing for example primary neurons, astrocytes, oligodendrocytes, microglia and neural progenitor cells as source cells. The source cells can be allogeneic, autologous or even xenogeneic in nature to the patient to be treated, i.e. the cells can be from the patient themselves or from an unrelated, matched or unmatched donor. In certain cases, allogeneic cells can be preferable from a medical point of view, as they can provide an immunomodulatory effect that can not be available from autologous cells of a patient suffering from a certain indication. For example, in the context of treating inflammatory or degenerative diseases, allogeneic MSC or AE as source of exosome-producing cells can be highly beneficial due to their inherent immunomodulatory effect of their membrane structures and in particular of their exosomes.Particularly interesting cell lines include human umbilical cord endothelial cells (HUVEC), human embryonic kidney (HEK) cells (such as HEK293 cells, HEK293T cells, serum-free HEK293 cells, suspension HEK293 cells), endothelial cell lines (such as microvascular or lymphatic endothelial cells), red blood cells, erythroid progenitor cells, chondrocytes, MSCs of different origins, amniotic cells, amniotic epithelial (AE) cells, any cells obtained by amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, epithelial cells, etc.

[0010] The tissue-targeting protein can specifically target the tissue by binding to a receptor on the cells of the tissue.

[0011] Common tissue-targeting proteins include: cyclic peptide (RGDyK) targeting ischemic areas of the brain, RVG targeting neurons, CSTSMLKAC peptide targeting ischemic myocardium, and WLSEAGPVVTVRALRGTGSW peptide targeting cardiomyocytes.

[0012] In a specific embodiment of the application, the tissue-targeting protein is RVG, which can specifically target neural cells and brain vascular endothelial cells by binding to nicotinic acetylcholine receptors.

[0013] Preferably, the genetically modified cell is a genetically modified EPC cell.

[0014] Preferably, the miR-3059-5p comprises pri-miR-3059-5p, pre-miR-3059-5p, mature miR-3059-5p.

[0015] The application provides a membrane structure comprising the aforementioned miR-3059-5p.

[0016] Further, the membrane structure comprises the aforementioned tissue-targeting protein and the aforementioned miR-3059-5p.

[0017] The tissue-targeting protein is connected to a transmembrane protein of the membrane structure.

[0018] Transmembrane proteins can include, but are not limited to, CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, AARDC1, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, syndecan-1, syndecan-2, syndecan-3, syndecan-4, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d / ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptor, interleukin receptor, immunoglobulin, MHC-I or MHC-II component, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, other exosomal polypeptides, and any combination thereof.

[0019] The membrane structure of the present application includes an exosome, a microvesicle (MV), or any other type of vesicle secreted from an endosome, endolysosome, and / or lysosomal pathway or from the plasma membrane of a parent cell. In general, the present application relates to any type of vesicular structure secreted, produced, and / or derived from a cell, including but not limited to an exosome, a vesicle, a microvesicle, a microparticle, an endosome-derived vesicle, a multivesicular body, or an apoptotic body.

[0020] In a particular embodiment of the present application, the membrane structure is an exosome.

[0021] The present application provides a method for preparing the genetically modified cell as described above, the method comprising introducing into the cell a vector expressing the tissue-targeting protein as described above and a vector expressing the miR-3059-5p as described above.

[0022] The present application provides a method for preparing the membrane structure as described above, the method comprising the method for preparing the genetically modified cell as described above.

[0023] Further, the method further comprises culturing the genetically modified cell obtained by the method as described above, obtaining the supernatant thereof, and isolating and purifying the membrane structure.

[0024] The skilled person will understand how to isolate the exosomes prepared according to the present application. Typically, exosomes are collected from cell supernatant and can be isolated by differential centrifugation or density centrifugation according to well-known protocols. Exosomes comprising exogenous material can be separated from exosomes not comprising exogenous material and subpopulations of exosomes can be obtained. The exogenous material of the present application can be isolated from the exosomes using standard methods.

[0025] A "vector" of the present application is an oligonucleotide molecule (DNA or RNA) used to transfer foreign genetic material into a cell. The vector can be an expression vector for expressing the foreign genetic material in a cell. Such a vector can include a promoter sequence operably linked to a nucleotide sequence encoding a gene sequence to be expressed. The vector can also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer and stop codon known in the art can be used in the present application. Suitable vectors include plasmids, binary vectors, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes).

[0026] The vector as described above can be introduced into a cell by using any suitable technique, examples of which include but are not limited to electroporation, incubation, cell activation and transfection, lipofection, lipid delivery, liposome delivery, polymer transfection, polymer delivery, delivery by peptides (i.e. but not limited to cationic peptides, amphipathic peptides, cell penetrating peptides), calcium or magnesium precipitation and ion precipitation (also known as DNA-calcium phosphate precipitation).

[0027] Viruses that can be used in the present application include but are not limited to adenovirus, adeno-associated virus, lentivirus, retrovirus.

[0028] Further, the virus is a lentivirus.

[0029] The present application provides the use of the genetically modified cell as described above, or the membrane structure as described above in the preparation of a medicament for preventing or treating a stroke.

[0030] The application provides the use of the genetically modified cell or the membrane structure in the preparation of a drug for preventing or treating brain damage induced by cerebral stroke.

[0031] The application provides the use of the genetically modified cell or the membrane structure in the preparation of a drug for improving the prognosis of a patient with cerebral stroke.

[0032] The application provides the use of the genetically modified cell or the membrane structure in the preparation of a drug for improving the motor coordination function of a patient with cerebral stroke in a recovery period.

[0033] The application provides the use of the genetically modified cell or the membrane structure in the preparation of a drug for improving the hippocampal synaptic plasticity of a patient with cerebral stroke.

[0034] The application provides the use of the genetically modified cell or the membrane structure in the preparation of a drug for improving the neurocognitive function of a patient with cerebral stroke in a recovery period.

[0035] Further, the cerebral stroke is an ischemic cerebral stroke.

[0036] The film structures and compositions described herein can be administered or formulated for administration by a variety of routes, including, but not limited to, aural, buccal, conjunctival, cutaneous, dental, electroporation, intra-cervical, intra-dental, intra-tracheal, intra-intestinal, intra-epidural, intra-amniotic, in vitro, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intra-biliary, intra-bronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracerebroventricular, intracisternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intradural, intradermal, intraesophageal, intra-gastric, intra-gingival, intra-ileal, intralesional, intraluminal, intralymphatic, intramedullary, intramembranous, intramuscular, intraocular, intra-ovarian, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intra-sinus, intra-spinal, intra-synovial, intra-tendinous, intra-testicular, intrathecal, intrathoracic, intracanalicular, intratumoral, intratympanic, intra-uterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing techniques, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, peri-articular, peri-dural, peri-neural, peri-odontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the foregoing routes of administration. The compositions of the present application can be formulated in liquid or solid form. Fluid formulations can be administered by injection into selected areas of the human or animal body. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The results of constructing and characterizing RVG-Exo-miR-3059-5p are shown in the result graphs, wherein (A) is a flow chart of constructing RVG-Exo-miR-3059-5p; (B) is a TEM observation of the morphological characteristics of RVG-Exo-miR-3059-5p, Scale bar = 100 nm; (C) is an NTA determination of the particle size of RVG-Exo-miR-3059-5p; (D) is a qPCR detection of the expression level of miR-3059-5p in the engineered EPCs and RVG-Exo-miR-3059-5p; (E) is a Western blot detection of the expression of the tag protein FLAG; Note: ****P < 0.0001;

[0038] Figure 2Figure showing the results of RVG-Exo-miR-3059-5p brain tissue targeting, wherein (A) small animal imaging detects the tissue distribution of RVG-Exo-miR-3059-5p; (B) qPCR detects the level of miR-3059-5p; (C) Western blot detects the expression level of TNFRSF1A in the brain after tail vein injection of RVG-Exo-miR-3059-5p; Note: ***P<0.001, ****P<0.0001;

[0039] Figure 3 Figure showing the results of the effect of RVG-Exo-miR-3059-5p on the brain infarction area and inflammatory factor levels of MCAO / R mice, wherein (A) TTC staining to observe the brain infarction area of each group; (B) TTC staining statistical analysis; (C) TNF-α, (D) IL-1β, (E) IL-6 levels; Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0040] Figure 4 Figure showing the results of the effect of RVG-Exo-miR-3059-5p on the brain tissue water content and BBB permeability of MCAO / R mice, wherein (A) brain tissue water content; (B) EB dye permeability quantitative analysis; (C) Western blot results of ZO-1 and Occludin; (D) relative quantitative analysis of ZO-1 expression; (E) relative quantitative analysis of Occludin expression; Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0041] Figure 5 Figure showing the results of the effect of RVG-Exo-miR-3059-5p on the expression of brain tissue apoptosis-related proteins and Parthanatos-related proteins of MCAO / R mice, wherein (A) Western blot results of cell apoptosis-related proteins; (B) relative quantitative analysis of Bcl-2 / Bax expression; (C) relative quantitative analysis of cleaved-Caspase-3 expression; (D) Western blot results and relative quantitative analysis of PARP-1; (E) Western blot results and relative quantitative analysis of AIF in mitochondria; (F) Western blot results and relative quantitative analysis of AIF in the nucleus; Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0042] Figure 6Figure showing the results of TEM observation of the effect of RVG-Exo-miR-3059-5p on the ultrastructure of mitochondria in the hippocampus of MCAO / R mice;

[0043] Figure 7 Figure showing the results of the effect of RVG-Exo-miR-3059-5p on neural plasticity in MCAO / R mice, wherein (A) is a graph of Golgi staining of apical and basal secondary dendritic spines of pyramidal neurons in the dorsal hippocampal CA1 region; (B) is a graph of apical secondary dendritic spine density; (C) is a graph of basal secondary dendritic spine density; (D) is a Western blot detection of the effect of RVG-Exo-miR-3059-5p on PSD95 protein expression; (E) is a relative quantification analysis of PSD95 protein expression; Note: *P<0.05, ***P<0.001, ****P<0.0001;

[0044] Figure 8 Figure showing the results of the effect of RVG-Exo-miR-3059-5p on the recovery of motor and cognitive function in MCAO / R model mice, wherein (A) is a schematic diagram of behavioral studies in mice; (B) is a mNSS score; (C) is a rotarod test of the fall latency of mice; (D) is a water maze test of the escape latency of mice; (E) is a water maze test of the number of times mice cross the target platform; Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0045] The present application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0046] The experimental methods in the following examples are all routine methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all routine biochemical reagents unless otherwise specified, and can be obtained commercially. Example RVG-Exo-miR-3059-5p effect on brain injury and recovery of nerve function in MCAO / R mice

[0047] Table 1

[0048]

[0049] I. Experimental procedures

[0050] 1. Animals and cell lines used in the experiment

[0051] The mice used in this study were all SPF (Specific-pathogens free) level, healthy male C57BL / 6J, 6-8 weeks old, weighing 20-25g, purchased from Beijing Huafukang Biotechnology Co., Ltd. (License No.: SCXK 2019-0008). All mice were housed in cages at 4-6 per cage in the animal housing room of Tianjin Institute of Anesthesiology, and were allowed to freely ingest food and drink water. The feeding environment was maintained at 20-24℃, humidity 40-60%, 12 hours of day and night alternation. Before the experiment, the mice were adaptively fed for one week. All animal experiments in this study were approved by the Tianjin Medical University Experimental Animal Management Committee.

[0052] The mouse hippocampal neuron cell line HT-22 used in this study was purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd. The culture medium was DMEM medium containing 10% fetal bovine serum (FBS) and 1% P / S (P: penicillin 10000 U / ml, S: streptomycin 10000 U / ml), cultured in a cell incubator at 37℃, 5% CO2.

[0053] 2. Experimental method

[0054] 2.1 Experimental grouping

[0055] A total of 232 healthy male C57BL / 6J mice were randomly divided into the following 4 groups (n=58):

[0056] ① Sham operation group (Sham group);

[0057] ② MCAO / R group (200 μl of sterile PBS solution was injected into the mouse tail vein at the moment of reperfusion, 24h after reperfusion and 48h after reperfusion, respectively);

[0058] ③ MCAO / R+RVG-Exo-NC group (RVG-Exo-NC group, 0.5 μg / μl RVG-Exo-NC 200 μl was injected into the mouse tail vein at the moment of reperfusion, 24h after reperfusion and 48h after reperfusion, respectively);

[0059] MCAO / R+RVG-Exo-miR-3059-5p group (RVG-Exo-miR-3059-5p group, 0.5 μg / μl RVG-Exo-NC 200 μl was injected into the mouse tail vein at the moment of reperfusion, 24h after reperfusion and 48h after reperfusion, respectively).

[0060] 2.2 Preparation of MCAO / R model

[0061] MCAO / R model was established using male C57BL / 6J mice (20-25g, 8 weeks old).

[0062] (1) Preoperative preparation: 12 hours before operation, mice were deprived of water and food, weighed and marked groups.

[0063] (2) Specific modeling steps

[0064] a, by inhalation of isoflurane anesthesia (induction concentration of 4%, maintenance concentration of 1%-1.5%), mice supine position fixed on the operating table. Pay attention to the mouse during modeling process.

[0065] Skin preparation from the chest to the neck, disinfection, make a longitudinal incision of about 1 cm along the median line of the neck, bluntly separate the muscles, fascia and nerves under the stereomicroscope with ophthalmic forceps, free the left common carotid artery (CCA), and tie a slipknot with 6-0 silk thread.

[0066] b, separate along the CCA, free the internal carotid artery (EAC) and external carotid artery (ICA). Ligate the distal end of ECA with the thyroid artery, and tie a virtual knot between the ligation site and the CCA bifurcation with a 6-0 suture.

[0067] c, between the virtual knot and the ligation site, cut a small hole with a microscopical scissors obliquely, insert the wire plug and tighten the virtual knot, the force should be just enough to make the wire plug slide in the blood vessel without blood flowing out of the hole.

[0068] d, cut the ECA close to the ligation site, adjust the angle to insert the wire plug into the ICA, and continue to push it forward along the ICA until you feel slight resistance, stop pushing the wire plug, at this time the front end completely blocks the origin of the middle cerebral artery, tie a slipknot at the ECA stump to fix the wire plug.

[0069] e, suture subcutaneous tissue and skin layer by layer, disinfect. After 1 hour of ischemia, pull out the wire plug outward, so that its head end retreats to the ECA, to ensure that the CCA blood flow reperfusion to the middle cerebral artery. Re-suture and disinfect, intraperitoneal injection of 1 ml of normal saline.

[0070] f, the sham group of mice, the operation was the same as above except that the wire plug was inserted into the ICA without embolizing the middle cerebral artery. After operation, mice were fed in separate cages, and attention was paid to maintain body temperature and timely rehydration.

[0071] 2.3 Lentivirus infection of EPCs

[0072] (1) Isolation and culture of mouse bone marrow-derived EPCs

[0073] 6-8 weeks old, body weight 20-25 g of male C57BL / 6J mice were sacrificed under deep anesthesia by cervical dislocation method, 75% alcohol complete immersion disinfection for 15-20 min and then transferred to a clean bench. According to the aseptic operation principle, the bilateral femur and tibia of the mouse were separated in turn, and the residual muscle tissue on the bone surface was removed. The bone metaphyseal ends on both sides were cut off, and the bone marrow cavity was repeatedly washed with PBS solution containing 10% FBS until the bone marrow cavity turned white, and the washing liquid was collected. Take a 15 ml centrifuge tube, add an equal amount of lymphocyte separation medium to the washing liquid, then slowly add the washing liquid to the surface of the separation liquid, centrifuge at 2500 rpm at room temperature for 20 min, and then absorb the annular milky white layer (single nuclear cell layer), wash with PBS for 2 times, and resuspend the cell pellet with EGM-2MV complete culture medium. Count under a microscope. Adjust the cell density to 5×10 6 / ml, and inoculate into a cell culture dish coated with Matrigel matrix glue in advance, and culture in a 37℃, 5% CO2 incubator. Change the liquid for the first time after 24 h, remove the unattached cells, and then change the liquid every 2 days. When the cells are fused to 80%-90%, the cells are passaged. Early passage EPCs (p2-6) can be used for subsequent experiments.

[0074] (2) Construction of RVG-Lamp2b (catalog number: #71294, purchased from Addgene) and miR-3059-5p (catalog number: miRB0014811-2-1, purchased from Guangzhou Ribobio Biotechnology Co., Ltd.) and miR-NC (catalog number: miR3N0000001-4-5, purchased from Guangzhou Ribobio Biotechnology Co., Ltd.) overexpression lentivirus.

[0075] (3) Lentivirus infection of EPCs

[0076] ① Adjust the density of EPCs, inoculate 2×10 5 cells / well into a cell culture 6-well plate, and culture at 37℃ for 16-24 h until the cell fusion degree is 20%-30%.

[0077] ② According to the multiplicity of infection (MOI) = 50, the number of inoculated cells (2×10 5 and the virus titer (1×10 9 TU / ml), the amount of lentivirus is calculated. The calculation formula is: virus volume = (MOI × cell number) / virus titer. The amount of lentivirus and negative control lentivirus is 10 μl per well.

[0078] ③Aspirate the supernatant from each well, and add 1 ml of EGM-2MV complete medium to each well. Add 10 μl of virus and HitransG P infection enhancer to two wells, and add 10 μl of negative control virus and HitransG P infection enhancer to the other two wells. Add nothing to the remaining two wells as blank controls. Continue to culture.

[0079] ④At 16 h after infection, replace the medium with 2 ml of complete medium, and continue to culture. Replace the medium as needed to maintain cell viability.

[0080] ⑤At about 72 h after infection, observe the infection efficiency under a fluorescence microscope. If the green fluorescence abundance is more than 80%, it indicates that the lentivirus infection is successful.

[0081] ⑥When the cell confluence is about 60%, discard the original culture supernatant, wash the cells with PBS, and add 2 ml of complete medium containing 8 μl of puromycin (1 μg / ml) to screen the EPCs cells successfully infected with lentivirus. Continue to screen for 1 week, and then perform subsequent experiments after the cell growth state is stable.

[0082] 2.4 Extraction and identification of EPCs-derived exosomes (EPCs-Exo)

[0083] 2.4.1 Extraction of EPCs-derived exosomes (EPCs-Exo)

[0084] ①Prepare the ultracentrifugation culture solution in advance: replace the FBS in the prepared EGM-2MV complete medium with ultracentrifugation FBS (4℃, 100 000g, centrifugation for 16 h) that does not contain exosomes.

[0085] ②After the EPCs are cultured in the ultracentrifugation culture solution for 48 h, collect the cell culture supernatant, i.e., EPCs-conditioned medium (EPCs-CM).

[0086] ③After the preparation, sequentially perform gradient centrifugation at 300g for 10 min, 2 000g for 10 min, and 10 000g for 30 min to remove the cell components and cell debris. Discard the precipitates after each centrifugation, and recover the supernatant.

[0087] ④Filter the supernatant through a 0.22 μm filter to a clean ultracentrifugation tube to remove large vesicle particles and protein aggregates. ⑤After strict preparation, ultracentrifuge the supernatant at 100 000g for 70 min, resuspend the precipitate in sterile PBS, and ultracentrifuge again at 100 000g for 70 min. The precipitate is the exosome. Resuspend the precipitate in sterile PBS, and store at -80℃ for long-term preservation.

[0088] 2.4.2 Identification of EPCs-derived exosomes (EPCs-Exo)

[0089] (1) Transmission electron microscope (TEM) observation of exosome morphology

[0090] 1% glutaraldehyde: 2.5% glutaraldehyde + ultrapure water diluted 2.5 times, prepared fresh.

[0091] 2% paraformaldehyde: 4% paraformaldehyde + PBS diluted in equal volume.

[0092] 2% methyl cellulose: ultrapure water 196 ml heated to 90°C, 4 g of methyl cellulose was added and dissolved quickly by stirring, quickly cooled to 10°C, stirred uniformly at 4°C for 3 days, and then diluted to 200 ml. 100000g, centrifuged for 95 min, collect supernatant, 4°C storage.

[0093] 0.15M oxalic acid: oxalic acid 0.945g + ultrapure water 50ml, fully dissolved uniformly, 4°C storage.

[0094] 4% uranyl acetate (PH=4): uranyl acetate 2g + ultrapure water 50ml, 4°C storage.

[0095] Methyl cellulose-uranyl acetate: prepared according to the ratio of methyl cellulose: uranyl acetate = 9:1.

[0096] Uranium oxalate (PH=7): mix equal volume of prepared 0.15M oxalic acid and 4% uranyl acetate, adjust PH=7 by adding 25% NaOH dropwise.

[0097] ② Resuspend the fresh exosome pellet after ultracentrifugation with 100μl 2% paraformaldehyde, if the exosomes are stored frozen at -80°C, add equal volume of 4% paraformaldehyde and mix well, fix at room temperature for 30min.

[0098] ③ Cut a piece of hole sealing film and lay it flat on the table, place the electron microscope copper mesh with the front face upwards, drop 5-10μl of exosomes on the front face of the copper mesh, and let it stand at room temperature for 20min.

[0099] ④ Add 100μl PBS on the hole sealing film, use a sharp forceps to pick up the copper mesh with adsorbed exosomes, make the front face contact with PBS, and wash at room temperature for 2min. Note that during the operation, the tip of the forceps always gently holds the edge of the copper mesh to avoid breaking the internal part of the copper mesh.

[0100] ⑤ Add 50μl 1% glutaraldehyde, cover the copper mesh with the 1% glutaraldehyde drop, and fix at room temperature for 5min.

[0101] ⑥Cover the copper mesh onto 100 μl of the drop of ultrapure water, and wash for 2 min at room temperature. Repeat this step for 8 times.

[0102] ⑦Cover the copper mesh onto 50 μl of the drop of uranyl oxalate, and stain for 5 min at room temperature.

[0103] ⑧Cover the copper mesh onto 50 μl of the drop of methylcellulose-uranyl acetate, and transfer to ice, and stain for 10 min.

[0104] ⑨Absorb the liquid at the edge of the copper mesh with filter paper, and dry naturally at room temperature.

[0105] ⑩Observe and take photos of the sample on the machine.

[0106] (2) Nanoparticle tracking analysis (NTA) for detecting the concentration and particle size distribution of exosomes

[0107] ①To avoid the interference of background impurities on the detection results, repeated tests were performed to ensure that the ultrapure water used for washing the pipeline and the PBS used for diluting the exosomes were free of aggregated large particles.

[0108] ②Rinse the machine pipeline with ultrapure water and PBS in turn.

[0109] ③Take 5 μl of exosomes, dilute with 1 ml of PBS, and then transfer to a 1 ml syringe for sampling. Detect and analyze the particle size distribution and concentration information of the exosomes.

[0110] 2.5 Real-time fluorescent quantitative PCR to verify the overexpression efficiency of miR-3059-5p

[0111] 2.5.1 Extraction and quality control of total RNA in EPCs and EPCs-Exo

[0112] (1) Sample processing

[0113] EPCs cell lysis: when the cell growth confluence in the 6-well plate reaches 80%-90%, discard the cell culture supernatant, wash twice with enzyme-free PBS solution, add 1 ml of TRIzol to gently blow the cells, and then transfer the lysate to a 1.5 ml EP tube after 5 min of room temperature standing. Centrifuge at 12000 rpm for 5 min, and recover the supernatant.

[0114] EPCs-Exo lysis: after centrifugation and precipitation of exosomes, add 1 ml of TRIzol to gently blow the cells, and then transfer the lysate to a 1.5 ml EP tube after 5 min of room temperature standing. Centrifuge at 12000 rpm for 5 min, and recover the supernatant.

[0115] (2) RNA extraction

[0116] ①Add 200 μl chloroform, tightly cover the EP tube cap, shake vigorously, and stand at room temperature for 10-15 min.

[0117] ②Centrifuge at 12000 rpm for 15 min at 4°C. Carefully aspirate the supernatant containing RNA (about 500 μl) without touching the middle white protein layer, and transfer it to a new EP tube.

[0118] ③Add an equal volume of isopropanol (about 500 μl) to the EP tube, mix well, and stand at room temperature for 10 min.

[0119] ④Centrifuge at 12000 rpm for 15 min at 4°C. The white precipitate at the bottom of the centrifuge tube is RNA, and the supernatant is aspirated as much as possible.

[0120] ⑤Add 1 ml of 75% ethanol (4°C pre-cooled) to the RNA precipitate, mix slowly, and wash the RNA precipitate. Centrifuge at 4°C, 12000 rpm for 5 min. Discard the supernatant and dry the RNA precipitate under the open cap.

[0121] ⑥Dissolve the RNA in 50 μl of DEPC water, stand for 5 min, and blow gently to dissolve completely.

[0122] ⑦RNA integrity test: identify the integrity of RNA by 1.5% agarose gel electrophoresis. The brightness ratio of 28s RNA to 18s RNA of eukaryotic organisms is usually 2:1.

[0123] ⑧RNA purity and concentration test: NanoDrop2000 detects the OD value and concentration of RNA, and the purity is determined according to the OD260 / OD280 ratio. RNA with a value in the range of 1.8-2.0 is considered to be of good quality and can be used for subsequent experiments.

[0124] ⑨The RNA sample can be stored at -80°C for a long time.

[0125] 2.5.2 Real-time quantitative PCR detection

[0126] a. In vitro reverse transcription of RNA to synthesize cDNA

[0127] Add the reagents in Table 2 to the RNase / DNase-free PCR tube in order, and operate on ice.

[0128] Table 2 Reverse transcription system 1

[0129]

[0130] Mix gently, centrifuge briefly, incubate at 65°C for 5 min, and cool on ice.

[0131] The reagents in Table 3 were added into the above PCR tube in turn, and the operation was performed on ice.

[0132] Table 3 Reverse transcription system 2

[0133]

[0134] Gently mix, incubate at 42°C for 60 min, heat at 70°C for 5 min, and terminate the reaction. The reaction product can be directly used for subsequent experiments or stored in a -80°C refrigerator after aliquoting.

[0135] 2.5.3 Real-time fluorescent quantitative PCR detection steps

[0136] ①The primer sequences used are shown in Table 4.

[0137] Table 4 Primer sequences

[0138]

[0139]

[0140] ②Diluted cDNA (100 ng / μl) was used as a template, and the reaction system is shown in Table 5.

[0141] Table 5 Real-time fluorescent quantitative PCR detection system

[0142]

[0143] ③Set the reaction program, 95°C pre-denaturation for 30 s; PCR reaction: 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles; melting curve: 95°C for 15 s, 60°C for 1 min, every 15 s, the temperature was increased by 0.3°C, 95°C

[0144] 15 s.

[0145] ④Data analysis: use the fluorescence quantitative PCR instrument to detect the Ct value of each template, and use 2 -ΔΔCt to calculate the relative expression of the target gene in the experimental group and the control group, ΔCt = Ct (target gene) - Ct (internal

[0146] reference), ΔΔCt = ΔCt (experimental group) - ΔCt (control group).

[0147] 2.6 Western blot method

[0148] (1) Verify the RVG-Lamp2b overexpression efficiency

[0149] Western blot was used to detect the expression of FLAG-Lamp2b in RVG-Lamp2b lentivirus infected EPCs and their secreted exosomes, and then the infection efficiency was verified. Western blot was performed according to the conventional operation steps. The primary antibody and dilution ratio used in this part of the experiment were as follows: Lamp2b 1:1000, FLAG 1:2000.

[0150] (2) Changes in expression of PSD95, apoptosis and Parthanatos death related proteins in brain tissue

[0151] The primary antibody and dilution ratio used in this part of the experiment were as follows: PSD95 1:1000, Bax 1:1000, Bcl-2 1:1000, cleaved-Caspase-3 1:1000, PARP-1 1:1000, AIF 1:1000, Histone H3 1:5000, COX IV 1:5000, GAPDH 1:5000.

[0152] 2.7 Detection of brain tissue distribution of exosomes

[0153] (1) DiR dye labeled exosomes

[0154] ① Add 1 ml DMSO to 10 mg DiR dye powder, heat to fully dissolve, concentration is 10 mg / ml, aliquot into EP tubes (100 μl / tube), wrap with tin paper to avoid light, store at -20°C. When used, dilute with ultrapure water to a final concentration of 1 μg / μl.

[0155] ② Take the exosomes, add DiR dye, and make the DiR concentration in the system 1 μg / ml.

[0156] ③ After mixing well, incubate at 37°C in the dark for 30-45 min, shake the EP tube bottom every 5 min during the incubation process to avoid exosomes and dye precipitation.

[0157] ④ Add the labeled exosomes to a 41Ti ultracentrifuge tube, add 10 ml PBS solution to make up the liquid, centrifuge at 4°C, 100000g for 70 min.

[0158] ⑤ Remove the supernatant, add 100 μl PBS, resuspend by blowing in the dark, and recover DiR labeled exosomes (DiR-Exo).

[0159] (2) In vivo distribution detection of DiR-Exo

[0160] ① Measure the concentration of DiR-Exo, take 80 μg DiR-Exo, supplement with PBS solution to 200 μl, and inject into the mouse body through the tail vein.

[0161] 2h after injection, the mice were sacrificed under deep anesthesia, and the heart (H), liver (Li), spleen (S), lung (Lu), kidney (K) and brain (B) were collected, respectively.

[0162] 3. Distribution of DiR in different tissues was detected by small animal imaging system. The excitation and emission wavelengths of DiR were 745 nm and 800 nm, respectively.

[0163] 2.8 TTC staining

[0164] 1. After 72 h of MCAO / R modeling, the mice were sacrificed under deep anesthesia, and the whole brain was dissected out and frozen at -20℃ for 20 min.

[0165] 2. The mouse brain mold was used to cut the brain tissue into 1 mm thick coronal sections.

[0166] 3. The brain tissue sections were immersed in 2% TTC staining solution and incubated at 37℃ in the dark for 20 min. During the incubation process, the sections were turned over to ensure uniform staining. The stained sections were then immersed in 4% paraformaldehyde for 24 h. 4. Photographs were taken, and image analysis was performed using ImageJ software. The infarct volume calculation formula was: Infarct volume percentage = (ischemic contralateral hemisphere volume - ischemic hemisphere non-infarct area volume) / ischemic contralateral hemisphere volume x 100%.

[0167] 2.9 Brain water content determination

[0168] 1. After 72 h of MCAO / R modeling, the mice were sacrificed under deep anesthesia, and the whole brain was dissected out and frozen at -20℃ for 20 min.

[0169] 2. The brain was cut into left and right hemispheres along the sagittal line, with the left hemisphere being the ischemic hemisphere. The left hemisphere was immediately weighed on an electronic balance and recorded as the wet weight.

[0170] 3. The brain was quickly placed in a 100℃ oven, and weighed again after 24 h, recorded as the dry weight.

[0171] 4. The brain water content was calculated using the following formula: Brain water content = (wet weight - dry weight) / wet weight x 100%.

[0172] 2.10 Evans blue (EB) extravasation method to detect BBB permeability

[0173] 1. 2% EB dye (4 ml / kg) was injected into the tail vein of the mice. 2 h after injection, the mice were anesthetized with 3% isoflurane, and perfusion was performed through the left ventricle with 50 ml of pre-cooled PBS solution. When the liquid flowing out of the right atrium became clear, the perfusion was stopped.

[0174] ②Quickly decapitate and cut the ischemic hemisphere along the sagittal line. After weighing, 100 mg of brain tissue was added to 1 ml of formamide and homogenized using an ultrasonic tissue cell disrupter.

[0175] ③Incubate at 37℃ for 48 h, centrifuge at 1500 rpm for 10 min, and collect the supernatant. Detect the absorbance value at 632 nm using a multifunctional microplate reader.

[0176] ④Draw the EB standard curve and calculate the EB dye content in the mouse brain tissue.

[0177] 2.11 Golgi Staining

[0178] (1) Reagent preparation

[0179] ①24 h before sampling, equal volumes of A and B solutions were taken and placed in an EP tube wrapped in tin foil in a dark environment to avoid vigorous shaking or stirring, and were kept in the dark.

[0180] ②Mix D, E, and distilled water in a ratio of 1:1:2 and use immediately.

[0181] (2) Staining steps

[0182] ①After deeply anesthetizing the mouse, decapitate and quickly remove the brain. Clean the surface of the brain tissue of hair, blood, and other substances with distilled water. Then immerse the brain tissue in an EP tube containing the A and B mixture. The next day, replace the new A and B mixture prepared 24 h in advance. The brain tissue is immersed in the A and B mixture for at least two weeks, and the light and static state are maintained during this period.

[0183] ②After two weeks, transfer the brain tissue to Golgi staining solution C, and replace the new C solution the next day. The brain tissue is immersed in C solution for at least 72 h.

[0184] ③Coat the slides with gelatin in advance, immerse the slides in the coating solution for 30 s, and air dry at room temperature for 12 h before use.

[0185] ④Under the condition of Golgi C solution immersion at -20℃, use a vibrating microtome to cut the brain tissue into slices with a thickness of 100 μm, and transfer the slices to the gelatin-coated slides.

[0186] ⑤Recover the C solution, and store the slides at room temperature in the dark until they are naturally dried.

[0187] ⑥Rinse the slides with distilled water for 4 min x 2 times to elute the residual Golgi staining solution C. Then immerse the slides in the mixed solution of prepared D, E, and distilled water for 10 min.

[0188] ⑦Distilled water rinse 4 min x 2 times, in turn in 50%, 75%, 90% ethanol solution to the section dehydration, each concentration of 4 min dehydration. Dehydration in anhydrous ethanol 4 min x 4 times.

[0189] ⑧The section was immersed in xylene, 4 min x 3 times, to make the background transparent. Resin mounting agent was used for mounting, and the sample was stored in the dark. The sample was observed under a microscope with 100x oil immersion lens and photographed.

[0190] 2.12 Modified neurological severity score (mNSS)

[0191] The neurological function of mice in each group was evaluated by mNSS before and at different time points (1st, 3rd, 7th, 14th day) after MCAO / R modeling. The content included motor, sensory, reflex, and balance tests, and the cumulative score of all tests reflected the severity of neurological injury in mice. Scores of 13-18 indicated severe injury, 7-12 indicated moderate injury, and 1-6 indicated mild injury.

[0192] 2.13 Rotarod test

[0193] The rotarod test was designed to test the motor ability of mice. Mice were placed on a rotating rod, and the speed was increased from 4 to 40 revolutions per minute within 300 seconds. The time for the mouse to fall off the rotating rod was recorded as the fall latency. Mice in each group were trained 3 times a day for 3 days before modeling, with an interval of 15 minutes each time. The average of the results of 3 experiments on the day before modeling was taken as the baseline value (Pre). On the 1st, 3rd, 7th, and 14th day after modeling, the same method was used for experiment, 3 times a day, and the average of 3 experimental data was calculated as the data of that day.

[0194] 2.14 Water maze test

[0195] The water maze test was designed to test the spatial learning and memory ability of mice.

[0196] (1) Experimental instruments

[0197] The water maze test system consisted of an infrared camera, an image automatic acquisition and processing system, and a cylindrical pool with a diameter of 150 cm and a height of 50 cm. The pool was artificially divided into four quadrants (I, II, III, IV) in a clockwise direction, and a circular platform with a diameter of 8 cm was set in the first quadrant. Clean water was added to the pool until it was 2 cm higher than the platform, and white paint was added to make the pool opaque and milky white, so that the platform was not visible to the naked eye. Circular, square, triangular, and pentagonal plastic plates were hung in the four quadrants to facilitate the positioning of the mice. The water temperature was maintained at 25°C at all times.

[0198] (2) Positioning navigation experiment

[0199] From the 8th day after modeling, the mice were made to face the pool wall and enter the water from the entry point of the four quadrants for 7 consecutive days. The time required for the mice to find the underwater hidden platform and stand on the platform was recorded as the escape latency. Each time was limited to 90 s. If the mouse failed to successfully land on the platform within 90 s, the time was recorded as 90 s, and the mouse was guided to find the platform and stand on it for 10 s.

[0200] (3) Spatial exploration experiment

[0201] After completing the place navigation experiment on the 7th day, the platform was removed, the mice were made to face the pool wall and enter the water from the entry point of the first quadrant, and the mice were allowed to swim freely in the pool within 90 s. The number of times each mouse crossed the original platform area was recorded.

[0202] II. Experimental results

[0203] 1. Construction of engineered exosomes RVG-Exo-miR-3059-5p

[0204] We used RVG-Lamp2b overexpression lentivirus and miR-3059-5p overexpression lentivirus to co-infect EPCs (obtained EPCs are called genetically modified EPCs). After obtaining stable expression, the cell culture supernatant was collected, and the exosomes were isolated and purified ( Figure 1 A). RVG-Exo-miR-3059-5p was characterized by TEM and NTA, and the results showed that RVG-Exo-miR-3059-5p had a typical double-membrane cup structure, with a particle size of 119.8 ± 3.3 nm, which was not significantly different from the unmodified natural EPCs-Exo ( Figure 1 B, C). This suggests that the modification of RVG-Lamp2b and miR-3059-5 will not affect the morphology and particle size of the exosomes. qPCR results showed that the expression level of miR-3059-5p in genetically modified EPCs and RVG-Exo-miR-3059-5p was significantly increased compared with the negative control virus group (P < 0.0001, Figure 1 D). The RVG-Lamp2b overexpression plasmid carries a FLAG-tag protein, which can be used to determine the expression of RVG-Lamp2b by detecting the expression of the FLAG-tag protein. Western blot results showed that both genetically modified EPCs and RVG-Exo-miR-3059-5p expressed the protein FLAG ( Figure 1 E). The above results show that RVG-Exo-miR-3059-5p is successfully constructed.

[0205] 2. Evaluation of brain tissue targeting of RVG-Exo-miR-3059-5p

[0206] We injected DiR-labeled EPCs-Exo and RVG-Exo-NC into mice in the Sham and MCAO / R groups via the tail vein system, respectively, and used a small animal fluorescence imaging system to detect the in vivo distribution of exosomes. The results showed ( Figure 2 A) Exosomes were mainly distributed in the liver. No fluorescent signal of native EPCs-Exo was detected in the brains of Sham group mice, while a very weak fluorescent signal was detected in the brains of MCAO / R group mice, possibly related to BBB disruption after MCAO / R treatment. Fluorescent signals of RVG-Exo-NC were detected in the brains of both Sham and MCAO / R group mice. Furthermore, fluorescent signals were also detected in the brains of MCAO / R mice after RVG-Exo-miR-3059-5p was injected via the tail vein, indicating that miR-3059-5p modification does not affect the brain-targeting effect of RVG.

[0207] qPCR results showed ( Figure 2 B), compared with the RVG-Exo-NC group, the expression level of miR-3059-5p in the brain tissue of mice in the RVG-Exo-miR-3059-5p group was significantly increased (P<0.0001); Western blot results showed that ( Figure 2 C) Compared with the RVG-Exo-NC group, the expression level of TNFRSF1A protein in the brain tissue of mice in the RVG-Exo-miR-3059-5p group was decreased (P<0.01). These results indicate that RVG-Exo-miR-3059-5p can successfully deliver miR-3059-5p to brain tissue.

[0208] 3. Effects of RVG-Exo-miR-3059-5p on cerebral infarction area and inflammatory response in mice

[0209] 72 hours after reperfusion, TTC-stained brain slices of mice in each group are shown below. Figure 3 As shown in Figure A, the red area represents the non-infarcted area, and the white area represents the ischemic infarcted area. Quantitative data indicate that ( Figure 3 B) The cerebral infarction area of ​​mice in the RVG-Exo-NC group and the RVG-Exo-miR-3059-5p group was significantly smaller than that of mice in the MCAO / R group (P<0.0001), but there was no statistical difference between the two groups (P>0.05).

[0210] The levels of inflammatory factors in the brain tissue of mice in each group were detected by ELISA. The results showed that ( Figure 3C-E), the infiltration levels of pro-inflammatory factors TNF-a, IL-1b and IL-6 in the brain tissue of mice after MCAO / R modeling were significantly increased (P < 0.0001); tail vein injection of RVG-Exo-NC and RVG-Exo-miR-3059-5p could significantly inhibit the inflammatory response of brain tissue, which was manifested in the significant decrease of the levels of pro-inflammatory factors TNF-a, IL-1b and IL-6 compared with the MCAO / R group (P < 0.0001); in addition, compared with the RVG-Exo-NC group, the levels of TNF-a, IL-1b and IL-6 in the RVG-Exo-miR-3059-5p group were further reduced.

[0211] 4, Effect of RVG-Exo-miR-3059-5p on brain water content and BBB permeability of mice

[0212] After 72 h of reperfusion, the brain tissue water content of the ischemic hemisphere of each group of mice was detected to evaluate the brain edema. As shown in Figure 4 A shows that compared with the Sham group, the brain water content of the MCAO / R group of mice was significantly increased (P < 0.0001); both RVG-Exo-NC and RVG-Exo-miR-3059-5p treatment could significantly reduce the brain water content, and the effect of RVG-Exo-miR-3059-5p treatment on reducing the brain water content of MCAO / R mice was more significant (P < 0.05 compared with the RVG-Exo-NC group).

[0213] The EB dye infiltrated in the brain tissue of each group of mice was quantitatively analyzed to evaluate the degree of damage to the integrity of the BBB, and the results showed that the EB dye content in the brain of the MCAO group of mice was significantly higher than that of the Sham group (P < 0.0001); tail vein injection of RVG-Exo-NC and RVG-Exo-miR-3059-5p could significantly reduce the EB dye infiltration in the brain of MCAO / R mice (P < 0.0001), but there was no significant difference between the two groups Figure 4 B). Further, Western blot was used to detect the expression of tight junction proteins ZO-1 and Occludin. The results showed that compared with the Sham group, the expression levels of ZO-1 and Occludin proteins in the brain tissue of the MCAO / R group of mice were significantly reduced; compared with the MCAO / R group, the expression amounts of ZO-1 and Occludin in the RVG-Exo-NC group and the RVG-Exo-miR-3059-5p group were increased, but there was no significant difference between the two groups Figure 4 C-E).

[0214] 5, Effect of RVG-Exo-miR-3059-5p on neuronal apoptosis and Parthanatos of mice

[0215] As Figure 5 A-C show that compared with the Sham group, the expression of pro-apoptotic protein Bax and cleaved-Caspased-3 in the brain tissue of the MCAO / R group was up-regulated, and the expression of anti-apoptotic protein Bcl-2 was decreased; compared with the MCAO / R group, the expression of Bax and cleaved-Caspased-3 in the RVG-Exo-NC group and the RVG-Exo-miR-3059-5p group was significantly reduced, and the expression of Bcl-2 was significantly increased; compared with the RVG-Exo-NC group, the above effects were more significant in the RVG-Exo-miR-3059-5p group.

[0216] As Figure 5 D-F show that compared with the Sham group, the expression level of PARP-1 in the brain tissue of the MCAO / R group was significantly increased (P<0.001), the expression of AIF in the mitochondria was significantly decreased (P<0.01), and the expression of AIF in the nucleus was increased (P<0.001); compared with the MCAO / R group, after the tail vein injection of RVG-Exo-miR-3059-5p, the expression level of PARP-1 in the brain of the mice was significantly reduced (P<0.001), the expression of AIF in the mitochondria was up-regulated (P<0.05), and the expression of AIF in the nucleus was down-regulated (P<0.05); and after the injection of RVG-Exo-NC, there was no statistically significant difference in the protein expression changes of PARP-1 and AIF (P<0.05).

[0217] 6, Effect of RVG-Exo-miR-3059-5p on the ultrastructure of mitochondria in the brain tissue of mice

[0218] The ultrastructure of mitochondria in the hippocampal tissue was observed by TEM, as shown in Figure 6 , the mitochondrial membrane of neurons in the hippocampal tissue of the Sham group was complete, and the structure of the ridge was clear; the mitochondrial membrane of the MCAO / R group was ruptured, and the structure was disintegrated, the ridge structure was dissolved and disappeared, and vacuolization occurred; the mitochondrial membrane structure of the RVG-Exo-NC group was clear, and the structure was relatively complete, part of the mitochondrial ridge structure was dissolved and disappeared or appeared vacuolization; the outer membrane structure of the mitochondria in the RVG-Exo-miR-3059-5p group was clear, and there was rupture in some areas, and the ridge structure was visible.

[0219] 7, Effect of RVG-Exo-miR-3059-5p on synaptic plasticity in mice

[0220] On the 14th day after modeling, the dendritic spines of the apical and basal secondary dendrites of the pyramidal neurons in the CA1 region of the dorsal hippocampus of the mice were observed by Golgi staining, and the staining results are shown in Figure 7 A. The density of dendritic spines was calculated and expressed as the number of dendritic spines per 10 μm of dendritic length. The histogram of the density of apical secondary dendritic spines is shown inFigure 7 B, 10.44 ± 1.02 / 10 pm in the Sham group, 5.69 ± 0.65 / 10 pm in the MCAO / R group, 7.39 ± 0.49 / 10 pm in the RVG-Exo-NC group, and 9.14 ± 0.43 / 10 pm in the RVG-Exo-miR-3059-5p group. The graph of the density of basal dendritic spines in the apical dendrites of the pyramidal neurons in the CA1 region of the dorsal hippocampus is shown in Figure 7 C, 9.95 ± 0.92 / 10 pm in the Sham group, 5.05 ± 0.75 / 10 pm in the MCAO / R group, 7.47 ± 0.56 / 10 pm in the RVG-Exo-NC group, and 8.33 ± 0.86 / 10 pm in the RVG-Exo-miR-3059-5p group. Compared with the Sham group, the density of apical and basal dendritic spines of the pyramidal neurons in the CA1 region of the dorsal hippocampus was significantly reduced in the MCAO / R group (P < 0.001). Compared with the MCAO / R group, the density of apical and basal dendritic spines of the pyramidal neurons in the CA1 region of the dorsal hippocampus was significantly increased in the RVG-Exo-NC and RVG-Exo-miR-3059-5p groups (P < 0.001), and the increase was more significant in the RVG-Exo-miR-3059-5p group.

[0221] The expression of PSD95 was detected by Western blot, and the results are shown in Figure 7 D-E, compared with the Sham group, the expression of PSD95 in the brain tissue of the MCAO / R group was significantly down-regulated (P < 0.0001); compared with the MCAO / R group, the expression of PSD95 in the brain tissue of the RVG-Exo-miR-3059-5p group was significantly up-regulated (P < 0.001), and there was no statistically significant difference in the RVG-Exo-NC group (P > 0.05). The above results show that RVG-Exo-miR-3059-5p can promote synaptic plasticity after IS.

[0222] 8. Effect of RVG-Exo-miR-3059-5p on the recovery of motor and cognitive functions in mice

[0223] To further explore the therapeutic effect of EPCs-Exo-miR-3059-5p on the recovery of IS mice, the neurological, motor, and cognitive functions of mice were evaluated at different time points after modeling, as shown in 8A. The mNSS scores of mice in each group were measured at 1, 3, 7, and 14 days after modeling, and the results showed that compared with the Sham group, the mNSS score of mice in the MCAO / R group was significantly increased; compared with the MCAO / R group, the mNSS scores of mice in the RVG-Exo-NC and RVG-Exo-miR-3059-5p groups were significantly improved from the 3rd day after modeling, but there was no significant difference between the two groups (Figure 8 B).

[0224] In the rotarod test, the fall latency of each group of mice was recorded as an evaluation index of the recovery of motor coordination function. Compared with the Sham group of mice, the fall latency of the MCAO / R group of mice was significantly shortened at each test time point. Compared with the MCAO / R group, the fall latency of the RVG-Exo-NC group of mice was prolonged at 1, 3, 7 and 14 days, but the difference was not statistically significant; while the mice treated with RVG-Exo-miR-3059-5p, the fall latency was significantly prolonged from the 3rd day after modeling, suggesting that RVG-Exo-miR-3059-5p can significantly improve the motor coordination ability of MCAO / R mice Figure 8 C).

[0225] From the 8th day after modeling, the water maze test was performed for 7 days to test the spatial learning and memory ability of mice. The results are shown in Figure 8 D, compared with the Sham group, the escape latency of the MCAO / R group of mice was significantly prolonged, and the number of crossing the platform in the spatial exploration stage was significantly reduced, suggesting that the spatial learning and memory ability was severely impaired. From the 10th day after modeling, in the positioning navigation experiment stage, the RVG-Exo-NC group and the RVG-Exo-miR-3059-5p group of mice showed a significant shortening of the escape latency; while in the spatial exploration stage on the 14th day, the number of crossing the platform of the RVG-Exo-miR-3059-5p group of mice was increased compared with the MCAO / R group and the RVG-Exo-NC group Figure 8 E).

[0226] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Use of exosomes secreted by genetically modified EPC cells in the preparation of a drug for treating ischemic stroke, wherein, the genetically modified EPC cells express tissue-targeting protein RVG and miR-3059-5p; the exosomes comprise miR-3059-5p and tissue-targeting protein RVG, which is connected with the transmembrane protein Lamp2b of the exosomes; and the miR-3059-5p comprises pri-miR-3059-5p, pre-miR-3059-5p or mature miR-3059-5p.

2. Use of exosomes secreted by genetically modified EPC cells in the preparation of a drug for treating brain injury induced by ischemic stroke, wherein, the genetically modified EPC cells express tissue-targeting protein RVG and miR-3059-5p; the exosomes comprise miR-3059-5p and tissue-targeting protein RVG, which is connected with the transmembrane protein Lamp2b of the exosomes; and the miR-3059-5p comprises pri-miR-3059-5p, pre-miR-3059-5p or mature miR-3059-5p.

3. Use of exosomes secreted by genetically modified EPC cells in the preparation of a drug for improving the prognosis of patients with ischemic stroke, wherein, the genetically modified EPC cells express tissue-targeting protein RVG and miR-3059-5p; the exosomes comprise miR-3059-5p and tissue-targeting protein RVG, which is connected with the transmembrane protein Lamp2b of the exosomes; and the miR-3059-5p comprises pri-miR-3059-5p, pre-miR-3059-5p or mature miR-3059-5p.

4. Use of exosomes secreted by genetically modified EPC cells in the preparation of a drug for improving the motor coordination function or neurocognitive function of patients in the recovery period of ischemic stroke, wherein, the genetically modified EPC cells express tissue-targeting protein RVG and miR-3059-5p; the exosomes comprise miR-3059-5p and tissue-targeting protein RVG, which is connected with the transmembrane protein Lamp2b of the exosomes; and the miR-3059-5p comprises pri-miR-3059-5p, pre-miR-3059-5p or mature miR-3059-5p.

5. Use of exosomes secreted by genetically modified EPC cells in the preparation of a drug for improving hippocampal synaptic plasticity of patients with ischemic stroke, wherein, the genetically modified EPC cells express tissue-targeting protein RVG and miR-3059-5p; the exosomes comprise miR-3059-5p and tissue-targeting protein RVG, which is connected with the transmembrane protein Lamp2b of the exosomes; and the miR-3059-5p comprises pri-miR-3059-5p, pre-miR-3059-5p or mature miR-3059-5p. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The miR-3059-5p includes pri-miR-3059-5p, pre-miR-3059-5p, or mature miR-3059-5p. The miR-3059-5p includes pri-miR-3059-5p, pre-miR-3059-5p, or mature miR-3059-5p.

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