CN114366763A

CN114366763A - Gynecological gel and preparation method thereof

Info

Publication number: CN114366763A
Application number: CN202210036023.2A
Authority: CN
Inventors: 曾鹏辉; 李熙阳; 浣溢帆; 周英杰; 欧阳贵; 熊智缘
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-01-10
Filing date: 2022-01-10
Publication date: 2022-04-19

Abstract

The application provides a gynecological gel and a preparation method thereof, the gynecological gel comprises an exosome, a gel, matrine, berberine and a fructus cnidii extracting solution, the exosome is obtained by cell secretion, contains various cell factors such as EGF and the like, can play a composite role in cell repair, and can repair damaged vaginal epithelial cells, and the matrine, the berberine and the fructus cnidii are antibacterial and anti-inflammatory traditional Chinese medicines. The gel has no pungent odor, is viscous, has good water solubility and adhesiveness, can be well adsorbed in female vagina, has neutral pH value, slowly decreases viscosity under acidic environment of vagina, gradually releases coated exosome and Chinese medicinal components, and covers damaged vaginal tissue. The traditional Chinese medicine composition can effectively control and treat the vagina inflammation, can act on damaged vagina epithelium through exosome to promote the repair of epithelial cells, and is adhered to a pathological change part through gel, so that the effect of protecting a wound surface is achieved.

Description

Gynecological gel and preparation method thereof

Technical Field

The application relates to the technical field of biological medicines, in particular to a gynecological gel and a preparation method thereof.

Background

Atrophic vaginitis, one of the diseases to which menopausal women are susceptible, is common in women after natural menopause and ovarian castration, and its main pathological change is atrophy of vaginal squamous epithelium due to estrogen reduction, often accompanied by glycogen reduction and increase of vaginal pH. The thinned epithelium has a reduced resistance to the vaginal flora, making it susceptible to inflammation caused by pathogenic bacteria invading the vagina, common pathogenic bacteria including: streptococcus, staphylococcus, escherichia coli and the like, and the main disease symptoms are vaginal bleeding, pruritus, dysuria, dyspareunia or dyspareunia. Vaginal atrophy is manifested by pale mucous membrane, ecchymosis and disappearance of plica, the reduction or disappearance of superficial and middle layer cells can be seen under the mirror, small ulcers with acute inflammation and granulation tissue are distributed in intact epithelium, and infiltration of submucosal lymphocytes and plasma cells can also be seen.

Current treatments for atrophic vaginitis are divided into hormonal and non-hormonal treatments depending on the severity of the condition. Hormone therapy mainly compensates for estrogen reduction caused by ovarian hypofunction by means of estrogen supplementation, and can be divided into systemic hormone therapy and local hormone therapy according to the administration mode, because the use of systemic hormone can cause the probability of hormone-related tumors to increase, so that the local hormone therapy, namely vaginal estrogen therapy, is generally adopted when no obvious contraindication exists. Vaginal estrogen therapy can effectively alleviate symptoms such as vaginal atrophy, but studies also show that local hormone therapy can also cause risks of tumors, cerebral apoplexy and venous embolism, so hormone therapy is forbidden for patients with related risks. Therefore, there is an urgent need for a vaginitis medicament that does not require the use of hormones.

Disclosure of Invention

The application aims to provide a gynecological gel, and aims to solve the problem that the existing hormone for treating atrophic vaginitis has side effects.

To achieve the above objects, the present application provides a gynecological gel comprising: exosome, gel, matrine, berberine and fructus cnidii extract.

Preferably, the composite material comprises the following components in percentage by mass:

20 to 40 percent of 0.5mg/ml to 1mg/ml exosome solution;

50 to 70 percent of gelling agent;

3 to 10 percent of matrine;

3-10% of berberine;

3 to 10 percent of fructus cnidii extract with 10 percent of weight as active ingredients.

Preferably, the composite material comprises the following components in percentage by mass: 60% of gel, 30% of exosome solution with the concentration of 0.5mg/ml-1mg/ml, 5% of berberine, 5% of matrine and 5% of fructus cnidii extract with the effective component of 10% by weight.

Preferably, the gelling agent is selected from one or a mixture of more of hydroxyethyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, carbomer, xanthan gum, sodium alginate, carrageenan and guar gum;

preferably, the gelling agent is carbomer.

Preferably, the exosome is from any one or a mixture of several of adipose mesenchymal stem cells, amnion mesenchymal stem cells, umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells;

preferably, the exosomes are from amniotic mesenchymal stem cells.

The application also provides a preparation method of the gynecological gel, which comprises the following steps:

adding an exosome solution and a common cnidium fruit extracting solution into the gel solution to carry out first-stage stirring;

adding berberine and matrine into the mixed solution, stirring at the second stage, and stirring to obtain the gynecological gel.

Preferably, the exosomes are extracted from stem cells treated with hypoxia, and the concentration of the hypoxia-treated oxygen is 5% -10%;

preferably, the oxygen concentration of the low oxygen treatment is 5%.

Preferably, the method for extracting exosomes comprises the following steps: any one of differential high-speed centrifugation, density gradient centrifugation, polymer-based precipitation technology extraction, size exclusion chromatography extraction, and immune separation technology extraction;

preferably, the method for extracting the exosome is differential speed high-speed centrifugation.

Preferably, the berberine and the matrine are added while stirring in the second stage of stirring.

Preferably, the first stage agitation is: stirring for 1-3 min at a speed of 100-300 rmp/min; the second-stage stirring comprises the following steps: stirring at 400-600 rpm/min for 15-20 min.

Compared with the prior art, the beneficial effect of this application includes:

the application provides a gynecological gel comprising: exosome, gel, matrine, berberine and fructus cnidii extract. The exosome is obtained by cell secretion, contains various cell factors such as EGF and the like, can play a composite role in cell repair, can repair vaginal epithelial cells and increase the resistance of vaginal epithelium, and the matrine, the berberine and the fructus cnidii are antibacterial and anti-inflammatory traditional Chinese medicines and achieve the healing effect by bacteriostasis of the traditional Chinese medicine components and repair of the exosome. The gel is viscous without pungent odor, has good water solubility and adhesiveness, can be well adsorbed in female vagina, has neutral pH value, slowly decreases viscosity under acidic environment of vagina, gradually releases coated substances and covers damaged vaginal tissue.

When in use, the gynecological gel is extruded into the deep vagina to cover the cervix or the part of the canker-house, and is naturally dissolved to release the coated exosome and the traditional Chinese medicine component, so that the vagina inflammation can be effectively controlled and treated through the traditional Chinese medicine component, the exosome can act on the damaged vaginal epithelium to promote the repair of epithelial cells, and the gynecological gel is attached to the pathological change part to achieve the effect of protecting the wound surface. Through the combined action of the traditional Chinese medicine components, exosomes and gel, the resistance of the vaginal epithelium at the pathological change part to pathogenic microorganisms is improved, the damaged vaginal epithelium is repaired, and the stable state of the vaginal microenvironment is restored, so that the effects of treating female vaginitis, cervical erosion and repairing the damaged vagina are achieved.

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments are briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope of the present application.

FIG. 1 is a diagram showing the results of flow cytometry on exosomes prepared in example 1 of the present invention;

FIG. 2 is a transmission electron microscopy imaging result chart of exosomes prepared in example 1 of the present invention;

FIG. 3 is a graph showing the results of particle size analysis of exosomes prepared in example 1 of the present invention;

FIG. 4 is a graph showing the results of an effect evaluation experiment of the exosome of the present invention;

FIG. 5 is a graph showing the result of the experiment of the antibacterial effect of the gynecological gel of the present invention;

FIG. 6 is a graph showing the results of the experiment of the gynecological gel of the present invention for treating atrophic vaginitis.

Detailed Description

The terms as used herein:

"prepared from … …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of … …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 ~ 5" is disclosed, the ranges described should be construed to include the ranges "1 ~ 4", "1 ~ 3", "1 ~ 2 and 4 ~ 5", "1 ~ 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

In these examples, the parts and percentages are by mass unless otherwise indicated.

"part by mass" means a basic unit of measure indicating a mass ratio of a plurality of components, and 1 part may represent any unit mass, for example, 1g or 2.689 g. If we say that the part by mass of the component A is a part by mass and the part by mass of the component B is B part by mass, the ratio of the part by mass of the component A to the part by mass of the component B is a: b. alternatively, the mass of the A component is aK and the mass of the B component is bK (K is an arbitrary number, and represents a multiple factor). It is unmistakable that, unlike the parts by mass, the sum of the parts by mass of all the components is not limited to 100 parts.

"and/or" is used to indicate that one or both of the illustrated conditions may occur, e.g., a and/or B includes (a and B) and (a or B).

The present application provides a gynecological gel comprising: exosome, gel, matrine, berberine and fructus cnidii extract.

Specifically, exosomes are secreted by cells, arising from the endocytic pathway of the cell, and are a population of nano-sized vesicles (30-150 nm in diameter). Exosomes are composed of lipid bilayers and carry a variety of biomolecules, including proteins, glycans, lipids, metabolites, RNA, and DNA. Exosomes, as mediators of intercellular transfer of signals, can play a role in a variety of biological pathways. When the exosome contacts the target cell, the exosome and the biomolecule carried by the exosome are received in an endocytosed form, released and acted on in the target cell. The lipid bilayer molecular membrane of the exosome can not only protect active substances from being degraded or diluted in an extracellular environment, but also utilize specific ligands on the surface of the membrane to identify specific cells and directionally gather, and the specific ligands are efficiently combined with receptor cells, the nanoscale volume enables the exosome to pass through a biological barrier without obstacles along with blood or other body fluids, reach and act on various target cells in a long distance, and the exosome is compatible with the cell membrane so as to transport nutrient substances coated in the exosome to the interior of the cells, thereby achieving the treatment effect.

Preferably, the exosome is derived from any one or a mixture of several of adipose mesenchymal stem cells, amniotic mesenchymal stem cells, umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells.

More preferably, the exosome is derived from an amniotic mesenchymal stem cell, and the amniotic mesenchymal stem cell is convenient to source and has more excellent repair capacity.

Specifically, matrine has wide biological activity, has various pharmacological activities such as cardiotonic, anti-inflammatory, anti-tumor, antibacterial and immunosuppressive effects, and berberine is an alkaloid extracted from plants such as coptis chinensis and phellodendron amurense, has a remarkable antibacterial effect, and has an obvious anti-inflammatory and anti-cancer effect. The fructus Cnidii contains various chemical components, such as coumarins, volatile oils, saccharides and trace elements. Osthole is one of the main components of fructus cnidii playing the drug effect, and the main effect of fructus cnidii is used for treating cold womb, cold-damp leucorrhea, damp arthralgia and lumbago, and externally treating vulvar eczema, pruritus vulvae and trichomonas vaginitis of women.

Specifically, the gel is used for wrapping exosomes and traditional Chinese medicine components to form gel, and is selected from any one or a mixture of more of hydroxyethyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, carbomer, xanthan gum, sodium alginate, carrageenan and guar gum; preferably, the gelling agent is carbomer.

The gel is viscous without pungent odor, has good water solubility and adhesiveness, can be well adsorbed in female vagina, has neutral pH value, slowly decreases viscosity under acidic environment of vagina, gradually releases coated substances and covers damaged vaginal tissue.

In a preferred embodiment, the gynecological gel comprises, in mass percent: 20 to 40 percent of 0.5mg/ml to 1mg/ml exosome solution; 50 to 70 percent of gelling agent; 3 to 10 percent of matrine; 3-10% of berberine; 3 to 10 percent of fructus cnidii extract with 10 percent of weight as active ingredients.

The gelling agent may be, for example, (50, 53, 55, 58, 60, 62, 64, 65, 68, or 70)%, or any value between 50% and 70%; the matrine can be (3, 4, 5, 6, 7, 8, 9 or 10)%, or any value between 3% and 10%; berberine may be, for example, (3, 4, 5, 6, 7, 8, 9 or 10)%, or any value between 3% and 10%; the cnidium fruit extract may be, for example, (3, 4, 5, 6, 7, 8, 9, or 10)%, or any value between 3% and 10%.

In a more preferred embodiment, the gynecological gel comprises, in mass percent: 60% of gel, 30% of exosome solution with the concentration of 0.5mg/ml-1mg/ml, 5% of berberine, 5% of matrine and 5% of fructus cnidii extract with the effective component of 10% by weight.

Preferably, the exosomes are extracted from hypoxic-treated stem cells, the hypoxic-treated oxygen concentration being between 5% and 10%.

Preferably, the oxygen concentration of the low oxygen treatment is 5%.

The stem cells are stimulated to secrete more exosomes through the hypoxia treatment, and the stem cells can secrete more cytokines, so that the number of the cytokines contained in the exosomes is increased, and the exosomes have better treatment effect.

Preferably, the method for extracting exosomes comprises the following steps: differential high-speed centrifugation, density gradient centrifugation, polymer-based precipitation technology extraction, size exclusion chromatography extraction, and immune separation technology extraction.

Embodiments of the present application will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

1.1 exosome preparation

1.11 isolation and culture of human amniotic mesenchymal Stem cells

The amniotic membrane after cesarean section of the parturient was taken, washed with DPBS containing 1% double antibody (DPBS solution containing 100U/ml penicillin and 0.1mg/ml streptomycin), and the coagulated blood clot and excess tissue were removed using tissue scissors and forceps. The amniotic membrane was cut into pieces by tissue scissors, 0.5% of pancreatin was added to digest the pieces at 37 ℃ for 1 hour with shaking, and then the digested tissue was filtered through a 200-mesh sieve, washed thoroughly with the above-mentioned DPBS containing 1% of diabase, and then the pieces of tissue were collected and digested at 37 ℃ for 1 hour with 1mg/ml of collagenase 1 with shaking. Filtering the digested tissue with 200 mesh sieve, collecting filtrate, centrifuging at 1500rpm/min for 5min, and collecting cell precipitate. The cell pellet was resuspended in DMEM/F12 medium and cultured at 37 ℃ with 5% CO₂And in the cell culture box, periodically filling stem cells between the sheep membranes and changing the liquid.

1.12 hypoxia-treated human amniotic mesenchymal stem cells

Collecting well-grown third generation human amniotic mesenchymal stem cells, removing cell culture medium, washing with PBS for 2-3 times, adding DMEM/F12 culture medium, and standing at 37 deg.C and 5% CO₂，5％O₂And culturing for 48h in an environment.

1.13 extraction of exosomes and exosome solution preparation

Collecting the human amniotic mesenchymal stem cell culture medium subjected to hypoxia treatment, and centrifuging the collected cell culture medium at 4 ℃ for 10min at 300 Xg to remove cell debris; centrifuging at 4 deg.C and 2000 Xg for 10min to remove impurities such as dead cells; filtering the supernatant with a sterile filter membrane of 0.22 mu m to further remove impurities; ultracentrifuging at 4 deg.C for 120min to obtain exosome precipitate, and adding 200uL PBS to obtain exosome solution derived from human amniotic mesenchymal stem cells. And (3) determining the protein concentration of the exosome by using a BCA method, and adding deionized water into the exosome according to the determination result so that the protein concentration of the exosome concentration is 0.5mg/ml-1.0 mg/ml.

1.2 preparation of gynecological gel

Preparation of 1% carbomer solution: one part of carbomer powder was dissolved in 99 parts of deionized water to make a 1% mass fraction solution of carbomer.

Fructus cnidii extracting solution: wherein the effective components account for 10% of the total mass, and the organic solution accounts for 90% of the total mass.

Preparation of gynecological gel:

adding 30% by mass of exosome solution and 5% by mass of fructus cnidii extract into 60% by mass of carbomer solution, stirring at 200rmp/min for 1 min. Then weighing 5% (wt) berberine and 5% (wt) matrine powder, stirring at 500rpm/min for 15-20min, adding under stirring to obtain gynecological gel of example 1.

Example 2

The difference from example 1 is: exosomes of the gynecological gel of example 2 were derived from normally cultured human amniotic mesenchymal stem cells without hypoxia treatment, and the rest of the steps were the same as in example 1 and will not be described herein again.

Comparative example 1

The difference from example 1 is: the gynecological gel of comparative example 1 has no exosomes.

Test example 1 identification of exosomes

1.1 flow cytometry assay

(1) The suspension of exosomes prepared in example 1 was taken and the suspended exosomes were captured overnight in the dark at room temperature using magnetic beads to allow the exosomes and magnetic beads to bind well.

(2) anti-CD 9 antibody [ VJ1/20] (PE) was added to the exosome suspension and incubated at 4 ℃ for 60 minutes in the dark.

(3) The sample was washed by adding 1mL of 1 × assay buffer.

(4) The magnetic beads are collected using a magnetic holder or centrifugation and the supernatant is removed.

(5) The pellet was resuspended in 350. mu.L of 1 × assay buffer (stored in the dark for up to 2 hours) and collected on a flow cytometer to identify the surface markers of the exosomes, as shown in FIG. 1, where P1 in FIG. 1 shows the forward scattered and side scattered light data of the instrument for all the substances in the sample, and the boxes in the figure are the data we need; p2 is the measurement of fluorescence signal for the data in the P1 box; p3 is the positive data selected after counting the data in the P2 box. FIG. 1 illustrates that the measured exosome expression was CD63 and CD 9.

1.2 Transmission Electron microscopy of exosome samples

(1) 10. mu.L of the exosome prepared in example 1 was taken out.

(2) 10 mu L of the sample is sucked and dropped on a copper net for precipitation for 1min, and the floating liquid is sucked off by filter paper.

(3) 10 mu L of uranyl acetate is dripped on a copper net for precipitation for 1min, and the floating liquid is absorbed by filter paper.

(4) Drying for several minutes at normal temperature.

(5) The imaging result of the transmission electron microscope is obtained by performing electron microscope detection imaging at 100kv and is shown in figure 2, and the exosomes are uniform in size.

1.3 exosome particle size analysis

(1) The frozen exosomes prepared in example 1 were thawed in a water bath at 25 ℃ and placed on ice.

(2) The exosome sample was diluted with 1 × PBS and directly used for NTA detection, and the result is shown in fig. 3, and the average particle size of the exosome prepared in example 1 was 148.3nm, and the concentration was 1.3E +10 Particles/mL.

Test example 2 evaluation of exosome effect

1. The protein concentrations of the exosome prepared in example 1 and the exosome prepared in example 2 were measured, respectively.

2. Vaginal epithelial cells (VK2) were taken to adjust cell concentration to 5X10³The content of the active carbon is one/ml,DMEM high-glucose + 10% FBS was used for culture and seeded in A, B, C three 96-well plates, respectively. The group A was not treated (control group), and the normally cultured amniotic mesenchymal stem cell exosomes (exosome group) of example 2 were added to the group B at a final concentration of 1 mg/ml. Group C the hypoxia-treated amniotic mesenchymal stem cell exosomes of example 1 (hypoxia-treated group) were added to a final concentration of 1 mg/ml.

3. The cck-8 reagent was added at 36 hours to measure cell activity.

4. The results are shown in fig. 4, in which the cell activities of both the exosome group and the hypoxia-treated group were higher than those of the control group, and the cell activity of the hypoxia-treated group was higher than that of the normally cultured exosome group.

Test example 3 antibacterial Effect of gynecological gel

1. A standard strain of staphylococcus was used and the medium was set to 0.5 McLeod (1.5X 10)⁸CFU/ml) was evenly spread on blood agar medium.

2. The paper sheet a was infiltrated with the exosome hydrogel prepared in example 1, the paper sheet B was infiltrated with normal saline, the paper sheet was air-dried, and was attached to a culture medium inoculated with the strain, and the results were observed after 24.

3. As a result, as shown in FIG. 5, the bacteriostatic ring of the sheet A was significantly larger than that of the sheet B.

Experimental example 4 gynecological gel effect on atrophic vaginitis in rats

1. 30 SD female rats (200-250g) were randomly assigned to A, B, C groups, A, B groups were castrated and group C was left untreated.

2. By performing vaginal smears on the vaginas of rats, when the vaginas are smeared in the estrus interval for five consecutive days, the model is successfully made.

The exosome gel prepared in example 1 (experimental group), the gel without exosomes prepared in comparative example 1 (control group), and no treatment (blank group) were administered to rats in A, B, C groups, respectively, by vaginal administration, with a dosing cycle of 0.5ml once a day for two consecutive weeks.

4. After the administration, the rats were euthanized and the vaginal tissue of the rats was taken to observe the thickness of the vaginal epithelium of the rats.

5. The results are shown in fig. 6, and show that the vaginal epithelium thickness of rats in the experimental group administered with the exosome gel is significantly thicker than that in the control group administered with the gel without exosome, but thinner than that in the normal blank group.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.

Moreover, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the application and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Claims

1. A gynecological gel comprising: exosome, gel, matrine, berberine and fructus cnidii extract.

2. The gynecological gel according to claim 1, characterized by comprising, in mass percent:

20 to 40 percent of 0.5mg/ml to 1mg/ml exosome solution;

50 to 70 percent of gelling agent;

3 to 10 percent of matrine;

3-10% of berberine;

3. The gynecological gel according to claim 2, characterized by comprising, in mass percent: 60% of gel, 30% of exosome solution with the concentration of 0.5mg/ml-1mg/ml, 5% of berberine, 5% of matrine and 5% of fructus cnidii extract with the effective component of 10% by weight.

4. The gynecological gel according to any one of claims 1 to 3, wherein the gelling agent is selected from one or a mixture of hydroxyethyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, carbomer, xanthan gum, sodium alginate, carrageenan and guar gum;

preferably, the gelling agent is carbomer.

5. The gynecological gel according to any one of claims 1 to 3, wherein the exosomes are derived from any one or a mixture of adipose mesenchymal stem cells, amnion mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells;

preferably, the exosomes are from amniotic mesenchymal stem cells.

6. A process for the preparation of a gynaecological gel according to any one of claims 1 to 5, comprising:

7. The method for preparing the recombinant cell line of claim 6, wherein the exosomes are extracted from stem cells treated with hypoxia, and the concentration of the hypoxia-treated oxygen is 5% -10%;

preferably, the oxygen concentration of the low oxygen treatment is 5%.

8. The method for preparing the exosome according to claim 6, wherein the method for extracting the exosome comprises: any one of differential high-speed centrifugation, density gradient centrifugation, polymer-based precipitation technology extraction, size exclusion chromatography extraction, and immune separation technology extraction;

9. The method according to claim 6, wherein the berberine and the matrine are added while stirring during the second-stage stirring.

10. The method of claim 6, wherein the first stage agitation is: stirring for 1-3 min at a speed of 100-300 rmp/min; the second-stage stirring comprises the following steps: stirring at 400-600 rpm/min for 15-20 min.