CN117210395A - Application of nocardia rubra cell wall skeleton in skin flap transplantation - Google Patents

Abstract

The present disclosure relates to the use of nocardia rubra cell wall scaffold in flap transplantation. The present disclosure provides a mesenchymal stem cell cultured via nocardia rubra cell wall skeleton, an exosome extracted therefrom, and a pharmaceutical composition comprising the exosome. The exosome or the pharmaceutical composition can enhance the angiogenesis of the transplanted skin flap, so that the survival rate of the skin flap is higher, the appearance of the skin flap after survival is improved, and the success rate of the skin flap transplantation is improved.

Description

Application of nocardia rubra cell wall skeleton in skin flap transplantation

Technical Field

The present disclosure relates to the field of medicine and biopharmaceuticals. In particular, the present disclosure relates to the use of nocardia rubra cell wall scaffold in flap transplantation.

Background

Nocardia rubra (Nocardia rubra) is one of Nocardia rubra. The nocardia rubra cell wall skeleton can be prepared by fermenting, cell disruption and protease degradation of nocardia rubra cells.

In the prior art, the nocardia rubra cell wall skeleton is commercially available, specifically, the commercial product (Nr-CWS) produced by the company of biopharmaceutical limited, garter, garrisite. Nocardia rubra cell wall skeleton has been used to treat cervical erosion, cervical precancerous lesions (CN 101073583 a), anti-human papilloma virus (CN 1935262A), skin lesions or ulcers (CN 101209267 a), fungal infections, herpes simplex, shingles (CN 1879661 a). The red nocardia cell wall skeleton has the advantages of reducing inflammatory reaction and promoting skin tissue regeneration.

Currently, flap implantation is one of the important and routine clinical treatments for repairing various types of skin defects. However, after skin flap implantation, problems such as skin flap ischemic necrosis, skin flap contracture, pigmentation, etc. often occur. How to solve these problems remains a great challenge.

In recent years, a number of cellular products have gained clinical attention. Research into exosomes is becoming more and more important. Exosomes are mature in wound repair and skin tissue regeneration with low immunogenicity and high-efficiency bioactivity. However, the complex exosome extraction procedure and low yield limit the wide clinical application.

However, the application of nocardia rubra cell wall skeleton combined exosome in flap transplantation has not been reported in the prior art.

Disclosure of Invention

In a first aspect, the present disclosure provides an exosome of mesenchymal stem cells.

Exosomes (exosomes) refer to vesicles comprising RNA and proteins and having a diameter of 40 to 100 nm.

A variety of cells secrete exosomes under normal and pathological conditions. Exosomes are mainly derived from multivesicular bodies formed by the invagination of intracellular lysosome particles, and are released into the extracellular matrix after fusion with the cell membrane via the outer membrane of the multivesicular bodies. Cell types cultured in vitro can also secrete exosomes, and exosomes naturally occur in body fluids (including blood, saliva, urine, cerebrospinal fluid).

Exosomes of mesenchymal stem cells refer to: exosomes secreted during mesenchymal stem cell culture.

In some embodiments, the mesenchymal stem cells are selected from any one of the following: umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, liver mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, pancreatic mesenchymal stem cells, dental pulp mesenchymal stem cells.

In some embodiments, the mesenchymal stem cells are mesenchymal stem cells cultured via nocardia rubra cell wall scaffold.

Nocardia rubra refers to Nocardia rubra (Nocardiarubra) of Nocardia.

Identification of nocardia rubra: based on known or future microbiological identification techniques, the skilled artisan can perform taxonomic identification of a strain of bacteria, for example, available identification techniques include morphology, physiological and biochemical characteristics, 16S rRNA, and the like. The skilled artisan understands that as technology evolves, identification techniques involve different approaches, mainly morphological and biochemical identification approaches have been used in earlier times, but this approach is less reliable. After the advent of sequencing technology, the skilled artisan can identify strains in a more reliable manner. For example, when the DNA sequence of 16S rRNA was identified as having a similarity of 97% or more, it was judged that two bacteria belong to the same species. In the case of nocardia rubra, known strains deposited in the international (or national grade) collection of strains are used as model strains and compared therewith.

In this disclosure, "nocardia rubra cell wall" can be understood as either a complete cell wall or an incomplete cell wall (e.g., broken, or partially degraded). Given the teachings of the present disclosure, the skilled artisan will appreciate that the ingredient exhibiting the desired activity is derived from (e.g., is the cell wall itself or a constituent thereof) the cell wall of nocardia rubra. Thus, it is within the scope of the present disclosure to allow for the use of intact cell walls, disrupted cell walls, incomplete degradation products of cell walls, constituents of cell walls, extracts of cell walls, and the like in clinical applications.

The cell wall scaffold of the present disclosure is not to be understood as merely representing a crosslinked network entity among cell walls, and the skilled artisan will appreciate that the term does not exclude other cell wall components adsorbed, bound, carried on the crosslinked network entity.

In a specific example, the cell wall scaffold of the present disclosure is the product of bacteria after disruption, removal of impurities (proteins, nucleic acids, cell membranes, lipids).

In a specific embodiment, the cell wall scaffold is nocardia rubra cell wall scaffold corresponding to national drug standard S20030009 or its renewal number.

The skilled artisan will appreciate that S20030009 is an administrative license number issued by the administration authority, which number will vary as credentials are updated, laws, and numbering rules are adjusted. However, the product standard, product parameters, production process, quality requirements represented by the number change are unchanged. Thus, S20030009 in this disclosure should be understood as S20030009 and its equivalent numbering.

In a second aspect, the present disclosure provides a method for preparing exosomes of mesenchymal stem cells, comprising the steps of:

1) Providing mesenchymal stem cells;

2) Incubating the mesenchymal stem cells with a culture medium comprising nocardia rubra cell wall scaffold;

3) Collecting the supernatant from the cell culture;

4) Carrying out ultra-high speed centrifugation on the supernatant;

5) And collecting exosomes.

In some embodiments, the culture medium is a serum-free MSC culture medium.

In some specific embodiments, the culture medium comprises other ingredients known in the art suitable for culturing mesenchymal stem cells. In order to provide safer cells when applied to humans, it is recommended to have no heterologous animal component during the culture process; such as serum-free culture medium.

In some specific embodiments, the skilled artisan can add cytokines, such as one or a combination of FGF, PDGF, TGF- β, HGF, EGF, CTGF, VEGF, insulin-like growth factors, to the cell culture medium as desired (e.g., to maintain dryness, or to promote differentiation).

In some specific embodiments, the amount of FGF (in final concentration) is preferably 0.1 to 100ng/ml. FGF refers to a growth factor in the fibroblast growth factor family, preferably FGF-1, bFGF.

In some specific embodiments, the PDGF content (in final concentration) is preferably 0.5 to 100ng/ml. PDGF refers to a growth factor in the platelet-derived growth factor family, preferably PDGF-BB or PDGF-AB.

In some specific embodiments, the TGF- β content (in final concentration) is preferably 0.5 to 100ng/ml. TGF-beta refers to a growth factor in the transforming growth factor-beta family, preferably TGF-beta 3.

In some specific embodiments, the HGF content (in terms of final concentration) is preferably 0.1 to 50ng/ml.

In some specific embodiments, the amount of EGF (in final concentration) is preferably 0.5 to 200ng/ml.

In some specific embodiments, the culture medium further comprises at least one phospholipid, and/or at least one fatty acid.

Phospholipids can be exemplified by, for example: phosphatidic acid, lysophosphatidic acid, phosphatidyl-inositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol. The total content of phospholipids (in final concentration) is preferably 0.1 to 30 μg/ml.

Examples of fatty acids include: linoleic acid, oleic acid, linolenic acid, arachidonic acid, tetradecanoic acid, palmitoleic acid, palmitic acid, stearic acid, and the like. The total content of fatty acids is preferably 1/1000 to 1/10 of the medium.

In some specific embodiments, the culture medium further comprises cholesterol.

In some specific embodiments, the culture medium further comprises ascorbic acid.

In some specific embodiments, the culture medium further comprises an antioxidant. Antioxidants can be exemplified by, for example: vitamin E.

In some specific embodiments, the culture medium further comprises transferrin.

In some specific embodiments, the culture medium further comprises selenate.

In some specific embodiments, the culture medium further comprises amino acids, nucleotides, trace elements required to maintain the cells.

In one specific example, the cell wall scaffold of the present disclosure is added to known commercially available mesenchymal stem cell culture media. Commercially available mesenchymal stem cell culture medium is selected, for example, from: mesenPRO RS ^TM 、MSC SFM、MSC SFMXenoFree、/>Human adipose-derived stem cell medium.

In some embodiments, the concentration of the cell wall scaffold in the culture medium is 5 to 20mg/L, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20mg/L, preferably 10mg/L.

In some embodiments, the incubation is 24 to 72 hours, e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 hours; preferably 36 to 60 hours; preferably 40 to 50 hours.

Methods for extracting exosomes from cell cultures are well known in the art, for example by ultra-high speed centrifugation to obtain vesicles of the desired size.

In a specific embodiment, the ultra-high speed centrifugation is performed by: centrifuging at 300 Xg for 10min, centrifuging at 2000 Xg for 10min, and centrifuging at 10000 Xg for 30min to remove cell particles and/or cell debris; the supernatant after centrifugation is passed through a filter to remove large vesicles; centrifuge 1 to 2 times at 100000 Xg for 90min each time, discard supernatant.

In some embodiments, the nocardia rubra cell wall scaffold is a nocardia rubra cell wall scaffold corresponding to national drug standard S20030009 or its renewal number.

In other embodiments, the nocardia rubra cell wall scaffold is obtained by a method comprising or consisting of the steps of:

1) Providing nocardia rubra;

2) Crushing the nocardia rubra to obtain a crushed product;

3.1 Removing lipids from the disrupted product;

3.2 Removing nucleic acid from the disrupted product;

3.3 Protein removal of the disrupted product;

3.4 Removing cell membranes from the disrupted product;

3.5 Obtaining nocardia rubra cell wall skeleton;

4) Optionally, sub-packaging;

5) Optionally, freeze-drying the nocardia rubra cell wall scaffold;

steps 3.1), 3.2), 3.3), 3.4) can be interchanged or parallel, and steps 4) and 5) can be interchanged.

The purpose of the disruption of nocardia rubra is to remove intracellular material. Therefore, the ultrasonic crushing, the high-pressure homogenizer crushing, the lysozyme and other technologies can be adopted. The skilled artisan will appreciate that any known or future method suitable for disrupting gram positive bacteria is suitable for use in the presently disclosed embodiments.

The skilled person has the ability to adapt the specific parameters and equipment of cultivation, crushing, separation, collection, impurity removal, sub-packaging to the subsequent application (e.g. topical application) of the active ingredient (cell wall and its constituent components) in order to avoid introducing factors affecting the subsequent application during the preparation step.

In some embodiments, the lipids in the broken product are removed using an organic solvent. In some embodiments, nucleases are used to remove DNA and RNA from the disrupted product. In some embodiments, the protein in the disruption product is degraded using a hydrolase. In some embodiments, a surfactant is used to remove cell membranes from the disrupted product.

In some embodiments, the average particle size of the crush is from 10nm to 1000nm; mention may be made of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 nm.+ -. 10nm, and ranges between any two of the values mentioned above. Particle size testing methods are well known in the art.

In some specific embodiments, the average particle size of the crush is from 10nm to 800nm.

In other specific embodiments, the average particle size of the crush is from 10nm to 500nm.

In particular embodiments, the dispensing refers to dispensing into bottles or ampoules. Just prior to use, a solvent (e.g., sterile water) is added to the bottle or ampoule. As an example, the bottle refers to a penicillin bottle (a real, made of borosilicate glass or soda lime glass).

In a third aspect, the present disclosure also provides an exosome prepared by the above method.

In a fourth aspect, the present disclosure also provides the use of the aforementioned exosomes in the preparation of a medicament, wherein the medicament is for flap implantation.

The present disclosure also provides the use of the aforementioned exosomes in the preparation of a medicament, wherein the medicament is for use in any one selected from the group consisting of: enhancing the angiogenesis of the graft flap, improving the survival rate of the graft flap and improving the appearance of the graft flap.

In some embodiments, the medicament is prepared in a dosage form selected from any one of the following: injection, ointment, cream, emulsion, suspension, paste, gel, lotion, tablet, aerosol, spray, liniment, powder, dressing, bandage, film, patch, and suppository.

In a fifth aspect, the present disclosure provides a pharmaceutical composition for flap implantation comprising: pharmaceutically acceptable carriers and exosomes of mesenchymal stem cells of the present disclosure.

The present disclosure provides a pharmaceutical composition for use in any one selected from the group consisting of: enhancing the angiogenesis of the graft flap, improving the survival rate of the graft flap and improving the appearance of the graft flap; and comprises: pharmaceutically acceptable carriers and exosomes of mesenchymal stem cells of the present disclosure.

The pharmaceutical compositions of the present disclosure may be prepared in unit dosage (or unit formulation) form.

In some embodiments, the pharmaceutical composition may be prepared in a liquid state (liquid formulation).

In other embodiments, the pharmaceutical composition may be prepared as a solid (dry powder formulation or lyophilized powder formulation).

The skilled artisan will appreciate that liquid formulations and dry powder formulations (or lyophilized powder formulations) may be converted to each other, differing only in water content. Removing most or all of water in the liquid preparation to obtain dry powder preparation (or lyophilized powder preparation). Dissolving (or re-dissolving) the dry powder preparation (or freeze-dried powder preparation) to obtain a liquid preparation.

In some embodiments, the pharmaceutically acceptable carrier is selected from, but is not limited to: fillers, stabilizers (e.g. trehalose, glycine), flavours (e.g. xylitol), disintegrants (e.g. sodium carboxymethylcellulose), binders (e.g. gelatin), lubricants (e.g. magnesium stearate).

In some embodiments, the stabilizer is selected from one or a combination of the following: glycine, lysine, arginine, hydroxyethyl starch, hydroxymethyl starch, trehalose, dextran.

In some embodiments, the flavoring agent is selected from one or a combination of the following: sucrose, monosaccharide, saccharin sodium, aspartame, sorbitol, xylitol, mannitol.

In some embodiments, the binder is selected from one or a combination of the following: sodium carboxymethyl cellulose, hypromellose, gelatin.

In some embodiments, the lubricant is selected from one or a combination of the following: comprises talcum powder, magnesium stearate and micro powder silica gel.

In some specific embodiments, pharmaceutically acceptable carriers suitable for use in the present disclosure may also be mentioned, for example, but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythritol, gelatin, magnesium stearate, propellant, humectant, solvent, solubilizer, emulsifier, antioxidant, pH regulator, and antiseptic. Specifically, non-limiting examples also include: white petrolatum, carbomer, hypromellose, methylcellulose, sodium hydroxymethyl cellulose, chitosan, thioaluminum chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerin, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, para-aminobenzoic acid, acetamide, isopropyl alcohol, tween, polyoxyethylated hydrogenated castor oil, stearic acid, glyceryl monostearate, sucrose fatty acid ester, sucrose acetate isobutyrate, sorbitan tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol ester, sulfite, cysteine, di-t-butylhydroxytoluene, potassium sorbate, phosphate buffer solution, triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, nipagin, thimerosal, chlorocresol, trichlorot-butanol, benzoic acid, and sodium salts thereof.

In a sixth aspect, the present disclosure provides a method of enhancing angiogenesis of a graft flap, increasing the survival rate of a graft flap, or improving the appearance of a graft flap, comprising the steps of: administering to a subject a therapeutically effective amount of an exosome, or a drug, or a pharmaceutical composition of the present disclosure.

"administering," "providing to," "treating," when applied to an animal, human, cell, tissue, organ, or biological sample, means that the drug or medical device is in contact with the animal, human, cell, tissue, organ, or biological sample.

By "treating" is meant administering an internal or external drug (therapeutic agent, active ingredient, or composition) (e.g., exosomes or pharmaceutical compositions of the present disclosure) or medical device to a subject to alleviate (reduce, delay, ameliorate, cure) one or more symptoms of a disease in the subject (or population) being treated, such that a clinically measurable degree is achieved, wherein the subject has suffered from, is suspected of suffering from, or is susceptible to one or more diseases or symptoms thereof.

The amount of a drug (therapeutic agent, active ingredient or composition) effective to alleviate any symptom of the disease is referred to as a therapeutically effective amount. May vary depending on a variety of factors, such as the disease state, age, and weight of the subject. It will be appreciated that the drug (therapeutic, active ingredient or composition) may be ineffective in alleviating the target disease or symptoms thereof in a single subject, but is statistically effective against the target disease or symptoms thereof as determined according to any statistical test method known in the art (e.g., student T test, chi-square test, U test according to Mann and Whitney).

In some specific embodiments, the subject is an animal other than a human, e.g., for farm animals, pets, working animals, ornamental animals, production animals, laboratory animals (e.g., rats, mice, guinea pigs, rabbits, dogs, primates).

In some specific embodiments, the subject is a human. In some specific embodiments, the subject is suspected of having, diagnosed with, already having, or susceptible to a disease of interest or a symptom thereof.

In some embodiments, the administration is 1-3 times a day, or once a day. Different doses are used depending on the area and extent of the lesion in the patient.

In some embodiments, the period of administration lasts for 2 days to 6 months, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks or more, and ranges between any two of the foregoing.

"optional" means that the subsequently described event may, but need not, occur; as the case may be. For example, "optionally" means that the product is allowed to be dispensed, but not necessarily.

"a", "an", "single", "the", if not explicitly stated, also include plural forms.

When referring to a range of values (e.g., 60 μg to 120 μg), this is a shorthand way of referring specifically to each value within the range, including fractional and integer numbers.

Detailed Description

The disclosure is further described below in connection with the examples. But these examples do not limit the scope of the present disclosure. When specific conditions are not noted, the operation is performed according to conventional conditions, according to the conditions recommended by the raw material supplier. The reagents of specific origin are not noted and are commercially available conventional reagents.

Examples

Example 1 commercially available nocardia rubra cell wall skeleton

The nocardia rubra cell wall skeleton (Nr-CWS) is purchased from Liaoning Ruishite biopharmaceutical Co., ltd., national drug standard S20030009 (the solid content of the cell wall skeleton in each bottle should be not less than 60. Mu.g, the cell wall acid content is not less than 1.0. Mu.g, the sugar content is not less than 4.0. Mu.g, and the redissolution volume is 2.0 ml).

Example 2 preparation of nocardia rubra cell wall skeleton

The cell wall skeleton of the national drug standard S20030009 has no obvious difference from the following steps in nature, but can be adjusted because of different production scales.

1. The cells were cultured according to a known method and collected. Cells are disrupted (e.g., sonicated or high pressure homogenizer disrupted). The cells may also be allowed to be disrupted by any suitable method known in the art. The breaking condition is checked under a microscope, the number of tangible bacteria in each field of view is not more than 5, and a plurality of (10 to 30) fields of view are checked to meet the standard.

2. Removing nucleic acid: the disrupted supernatant was centrifuged, and DNase and RNase were added to the obtained precipitate to remove nucleic acid according to the procedure recommended by the enzyme supplier.

3. Protein removal: the precipitate is added with a common protease (e.g., trypsin) and the protein is removed according to the procedure recommended by the enzyme supplier.

4. Lipid removal: adding organic reagent (such as one or a combination of acetone, diethyl ether and ethanol) into the precipitate, and removing lipid according to conventional operation in the field.

5. Removal of cell membranes: adding TritonX-100 into the precipitate, centrifuging to collect precipitate, and rinsing with PBS.

It should be appreciated that between the above steps for removing impurities, the skilled person may adjust the sequencing so that the steps are compatible. After removing non-cell wall components, the precipitate was redissolved in water for injection for use. Optionally, sterilization can be performed at 115℃for 20-30 minutes as a stock solution for the cell wall skeleton.

EXAMPLE 3 preparation of exosomes

Human umbilical cord mesenchymal stem cell exosomes (hereinafter referred to as control exosomes), exosomes extracted from human umbilical cord mesenchymal stem cells cultured using red nocardia cell wall scaffolds (hereinafter referred to as: cell wall scaffold-exosomes): the 4 th generation MSC is taken and density gradient centrifugation is adopted to extract exosomes.

1. Extraction of control exosomes:

when the cell fusion reached 80%, the culture was continued for 48 hours with serum-free MSC medium instead of DMEM/F12, and the cell culture supernatant was collected.

Cell particles, dead cells and cell debris were separated and removed from the supernatant by ultra-high speed centrifugation (300 Xg centrifugation for 10min,2000 Xg centrifugation for 10min,10000 Xg centrifugation for 30 min).

The supernatant after centrifugation was filtered through a 0.22 μm filter (Millipore, usa) to remove larger extracellular vesicles. And (3) performing ultracentrifugation for 90min for 2 times at 100000 Xg, discarding supernatant, and re-suspending with PBS to obtain precipitate.

2. Extraction of cell wall skeleton-exosome:

the only difference compared to the extraction of control exosomes is that 10mg/L of the cell wall scaffold of example 1 is also included in the serum-free MSC culture broth.

3. The concentration of exosomes was measured using BCA protein concentration measurement kit and stored in-80 ℃ refrigerator.

4. Identification of control exosomes and cell wall scaffold-exosomes: observing the shape by using a transmission electron microscope; western blot detection of membrane surface marker proteins (CD 63, TSG 101); the nanoparticle tracking analyzer measures its particle size distribution.

Example 4 exemplary preparation of pharmaceutical compositions

1. The product obtained in example 3 was coated on a dressing (e.g., sterile gauze) to prepare a medicament for external use.

2. The product obtained in example 3 was prepared as a lyophilized powder.

3. Lotion preparation methods well known in the art may also be employed, for example: the lotion takes water and alcohol as dispersion medium; is prepared from active component, electrolyte, isotonic regulator, etc. in dispersing medium.

4. The product obtained in example 3 was prepared into capsules.

5. The product obtained in example 3 was dissolved in water for injection to prepare an injection.

Test case

Test example 1. Effect on proliferation, migration and differentiation of human umbilical vein vascular endothelial cells (HUVECs)

The influence of the cell wall skeleton-exosome on the activity of human vascular endothelial cells is detected, and the effect of promoting the growth of the cells is objectively evaluated.

1. Grouping:

group 1: control exosomes;

group 2: cell wall scaffold-exosome group;

group 3: cell wall skeleton group (nocardia rubra cell wall skeleton (trade name: nakejia) was purchased from Liaoning Geruishite biopharmaceutical Co., ltd., approved national drug standard number S20030009 (2 ml/ampoule; lyophilized powder) containing 60 μg of active ingredient and 15mg of dextran 40).

2. Co-culture with HUVEC:

(1) After co-culturing the two groups of exosomes fluorescently labeled with PKH26 with HUVECs, respectively, confocal microscopy was used to observe whether the two groups of exosomes could enter HUVECs. PKH26 fluorescently labeled exosomes fluoresce in red.

(2) Three groups were co-cultured with HUVECs 1, 2, 3, 4, 5d, respectively, and the CCK-8 method examined for differences in the effect on HUVEC proliferation. When the cells were co-cultured for 12 hours, the expression level of VEGF protein in the cell supernatant was measured by ELISA, and the difference between the exosomes of each group was compared.

(3) Three groups of differences in the effect of Transwell migration experiments on HUVEC migration capacity were examined.

(4) Three groups of differences in the effect of HUVEC tubular structure formation were observed by in vitro Matrigel experiments.

The experiments described above all used an equal amount of PBS as a blank.

3. The statistical method comprises the following steps:

(1) Checking the counting data by using a chi-square;

(2) The metering data is tested by F test or t test;

(3) The grade data is analyzed or checked by the Ridit;

(4) When multi-center effect is considered, the counting data or the grade data is compared and checked by using a chi-square;

(5) Statistical software package: data management used EpiData3.0 and statistical analysis used SPSS26.0 software programming analysis.

Test example 2 cell wall skeleton-exosomes promote survival of Ultra-long random flaps (Ultra-long Random Skin Flaps) in rats

The cell wall skeleton-exosome is used for treating the overlength random skin flap of the rat, and the effectiveness of the overlength random skin flap in promoting survival of the skin flap is objectively evaluated.

1. Preparation of rat ultralong random skin flap model

Animal feeding: animal experiments were approved and approved by the ethics committee. The experiment selects 20 SD male rats with weight of 250-300g, and randomly divides the rats into four groups of 5 rats; animals were kept for more than one week in the university of Xuzhou medical university animal experiment center after purchase.

Grouping: control exosome group, cell wall skeleton-exosome group, PBS group.

Feeding conditions: the temperature is 20-25 ℃, the relative humidity is 40-70%, and the special feed and purified water for rats are fed.

And (3) model making: referring to the preparation method of the improved McFarland skin flap model, the back parts of four groups of rats are manufactured into random skin flaps with the area of 9cm multiplied by 3cm along the long axis direction; control exosomes, cell wall scaffolds, cell wall scaffold-exosomes or PBS were injected at the proximal, intermediate and distal regions, respectively.

2. Evaluation index and method for skin flap survival

(1) Integral observation

And (5) observing survival of the rat and healing of the skin flap after operation. Skin tissue, blood vessels and other accessory structures are typically formed 7d after the flap implantation procedure, so observations were selected at day 7 post-procedure.

On the 7 th day after operation, 4 groups of rats were anesthetized, the back flap survival area was depicted, photographed and marked on a ruler, then the ruler was introduced into a computer, the area was measured using Image Proplus 6.0 software (MediaCybernetics), and the flap survival rate (survival flap area/total flap area×100%) was calculated.

(2) Histological observations

Skin tissues in the middle of the 3 areas of the skin flap are cut out respectively from each group, and the skin tissues are cut into slices after being fixed, dehydrated and embedded, wherein the thickness of each slice is 3 mu m. A portion of the sections were routinely HE stained and examined for tissue morphology at various levels of skin.

(3) Immunohistochemical staining observations

Taking 4 groups of partial slices, dewaxing by conventional xylene, hydrating by ethanol with various levels of concentration, repairing antigen by citrate buffer solution, blocking by 3% H2O2, and dropwise adding anti-CD 31 (1:100) overnight at 4 ℃; and dripping horseradish peroxidase-labeled secondary antibody, and incubating for 30min at 4 ℃. Each slice is dripped with DAB liquid for 20min, ethanol with different levels of concentration is rapidly water, xylene is transparent, and neutral resin is sealed. After air drying, the shape of the tube sample is observed under a mirror, and the tube sample is positively dyed with brown. 10 sections were taken per group, 5 fields were randomly selected per section under a 100-fold mirror, CD31 positive blood vessels were counted, and the mean was taken as microvascular density (mean blood vessel density, MVD).

3. Statistical method

(1) Checking the counting data by using a chi-square;

(2) The metering data is tested by F test or t test;

(3) The grade data is analyzed or checked by the Ridit;

(4) When multi-center effect is considered, the count data or the grade data is compared and checked by using a CMH chi-square;

(5) Statistical software package: data management used epidata3.0 and statistical analysis used SPSS26.0 software programming analysis.

The combination of exosomes and red nocardia cell wall skeleton not only maintains the bioremediation effect, but also makes up the limitation of complex exosome extraction procedure and low yield. Therefore, the two are combined to enhance the angiogenesis of the graft flap, so that the survival rate of the flap is higher, the appearance (more beautiful graft area) of the graft flap after survival is improved, the patient compliance is improved, and the success rate of the flap transplantation is improved.

Claims (13)

1. An exosome of mesenchymal stem cells, wherein:

the mesenchymal stem cells are mesenchymal stem cells cultured by nocardia rubra cell wall skeleton;

the mesenchymal stem cells are selected from any one of the following: umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, liver mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, pancreatic mesenchymal stem cells, dental pulp mesenchymal stem cells;

preferably, the nocardia rubra cell wall skeleton is a nocardia rubra cell wall skeleton corresponding to national drug standard S20030009 or its update number.

2. Use of exosomes of mesenchymal stem cells in the preparation of a medicament, wherein:

the medicine is used for skin flap transplantation;

the mesenchymal stem cells are mesenchymal stem cells cultured by nocardia rubra cell wall skeleton.

3. The use according to claim 2, wherein the mesenchymal stem cells are selected from any one of the following: umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, liver mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, pancreatic mesenchymal stem cells, dental pulp mesenchymal stem cells.

4. Use according to claim 2 or 3, the medicament being prepared in a dosage form selected from any one of the following: injection, ointment, cream, emulsion, suspension, paste, gel, lotion, tablet, aerosol, spray, liniment, powder, dressing, bandage, film, patch, and suppository.

5. The use according to any one of claims 2 to 4, the medicament being capable of achieving a therapeutic effect selected from any one of the following: enhancing the angiogenesis of the graft flap, improving the survival rate of the graft flap and improving the appearance of the graft flap.

6. The use according to any one of claims 2 to 5, wherein the nocardia rubra cell wall scaffold is a nocardia rubra cell wall scaffold corresponding to national drug standard S20030009 or its renewal number.

7. The use according to any one of claims 2 to 5, wherein the nocardia rubra cell wall scaffold is obtained by a method comprising or consisting of the steps of:

1) Providing nocardia rubra;

2) Crushing the nocardia rubra to obtain a crushed product;

3.1 Removing lipids from the disrupted product;

3.2 Removing nucleic acid from the disrupted product;

3.3 Protein removal of the disrupted product;

3.4 Removing cell membranes from the disrupted product;

3.5 Obtaining nocardia rubra cell wall skeleton;

4) Optionally, sub-packaging;

5) Optionally, freeze-drying the nocardia rubra cell wall scaffold;

wherein,

steps 3.1), 3.2), 3.3), 3.4) can be interchanged or parallel,

step 4) and step 5) can be interchanged;

the average particle size of the break-up is from 10nm to 1000nm, preferably from 10nm to 800nm, more preferably from 10nm to 500nm.

8. Use according to any one of claims 2 to 7, wherein the mesenchymal stem cells are cultured by a method comprising or consisting of the steps of:

1) Providing mesenchymal stem cells;

2) Incubating the mesenchymal stem cells with a culture medium comprising nocardia rubra cell wall scaffold;

preferably, the culture medium is a serum-free MSC culture medium;

preferably, the incubation is from 24 to 72 hours; preferably 36 to 60 hours; preferably 40 to 50 hours;

preferably, the concentration of nocardia rubra cell wall skeleton in the culture medium is 5 to 20mg/L, preferably 10mg/L.

9. A method for preparing exosomes of mesenchymal stem cells, comprising the steps of:

1) Providing mesenchymal stem cells;

2) Incubating the mesenchymal stem cells with a culture medium comprising nocardia rubra cell wall scaffold;

3) Collecting the supernatant from the cell culture;

4) Carrying out ultra-high speed centrifugation on the supernatant;

5) Collecting exosomes;

preferably, the culture medium is a serum-free MSC culture medium;

preferably, the incubation is from 24 to 72 hours; preferably 36 to 60 hours; preferably 40 to 50 hours;

preferably, the concentration of nocardia rubra cell wall skeleton in the culture medium is 5 to 20mg/L, preferably 10mg/L;

preferably, the ultra-high speed centrifugation is performed by: centrifuging at 300 Xg for 10min, centrifuging at 2000 Xg for 10min, and centrifuging at 10000 Xg for 30min to remove cell particles and/or cell debris; the supernatant after centrifugation is passed through a filter to remove large vesicles; centrifuging at 100000 Xg for 1-2 times, each for 90min, and discarding supernatant;

the mesenchymal stem cells are selected from any one of the following: umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, liver mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, pancreatic mesenchymal stem cells, dental pulp mesenchymal stem cells.

10. The method of claim 9, wherein the nocardia rubra cell wall scaffold is a nocardia rubra cell wall scaffold corresponding to national drug standard S20030009 or its renewal number.

11. The method of claim 9, wherein the nocardia rubra cell wall scaffold is obtained by a method comprising or consisting of:

1) Providing nocardia rubra;

2) Crushing the nocardia rubra to obtain a crushed product;

3.1 Removing lipids from the disrupted product;

3.2 Removing nucleic acid from the disrupted product;

3.3 Protein removal of the disrupted product;

3.4 Removing cell membranes from the disrupted product;

3.5 Obtaining nocardia rubra cell wall skeleton;

4) Optionally, sub-packaging;

5) Optionally, freeze-drying the nocardia rubra cell wall scaffold;

wherein,

steps 3.1), 3.2), 3.3), 3.4) can be interchanged or parallel,

step 4) and step 5) can be interchanged;

the average particle size of the break-up is from 10nm to 1000nm, preferably from 10nm to 800nm, more preferably from 10nm to 500nm.

12. An exosome of mesenchymal stem cells prepared by the method of any one of claims 9 to 11.

13. A pharmaceutical composition for flap implantation comprising:

a pharmaceutically acceptable carrier; and

the exosome of the mesenchymal stem cell of claim 1 or 12.