阅读说明：本技术 制备外泌体方法及其药物组合物在组织修复中的应用 (Method for preparing exosome and application of pharmaceutical composition thereof in tissue repair ) 是由 李少波 亓爱杰 于 2021-08-16 设计创作，主要内容包括：本发明涉及制备外泌体方法及其药物组合物在组织修复中的应用。本发明提供了制备外泌体的方法以及获得了针对天冬氨酸内肽酶的单克隆抗体,将外泌体、bFGF和单克隆抗体三者联合使用之后,可以有效的用于脊髓损伤的治疗中去,并且效果良好,比单独使用外泌体和单抗的效果都要显著。具有很好的市场应用价值。(The invention relates to a method for preparing exosomes and application of a pharmaceutical composition thereof in tissue repair. The invention provides a method for preparing an exosome and a monoclonal antibody for aspartic endopeptidase, and the exosome, bFGF and the monoclonal antibody are combined for use, so that the exosome, bFGF and the monoclonal antibody can be effectively used for treating spinal cord injury, and the effect is good and is more obvious than that of singly using the exosome and the monoclonal antibody. Has good market application value.)

1. A pharmaceutical combination consisting of an exosome, bFGF and monoclonal antibody; the combination is for use in the treatment of spinal cord injury; wherein the exosome is prepared by adopting the following method: taking 1mL serum, adding 19mLPBS, 300g, centrifuging at 4 ℃ for 10 min; taking the supernatant, transferring the supernatant into a new centrifuge tube, and centrifuging the supernatant at 2000g and 4 ℃ for 10 min; transferring the supernatant to a new centrifuge tube, and adding 8% 2X PEG6000 into serum, wherein the specific formula comprises 16g PEG6000+5.844g NaCl +100mL ultrapure water to prepare 8% 2X PEG6000 solution; standing at 4 deg.C, incubating for 30min, centrifuging at 4 deg.C for 1 hr at 10000g, removing supernatant, resuspending with 20ml PBS, centrifuging at 4 deg.C for 70min, and precipitating at bottom to obtain exosome;

the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO: 1 is shown in the specification; the heavy chain variable region sequence is shown as SEQ ID NO: 2, respectively.

2. Use of an exosome, bFGF and monoclonal antibody in the preparation of a combination for treating spinal cord injury; wherein the exosome is prepared by adopting the following method: taking 1mL serum, adding 19mLPBS, 300g, centrifuging at 4 ℃ for 10 min; taking the supernatant, transferring the supernatant into a new centrifuge tube, and centrifuging the supernatant at 2000g and 4 ℃ for 10 min; transferring the supernatant to a new centrifuge tube, and adding 8% 2X PEG6000 into serum, wherein the specific formula comprises 16g PEG6000+5.844g NaCl +100mL ultrapure water to prepare 8% 2X PEG6000 solution; standing at 4 deg.C, incubating for 30min, centrifuging at 4 deg.C for 1 hr at 10000g, removing supernatant, resuspending with 20ml PBS, centrifuging at 4 deg.C for 70min, and precipitating at bottom to obtain exosome;

the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO: 1 is shown in the specification; the heavy chain variable region sequence is shown as SEQ ID NO: 2, respectively.

3. The use according to claim 2, further comprising a pharmaceutically acceptable carrier.

4. Use according to claim 3, characterized in that the carrier is a variety of excipients.

5. Use according to claim 4, characterized in that the excipients comprise diluents, binders, wetting agents, disintegrants, lubricants, glidants.

6. Use according to claim 3, characterized in that it further comprises a diluent which is starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulphate, dibasic calcium phosphate, or calcium carbonate.

Technical Field

The invention relates to the field of biology, in particular to a method for preparing exosomes and application of pharmaceutical compositions of the exosomes in tissue repair.

Background

Exosomes were first discovered in 1983 by Harding et al in rat reticulocytes, and then in 1985 Pan et al observed with an endoscope during the development of sheep reticulocytes into mature erythrocytes, and these vesicles were also discovered in Johnstone et al formally defined as "exosomes", exosomes, in 1987. At first, exosomes are considered to be only some unnecessary cellular components for cell clearance, until 2007, Valadi et al found genetic materials such as microRNA and mRNA in exosomes for the first time, so that people have further knowledge on the composition of exosomes, and at the same time, people also realize that exosomes can be a new mechanism for mediating intercellular communication, and therefore people have conducted deeper research on the components, formation mechanism, action mechanism and function of exosomes.

The exosome is an endosome (ILVs) formed by invagination of an endosome in the process of changing an early endosome to a later endosome/multivesicular body (MVBs), the endosome is a subcellular bilayer membrane structure, the membrane structure is rich in components such as sphingomyelin, cholesterol and ceramide, and simultaneously contains a small amount of lipid rafts and the like, phosphatidylserine and the like are also found in some membrane structures of the exosome, when the endosome is formed, some specific cytoplasmic proteins, micrornas and mrnas, even including some small soluble biological factors such as chemokines, growth factors, transcription factors, cytokines and the like are contained in the membrane structure to form a closed space with the diameter of 40-100nm, and when the endosome/multivesicular body is fused with the late endosome, the endosome is an exocytosis membrane vesicle. Thus, there are two possible routes for exosomes to enter target cells: first, it is captured into cells via cellular diapause; secondly, the fusion is carried out with the target cell membrane in a membrane fusion mode, and substances and information contained in the fusion are directly released to target cells. The biological functions performed by different cells vary due to the differences in proteins and genetic material secreted by them.

Tissue damage caused by various diseases, such as myocardial ischemia-reperfusion injury, spinal cord injury, neuron injury, acute kidney injury, brain injury, skin mucosa and vascular endothelium injury, skin injury caused by burn, and the like, is always the most main cause of high morbidity and high mortality. The treatment effect is not very satisfactory. As the most common treatment for burns, skin grafting, is not only limited in its source of skin but also susceptible to immunological rejection. Particularly, the organ displacement deformity caused by scar tissues in a certain area caused by facial trauma can bring huge psychological damage to patients. A large amount of evidence shows that the exosome is a vesicle with the diameter of 30-100 nm and secreted by various tissue cells, has wide sources and basically exists in all organisms. In addition, the protein-rich recombinant human immunodeficiency virus (mRNA), microRNA, protein and other components are contained in the protein-rich recombinant human immunodeficiency virus (mRNA), can exchange information with target cells, promotes the proliferation and migration of the cells or inhibits the apoptosis of the cells, and further achieves the aim of promoting the tissue damage repair.

The pathophysiological mechanisms of spinal cord injury are complex and include apoptotic inflammatory responses, vascular injury, excitotoxicity, fluid-electrolyte disorders, mitochondrial dysfunction, calcium, and other processes. Where apoptosis and inflammatory responses are the major events of secondary injury to spinal cord injury. Apoptosis is mainly regulated by an upstream Bcl-2 family and a downstream caspase family, wherein an anti-apoptotic protein Bcl-2 and a pro-apoptotic protein Bax are the most common apoptosis markers of apoptosis. 100 μ g total exosome protein was implanted into spinal cord injured rats in the T10 shock model by tail vein injection and MSCs-exosomes treatment was found to reduce the number of TUNEL positive cells in the spinal cord of rats after spinal cord injury. At the same time, the level of pro-apoptotic Bax is significantly inhibited, while the level of anti-apoptotic Bcl-2 is up-regulated. The results indicate that MSCs-exosomes play an anti-apoptotic role in spinal cord injury.

Following spinal cord injury, the blood-brain barrier is damaged and the injured spinal cord can be rapidly infiltrated with neutrophils from the blood. TNF-alpha positive and IL-1 beta positive cells were strongly upregulated in the perispinal cord area following spinal cord injury. TNF-alpha and IL-1 beta are important inflammatory mediators that potentiate neuronal cell death in the spinal cord. MSCs-exosomes injected into tail vein can obviously reduce protein level of proinflammatory cytokines (TNF-alpha and IL-1 beta) and up-regulate protein level of anti-inflammatory cytokines (IL-10), and play an anti-inflammatory role after spinal cord injury. Aspartic endopeptidase inhibitor treated spinal cord injured mice are capable of improving spinal cord injury repair.

The exosome is used as a new treatment strategy and shows a certain curative effect on the treatment of spinal cord injury. MSCs-exosomes have the advantage of longer survival in vivo, low carcinogenicity, high delivery efficiency compared to transplanted MSCs. Exosomes can enter the central nervous system not only through the blood brain barrier, but also through gene modification, improving the treatment efficiency. Although few direct reports of exosomes for spinal cord injury treatment are available at present, various characteristics show that exosomes have great prospects for treatment and research of spinal cord injury. Therefore, the study of exosomes for tissue injury such as spinal cord injury becomes particularly urgent.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition for promoting spinal cord injury repair, and provides a preparation method of a serum-extracted exosome, so as to finally realize the recovery of spinal cord injury function.

In one aspect of the present invention, there is provided a method for serum extraction of exosomes, the method comprising the steps of:

1mL of serum was added to 19mL of PBS and centrifuged at 300g and 4 ℃ for 10 min. Taking the supernatant, transferring the supernatant into a new centrifuge tube, and centrifuging the supernatant at 2000g and 4 ℃ for 10 min; transferring the supernatant to a new centrifuge tube, and adding 8% PEG6000(2X) into serum, wherein the specific formula comprises 16g PEG6000+5.844g NaCl +100mL ultrapure water to prepare 8% PEG6000(2X) solution; standing at 4 deg.C for incubation for 30min, centrifuging at 4 deg.C for 1 hr at 10000g, removing supernatant, resuspending with 20ml PBS, centrifuging at 4 deg.C for 70min, and precipitating at bottom to obtain exosome.

In another aspect of the present invention, there is provided a monoclonal antibody specific for aspartic endopeptidase, wherein the sequence of the monoclonal antibody is 3C 7.

Light chain variable region (SEQ ID NO: 1):

DIVITQRPAMMAASPGEKVTITCRQFNMDKGFFWKWYQQKSGISPKPWIYMQCNQEQGVPARFSGSGSGTSYSLTITSMEAEDAATYYCCRGIPWQTCFGAGTKLELK

heavy chain variable region (SEQ ID NO: 2):

EVQLEESATELSRPGASVKLSCKASGYIFSWDKQCWIKQRPGQGLEWIGWTRIHRFLRPKNHHLHGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGDKHDQTQWGLGTTLAVSS。

in one aspect of the invention, a kit is provided, the kit consisting of an exosome, bFGF and a monoclonal antibody; the kit can be used for treating spinal cord injury. Wherein the exosome is prepared by adopting the following method: 1mL of serum was added to 19mL of PBS and centrifuged at 300g and 4 ℃ for 10 min. Taking the supernatant, transferring the supernatant into a new centrifuge tube, and centrifuging the supernatant at 2000g and 4 ℃ for 10 min; transferring the supernatant to a new centrifuge tube, and adding 8% PEG6000(2X) into serum, wherein the specific formula comprises 16g PEG6000+5.844g NaCl +100mL ultrapure water to prepare 8% PEG6000(2X) solution; standing at 4 deg.C, incubating for 30min, centrifuging at 4 deg.C for 1 hr at 10000g, removing supernatant, resuspending with 20ml PBS, centrifuging at 4 deg.C for 70min, and precipitating at bottom to obtain exosome;

the monoclonal antibody is an antibody with the sequence as shown in the specification:

light chain variable region (SEQ ID NO: 1):

DIVITQRPAMMAASPGEKVTITCRQFNMDKGFFWKWYQQKSGISPKPWIYMQCNQEQGVPARFSGSGSGTSYSLTITSMEAEDAATYYCCRGIPWQTCFGAGTKLELK

heavy chain variable region (SEQ ID NO: 2):

EVQLEESATELSRPGASVKLSCKASGYIFSWDKQCWIKQRPGQGLEWIGWTRIHRFLRPKNHHLHGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGDKHDQTQWGLGTTLAVSS。

in another aspect, the kit further comprises a pharmaceutically acceptable carrier.

Further, the kit of the present invention may be prepared according to methods well known in the art. For this purpose, if desired, the two pharmaceutical ingredients of the invention can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to form suitable administration forms or dosage forms for use as human or veterinary medicine.

The kit of the present invention can be administered in unit dosage form, and the administration route can be intestinal or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung, skin, vagina, peritoneum, rectum, etc., preferably intraperitoneal injection.

To form the unit dosage form into a tablet, a wide variety of excipients well known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the humectant can be water, ethanol, isopropanol, etc.; the binder can be starch slurry, dextrin, syrup, Mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene dipropyl alcohol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethylcellulose, sodium carboxymethyl starch, sodium bicarbonate, citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, and sodium dodecyl sulfate; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

For making the administration units into pills, a wide variety of carriers well known in the art can be used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, glycerol laureth glycol, kaolin, talc and the like; binding agent such as acacia, xanthan gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc.

In another aspect, the use of exosomes, bFGF and monoclonal antibodies in the preparation of a kit for the treatment of spinal cord injury is provided.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a method for preparing an exosome and a monoclonal antibody aiming at aspartic endopeptidase, and after the exosome, bFGF and the antibody are used in combination, the exosome, bFGF and the antibody can be effectively used for treating spinal cord injury, and the effect is good and is more obvious than that of singly using the exosome or the monoclonal antibody. Has good market application value.

Drawings

FIG. 1 is a graph showing the results of determination of the specificity of an antibody

FIG. 2BBB score results chart

FIG. 3 BBB score results graph used in combination

FIG. 4 is a graph showing the results of Caspase-3 activity assay

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation and characterization of serum exosomes

1mL of rat serum was added to 19mL of PBS, 300g of the serum, and centrifuged at 4 ℃ for 10 min. The supernatant was transferred to a new centrifuge tube and centrifuged at 2000g at 4 ℃ for 10 min. Transferring the supernatant to a new centrifuge tube, and adding 8% PEG6000(2X) into serum, wherein the specific formula comprises 16g PEG6000+5.844g NaCl +100mL ultrapure water to prepare 8% PEG6000(2X) solution; after incubation at 4 ℃ for 30min, 10000g was centrifuged at 4 ℃ for 1 h. The supernatant was removed, resuspended with 20ml PBS, 100000g, centrifuged at 4 ℃ for 70min, and the bottom pellet was exosome.

Taking a small amount of exosome to crack and extract total protein, running 10% acrylamide gel electrophoresis at the same protein concentration, transferring to a PVDF membrane after the electrophoresis is finished, sealing for 2h by 5% skimmed milk powder, incubating HSP70, CD63 and TSG101 for one night at 4 ℃, adding corresponding secondary antibody to incubate for 1h at room temperature, carrying out exposure imaging in a protein imaging system, and taking pictures. And meanwhile, the exosome is taken out and placed on a copper mesh, and the form of the exosome is observed under a transmission electron microscope after negative staining.

The result shows that the expression of exosome marker proteins HSP70, CD63 and TSG101 is detected in the extracted exosomes, the structure of a saucer shape can be seen under an electron microscope, the particle size is about 90 +/-10 nm, and the purity is high.

Example 2 preparation and characterization of aspartic acid endopeptidase monoclonal antibody

Recombinant mouse aspartic acid endopeptidase is used as immunogen (the cargo number is JN1806, Beijing Baiolai Boke technology, Inc.), 80 mug/L of recombinant mouse aspartic acid endopeptidase is taken and dissolved in 250 mug of physiological saline and is fully emulsified with equal volume of Freund's complete adjuvant, a BALB/c female mouse of 6 weeks old is taken, and the subcutaneous multipoint immunization method is adopted, wherein the immunization dose is 50 mug/mouse. 4 weeks after the first immunization, a second immunization was performed, with half the immunogen dose, and emulsified with Freund's incomplete adjuvant. The 2 nd immunization 7d, from the tail vein, serum antibody titers were detected by ELISA. Selecting a mouse with the highest titer, finally screening 1 strain of the strongest positive cell strain 3C7 by adopting a conventional cell fusion and screening method, and preparing an ascites type antibody by adopting a mouse in vivo induction method; and purifying the ProteinG affinity chromatography column for later use.

Identification of mAb Ig class and subclass the selected antibody Ig class and subclass were determined using SBA Clonotyping System-HRP kit, all procedures were performed exactly according to the kit instructions.

Determination of mAb antibody Affinity constant the Affinity constant (Affinity constant), Ka value, was determined in a non-competitive enzyme immunoassay. With reference to the method of Raghava et al, according to the formula Ka ═ n-1-

2(n [ Ab' ] t- [ Ab ] t) the value of the affinity constant Ka is calculated. The results are shown in Table 1.

TABLE 1 identification of antibodies

mAb	Subtype of cell	Titer of the product	Affinity (L/mol)
				3C7	IgG1	1:2.56×106	1.07×109

As can be seen from the results in Table 1, the subtype of the 3C7 monoclonal antibody was IgG1, and its titer reached 1: 2.56X 10⁶Affinity reaches 1.07 x 10⁹L/mol has better effect.

Identification of 3C7 mAb antibody specificity:

murine aspartic endopeptidase protein, BSA and diluted to 1. mu.g/ml were added to the microplate in 100. mu.l/well and incubated overnight at 4 ℃. Washing with PBST containing 0.05% Tween for 3 times (3 min each time), blocking with 5% skimmed milk powder at 37 deg.C for 1h, and washing with PBST for 3 times (3 min each time). The purified antibody was diluted in two-fold ratio starting from 1: 1000. Mu.l of the suspension was added to each well and incubated at 37 ℃ for 1 h. PBST was washed 3 times for 3min each, then 100. mu.l of HRP-labeled goat anti-mouse enzyme-labeled secondary antibody diluted 8000 times was added, and incubation was carried out at 37 ℃ for 40 min. PBST is washed for 5 times, TMB single-component developing solution is added, reaction is carried out for 10min in a dark place at room temperature, 50 mu l of concentrated sulfuric acid stop solution is added to stop the reaction, and an enzyme-linked immunosorbent assay (OD 450) value is read by an enzyme-linked immunosorbent assay (ELISA) instrument to carry out result analysis.

The results are shown in FIG. 1. As can be seen from FIG. 1, the 3C7 mAb antibody is highly specific and binds only to aspartic endopeptidase but not to BSA.

The sequence of the 3C7 monoclonal antibody obtained by the antibody sequence identification method commonly used in the art is shown below.

Light chain variable region (SEQ ID NO: 1):

DIVITQRPAMMAASPGEKVTITCRQFNMDKGFFWKWYQQKSGISPKPWIYMQCNQEQGVPARFSGSGSGTSYSLTITSMEAEDAATYYCCRGIPWQTCFGAGTKLELK

heavy chain variable region (SEQ ID NO: 2):

EVQLEESATELSRPGASVKLSCKASGYIFSWDKQCWIKQRPGQGLEWIGWTRIHRFLRPKNHHLHGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGDKHDQTQWGLGTTLAVSS

example 3 animal model test

(1) And (5) grouping the animals. The C57BL/6 mice were randomly divided into an exosome-treated group, an mab-treated group, an exosome + mab-treated group, a saline control group, and a normal mouse group, 10 mice per group. (2) And (5) constructing a mouse spinal cord injury model. The mice were anesthetized with 5% pentobarbital sodium by intraperitoneal injection, the backs were dehaired, and the mice were fixed on an operating table. Mice, except for the normal group, were subjected to a surgical microscope to dissect the skin in a longitudinal direction to remove the T9 vertebral lamina, and after fully exposing the T9 spinal segment, the spinal cord was clamped with a modified artery clamp for 3s to create an acute spinal cord injury model. The normal mouse group was laminectomy alone, and no spinal cord clamping was performed. Suturing muscles and skin layer by layer, and putting the postoperative mouse on a body temperature maintaining mat for reviving; postoperative adjuvant mice urinate 2 times a day, and after each urination, the urethra was disinfected with iodophor. (3) Methods of administration. The medicine is administered for 30min after postoperative recovery.

Exosome treatment group: 500. mu.l of exosome was injected into the tail vein of the exosome prepared in example 1 (the concentration of exosome protein was 200. mu.g/ml, i.e., the amount of exosome tail vein transplantation was 100. mu.g);

monoclonal antibody treatment group: the 3C7 monoclonal antibody is administrated by intraperitoneal injection at the concentration of 10 mg/kg;

exosome + mab treatment group: 500. mu.l of exosome was injected into the tail vein of the exosome prepared in example 1 (the concentration of exosome protein was 200. mu.g/ml, i.e., the amount of exosome tail vein transplantation was 100. mu.g); simultaneously carrying out intraperitoneal injection with 10mg/kg of 3C7 monoclonal antibody;

saline control model group: normal saline 10mg/kg is administrated by intraperitoneal injection;

normal mouse group: normal saline 10mg/kg is administrated by intraperitoneal injection;

the administration is 1 time per day for 7 days; saline controls were injected intraperitoneally at the same time points daily for 14 days.

Assessment of the locomotor ability of the rats: the inclined plate test standard is that the inclined plate test is carried out according to an improved Rivlin method, the body axes of rats of different treatment groups are placed in a vertical position with the longitudinal axis of the inclined plate, the inclined plate is raised by 5 degrees every time, and the maximum angle at which the rats can stay for 5 seconds is taken as a functional value. The results are shown in Table 2.

As can be seen from the results in table 2, after 2 weeks, the angle of the spinal cord injured rats of the exosome and mab co-treatment group kept on the inclined plate was gradually increased, and compared with the injured group, the difference was significant (P < 0.01), and the level was close to that of the normal mice group, which fully indicates that the combined treatment of exosome and mab has a more protective effect on the spinal cord injured rats. .

Meanwhile, the behavior of rats in different treatment groups is recorded according to a BBB scoring method by adopting a BBB scoring standard. The results are shown in FIG. 2.

From the results in fig. 2, it was shown that BBB motor function scores were significantly restored in mice treated with mab and exosome treatment groups compared to saline control model animals. In addition, the study found that the effect of the combination therapy of the exosome and the monoclonal antibody is more remarkable than that of the combination therapy of the exosome or the monoclonal antibody alone.

Example 4 synergistic therapeutic experiments

(1) And (5) grouping the animals. The C57BL/6 mice were randomly divided into exosome + mab treated group, normal saline control group, normal mouse group, 10 per group. (2) And (5) constructing a mouse spinal cord injury model. The mice were anesthetized with 5% pentobarbital sodium by intraperitoneal injection, the backs were dehaired, and the mice were fixed on an operating table. Mice, except for the normal group, were subjected to a surgical microscope to dissect the skin in a longitudinal direction to remove the T9 vertebral lamina, and after fully exposing the T9 spinal segment, the spinal cord was clamped with a modified artery clamp for 3s to create an acute spinal cord injury model. The normal mouse group was laminectomy alone, and no spinal cord clamping was performed. Suturing muscles and skin layer by layer, and putting the postoperative mouse on a body temperature maintaining mat for reviving; postoperative adjuvant mice urinate 2 times a day, and after each urination, the urethra was disinfected with iodophor. (3) Methods of administration. The medicine is administered for 30min after postoperative recovery.

Exosome + mab + bFGF treatment group: 500. mu.l of exosome was injected into the tail vein of the exosome prepared in example 1 (the concentration of exosome protein was 200. mu.g/ml, i.e., the amount of exosome tail vein transplantation was 100. mu.g); simultaneously, the injection is carried out by intraperitoneal injection together with 10mg/kg of monoclonal antibody 3C7 and 80 mu g/kg of bFGF;

group of bFGF: 80 mu g/kg of bFGF is administrated by intraperitoneal injection;

exosome + mab treatment group: 500. mu.l of exosome was injected into the tail vein of the exosome prepared in example 1 (the concentration of exosome protein was 200. mu.g/ml, i.e., the amount of exosome tail vein transplantation was 100. mu.g); simultaneously carrying out intraperitoneal injection with 10mg/kg of 3C7 monoclonal antibody;

saline control model group: normal saline 10mg/kg is administrated by intraperitoneal injection;

normal mouse group: normal saline 10mg/kg is administrated by intraperitoneal injection;

the administration is 1 time per day for 7 days; saline controls were injected intraperitoneally at the same time points daily for 14 days.

Assessment of the locomotor ability of the rats: the inclined plate test standard is that the inclined plate test is carried out according to an improved Rivlin method, the body axes of rats of different treatment groups are placed in a vertical position with the longitudinal axis of the inclined plate, the inclined plate is raised by 5 degrees every time, and the maximum angle at which the rats can stay for 5 seconds is taken as a functional value. The results are shown in Table 3.

Each group of	Scoring
		Group of Normal mice	72.29±0.02
Physiological saline control model group	49.51±2.10
		Exosome + monoclonal antibody treatment group	68.49±2.17*
bFGF group	57.81±2.08*
		Exosome + monoclonal antibody + bFGF group	71.30±1.59*

As can be seen from the results in table 3, after 2 weeks, the angle of the spinal cord injured rats of the bFGF + exosome and mab co-therapy group kept on the inclined plate was gradually increased, and the difference was significant compared with the injured group (P < 0.01), and was closer to the level of the normal mice than the exosome and mab co-therapy group, which fully indicates that the bFGF and exosome and mab co-therapy had more protective effect on the spinal cord injured rats.

Meanwhile, the behavior of rats in different treatment groups is recorded according to a BBB scoring method by adopting a BBB scoring standard. The results are shown in FIG. 3.

From the results in fig. 3, it was shown that the BBB motor function score was significantly restored in mice treated with bFGF and mab and exosome treated groups compared to the saline control model group animals. In addition, the study found that the combination therapy of exosome and bFGF has a remarkable effect compared with the single use of exosome and monoclonal antibody.

After scoring, spinal cord tissue from the injury area was taken. The procedure was followed in accordance with the Caspase-3 activity assay kit instructions, i.e., 100. mu.l of lysate per 5mg of tissue was added and homogenized in a glass homogenizer on an ice bath. The homogenate was then transferred to a centrifuge tube and lysed in an ice bath for 5 min. Centrifuge at 20000g for 15min at 4 ℃. The supernatant was transferred to an ice-bath pre-cooled centrifuge tube. Caspase 3 activity was measured and the results are shown in FIG. 4.

The results of activity detection of Caspase 3 from FIG. 4 show a significant decrease in the treated group compared to the control model group.

This fully indicates that treatment with bFGF together with monoclonal antibodies and exosomes has a significant protective effect on neuronal damage to the spinal cord.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

<110> Nosai Union (Beijing) biomedical science and technology Co., Ltd

<120> method for preparing exosome and application of pharmaceutical composition thereof in tissue repair

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115