A kind of exosome aloe preparation and its application

Abstract

The present invention discloses an exosome aloe preparation and its application, belonging to the field of biomedical technology, wherein the exosome preparation comprises exosomes separated from mesenchymal stem cells and a pharmaceutically acceptable carrier, wherein the carrier is an aloe hydrogel prepared from a concentrated extract of aloe juice, and the exosomes are stably and continuously transported to the damaged part by utilizing the physical properties of the hydrogel, thereby solving the problem of short storage time of the exosomes in clinical application, and the carrier itself retains the complete biological function of aloe, and can cooperate with the exosomes to produce a therapeutic effect. The present invention is verified by parallel control experiments and multiple groups of animal models, and the prepared exosome aloe preparation can resist inflammation and inhibit bacteria, promote the repair of damaged endometrial tissue, and improve pregnancy outcomes.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an exosome aloe preparation and application thereof. Background Art

[0002] The endometrium is one of the important components of the uterus, providing the necessary internal environment for embryo implantation and pregnancy maintenance. Uterine cavity manipulation, inflammation and infection are important causes of endometrial injury (EMI). Severe endometrial injury that reaches the basal layer of the endometrium can cause endometrial repair disorders, manifested as intrauterine adhesions (IUA) with partial or complete occlusion of the uterine cavity, leading to reduced menstrual volume, amenorrhea, recurrent miscarriage and infertility. Clinically, conventional treatments for endometrial injury include both oral estrogen, aspirin and traditional Chinese medicine, as well as local application of biomaterials such as amniotic membrane and hyaluronic acid to reduce the incidence of adhesions after endometrial injury, promote endometrial repair, and improve menstruation and pregnancy outcomes. However, for patients with severe, refractory and recurrent IUA, the difficulty of endometrial regeneration, high adhesion recurrence rate and low pregnancy rate are still major clinical problems. The treatment of endometrial damage should not only control the inflammatory response and restore the normal uterine cavity morphology, but also improve the endometrial receptivity to facilitate implantation and pregnancy. In this regard, stem cells have shown great potential.

[0003] Mesenchymal stem cells (MSCs) are multipotent stem cells derived from mesoderm and ectoderm. They have the functions of inhibiting inflammation, immune regulation, and injury repair. They have the advantages of low immunogenicity, easy access, high in vitro expansion efficiency, and no toxic side effects. They have attracted much attention in the field of tissue engineering. However, their low survival rate, transplant rejection, limited sources, and potential tumorigenicity limit their application.

[0004] With further in-depth research, it was found that when MSCs are involved in disease treatment, only a very small number of stem cells can actually reach the target site. More importantly, stem cells need to function through the "paracrine factors" produced by autocrine or paracrine mechanisms.

[0005] In order to maintain and perform normal biochemical functions, cells release various types of extracellular vesicles. Exosomes are an important component of extracellular vesicles. They are vesicular vesicles secreted by living cells and have a lipid bilayer membrane structure. Exosomes can be released by various types of cells and exist in various body fluids. Exosomes carry a variety of bioactive substances, including gDNA, mRNA, circRNA, lncRNA, miRNA, proteins and lipids. As a carrier that mediates intercellular communication, exosomes transport bioactive molecules from donor cells to nearby or distant recipient cells, and participate in the pathophysiological regulation of recipient cells. Compared with cell therapy, exosomes are a safe, effective and easy-to-control treatment method that is more convenient for large-scale manufacturing, storage and transportation.

[0006] Although exosomes have been initially studied and applied in the treatment of endometrial damage, there are still many shortcomings. From the perspective of exosome preparation, the existing exosome separation and purification technologies are mostly based on differential ultracentrifugation, gradient density, direct current filtration, adsorption precipitation and other principles. These methods have the defects of causing damage to exosomes, high loss rate (more than 50%), huge time consumption (usually dozens of hours), and difficulty in purification, making it difficult to obtain sufficient quality and quantity of exosomes. From the perspective of preparation, a simple cell exosome suspension preparation does not have the ability to stably remain and release in the uterine cavity.

[0007] In order to make exosomes play a better therapeutic role, the existing technology also uses various hydrogels as carriers, but usually in this combination, the carrier itself does not have a therapeutic effect. Therefore, the current exosome preparations are often difficult to achieve the expected therapeutic effect in actual clinical applications.

[0008] Aloe vera is one of the most famous medicinal plants in the world. It contains 99% water and 1% solid matter, covering more than 75 different compounds. Aloe vera can integrate into human tissues, promote natural healing and regeneration processes, have a mild effect, and can effectively resist oxidation, inhibit bacterial growth and reduce inflammation. In addition, the active compounds in aloe vera can stimulate the production of new cells and collagen. Although there is currently an application of a single ingredient extracted from aloe vera in exosome preparations, the therapeutic ability of aloe vera depends on the combined action of all its active phytochemical components. The extract of a single component does not have complete biological functions, and there is currently no sufficient scientific theory to support this method to exert the expected therapeutic ability. Summary of the invention

[0009] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides an exosome aloe preparation and its application. Exosomes of MSCs are extracted by in vitro cell culture, and exosomes are prepared into an exosome-aloe hydrogel preparation in a certain ratio with aloe hydrogel, so that the exosomes and aloe hydrogel act together, providing a new method for the treatment of endometrial damage.

[0010] First, as a substitute for MSC, exosomes have higher safety and are easier to store and transport while completing the main functions of MSC; secondly, aloe vera gel as a carrier solves the problem of short storage time of exosomes in clinical application, and can inhibit the growth of bacteria, provide a stable environment for exosomes and improve the effect of exosomes. In addition, the acetylated mannan contained in aloe vera can activate the protein translation mechanism mediated by the AKT / mTOR pathway, promote cell proliferation, induce cell migration, and work together with the "paracrine factors" carried by MSC exosomes to significantly improve angiogenesis at the site of endometrial injury, while inhibiting neutrophil infiltration by relieving inflammation, reshaping the anti-inflammatory phenotype of macrophages, and recruiting regulatory T cells to the site of injury to regulate the local immune microenvironment, thereby accelerating tissue repair.

[0011] On this basis, the present application designs and prepares a MSC exosome gel preparation derived from human umbilical cord, which can achieve the expected therapeutic and restorative effects, including treating damaged endometrium and improving infertility caused by endometrial damage after treatment.

[0012] However, the existing extraction methods of exosomes are prone to damage to exosomes, and there are problems such as time-consuming and high loss rate. In addition, the aloe vera generally used in the treatment of endometrial damage in the prior art is a single-component extract and does not have complete biological functions. In this application, based on the above problems, this application uses sine wave negative pressure oscillation and double coupled resonance technology to prepare high-purity, complete MSC exosomes, and obtains aloe vera juice concentrated extract with complete biological functions through molecular weight cutoff membrane (ultrafiltration membrane) fractionation, and prepares high-concentration aloe vera hydrogel, which acts together with exosomes on the endometrial damage site.

[0013] Based on the above analysis and preparation method, quantitative experiments were conducted to verify that aloe vera hydrogel can cooperate with exosomes to repair endometrial damage, and the optimal concentration of exosome aloe vera preparations was determined. Furthermore, comparative experiments were conducted to verify that the exosome aloe vera preparations prepared by the present invention have better therapeutic effects than hyaluronic acid gel and simple exosome preparations. For a specific technical roadmap, please refer to the attached Fig.16 .

[0014] Specifically, the present invention is achieved through the following scheme:

[0015] In a first aspect, the present invention provides an exosome aloe preparation, comprising exosomes separated from mesenchymal stem cells and a pharmaceutically acceptable carrier, wherein the carrier is an aloe hydrogel prepared from a concentrated extract of aloe juice.

[0016] Preferably, the mesenchymal stem cells (MSCs) are human umbilical cord-derived mesenchymal stem cells.

[0017] Preferably, the exosomes are prepared by the following method, comprising:

[0018] Step 1: Extracting exosomes from umbilical cord-derived mesenchymal stem cells: Using mesenchymal stem cells (MSCs) cultured to the 3rd to 5th passage to extract exosomes;

[0019] Step 2, purification of exosomes: including preliminary centrifugal separation and purification to remove large vesicles; and purifying the preliminarily purified exosome solution using the EXODUS exosome purification system.

[0020] Among them, the step one includes ① processing the collected umbilical cord tissue, digesting it and collecting the cell precipitate by centrifugation; ② resuspending the cell precipitate in MSC culture medium and placing it in a cell culture bottle for incubation and expansion; ③ splitting and passage; ④ using MSC cultured to the 3rd to 5th generation to collect the cell supernatant containing exosomes.

[0021] Among them, in the step 2, the EXODUS exosome purification system is used to purify and remove small molecules below 50nm through sine wave negative pressure oscillation and double coupling resonance technology, and the preliminary purified exosomes are further concentrated to 1.0×10 11 -1.0×10 12 Pieces / ml.

[0022] Preferably, the diameter of the exosomes is 50-150 nm. The exosomes prepared by the above method are fluorescently labeled and nano-flow cytometry detected. The results of the nano-flow cytometry test of the expression of exosome surface proteins refer to Table 1; the NTA test results of the exosome samples refer to Figure 1 ; Reference to the electron microscopy results of exosome samples Figure 2 Among them, in the samples tested by NTA, the content of particles with a particle size of 116 nm is the largest, and the content of particles with a particle size greater than 150 nm is relatively small. Combined with the results of electron microscopy detection and the results of the exosome surface protein expression experiment, it can be explained that the exosomes prepared by the preparation method in the present application are complete in morphology and high in purity. Compared with the existing purification and separation methods based on differential ultracentrifugation, gradient density, direct current filtration, adsorption precipitation and other technologies, the quality and quantity of the obtained exosomes are better.

[0023] Preferably, the concentrated extract of aloe vera juice is prepared by extracting a transparent gel from the inner leaf of a healthy, mature aloe vera leaf without any damage or discoloration, removing the yellow latex layer that may be irritating, and washing to remove impurities, and filtering out excess water through an ultrafiltration membrane to form a concentrated extract of juice.

[0024] The concentrated aloe juice extract prepared by the above method is identified to prepare a solution to be identified, and a standard aloe reference medicinal material is taken to prepare a reference solution. The prepared concentrated aloe juice extract is identified and analyzed for components by thin layer chromatography and high performance liquid chromatography to verify that the concentrated aloe juice extract retains the active ingredients and biological functions of the aloe medicinal material.

[0025] Preferably, the exosome aloe preparation is prepared by mixing aloe hydrogel at a concentration of 15% with 1.0×10 11 -1.0×10 12 The exosomes were mixed at a concentration of 100 / ml.

[0026] Preferably, the concentration of the aloe hydrogel is 15%, and the concentration of the exosomes is 1.0×10 11 Pieces / ml.

[0027] Among them, in the exosome quantification experiment conducted, the exosome concentration in the exosome aloe vera preparation was 1.0×10 11 and 1.0×10 12 When the concentration of MSC exosomes in the aloe vera preparation was 1.0×10 / ml, there was no significant difference in the mRNA expression levels of inflammatory factors and endometrial fibrosis markers in the serum of the two experimental groups. Therefore, based on the comprehensive considerations of preparation cost and animal equivalent dose conversion, the optimal concentration of MSC exosomes in the aloe vera preparation was 1.0×10 11 / ml, and in subsequent animal experiments, it was also verified that this concentration of exosome aloe preparation achieved the expected experimental effect.

[0028] Preferably, the exosome aloe preparation is a hydrogel preparation.

[0029] In the second aspect, the present invention also provides the use of the exosome aloe preparation proposed in the first aspect in the preparation of drugs related to the treatment of endometrial damage. In the comparative experiment conducted, the exosome aloe preparation was used on the established endometrial damage rat model (using aloe hydrogel as a biomaterial carrier), and the endometrial repair-related indicators were better than those of the sodium hyaluronate gel group and the simple exosome group of the same concentration, verifying that the exosome aloe preparation involved in the present application has a significant repair effect on rat endometrial damage.

[0030] In the third aspect, the present invention also provides the use of the exosome aloe preparation proposed in the first aspect in the preparation of drugs related to improving infertility caused by endometrial damage. A comparative experiment was conducted to evaluate whether the exosome aloe preparation can significantly improve the pregnancy outcome of the endometrial damage rat model. The experimental results showed that the pregnancy rate of the exosome aloe preparation group was significantly higher than that of the sodium hyaluronate gel treatment group and the model group (P < 0.01). At the same time, compared with the exosome preparation group that did not use aloe as a carrier and other treatment groups, the number of embryo implantation in the exosome aloe preparation group increased significantly, reaching the level of the sham operation group (P > 0.05). This shows that the exosome aloe preparation proposed in the first aspect can be used to repair the damaged endometrium and further improve the pregnancy outcome of the endometrial damage rat model.

[0031] According to the above technical solution, the beneficial effects of the present invention are as follows:

[0032] The exosome aloe preparation prepared by exosomes and aloe hydrogel proposed in this application uses high-concentration aloe hydrogel with complete biological functions as a biomaterial carrier and effective component, which not only solves the problem of short storage time in the application of existing exosome solution preparations, but also synergizes with the prepared exosome preparations to produce therapeutic effects, relieve inflammation, regulate the local immune microenvironment, and accelerate tissue repair. Related experiments have verified that the therapeutic effect of exosome aloe preparations is better than that of simple exosome preparations and more mature gel preparations. The key point is that exosome aloe preparations can repair the endometrium of uterine injury rat models and further improve the pregnancy outcomes of endometrial injury rat models. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0034] Figure 1 This is the NTA test result of the exosome sample in Example 1;

[0035] Figure 2 This is an electron microscope photo of the exosome sample in Example 1;

[0036] Figure 3 Comparison of serum inflammatory factor levels in rats of each group D4 in Example 4 ( ); unit: pg / mL;

[0037] Figure 4 Comparison of serum inflammatory factor levels in rats of each group D12 in Example 4 ( ); unit: pg / mL;

[0038] Figure 5is the change in the mRNA expression level of endometrial fibrosis marker (TGF-β) in each group of rats in Example 4 ( ); Unit: each index / internal reference, %;

[0039] Figure 6 is the change in the mRNA expression level of endometrial fibrosis marker (TIMP) in each group of rats in Example 4 ( ); Unit: each index / internal reference, %;

[0040] Figure 7 is the change in the mRNA expression level of endometrial fibrosis marker (PDGF-bb) in each group of rats in Example 4 ( ); Unit: each index / internal reference, %;

[0041] Figure 8 is the change in the mRNA expression level of the endometrial fibrosis marker (COL1A1) in each group of rats in Example 4 ( ); Unit: each index / internal reference, %;

[0042] Fig. 9 Comparison of serum inflammatory factor levels in rats of each group D4 in Example 5 ( )Unit: pg / mL;

[0043] Fig.10 Comparison of serum inflammatory factor levels in rats of each group D12 in Example 5 ( )Unit: pg / mL;

[0044] Fig.11 is the change in the mRNA expression level of endometrial fibrosis marker (TGF-β) in each group of rats in Example 5 ( ); Unit: each index / internal reference, %;

[0045] Fig.12 is the change in the mRNA expression level of endometrial fibrosis marker (TIMP) in each group of rats in Example 5 ( ); Unit: each index / internal reference, %;

[0046] Fig.13 is the change in the mRNA expression level of endometrial fibrosis marker (PDGF-bb) in each group of rats in Example 5 ( ); Unit: each index / internal reference, %;

[0047] Fig.14 is the change in the mRNA expression level of the endometrial fibrosis marker (COL1A1) in each group of rats in Example 5 ( ); Unit: each index / internal reference, %;

[0048] Fig.15 Comparison of the number of embryo implantation in each group of rats in Example 5;

[0049] Fig.16 The technical roadmap for this application;

[0050] Fig.17 It is the chromatogram of the test sample in Example 2; wherein, Peak 1: aloin B; Peak 2: aloin D; Peak 3 (S): aloin. Aloe vera (Aloe barbadensis) formula granules control characteristic spectrum. Chromatographic column: ACQUITYUPLC HSS T3, 2.1 mm×150 mm, 1.8 μm. DETAILED DESCRIPTION

[0051] The present invention discloses an exosome aloe preparation and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0052] Description of the technical route of the present invention: preparation of exosomes, preparation of aloe hydrogel → preparation of exosome aloe preparation → quantitative experiment (verifying the optimal concentration of exosome aloe preparation) → comparative experiment (verifying the effect of exosome aloe preparation in repairing endometrial damage and improving pregnancy).

[0053] Example 1: Exosome extraction, purification and identification.

[0054] Most of the existing technologies are based on separation and purification methods such as differential ultracentrifugation, gradient density, direct current filtration, and adsorption precipitation. These methods have the defects of mechanical force causing damage to exosomes, high loss rate (more than 50%), huge time consumption (usually dozens of hours), and difficulty in purification, making it difficult to obtain exosomes of sufficient quality and quantity. The exosome preparation method proposed in this application includes:

[0055] Step 1: Extracting exosomes from umbilical cord-derived MSCs: Using MSCs cultured to passage 3-5 to extract exosomes;

[0056] Step 2, purification of exosomes: including preliminary centrifugal separation and purification to remove large vesicles; using the EXODUS exosome purification system to purify the preliminarily purified exosome solution through sine wave negative pressure oscillation and double coupled resonance technology.

[0057] The exosome samples prepared by the above method were tested in three aspects, including exosome surface protein detection, NTA detection and electron microscopy detection:

[0058] (1) Detection of exosome surface proteins: using fluorescence labeling and nanoflow cytometry, the results of the single plate expression experiment on the surface of exosomes are shown in Table 1;

[0059] (2) NTA test: The exosome sample was enriched with particles with a particle size of 116 nm. The test results refer to Figure 1 ;

[0060] (3) Electron microscopy results of exosome samples, see Figure 2 ;

[0061] Refer to Table 1, Figure 1 and Figure 2 Based on the test results of the above three aspects, it can be concluded that the exosomes prepared by the above method are of sufficient quality and quantity, and the exosomes are separated by sine wave negative pressure oscillation and double coupled resonance technology, with high acquisition rate, high purity, no mechanical damage to the exosomes, fast separation speed, and better quantity and quality of the obtained exosomes.

[0062] In this embodiment, the specific steps of isolating exosomes are as follows:

[0063] Exosome extraction: Fresh umbilical cords were obtained, washed 3 times with 70% ethanol, and then washed 3-5 times with PBS to sterilize and remove blood. The umbilical cords were cut into small pieces and transferred to 50mL conical tubes, and then incubated with 500U type I collagenase solution at 37°C for 1 hour with shaking. After digestion, the solution was diluted in chilled PBS and centrifuged at 1400G for 10min at 4°C to collect the cell pellet. The cell pellet was resuspended in MSC culture medium and transferred to a cell culture flask coated with solution CTS TM CELLstart TM substrate (diluted in PBS at a ratio of 1:300) and incubated at 37°C and 5% CO2 for cell expansion. The cell pellet was seeded into 1 T25 cell culture flask per 5mg umbilical cord. The culture medium was changed every 2 days of culture. When the cells reached 80% confluence, the cells were split with 0.05% trypsin for the next passage. Exosomes were extracted from MSCs cultured to the 3rd to 5th passages. The original culture medium was discarded, the cells were washed three times with PBS, replaced with serum-free exosome culture medium, and cultured in a 37°C incubator for 48 h, after which the supernatant containing exosomes was collected.

[0064] Exosome purification: The collected exosome supernatant was centrifuged at 20000G to remove the remaining large vesicles. The preliminarily purified exosome solution was purified using the EXODUS exosome extraction system through sine wave negative pressure oscillation and double coupling resonance technology to control the exosome concentration at 1×10 12 / ml.

[0065] In this embodiment, the steps of fluorescence labeling and nanoflow cytometry detection of exosome samples are as follows:

[0066] ① Dilute 30uL of exosomes to 90uL, take 30uL of diluted exosomes and add 20uL of fluorescently labeled antibodies (CD9, CD63, CD81), mix well, and incubate at 37℃ for 30min in the dark.

[0067] ② Add 1 mL of pre-cooled PBS, select an ultraspeed rotor, and ultracentrifuge at 4°C, 110,000×g for 70 min.

[0068] ③ Carefully remove the supernatant, add 1 mL of pre-cooled PBS, select an ultraspeed rotor, and ultracentrifuge again at 4°C, 110,000×g for 70 min.

[0069] ④Carefully remove the supernatant and resuspend in 50uL pre-cooled 1× PBS.

[0070] ⑤ Use standard products to perform instrument performance tests first and then load exosome samples. Note that gradient dilution is required to avoid clogging of the injection needle by the sample.

[0071] ⑥ Once the sample is tested, the protein index results of the NanoFCM instrument can be obtained.

[0072] Table 1: Nanoflow cytometry results of the surface protein expression test of exosomes

[0073]

[0074] Example 2: Preparation of concentrated aloe vera juice extract.

[0075] The concentrated aloe vera juice extract prepared in this example, as a carrier of the exosomes prepared in Example 1, also completely retains the effective biological function and therapeutic ability, and can cooperate with the exosomes to exert therapeutic effects.

[0076] In this embodiment, the method for preparing the concentrated aloe juice extract is as follows: a transparent gel is extracted from the inner leaf of a healthy, mature aloe leaf without any damage or discoloration, the yellow latex layer that may be irritating is removed, and the gel is washed to remove impurities. Excess water is filtered out using an ultrafiltration membrane to form a concentrated juice extract.

[0077] Identification of aloe vera juice concentrated extract: (1) Test identification according to thin layer chromatography (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia); (2) Component analysis: Determined by high performance liquid chromatography (General Rules 0512 of the 2020 edition of the Chinese Pharmacopoeia) (characteristic spectrum).

[0078] Specifically, the identification steps are as follows: take 0.5g of concentrated extract of aloe vera juice, grind it, add 20ml of methanol, heat it to boiling in a water bath, shake it for several minutes, filter it, and take the filtrate as the test solution. Take another 0.5g of aloe vera (Aloe vera) reference medicinal material, and make a reference medicinal material solution in the same way. Take the aloin reference substance, add methanol to make a solution containing 3mg per 1ml, as the reference solution. Thin layer chromatography (Chinese Pharmacopoeia 2020 Edition General Principles 0502) test, take 5µl of each of the above three solutions, spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-water (100:17:13) as the developing agent, develop, take out, dry, spray with 10% potassium hydroxide methanol solution, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

[0079] Characteristic spectrum: Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0080] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler (column length 150mm, inner diameter 2.1mm, particle size 1.8μm) as chromatographic column; acetonitrile as mobile phase A, water as mobile phase B, gradient elution according to the provisions of Table 2; flow rate 0.3ml per minute, column temperature 40℃, detection wavelength 210nm. The theoretical plate number calculated based on the aloin peak should be no less than 8000.

[0081] Table 2

[0082]

[0083] Preparation of reference solution: Take 0.5g of aloe (Aloe vera) reference medicinal material, place it in a stoppered conical bottle, add 25ml of methanol, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take appropriate amounts of aloin reference substance, aloin D reference substance, and aloin B reference substance, add methanol to make solutions containing 50μg, 30μg, and 40μg per 1ml, respectively, as reference substance reference solutions.

[0084] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take 0.1g, put it in a stoppered conical flask, add 25ml of methanol, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0085] Determination method: Accurately pipette 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. Fig.17There should be 5 characteristic peaks in the chromatogram of the test sample, and the retention times of the 5 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Peak 1, Peak 2, and Peak 3 should correspond to the retention times of the corresponding reference peaks, respectively. The peak corresponding to the aloin reference peak is the S peak. The relative retention times of Peak 4, Peak 5 and S peak are calculated. The relative retention times should be within ±10% of the specified values, and the specified values ​​are: 1.06 (Peak 4), 1.19 (Peak 5).

[0086] Example 3: Preparation and preservation of exosome aloe vera preparation.

[0087] In this embodiment, the exosome aloe preparation includes exosomes separated from MSC and a pharmaceutically acceptable carrier. The MSC exosomes can be prepared by the preparation method in Example 1. The carrier is a hydrogel made from concentrated aloe juice extract, which can be prepared by the concentrated aloe juice extract in Example 2.

[0088] Preparation method of exosome aloe preparation: dissolve aloe juice concentrated extract at a high concentration of 15% (w / v) (preferably 15% aloe concentration) in 50°C deionized water, stir continuously until completely dissolved, and after the temperature drops to 37°C, add 1.0×10 11 ~1.0×10 12 / ml of exosomes (preferably 1×10 11 / ml) and incubate together, stirring constantly to mix thoroughly, and then cooling to room temperature to form exosome-aloe hydrogel. It is then refrigerated in a moisture-proof sealed container (such as a syringe) for storage.

[0089] Example 4: Quantitative experiment on the concentration of exosome aloe vera preparation.

[0090] The remodeling and regeneration during the endometrial repair process depends on the presence of adult stem cells and migratory resident cells, the influx of coordinated specific immune cells, controllable inflammatory response and angiogenesis, and related factors including transforming growth factor-β (TGF-β), tissue inhibitor of metalloproteinases (TIMP), platelet-derived growth factor (PDGF-bb) and type Ⅰ collagen (COL1A1) have been confirmed to be involved in the pathological process of promoting IUA fibrosis. Therefore, the exosome aloe preparation proposed in the present invention was used to experiment on the endometrial injury rat model, and the expression of inflammatory factors in the serum of the rat model and the mRNA expression level of endometrial fibrosis markers were detected to explore and verify the effective concentration and dosage of the exosome aloe preparation prepared by the preparation method of Example 3 in the endometrial repair.

[0091] Specifically, according to the preparation method in Example 3, exosome aloe preparations containing exosomes of different concentrations were prepared, and an endometrial injury rat model was established. The established rat model was randomly divided into five groups and administered drugs respectively for concentration quantitative experiments.

[0092] The experimental groups are:

[0093] ① Injury group (Model): After the model was constructed, only saline was given to treat the injured part, 500uL, n=6;

[0094] ② Exosome preparations 1: low concentration group, after the model was constructed, the exosomes were treated with aloe preparations: 500uL of preparations, with 1.0×10 10 , aloe vera concentration 15%, n=6;

[0095] ③ Exosome preparations 2: medium concentration group, after the model was constructed, the exosomes were treated with aloe preparations: 500uL of preparations, with 1.0×10 exosomes 11 , aloe vera concentration 15%, n=6;

[0096] ④ Exosome preparations 3: high concentration group, after the model was constructed, the exosomes were treated with aloe preparations: 500uL of preparations, with 1.0×10 12 , aloe vera concentration 15%, n=6;

[0097] ⑤ Sham operation group: the skin was incised to expose the uterus without damaging the uterus and endometrium, n=6.

[0098] Treatment time and method: On the 3rd and 5th day after modeling, vaginal infusion treatment is performed on the uterine cavity. The test includes the following two aspects:

[0099] (1) Detection of inflammatory factor expression in serum: On the 4th and 12th day of modeling, the levels of inflammatory factors (IL-17, TNF-α) in the serum of each group of rats were detected; the test results are shown in Tables 3, 4, Figure 3 and Figure 4 .

[0100] The test results showed that on D4, the first day after infusion treatment, the inflammatory factor IL-17 in all exosome aloe vera preparation treatment groups was significantly lower than that in the injury group; among the three exosome aloe vera preparations, the effect of reducing the inflammatory factor IL-17 in exosome aloe vera treatment groups 2 and 3 was better than that in exosome aloe vera treatment group 1.

[0101] On D12, one week after the second infusion treatment, compared with the injury group, the IL-17 and TNF-α in all exosome aloe preparation treatment groups decreased significantly, especially in exosome aloe preparation treatment groups 2 and 3, the pro-inflammatory cytokine TNF-α was basically reduced to the level of the sham operation group (P>0.05).

[0102] (2) Detection of mRNA expression levels of endometrial fibrosis markers: qRT-PCR was used to detect the mRNA expression of endometrial fibrosis-related factors after treatment 4-5 estrus cycles after treatment; the test results are shown in Table 6. Figure 5-8 The test results showed that the fibrosis-related factors TGF-β, TIMP and PDGF-bb in the exosome aloe preparation treatment group 1 were significantly lower than those in the injury group, and there was no significant difference in COL1A1 between the two groups; the fibrosis-related factors TGF-β, TIMP, PDGF-bb and COL1A1 in the exosome aloe preparation treatment groups 2, 3 and the sham operation group were significantly lower than those in the injury group.

[0103] Based on the above two experimental results, on the first day after treatment, the pro-inflammatory factor IL-17 in all exosome aloe preparation groups was significantly lower than that in the injury group, verifying that the exosome aloe preparation prepared in the present application has a significant correlation with the treatment of rat endometrium, and one week after the second treatment, the IL-17 and TNF-α of all exosome aloe preparation treatment groups were significantly decreased, especially the IL-17 level of exosome aloe preparation groups 2 and 3 was close to that of the sham operation group, and TNF-α was basically reduced to the sham operation group (refer to Tables 3 and 4). One week after the second administration, the exosome aloe preparation groups 2 and 3 maintained a sustained and significant therapeutic effect, and the endometrial fibrosis-related factors TGF-β, TIMP, PDGF-bb, and COL1A1 in the exosome aloe preparation groups 2 and 3 were significantly lower than those in the injury group, verifying that the exosome aloe preparation proposed by the present invention has a significant effect on endometrial repair in the rat model of endometrial injury and a sustained therapeutic effect.

[0104] In the above quantitative experiment, there was no significant difference in the test results between the exosome aloe vera preparation treatment groups 2 and 3 (P>0.05). Therefore, based on the experimental results and cost considerations, the optimal number of exosomes for rats with exosome aloe vera preparation was 1.0×10 11 , that is 5.0×10 11 / kg (rats weigh 0.2kg). Based on the dose per unit body weight, the equivalent dose for rats is 6.3 times that for humans. The optimal dose for humans is 7.9×10 10 / kg (the optimal dose for a 70kg human is 5.5×10 12 ).

[0105] In this example, the method for establishing the endometrial injury model animal is as follows:

[0106] Sprague-Dawley (SD) female rats weighing 200-230 g and aged 7 to 9 weeks were selected as the research targets. The rats were group housed with free access to food and water and maintained on a 12-hour light / dark cycle. Vaginal cytology performed in the morning was used to understand the frequency and regularity of the estrous cycle after 1-2 weeks of adaptation. Forty rats with the same estrous cycle were selected for the study.

[0107] After the rats were anesthetized by intraperitoneal injection of 3% chloral hydrate (1 ml / 100 g body weight), the abdominal cavity was opened under sterile conditions to expose the bilateral uterine horns. At the lower third of the junction of the middle and distal ends of the uterus, a 1.0 cm incision was made through the uterus, and the endometrium was scraped with a 2 mm scraper until the uterine wall became rough and the endometrium was obviously swollen. After flushing the uterus and the abdominal cavity with sterile saline, the uterus (6-0 absorbable suture) and the abdominal wall (4-0 absorbable suture) were sutured in sequence. All postoperative rats were given antibiotic treatment for three consecutive days (100,000 units of penicillin per rat intramuscularly).

[0108] In this embodiment, the method for detecting the expression of inflammatory factors in serum is as follows: On the 4th and 12th day of modeling, the content level of inflammatory factors (IL-17, TNF-α) in the serum of each group of rats was detected. Blood was collected through the rat tail vein with a blood collection needle. The obtained blood was placed in a sterile centrifuge tube and allowed to stand in a 4°C refrigerator for 30 minutes, then centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected to obtain serum. According to the operating instructions of the ELISA kit, the content levels of IL-17 and TNF-α in the serum of each group of animals were detected.

[0109] Table 3: Results of inflammatory factor detection in D4 serum

[0110]

[0111] Table 4: Results of inflammatory factor detection in serum at D12

[0112]

[0113] In this example, the mRNA expression level of endometrial fibrosis markers was detected as follows: transforming growth factor-β (TGF-β), tissue inhibitor of metalloproteinase (TIMP), platelet-derived growth factor (PDGF-bb) and type Ⅰ collagen (COL1A1) were confirmed to be involved in the pathological process of promoting IUA fibrosis. qRT-PCR was used to detect the expression of mRNA of endometrial fibrosis-related factors after treatment. Six rats were randomly selected from each group 4-5 estrus cycles after treatment, the abdominal cavity was opened, the uterus was exposed and separated, the blood and mucus on the surface of the uterus were cleaned with PBS, the uterine tissue was cut and quickly frozen with liquid nitrogen, and ground into homogenate with a grinder; the total RNA of the cells was extracted using an RNA extraction kit; the RNA was reverse transcribed into cDNA using a reverse transcription kit according to the operating instructions; the SYBR Green kit was used for real-time quantitative PCR, and the relative expression of the sample RNA was determined on the machine, and mouse GAPDH was used as an internal control. The differences in the expression of endometrial fibrosis indicators in rats in each group were compared.

[0114] Table 5: PCR primer sequences

[0115]

[0116] Table 6: Results of mRNA expression level detection of endometrial fibrosis markers

[0117]

[0118] In this embodiment, the statistical method is described as follows:

[0119] Data are presented as mean ± standard deviation (SD), and t-test was used for comparison between two groups. For comparison of multiple groups, statistical significance was estimated using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test or two-way analysis of variance (ANOVA) with Šídák's multiple comparison test. Data were analyzed using GraphPad-Prism 10.1.2 software. P value < 0.05 was considered statistically significant (Note: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, ns indicates P > 0.05).

[0120] Example 5: Comparative experiment on the efficacy of exosome aloe vera preparations.

[0121] The commonly used biological material for repairing endometrial damage is sodium hyaluronate gel, which stays in the uterine cavity for a long time and can prevent the recurrence of adhesions. In this embodiment, pure exosomes and sodium hyaluronate are used as comparative examples for comparative experiments to verify the therapeutic effect of the exocrine aloe vera preparation of the present application in repairing endometrial damage and improving pregnancy outcomes.

[0122] On this basis, a comparative experiment was designed, in which the modeling method, treatment time node and method of the comparative experiment in this embodiment were the same as those in Example 4. In addition to detecting the expression of inflammatory factors in the serum of the rat model and the mRNA expression level of endometrial fibrosis markers, the comparative experiment in this embodiment also included a pregnancy outcome assessment to verify that the exosome aloe preparation prepared by the preparation method of Example 3 can be used for endometrial repair and can also improve infertility caused by endometrial damage.

[0123] The comparative experiment groups are as follows:

[0124] ① Injury group (Model): After the model was constructed, only saline was given to treat the injury, 500uL, n=6;

[0125] ② Exosome preparations treatment group: After the model was constructed, the exosome preparations were used for treatment: 500uL of the preparations, with 1.0×10 11 , aloe vera concentration 15%, n=6;

[0126] ③ Exosome treatment group: After constructing the model, 1.0×10 11 Exosomes were treated with 500uL saline, n=6;

[0127] ④Sodium hyaluronate treatment group: after the model was constructed, 500uL medical sodium hyaluronate gel was used for treatment, n=6;

[0128] ⑤ Sham operation group: the skin was incised to expose the uterus without damaging the uterus and endometrium, n=6.

[0129] Treatment time and method: On the 3rd and 5th day after modeling, vaginal infusion treatment is performed on the uterine cavity. The detection indicators include the following three aspects:

[0130] (1) Inflammatory factors in serum: On the 4th and 12th day of modeling, the levels of inflammatory factors (IL-17, TNF-α) in the serum of each group of rats were detected; the test results are shown in Tables 7, 8, Fig. 9 and Fig.10 ;

[0131] The test results of the exosome aloe vera preparation treatment group showed that on the 4th day, the level of inflammatory factor IL-17 in serum was significantly lower than that in the injury group, and there was no significant difference between TNF-α and the sham operation group; on the 12th day, the level of inflammatory factor IL-17 in serum was significantly lower than that in the injury group, and there was no significant difference between TNF-α and the sham operation group;

[0132] The results of the sodium hyaluronate treatment group showed that on the 4th day, there was no significant reduction in the expression of inflammatory factors IL-17 and TNF-α in the serum; on the 12th day, the expression of inflammatory factor IL-17 in the serum was the worst, and there was a significant difference with the exosome preparation treatment group, and the expression of TNF-α was not significantly different from that of the injury group;

[0133] The test results of the pure exosome treatment group showed that on the 4th day, the level of inflammatory factor IL-17 in serum was not significantly different from that in the exosome preparation treatment group, and there was no obvious reduction effect on the expression of TNF-α; on the 12th day, the effect of reducing the level of IL-17 in serum was not significantly different from that in the sodium hyaluronate treatment group, and the reduction effect was worse than that in the exosome preparation treatment group, and there was no significant difference in the expression of TNF-α between the sodium hyaluronate treatment group and the group.

[0134] According to the test results, the exosome aloe preparation treatment group and the sodium hyaluronate treatment group were compared: the test results of the exosome aloe preparation treatment group showed that on D4, the exosome aloe preparation treatment group had a significant effect in reducing the expression of inflammatory factors. On D12, the inflammatory factor level of the exosome preparation treatment group had basically recovered to the inflammatory factor level of the sham operation group, while the inflammatory factor level of the sodium hyaluronate treatment group was not significantly different from that of the injury group. It can be seen that the exosome aloe preparation has achieved the expected effect of reducing the level of inflammatory factors, which is better than the effect of sodium hyaluronate on reducing the expression level of serum inflammatory factors.

[0135] According to the test results, the exosome aloe preparation treatment group and the pure exosome treatment group were compared: on D12, the expression levels of inflammatory factors IL-17 and TNF-α in the exosome aloe preparation treatment group were significantly different from those in the pure exosome treatment group (P < 0.0001 and P < 0.05).

[0136] According to the test results, the exosome aloe preparation treatment group, sodium hyaluronate treatment group and pure exosome treatment group were compared: D4, the first day after the first treatment, in terms of the effect of reducing inflammatory factors, the exosome aloe preparation treatment group was better than the pure exosome preparation treatment group and the sodium hyaluronate gel treatment group; D12, one week after the second treatment, the exosome aloe preparation treatment group was better than the pure exosome preparation treatment group and the sodium hyaluronate gel treatment group. The exosome aloe preparation treatment group had a fast onset of action and had a sustained and significant therapeutic effect (the sodium hyaluronate treatment group had the worst effect on reducing inflammatory factors, and there was no significant difference in the expression of TNF-α between the injury group and the injury group (P>0.05)).

[0137] Table 7: Results of inflammatory factor detection in serum at D4

[0138]

[0139] Table 8: Results of inflammatory factor detection in serum at D12

[0140]

[0141] (2) Fibrosis markers: qRT-PCR was used to detect the mRNA expression of endometrial fibrosis-related factors after treatment 4-5 estrus cycles; the test results refer to Table 9, Figure 11-14 .

[0142] The results of the exosome aloe preparation treatment group showed that the expression level of TGF-β had no significant difference with the sham operation group and was significantly lower than that of the injury group; the expression level of PDGF-bb was significantly lower than that of the injury group; the expression level of TIMP was significantly lower than that of the injury group; and the expression level of COL1A1 was significantly lower than that of the injury group.

[0143] Among them, the expression level of TGF-β in the exosome aloe preparation treatment group was lower than that in the pure exosome treatment group (P < 0.05), and there was an extremely significant difference compared with the sodium hyaluronate treatment group (P < 0.0001); the expression levels of PDGF-bb and TIMP in the exosome aloe preparation treatment group were extremely significantly different from those in the pure exosome treatment group and the sodium hyaluronate treatment group (P < 0.0001); there was no statistical difference in the expression level of the marker COL1A1 between the pure exosome treatment group and the sodium hyaluronate treatment group (P > 0.05).

[0144] Table 9: Results of mRNA expression level detection of endometrial fibrosis markers

[0145]

[0146] (3) Pregnancy function assessment: Further evaluation of whether the mesenchymal exosome aloe vera preparation can improve the pregnancy outcome of the endometrial injury rat model; the test results refer to Table 10 and Fig.15 The evaluation results showed that the pregnancy rate of rats in the sham operation group was 83.33%, while the pregnancy rate of the exosome aloe preparation treatment group was 66.67%, which was significantly higher than that of the sodium hyaluronate treatment group and the injury group (P < 0.01). The pregnancy rate of the exosome aloe preparation was 50%. The sham operation group had the largest number of embryo implantation, which were 13, 17, 14, 15, 13, and 0 embryos, respectively. The number of embryo implantation in the exosome group was 4, 9, 7, 0, 0, and 0 embryos, respectively. There was no embryo implantation in the injury group and the sodium hyaluronate treatment group. Compared with the injury group and other treatment groups, the number of embryo implantation in the exosome aloe preparation treatment group increased significantly, which was 14, 18, 13, 10, 0, and 0, reaching the level of the sham operation group (P > 0.05).

[0147] Table 10: Evaluation results of implantation number of rats in each group

[0148]

[0149] In this embodiment, the method for evaluating pregnancy function is as follows: 4 weeks after surgery, 6 rats from each group are caged with healthy male rats; the next morning, the vaginal plugs of the female rats are observed, and the rats with vaginal plugs are recorded as pregnant; on the 14th day of pregnancy, the abdominal cavity of the rats is opened under general anesthesia, the number of embryos in the uterus of each group of rats is calculated and compared, and the recovery of the pregnancy function of each group of rats is evaluated (in this embodiment, the statistical method is consistent with that in Example 4).

[0150] In summary, the exosome aloe preparation includes exosomes and aloe hydrogel prepared by the method of the above embodiment. The exosomes have a lipid bilayer membrane structure and can be transported to the target site through the chemotaxis of autologous cells. The aloe hydrogel retains its active ingredients that can be used for antibacterial and anti-inflammatory and stimulating the regeneration of new cells and collagen (collagen is an essential protein in the tissue regeneration process), and has the characteristics of a gel. When acting on the damaged endometrium of rats, the active ingredients of the exosomes and aloe hydrogel act on the damaged site to repair the endometrial damage. The repair effect is better than that of simple exosomes and sodium hyaluronate gel, and the effect of restoring pregnancy function is close to that of the sham operation group (although sodium hyaluronate gel has a certain repair and treatment effect on the damaged endometrium of rats, the rats after treatment are not ideal in improving the pregnancy outcome. At least under the same treatment cycle, the rats in the sodium hyaluronate treatment group are not ideal in improving the pregnancy outcome). The aloe hydrogel in the exosome aloe hydrogel preparation not only acts as a gel carrier on the exosomes, allowing the exosomes to stay in the site of endometrial damage for a long time, but also allows its active ingredients to work synergistically with the exosomes, utilizing the structural characteristics of the exosomes and their own unique ingredients to repair endometrial damage and improve pregnancy levels.

[0151] Therefore, in summary, exosome aloe vera preparations can repair the endometrium of rats with endometrial damage, and further improve the pregnancy function of rats with endometrial damage. They are superior to sodium hyaluronate gel in terms of treatment, sustained treatment effect and improvement of pregnancy outcomes. They can be used to prepare drugs related to the treatment of endometrial damage and drugs related to improving pregnancy, providing new ideas and methods for the treatment of endometrial damage and intrauterine adhesions.

[0152] Although the embodiments of the present invention have been shown and described, it is understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents. In short, if those skilled in the art are inspired by them and design structural modes and embodiments similar to the technical solution without creativity without departing from the purpose of the present invention, they shall all fall within the scope of protection of the present invention.

Claims

1. An exosome aloe preparation for treating endometrial damage, characterized in that: It comprises exosomes separated from mesenchymal stem cells and a pharmaceutically acceptable carrier, wherein the carrier is an aloe hydrogel prepared from concentrated aloe juice extract; the exosome has a diameter of 50-150 nm; The exosomes are prepared by the following method, comprising: Step 1: Extracting exosomes from umbilical cord-derived mesenchymal stem cells: Using mesenchymal stem cells cultured to the 3rd to 5th generation to extract exosomes: Step 2: Exosome purification: including preliminary centrifugal separation and purification to remove extracellular vesicles with larger particle size; using the EXODUS exosome purification system to purify through sine wave negative pressure oscillation and double coupled resonance technology, the exosome concentration is controlled at 1.0×10 11 -1×10 12 / ml; The exosome aloe preparation is composed of aloe hydrogel at a concentration of 15% and 1.0×10 11 -1×10 12 The exosomes of different concentrations were mixed to form; The exosome aloe preparation is a hydrogel preparation.

2. The exosome aloe preparation according to claim 1, characterized in that: The mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

3. Use of an exosome aloe preparation according to any one of claims 1-2 in the preparation of drugs for treating endometrial damage.

4. Use of an exosome aloe preparation according to any one of claims 1-2 in the preparation of a drug for improving infertility caused by endometrial damage.

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