A method for preparing and applying umbilical cord mesenchymal stem cells for treating cerebral infarction

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically relates to an umbilical cord mesenchymal stem cell for treating cerebral infarction, and a preparation method and application thereof. The exosomes of the umbilical cord mesenchymal stem cells provided by the present invention contain a fusion protein, which is formed by the fusion of a peptide targeting brain tissue and an exosome transmembrane protein CD63, and is anchored on the surface of the exosomes, thereby ensuring that the exosomes have a stronger ability to cross the blood-brain barrier, improve targeting and activity, and further treat cerebral infarction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to exosomes derived from umbilical cord mesenchymal stem cells and a preparation method and application thereof. Background Art

[0002] Stroke is a common neurological disease with high disability and mortality rates. It can be divided into ischemic stroke and hemorrhagic stroke, of which ischemic stroke is also called cerebral infarction. So far, the treatment of cerebral infarction is mainly thrombolysis and thrombectomy, but these two methods not only have a narrow effective treatment time window, but are also prone to various clinical complications. In addition, these two methods only reopen the occluded blood vessels, but fail to inhibit local tissue verification reactions and promote the repair of nerve function during blood reperfusion.

[0003] In recent years, stem cells and their secreted exosomes have become a strategy for treating the brain. Exosomes secreted by stem cells carry substances such as DNA, RNA, proteins, and lipids, which can transfer these contents from one cell to another and play a role in target cells. Studies have shown that exosomes may be involved in physiological processes such as inflammatory response, apoptosis, angiogenesis, maintenance of blood-brain barrier integrity, and oxidative stress, and can promote the reduction of infarct area and the repair of neurological function in rodent ischemic stroke models, including exosomes derived from mesenchymal stem cells. Not only that, because exosomes are natural carriers for transporting biological molecules between cells, they are also effective tools for drug therapy. They can improve the water solubility of drug systems, reduce the toxicity of drugs, and can be cultured in large quantities in vitro. They are very stable in themselves. Therefore, exosome transplantation for the treatment of cerebral infarction is very popular. However, exosomes have poor targeting specificity and their application is very limited. Therefore, the targeting of exosomes is an urgent problem to be solved. Summary of the invention

[0004] Based on the deficiencies in the prior art, the purpose of the present invention is to provide an umbilical cord mesenchymal stem cell exosome, the surface of which expresses a fusion protein of a brain tissue-targeting peptide and an exosome transmembrane protein CD63, and the exosome can improve targeting and improve the therapeutic effect.

[0005] One aspect of the present invention is to provide a peptide targeting brain tissue. Preferably, the peptide of the present invention is obtained by screening through phage display technology. Specifically, the present invention uses phage display technology to screen and obtain peptide segments with high binding specificity to brain tissue from a polypeptide library. Specifically, the amino acid sequence of the peptide of the present invention is shown in SEQ ID No. 1: CTHTGNSPALVC.

[0006] The steps for preparing the peptide targeting brain tissue are as follows:

[0007] (1) selecting a phage displayed random twelve-peptide library for screening;

[0008] (2) 4 rounds of panning to enrich positive clones;

[0009] (3) Sequencing the positive clones obtained;

[0010] (4) Screening for peptides with high binding specificity.

[0011] Specifically, the Ph.D-C12C from NEW ENGLAND Biolabs TM The peptide library was diluted to 1×10 10 pfu titer, the diluent is TBS buffer, and it is injected into the rat through the tail vein. The rat is killed after 60 minutes, and the heart is perfused with normal saline to remove non-specific phages in circulation. The rat brain tissue is taken in the clean bench, homogenized under sterile conditions, and TBS containing 1Mm pmsf, 20μg / ml protease inhibitor and 1μg / ml leupeptin is added at the same time. It is rinsed three times with TBST, and glycine elution buffer is added to elute and recover the specific phages that have entered the brain. 150μl TBS is added for neutralization, and the supernatant is collected, which is the phage obtained in the first round of screening, and then amplified for the next round of screening.

[0012] Specifically, in the amplification method, the E. coli culture was diluted 1:100 and inoculated into LB medium, cultured at 37°C in a shaking incubator for 5 hours, and after centrifugation of the culture solution, 500 μl of the supernatant was transferred into a clean centrifuge tube to obtain an amplified phage storage solution.

[0013] In the second round of screening, the concentration of Tween-20 in TBST in the washing solution was increased to 0.5%, the number of washes was increased to 5 times, and the other conditions were the same as the first round.

[0014] In the third round of screening, the concentration of Tween-20 in TBST in the washing solution was increased to 1%, the number of washes was increased to 8 times, and the other conditions were the same as the first round.

[0015] The fourth round of screening is the same as the third round.

[0016] Positive clones were collected from the fourth round of results for sequencing.

[0017] The most specific binding peptides were screened by immunohistofluorescence localization.

[0018] Specifically, the synthetic polypeptide was labeled with a Cy5.5 fluorescent group and injected into a rat cerebral infarction model via the tail vein. After circulating in vivo for 12 hours, the rats were killed, and the strongest brain tissue binding peptide CTHTGNSPALVC was screened based on the fluorescence intensity and co-localization of brain cells.

[0019] Another aspect of the present invention also provides an exosome transmembrane protein CD63, which binds to the CD63 receptor anchored on the surface of the exosome membrane, and its amino acid sequence is shown in SEQ ID No. 2: MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCIDVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM.

[0020] Another aspect of the present invention also provides a fusion protein, specifically, a peptide targeting brain tissue is directly connected to a CD63 protein to construct a fusion protein. The fusion protein of the present invention binds to CD63R on the surface of the exosome through CD63, anchors the surface of the exosome, so that the peptide targeting brain tissue is tightly connected to the exosome through the form of binding of CD63 to the CD63 receptor, and then targets the brain tissue through the polypeptide, bringing the exosome to the lesion area for treatment, improving the targeting of the exosome, prolonging the residence time of the exosome in the lesion area, and improving the effect of treating cerebral infarction.

[0021] The steps for preparing the fusion protein are as follows:

[0022] (1) Directly synthesize the DNA sequence of CTHTGNSPALVC-CD63 and construct a recombinant plasmid;

[0023] (2) Inserting a lentiviral vector to construct a recombinant expression vector;

[0024] (3) Transfect 293T cells to express the fusion protein.

[0025] Specifically, the DNA sequence of CTHTGNSPALVC-CD63 was directly synthesized

[0026] DNA sequence of CTHTGNSPALVC: (SEQ ID No. 3)

[0027] tgcacccataccggcaacagcccggcgctggtgtgc

[0028] DNA sequence of CD63: (SEQ ID No.4)

[0029] atggcggtgg aaggaggaat gaaatgtgtg aagttcttgc tctacgtcct cctgctggcc

[0030] tttttgcgcct gtgcagtggg actgattgcc gtgggtgtcg gggcacagct tgtcctgagt

[0031] cagaccataa tccaggggc tacccctggc tctctgttgc cagtggtcat catcgcagtg

[0032] ggtgtcttcc tcttcctggt ggcttttgtg ggctgctgcg gggcctgcaa ggagaactat

[0033] tgtcttatga tcacgtttgc catctttctg tctcttatca tgttggtgga ggtggccgca

[0034] gccattgctg gctatgtgtt tagagatag gtgatgtcag agtttaataa caacttccgg

[0035] cagcagatgg agaattaccc gaaaaaac caacactgctt cgatcctgga caggatgcag

[0036] gcagatttta agtgctgtgg ggctcctaac tacacagatt gggagaaaat cccttccatg

[0037] tcgaagaacc gagtccccga ctcctgctgc attgatgtta ctgtgggctg tgggattaat

[0038] ttcaacgaga aggcgatcca taggagggc tgtgtggaga agattggggg ctggctgagg

[0039] aaaaatgtgc tggtggtagc tgcagcagcc cttggaattg cttttgtcga ggttttggga

[0040] attgtctttg cctgctgcct cgtgaagagt atcagaagtg gctacgaggt gatg

[0041] The linearized pLVX-IRES-Puro plasmid was ligated with the above target gene using T4 ligase.

[0042] Take the recombinant plasmid and add it to the competent cell JM110, react in an ice bath for 20-30 minutes, then put the competent cells containing the plasmid into a 42°C water bath, heat shock for 90 seconds, and put them on ice again for 5 minutes. After the heat shock transformation is completed, add 1ml LB medium and mix it, and shake and culture it in a shaker at 37°C for 60 minutes at a speed of 150rpm. Spread the LB plate and pick the monoclonal strain. Expand the monoclonal strain and extract the plasmid to obtain the pLVX-targeting peptide-CD63 recombinant plasmid.

[0043] On the other hand, 293T cells were cultured to obtain stably passaged 293T cells.

[0044] On the other hand, for lentiviral packaging, the cell culture dish is coated with poly-lysine, and the above-mentioned 293T cells are inoculated in the culture dish. The cell culture medium is changed once a day until the cell density reaches about 70%-80%. Prepare a three-plasmid system, VSVG, psPAX2 and recombinant plasmid, with a mass ratio of 2:3:4, and the volume ratio of the three plasmids: transfection reagent lip8000 is 1:2. Replace the cell culture medium with fresh serum-free complete culture medium, and evenly add 750μl of opti-MEM, three plasmids and lip8000 mixture to the culture dish and mix gently. After 4-6 hours of culture, replace the cell culture medium. After 48 hours of continuous culture, the virus packaging is completed.

[0045] Another aspect of the present invention is to provide an exosome, specifically, the surface of the exosome is anchored with a fusion protein via a CD63 receptor, wherein the exosome of the present invention is derived from umbilical cord mesenchymal stem cells.

[0046] The steps for preparing exosomes are as follows:

[0047] Extract human umbilical cord tissue, rinse it with PBS solution, remove blood vessels, and cut it into pieces of about 1-3 mm. 3sized tissue blocks; put the tissue blocks into 1g / L collagenase IV solution and stir and digest for 20 minutes at 37°C; transfer the tissue blocks to 1g / L pancreatin solution and continue to stir and digest for 25 minutes; filter with a 200-mesh cell sieve and centrifuge the filtered cell suspension (1000rpm, 5 minutes); discard the supernatant after centrifugation, resuspend the cells with DMEM-F12 medium, and inoculate them in a culture bottle containing DMEM / F12 medium (containing 20% ​​fetal bovine serum, 25mmo l / L glutamine, and 1% double antibody) at a cell density of 1.0×106 / cm2; replace the medium after 4 days of culture, and then change the medium once every 3 days. After the first passage after 10 days, the culture is subcultured every 3 days at a ratio of 1:3 for expansion culture. The human umbilical cord mesenchymal stem cells isolated and cultured were revived to 2 150cm 2 Culture the cells in a 150 cm dish using a cell culture medium containing 10% fetal bovine serum for 2-3 days. When the cell density reaches 80%, subculture the cells to 20 150 cm 2 Continue to culture in the culture dish. When the cell density reaches more than 85%, discard the existing culture medium and wash 3 times with PBS. Replace the culture medium with a serum-free medium with clear ingredients. After 6 hours of culture, discard the cell supernatant, add a new serum-free medium and continue to culture for 48 hours before collecting the culture supernatant. The collected cell culture supernatant is centrifuged at 4°C and 500g for 10 minutes to remove dead cells; after collecting the supernatant, centrifuge at 4°C and 2000g for 20 minutes to remove cell debris and impurities; after collecting the supernatant, centrifuge at 4°C and 10000g for 30 minutes to remove apoptotic bodies; after collecting the supernatant, centrifuge at 4°C and 110000g for 80 minutes to separate exosomes; after discarding the supernatant, resuspend the precipitate with PBS, centrifuge again at 4°C and 110000g for 80 minutes, discard the supernatant, and the remaining is UC-MSC exosomes. After resuspending with sterile PBS, filter with a 0.22nm filter membrane for sterilization and store at -80°C for later use.

[0048] This also includes identifying the prepared mesenchymal stem cell exosomes.

[0049] (1) Western Blot (WB) was used to identify exosome surface markers: CD9, CD63, CD81, and internal markers: TSG101, ALIX, etc.

[0050] (2) Electron microscopy analysis of the morphological characteristics of exosomes: saucer-shaped or hemispherical with one side concave.

[0051] Another aspect of the present invention is to provide an application of umbilical cord mesenchymal stem cells for treating cerebral infarction. Specifically, the exosomes anchor the fusion protein through the surface of the CD63 receptor, and then target the brain tissue through the polypeptide, so that the exosomes are brought to the lesion area for treatment, thereby improving the targeting of the exosomes, prolonging the residence time of the exosomes in the lesion area, and improving the effect of treating cerebral infarction.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] 1. The present invention screened a peptide CTHTGNSPALVC that specifically targets brain tissue through phage display technology, and constructed a fusion protein with CD63. The property of CD63 binding to the CD63 receptor on the surface of the exosome membrane is used to anchor the fusion protein on the surface of the exosome, and the peptide is further used to bring the exosome to the brain tissue, and finally the exosome is used to treat cerebral infarction.

[0054] 2. Through the targeting of peptides and CD63 as a bridge, exosomes are brought to the lesion area, which not only improves the targeting of exosomes, but also increases the residence time of exosomes in the lesion area, thereby improving the treatment efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Electron micrograph of exosomes

[0056] Figure 2 WB expression of neuronal marker protein tubulin-β3 and glial cell marker protein GFAP DETAILED DESCRIPTION

[0057] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0058] The human umbilical cord mesenchymal stem cells used in the following embodiments were purchased from ATCC.

[0059] Example 1: Screening of peptides targeting brain tissue using phage display technology

[0060] Ph.D-C12C from NEW ENGLAND Biolabs TM The peptide library was diluted to 1×10 10pfu titer, the diluent is TBS buffer, and it is injected into the rat through the tail vein. The rat is killed 60 minutes later, and the heart is perfused with physiological saline to remove non-specific phages in circulation. The rat brain tissue is taken from the clean bench, homogenized under sterile conditions, and TBS containing 1Mm pmsf, 20μg / ml protease inhibitor and 1μg / ml leupeptin is added at the same time. It is rinsed three times with TBST, and glycine elution buffer is added to elute and recover the specific phages that have entered the brain. 150μl TBS is added to neutralize, and the supernatant is collected, which is the phage obtained in the first round of screening, and then amplified for the next round of screening. The amplification method is to dilute the Escherichia coli culture at 1:100 and inoculate it in LB medium, culture it on a shaker at 37℃ for 5h, and after the culture fluid is centrifuged, 500μl of the supernatant is transferred to a clean centrifuge tube to obtain the amplified phage storage solution. In the second round of screening, the concentration of Tween-20 in the TBST in the washing solution is increased to 0.5%, and the number of washes is increased to 5 times. The other conditions are the same as the first round. In the third round of screening, the concentration of Tween-20 in TBST in the washing solution was increased to 1%, the number of washes was increased to 8 times, and the other conditions were the same as the first round. The fourth round of screening was the same as the third round. Positive clones were collected from the results of the fourth round for sequencing. The above-synthesized peptides were labeled with the Cy5.5 fluorescent group and injected into the rat cerebral infarction model via the tail vein. After circulating in the body for 12 hours, the rats were killed, and the strongest brain tissue binding peptide CTHTGNSPALVC was screened based on the fluorescence intensity and co-localization of brain cells.

[0061] Example 2: Construction of fusion protein

[0062] DNA sequence of peptide targeting brain tissue: (SEQ ID No. 3)

[0063] tgcacccataccggcaacagcccggcgctggtgtgc

[0064] DNA sequence of CD63: (SEQ ID No.4)

[0065] atggcggtgg aaggaggaat gaaatgtgtg aagttcttgc tctacgtcct cctgctggcc

[0066] ttttgcgcct gtgcagtggg actgattgcc gtgggtgtcg gggcacagct tgtcctgagt

[0067] cagaccataa tccagggggc tacccctggc tctctgttgc cagtggtcat catcgcagtg

[0068] ggtgtcttcc tcttcctggt ggcttttgtg ggctgctgcg gggcctgcaa ggagaactat

[0069] tgtctttatga tcacgtttgc catctttctg tctcttatca tgttggtgga ggtggccgca

[0070] gccattgctg gctatgtgtt tagagataag gtgatgtcag agtttaataa caacttccgg

[0071] cagcagatgg agaattaccc gaaaaacaac cacactgctt cgatcctgga caggatgcag

[0072] gcagatttta agtgctgtgg ggctgctaac tacacagatt gggagaaaat cccttccatg

[0073] tcgaagaacc gagtccccga ctcctgctgc attgatgtta ctgtgggctg tgggattaat

[0074] ttcaacgaga aggcgatcca taaggagggc tgtgtggaga agattggggg ctggctgagg

[0075] aaaaatgtgc tggtggtagc tgcagcagcc cttggaattg cttttgtcga ggttttggga

[0076] attgtctttg cctgctgcct cgtgaagagt atcagaagtg gctacgaggt gatg

[0077] The above target gene was synthesized, and the linearized pLVX-IRES-Puro plasmid was ligated with the above target gene using T4 ligase.

[0078] Take the recombinant plasmid and add it to the competent cell JM110, react in an ice bath for 20-30 minutes, then put the competent cells containing the plasmid into a 42°C water bath, heat shock for 90 seconds, and put them on ice again for 5 minutes. After the heat shock transformation is completed, add 1ml LB medium and mix it, and shake and culture it in a shaker at 37°C for 60 minutes at a speed of 150rpm. Spread the LB plate and pick the monoclonal strain. Expand the monoclonal strain and extract the plasmid to obtain the pLVX-targeting peptide-CD63 recombinant plasmid.

[0079] 293T cells were cultured to obtain stably passaged 293T cells.

[0080] For lentiviral packaging, coat the cell culture dish with poly-lysine, inoculate the above 293T cells in the culture dish, change the cell culture medium once a day until the cell density reaches about 70%-80%. Prepare a three-plasmid system, VSVG, psPAX2 and recombinant plasmid, with a mass ratio of 2:3:4, and the volume ratio of the three plasmids: transfection reagent lip8000 is 1:2. Replace the cell culture medium with fresh serum-free complete culture medium, and evenly add 750μl of opti-MEM, three plasmids and lip8000 mixture to the culture dish and mix gently. After 4-6 hours of culture, replace the cell culture medium. After 48 hours of continuous culture, complete the virus packaging.

[0081] Example 3: Preparation of exosomes

[0082] Extract human umbilical cord tissue, rinse it with PBS solution, remove blood vessels, and cut it into pieces of about 1-3 mm. 3 sized tissue blocks; put the tissue blocks into 1g / L collagenase IV solution and stir and digest for 20 minutes at 37°C; transfer the tissue blocks to 1g / L pancreatin solution and continue to stir and digest for 25 minutes; filter with a 200-mesh cell sieve and centrifuge the filtered cell suspension (1000rpm, 5 minutes); discard the supernatant after centrifugation, resuspend the cells with DMEM-F12 medium, and inoculate them in a culture bottle containing DMEM / F12 medium (containing 20% ​​fetal bovine serum, 25mmo l / L glutamine, and 1% double antibody) at a cell density of 1.0×106 / cm2; replace the medium after 4 days of culture, and then change the medium once every 3 days. After the first passage after 10 days, the culture is subcultured every 3 days at a ratio of 1:3 for expansion culture. The human umbilical cord mesenchymal stem cells isolated and cultured were revived to 2 150cm 2 Culture the cells in a 150 cm dish using a cell culture medium containing 10% fetal bovine serum for 2-3 days. When the cell density reaches 80%, subculture the cells to 20 150 cm 2Continue to culture in the culture dish. When the cell density reaches more than 85%, discard the existing culture medium and wash 3 times with PBS. Replace the culture medium with a serum-free medium with clear ingredients. After 6 hours of culture, discard the cell supernatant, add a new serum-free medium and continue to culture for 48 hours before collecting the culture supernatant. The collected cell culture supernatant is centrifuged at 4°C and 500g for 10 minutes to remove dead cells; after collecting the supernatant, centrifuge at 4°C and 2000g for 20 minutes to remove cell debris and impurities; after collecting the supernatant, centrifuge at 4°C and 10000g for 30 minutes to remove apoptotic bodies; after collecting the supernatant, centrifuge at 4°C and 110000g for 80 minutes to separate exosomes; after discarding the supernatant, resuspend the precipitate with PBS, centrifuge again at 4°C and 110000g for 80 minutes, discard the supernatant, and the remaining is UC-MSC exosomes. After resuspending with sterile PBS, filter with a 0.22nm filter membrane for sterilization and store at -80°C for later use.

[0083] The prepared mesenchymal stem cell exosomes were identified: the morphological characteristics of the exosomes were analyzed by electron microscopy: saucer-shaped or hemispherical with a depression on one side.

[0084] Example 4: Application of exosomes in treating cerebral infarction

[0085] (1) Preparation of rat cerebral infarction model (animal model of middle cerebral artery occlusion (MCAO))

[0086] This experiment used SPF healthy male SD rats (weight 200-230g, age 7-8 weeks). Before the experiment, all rats were adapted to an environment with 12h light and dark alternation for 7 days, and they had free access to food and water. The rats were randomly divided into 3 groups, MCAO+PBS group, MCAO+EVs (without fusion protein) group and MCAO+EVs (with fusion protein) group, with 6 rats in each group. Within 1 hour after the completion of the MCAO animal model surgery, the tail vein of each group was injected with drugs, that is, 200μL PBS was injected into each rat in the MCAO+PBS group, once a day, for 3 consecutive days. The treatment method of the MCAO+EVs (without fusion protein) group was to prepare the exosomes derived from umbilical cord mesenchymal stem cells with PBS to 2mg / mL umbilical cord mesenchymal stem cells. Stem cell-derived exosomes, and then each rat was injected with 200 μL of 2 mg / mL umbilical cord mesenchymal stem cell-derived exosomes, once a day, for 3 consecutive days. The treatment method of the MCAO+EVs (containing fusion protein) group was to prepare the umbilical cord mesenchymal stem cell-derived exosomes prepared in Example 3 with PBS to prepare 2 mg / mL umbilical cord mesenchymal stem cell-derived exosomes, and then each rat was injected with 200 μL of 2 mg / mL umbilical cord mesenchymal stem cell-derived exosomes, once a day, for 3 consecutive days.

[0087] Construction of MCAO animal model: 200-230g healthy male SD rats were taken and fasted for 12 hours before surgery. Water was available freely. 1% sodium pentobarbital (5mL / kg) was used for intraperitoneal injection anesthesia. After the rats were fully anesthetized, the rats were placed in a supine position, the head and limbs were fixed, the hair on the neck was shaved, and the neck of the rats was disinfected with iodine. A midline incision was made in the neck with ophthalmic scissors, and a small retractor was used to pull open the skin and soft tissue to expose the blood vessels. The left common carotid artery (CCA) and the external carotid artery (ECA) were separated. A slipknot was tied in the CCA to avoid stimulating the vagus nerve and trachea accompanying the common carotid artery. A knot was tied at the distal end of the ECA, a slipknot was tied near the bifurcation at the proximal end of the ECA, and a slipknot was tied in the middle of the ECA vein with a suture. A slipknot was tied around the ECA near the bifurcation of the CCA, a knot was tied near the head of the distal end of the ECA and fixed on the tape to exert a slight pulling effect, and a slipknot was tied at the proximal end of the internal carotid artery (ICA). A thread plug that matched the weight of the rat was taken, and a small opening was made between the first and second threads at the distal end of the ECA with ophthalmic scissors at an angle of 45 degrees toward the bifurcation of the ECA, but it could not be cut off; the thread plug was inserted from the opening toward the bifurcation of the ECA, and the thread plug was fixed with the second thread to prevent slipping; the thread at the proximal end of the ECA was loosened, the distal end of the ECA was cut off, the thread plug was turned toward the ICA, and the thread plug was slowly inserted along the ICA. When the insertion encountered resistance, the insertion was stopped immediately, and the wound was sutured. During the operation, a constant temperature pad was used to maintain the rectal temperature of the rat at about 37°C. After the operation, the rat was placed in a warming blanket until it woke up.

[0088] (2) Detection of neuronal marker protein expression in brain tissue

[0089] Brain tissues of three rats in each group were used for protein extraction, and the expressions of neuronal marker protein tubulin-β3 and glial cell marker protein GFAP were quantified by WB.

[0090] (3) Three rats from each group were selected for behavioral evaluation.

[0091] Results and Analysis of Examples 1-4

[0092] Example 1: Through four rounds of enrichment, the strongest brain tissue binding peptide CTHTGNSPALV C was screened and obtained.

[0093] Example 2: By sequencing the fusion protein, it can be seen that the constructed fusion protein is expressed correctly.

[0094] Example 3: Exosomes were identified by electron microscopy and the exosomes were found to have a diameter of 30-100 nm and a typical cup-shaped shape ( Figure 1 ).

[0095] Embodiment 4:

[0096] (1) Through WB analysis, the neuron marker protein tubulin-β3 in the brain tissue of the exosome group containing the fusion protein increased significantly compared with the PBS treatment group, and also showed a better therapeutic effect compared with the exosome group without the fusion protein; the glial cell marker protein GFAP in the brain tissue of the exosome group containing the fusion protein decreased significantly compared with the PBS treatment group, and also showed a better therapeutic effect compared with the exosome group without the fusion protein. It can be seen that the exosomes containing the fusion protein of the present invention improve the effect of exosomes in treating cerebral infarction by utilizing the targeting of the peptide ( Figure 2 ).

[0097] (2) Behavioral analysis:

[0098] After 3 days of administration, the neurobehavioral scores of rats in each group were evaluated according to the Longa

[0099] Scoring method. Longa scoring standard is 0 points: no symptoms of nerve damage; 1 point: unable to fully extend the contralateral forelimb; 2 points: the body turns to the contralateral side when walking; 3 points: the body leans to the contralateral side when walking; 4 points:

[0100] Points: Unable to walk spontaneously, loss of consciousness.

[0101] The results showed that the neurological function scores of rats in the MCAO+PBS treatment group were significantly increased;

[0102] The neurological scores of the MCAO+EVs (without fusion protein) group and the MCAO+EVs (with fusion protein) group showed a significant downward trend, indicating that the injection of exosomes derived from umbilical cord mesenchymal stem cells significantly improved the neurobehavioral ability of rats.

[0103] The neurological score of the group (without fusion protein) was slightly lower, indicating that the targeting of the fusion protein improved the therapeutic effect (Table 1).

[0104] Table 1

[0105]

[0106] The above describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the description only describe the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A polypeptide targeting brain tissue, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. The polypeptide according to claim 1, characterized in that The polypeptide is obtained by screening through phage display technology.

3. A fusion protein, characterized in that The fusion protein is formed by directly fusing the polypeptide according to claim 1 with the CD63 protein, and the amino acid sequence of the CD63 is shown in SEQ ID No.

2.

4. The fusion protein according to claim 3, characterized in that The fusion protein is expressed by transfecting 293T cells with a lentiviral expression vector.

5. An exosome of umbilical cord mesenchymal stem cells, characterized in that: The fusion protein according to claim 3 is anchored on the membrane surface of the exosomes.

6. The exosome according to claim 5, characterized in that The fusion protein binds to the CD63 receptor on the membrane surface of the exosome through CD63, so that the fusion protein is anchored on the membrane surface of the exosome.

7. A use of the exosomes according to claim 5, characterized in that: The application is for preparing medicine for treating cerebral infarction.

8. The use according to claim 7, characterized in that: The treatment is to anchor the CD63 of the fusion protein on the membrane surface of the exosome by binding to the CD63 receptor on the membrane surface of the exosome, and then target the exosome to the brain tissue through the polypeptide, thereby using exosomes to treat cerebral infarction.

9. A drug for treating cerebral infarction, characterized in that: The medicine comprises the exosomes according to claim 5 or mesenchymal stem cells comprising the exosomes according to claim 5.

10. The medicament according to claim 9, characterized in that The medicine further comprises a pharmaceutically acceptable carrier.

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