Preparation method and experimental application of tea nano-vesicle transboundary modified mesenchymal stem cell exosome for treating rhinitis

Abstract

The invention relates to the technical field of mesenchymal stem cell exosome transformation, in particular to a preparation method and experimental application of a mesenchymal stem cell exosome transboundary-transformed from tea nano-vesicles for treating rhinitis. According to the invention, plant-derived tea exosome-like nano-vesicles are used for transboundary transformation of human mesenchymal stem cell exosomes, and a new method is explored for exosome therapy of allergic rhinitis; the influence of co-incubation of the tea exosome-like nano-vesicles and the human mesenchymal stem cells on the ultrastructure, particle size, potential, protein characterization and chemical inclusions of the human mesenchymal stem cell exosome is observed, and a basis is provided for ascertaining the transboundary transport mechanism and biological effect of the plant nano-vesicles; the ultrastructures, the particle sizes, the potentials, the lipidomics, the proteomics and the tea leaf contents of the two tea leaf exosome-like nano-vesicles are detected and contrastively analyzed, and the method has positive significance for exploring the change characteristics of the physicochemical structures and the biological activity of the tea leaf exosome-like nano-vesicles in the processing process.

Description

Technical Field

[0001] The present invention relates to the technical field of modifying mesenchymal stem cell exosomes, and particularly to a preparation method and experimental application of cross-border modified mesenchymal stem cell exosomes with tea nano vesicles for treating rhinitis. Background Art

[0002] Allergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa mainly mediated by immunoglobulin E (IgE) after atopic individuals are exposed to allergens. Epidemiological investigations show that the prevalence of AR has increased significantly in recent years and has become a major chronic inflammatory disease of the respiratory tract, seriously affecting the quality of life of patients and social economy.

[0003] Currently, the treatment strategies for allergic rhinitis mainly include patient education, avoiding contact with allergens and irritants, or symptomatic drug treatment. It is mainly symptomatic drug treatment, including glucocorticoid drugs, antihistamines, anti-leukotrienes, mast cell stabilizers, decongestants, anticholinergic drugs, traditional Chinese medicine, nasal saline irrigation, anti-IgE treatment, and combined drug treatment, etc. The use of a large number of glucocorticoid drugs and antihistamine drugs has great side effects, and the long-term symptom control rate is low and the recurrence rate is high. Therefore, it is of great significance to find a safer and more effective new scheme for treating allergic rhinitis. Summary of the Invention

[0004] Other features and advantages of the present invention will be described in the following specification, and will be partially obvious from the specification, or understood by implementing the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the structures specifically pointed out in the specification and other specification drawings.

[0005] The objective of the present invention is to overcome the above deficiencies and provide a preparation method and experimental application of cross-border modified mesenchymal stem cell exosomes with tea nano vesicles for treating rhinitis.

[0006] To achieve the above objective, the technical solution of the present invention is: a preparation method of cross-border modified mesenchymal stem cell exosomes with tea nano vesicles for treating rhinitis, comprising the following steps: (A) Extraction of tea exosome-like nano vesicles (ELNs): The tea is divided into fresh tea leaves (X) and finished tea (C), and two ELNs samples (X-ELNs and C-ELNs) are obtained from the fresh tea leaves (X) and the finished tea (C) respectively; (B)Preparation and quantification of tea exosome-like nanovesicles cross-border modified mesenchymal stem cell exosomes (ELNs-MSCs-exo): The tea nanovesicles X-ELNs and C-ELNs were co-incubated with human mesenchymal stem cells (MSCs) respectively to cross-border modify the mesenchymal stem cell exosomes (MSCs-exo). The mesenchymal stem cell exosomes X-ELNs-MSCs-exo and C-ELNs-MSCs-exo cross-border modified by tea exosome-like nanovesicles X-ELNs and C-ELNs were obtained respectively, and the two exosomes were quantified by protein content; (C)Measure the particle size, zeta potential and morphological characteristics of the obtained ELNs samples; (D)Perform lipidomics analysis on the obtained ELNs samples; (E)Perform protein extraction and proteomics determination on the obtained ELNs samples.

[0007] In some embodiments, before obtaining the ELNs sample (X-ELNs) in step (A), the fresh tea leaves (X) are processed. After weighing the fresh tea leaves (X), they are first washed with clean water, then put into 2 times the weight of cold PBS buffer at 4°C and homogenized with a tissue homogenizer at 20000 r / min for 2 min to make a tea slurry. After passing through a 300-mesh sieve, the tea juice is collected. After centrifugation at 1000 g for 10 min to take the supernatant, centrifugation at 5000 g for 20 min to take the supernatant, and centrifugation at 10000 g for 30 min to take the supernatant, then use a vertical flow ultrafiltration machine to ultrafilter through organic membranes with a pore size of 30 nm and 250 nm to separate out a suspension of fresh tea leaf nanoparticles with a particle size range of 30-250 nm. Then use an ultra-high speed and low temperature centrifuge to centrifuge at 100000 g for 1 h, discard the supernatant, suspend the precipitate in cold PBS buffer at 4°C, and then perform sucrose density gradient centrifugation for purification. Take the gradient band between 45% and 60% sucrose. Finally, wash with cold PBS at 4°C and centrifuge at 100000 g for 1 h with an ultra-high speed and low temperature centrifuge to remove the sucrose and impurities therein, and the fresh leaf ELNs sample (X-ELNs) is obtained.

[0008] In some embodiments, before obtaining the ELNs sample (C-ELNs) in step (A), the finished tea (C) is processed. The finished tea (C) is weighed and then frozen in a -80 °C low-temperature refrigerator for 2 hours, and then pulverized with a tissue pulverizer at 20000 r / min for 2 min. After passing through a 60-mesh sieve, the tea powder is stirred and extracted with 12 times its weight of PBS buffer at 4 °C for 24 hours. After the extract is filtered through a 300-mesh filter, the filtrate is centrifuged at 1000 g for 10 min to take the supernatant, centrifuged at 5000 g for 20 min to take the supernatant, and centrifuged at 10000 g for 30 min to take the supernatant. Then, it is ultrafiltered through organic membranes with pore sizes of 30 nm and 250 nm using a vertical flow ultrafiltration machine to separate out a suspension of tea finished tea nanoparticles with a particle size range of 30 - 250 nm. Then, it is centrifuged at 100000 g for 1 h using a high-speed refrigerated centrifuge, and the supernatant is discarded. The precipitate is suspended in cold PBS buffer at 4 °C, and then purified by sucrose density gradient centrifugation. The gradient band between 45% and 60% sucrose is taken. Finally, it is washed with cold PBS at 4 °C and centrifuged at 100000 g for 1 h using a high-speed low-temperature centrifuge to remove the sucrose and impurities therein, thus obtaining the finished tea ELNs sample (C-ELNs).

[0009] In some embodiments, in step (B), mesenchymal stem cells (MSCs) are prepared into a cell density of 2×10 5 cells / ml with a serum-free medium and inoculated in two batches of culture flasks. They are cultured at 37 °C and 5% CO2 saturated humidity for 24 hours. After discarding all the culture medium and washing the cells 3 times with PBS buffer, equal volumes of exosome-specific media containing 2 μg / mL X-ELNs and C-ELNs are added respectively, and they are continuously cultured at 37 °C and 5% CO2 saturated humidity for 48 hours. All the cell culture media are collected, centrifuged at 1000 g for 10 min to take the supernatant, centrifuged at 5000 g for 20 min to take the supernatant, and centrifuged at 10000 g for 30 min to take the supernatant. Then, it is ultrafiltered through organic membranes with pore sizes of 30 nm and 200 nm using a vertical flow ultrafiltration machine to separate out a mesenchymal stem cell exosome fluid with a particle size range of 30 - 200 nm. Then, it is centrifuged at 100000 g for 1 h using a high-speed low-temperature centrifuge, and the supernatant is discarded. The precipitate is suspended in cold PBS buffer at 4 °C, and then purified by sucrose density gradient centrifugation. The gradient band between 45% and 60% sucrose is taken. Finally, it is washed with cold PBS at 4 °C and centrifuged at 100000 g for 1 h using a high-speed low-temperature centrifuge to remove the sucrose and impurities therein, preparing mesenchymal stem cell exosomes X-ELNs-MSCs-exo and C-ELNs-MSCs-exo cross-transformed by tea exosome-like nanovesicles X-ELNs and C-ELNs. The BCA kit method is used to detect X-ELNs-MSCs-exo and C-ELNs-MSCs-exo, and a microplate reader is used to perform protein quantification at 405 nm.

[0010] In some embodiments, in step (C), the measurement method is as follows: Take 20 μg of the ELNs sample and dissolve it in 1 mL of PBS (pH 7.4). At 37 °C, use a dynamic light scattering instrument DLS to measure the average hydrated particle size, polydispersity index, and Zeta potential of X-ELNs and C-ELNs respectively; Drop the diluted X-ELNs and C-ELNs suspensions onto a copper mesh with an ultra-thin carbon film and a mica sheet, place them in an oven at 40 °C, and after the water is completely dried, observe the morphologies of the two types of ELNs under a high-resolution transmission electron microscope respectively.

[0011] In some embodiments, in step (D), the analysis method is as follows: Take 200 μL of the X-ELNs and C-ELNs samples respectively and add them to 800 μL of methanol and 600 μL of ddH2O, centrifuge for 10 min, take the supernatant and evaporate to dryness, add 400 μL of methanol / isopropanol 1:1 (v / v) to extract the supernatant, and analyze the lipid composition using a triple quadrupole mass spectrometer.

[0012] In some embodiments, in step (E), the analysis method is as follows: Separate the protein bands in X-ELNs and C-ELNs by SDS-PAGE electrophoresis, and use liquid chromatography-tandem mass spectrometry (LC-MS / MS) to identify and quantify the proteins.

[0013] The experimental application of tea-derived nanovesicles for rhinitis treatment to cross-transform mesenchymal stem cell exosomes includes: culturing, subculturing of mesenchymal stem cells (MSCs), observation experiments on morphology and growth characteristics, cytotoxicity experiments of X-ELNs and C-ELNs on MSCs, experiments on the effects of X-ELNs and C-ELNs on the proliferation and migration abilities of MSCs, experiments on the effects of X-ELNs and C-ELNs on the phenotypic characteristics of MSCs, experiments on the effects of X-ELNs and C-ELNs on the multi-differentiation abilities of MSCs, experiments on the effects of cross-border transport of X-ELNs and C-ELNs on mesenchymal stem cell exosomes (MSCs-exo), and experiments on the therapeutic effects of cross-transformed MSCs-exo (X-ELNs-MSCs-exo and C-ELNs-MSCs-exo) by X-ELNs and C-ELNs on allergic rhinitis.

[0014] By adopting the above technical solutions, the beneficial effects of the present invention are as follows: (1) Cross-transform human mesenchymal stem cell exosomes with plant-derived exosome-like nanovesicles, observe the therapeutic effects of the newly generated exosomes on allergic rhinitis model mice, and explore new methods for exosome therapy of allergic rhinitis; (2)The effects of co-incubating tea exosome-like nanovesicles with human mesenchymal stem cells on the ultrastructure, particle size, zeta potential, protein characterization, and chemical inclusions of human mesenchymal stem cell-derived exosomes (MSCs-exo) were observed, providing a basis for exploring the mechanism and biological effects of cross-kingdom transport of plant nanovesicles; (3)The cytotoxicity of tea exosome-like nanovesicles to human mesenchymal stem cells, as well as their effects on cell proliferation, migration, phenotype, osteogenic, and adipogenic differentiation abilities, were observed; (4)Fresh tea leaves and made tea were respectively used to prepare exosome-like nanovesicles (X-ELNs and C-ELNs), and the ultrastructure, particle size, zeta potential, lipidomics, proteomics, and tea inclusions of the two types of exosome-like nanovesicles were detected and comparatively analyzed, which is of positive significance for exploring the changes in the physicochemical structure and biological activity of tea extracellular vesicles during the processing process.

[0015] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and do not limit the present disclosure.

[0016] Undoubtedly, such objects of the present invention and other objects will become more apparent after the details of the preferred embodiments described below with multiple drawings and illustrations.

[0017] To make the above beneficial effects, other objects, features, and advantages of the present invention more obvious and understandable, one or several preferred embodiments are specifically given below, and in conjunction with the accompanying drawings, the detailed description is as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention, but do not constitute a limitation to the present invention.

[0019] In the drawings, the same components are denoted by the same reference numerals, and the drawings are schematic and not necessarily drawn to actual scale.

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only one or several embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on such drawings without creative efforts.

[0021] MAIN REFERENCE NUMERAL DESCRIPTIONS: Figure 1 It is a flow chart for the preparation of tea exosome-like nanovesicles of the present invention; Figure 2Flow chart of the effects of the tea ELNs of the present invention on the apoptosis, proliferation, migration and differentiation abilities of MSCs; Figure 3 Flow chart of the effects of the X-ELNs and C-ELNs of the present invention on human mesenchymal stem cell exosomes (MSCs-exo); Figure 4 Experimental flow chart of the therapeutic effect of ELNs-modified MSCs-exo (X-ELNs-MSCs-exo and C-ELNs-MSCs-exo) on allergic rhinitis; Figure 5 Characteristic diagram of the tea fresh tea exosome-like nanovesicles (X-ELNs) of the present invention under transmission electron microscopy; Figure 6 Characteristic diagram of the particle size distribution of the tea fresh tea exosome-like nanovesicles (X-ELNs) of the present invention; Figure 7 Characteristic diagram of the tea processed tea exosome-like nanovesicles (C-ELNs) of the present invention under transmission electron microscopy; Figure 8 Characteristic diagram of the particle size distribution of the tea processed tea exosome-like nanovesicles (C-ELNs) of the present invention. Detailed implementation mode

[0022] The following will combine the drawings and embodiments to detail the implementation mode of the present invention, so as to fully understand how the present invention uses technical means to solve technical problems and achieve the implementation process of technical effects and implement accordingly. It should be noted that as long as there is no conflict, each embodiment in the present invention and each feature in each embodiment can be combined with each other, and the formed technical solution is within the protection scope of the present invention.

[0023] At the same time, in the following description, many specific details are elaborated for the purpose of explanation to provide a thorough understanding of the embodiments of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without the specific details here or in the specific manner described.

[0024] Please refer to Figure 1-8 , the present invention provides a method for preparing cross-border modified mesenchymal stem cell exosomes with tea nanovesicles for treating rhinitis, including the following steps: (A) Extraction of tea exosome-like nanovesicles (ELNs): The tea is divided into fresh tea leaves (X) and processed tea (C), and two ELNs samples (X-ELNs and C-ELNs) are obtained from the fresh tea leaves (X) and processed tea (C) respectively; (B)Preparation and quantification of tea exosome-like nanovesicles cross-transformed mesenchymal stem cell exosomes (ELNs-MSCs-exo): The tea nanovesicles X-ELNs and C-ELNs were co-incubated with human mesenchymal stem cells (MSCs) respectively to cross-transform the exosomes of mesenchymal stem cells (MSCs-exo). The mesenchymal stem cell exosomes X-ELNs-MSCs-exo and C-ELNs-MSCs-exo cross-transformed by tea exosome-like nanovesicles X-ELNs and C-ELNs were obtained respectively, and the two exosomes were quantified by protein content; (C)Measure the particle size, zeta potential, and morphological characteristics of the obtained ELNs samples; (D)Perform lipidomics analysis on the obtained ELNs samples; (E)Extract proteins and perform proteomics determination on the obtained ELNs samples.

[0025] From the above, it can be known that ELNs were extracted and purified from fresh tea leaves (X) and made tea (C) of tea respectively to obtain X-ELNs and C-ELNs. And their ultrastructure, particle size, zeta potential, lipidomics, proteomics, and tea inclusions were detected and comparative analysis was made.

[0026] According to some embodiments of the present application, optionally, before obtaining the ELNs sample (X-ELNs) in step (A), the fresh tea leaves (X) were processed. After weighing the fresh tea leaves (X), they were first washed with clean water, then put into 4°C cold PBS buffer with 2 times the weight and homogenized with a tissue homogenizer at 20000 r / min for 2 min to make tea slurry. After passing through a 300-mesh sieve, the tea juice was collected. After centrifuging at 1000 g for 10 min to take the supernatant, centrifuging at 5000 g for 20 min to take the supernatant, and centrifuging at 10000 g for 30 min to take the supernatant, then using a vertical flow ultrafiltration machine to ultrafilter through organic membranes with pore sizes of 30 nm and 250 nm to separate out a suspension of tea fresh leaf nanoparticles with a particle size range of 30 - 250 nm. Then, it was centrifuged at 100000 g for 1 h using an ultra-high speed low-temperature centrifuge, the supernatant was discarded, the precipitate was suspended in 4°C cold PBS buffer, and then purified by sucrose density gradient centrifugation. The gradient band between 45% and 60% sucrose was taken, and finally, it was washed with 4°C cold PBS and centrifuged at 100000 g for 1 h using an ultra-high speed low-temperature centrifuge to remove the sucrose and impurities therein, thus obtaining the fresh leaf ELNs sample (X-ELNs).

[0027] According to some embodiments of the present application, optionally, before obtaining the ELNs sample (C-ELNs) in step (A), the finished tea (C) is processed. The finished tea (C) is weighed and frozen in a -80°C low-temperature refrigerator for 2 hours, and then pulverized with a tissue grinder at 20,000 r / min for 2 minutes. After passing through a 60-mesh sieve, the tea powder is stirred and extracted with 12 times its weight of PBS buffer at 4°C for 24 hours. After the extract is filtered through a 300-mesh filter, the filtrate is centrifuged at 1000 g for 10 minutes to take the supernatant, centrifuged at 5000 g for 20 minutes to take the supernatant, and centrifuged at 10,000 g for 30 minutes to take the supernatant. Then, it is ultrafiltered through organic membranes with pore sizes of 30 nm and 250 nm using a vertical flow ultrafiltration machine to separate out a suspension of tea finished tea nanoparticles with a particle size range of 30 - 250 nm. Then, it is centrifuged at 100,000 g for 1 hour using a super-high-speed refrigerated centrifuge, and the supernatant is discarded. The precipitate is suspended in cold PBS buffer at 4°C, and then purified by sucrose density gradient centrifugation. The gradient band between 45% and 60% sucrose is taken. Finally, it is washed with cold PBS at 4°C and centrifuged at 100,000 g for 1 hour using a super-high-speed low-temperature centrifuge to remove the sucrose and impurities therein, thus obtaining the finished tea ELNs sample (C-ELNs).

[0028] According to some embodiments of the present application, optionally, in step (B), the mesenchymal stem cells (MSCs) are formulated into 2×10 5Cells were inoculated in two batches of culture flasks at a cell density of

[0029] According to some embodiments of the present application, optionally, in step (C), the measurement method is as follows: Dissolve 20 μg of ELNs sample in 1 mL of PBS (pH 7.4), and use a dynamic light scattering instrument DLS to measure the average hydrated particle size, polydispersity index, and Zeta potential of X-ELNs and C-ELNs at 37°C; Drop the diluted X-ELNs and C-ELNs suspensions onto a copper mesh with an ultra-thin carbon film and a mica sheet, place them in an oven at 40°C, and observe the morphologies of the two types of ELNs under a high-resolution transmission electron microscope after the water is completely dried. Add X-ELNs and C-ELNs to the special medium for MSCs exosomes respectively, and co-incubate with MSCs for 48 hours. Compare with MSCs in the medium without adding X-ELNs and C-ELNs, and observe the changes in the ultrastructure, particle size, potential, protein characterization, and inclusions of the newly formed exosomes of MSCs modified by X-ELNs (X-ELNs-MSCs-exo) and the newly formed exosomes of MSCs modified by C-ELNs (C-ELNs-MSCs-exo).

[0030] According to some embodiments of the present application, optionally, in step (D), the analysis method is as follows: Add 200 μL of X-ELNs and C-ELNs samples to 800 μL of methanol and 600 μL of ddH2O respectively, centrifuge for 10 min, take the supernatant and evaporate to dryness, add 400 μL of methanol / isopropanol 1:1 (v / v) to extract the supernatant, and analyze the lipid composition using a triple quadrupole mass spectrometer.

[0031] According to some embodiments of the present application, optionally, in step (E), the analysis method is as follows: Separate the protein bands in X-ELNs and C-ELNs by SDS-PAGE electrophoresis, and use liquid chromatography-tandem mass spectrometry (LC-MS / MS) to identify and quantify the proteins.

[0032] In this example: (1) By comparing and observing the differences in the ultrastructure, particle size, potential, lipidomics, proteomics, and tea inclusions of exosome-like nanovesicles X-ELNs and C-ELNs derived from fresh tea leaves (X) and made tea (C), understand the effects of the tea processing process on the physicochemical structure and biological activity of extracellular vesicles of fresh tea leaves.

[0033] (2) Observe the cytotoxic effects of different doses of X-ELNs and C-ELNs on MSCs, and observe the effects of the two on the cell proliferation, migration, phenotype, osteogenic differentiation, and adipogenic differentiation abilities of MSCs at safe doses.

[0034] (3) By comparing the effects of X-ELNs and C-ELNs on the ultrastructure, particle size, potential, protein characterization and content changes of newly formed exosomes from MSCs, the cross-border transformation effect of tea ELNs from different sources on the extracellular vesicle characteristics of MSCs was explored.

[0035] In this example, the tea used, Tieguanyin, is a semi-fermented tea. The primary production process includes ten steps: sun-drying, air-drying, shaking, stir-frying, rolling, initial baking, wrapping and rolling, re-baking, re-wrapping and rolling, and drying. The shaking and repeated rolling before drying can cause a large number of cell wall disruptions, promoting microbial fermentation and enzymatic oxidation reactions, which are extremely beneficial for the formation of nanovesicles. Comparative studies of the differences in the content of ELNs between fresh Tieguanyin leaves and finished tea leaves have both scientific significance and economic value for exploring the formation mechanism of tea ELNs, and are also feasible.

[0036] Experimental application of tea nanovesicles cross-border transformation of mesenchymal stem cell exosomes for the treatment of rhinitis, including: experiments on MSCs (mesenchymal stem cells) culture, passaging, and observation of morphological and growth characteristics, experiments on the cytotoxicity of X-ELNs and C-ELNs on MSCs, experiments on the effects of X-ELNs and C-ELNs on the proliferation and migration ability of MSCs, experiments on the effects of X-ELNs and C-ELNs on the phenotypic characteristics of MSCs, experiments on the effects of X-ELNs and C-ELNs on the multiple differentiation capabilities of MSCs, experiments on the effects of X-ELNs and C-ELNs cross-border transport on MSCs exosomes (MSCs-exo), and experiments on the therapeutic effects of X-ELNs and C-ELNs cross-border transformation of MSCs-exo (X-ELNs-MSCs-exo and C-ELNs-MSCs-exo) on allergic rhinitis.

[0037] ①MSCs (mesenchymal stem cells) culture, passage, and morphology and growth characteristics observation experiment: (1) Culture and passaging: When the cells have grown to more than 80% to 90% of the bottom of the culture dish, remove the culture medium, wash the cells with PBS, and digest the adherent cells with 0.25% trypsin. Culture the cells with MSCs serum-free medium, terminate the digestion with aprotinin, pass the cells at a ratio of 1:2, and add new MDCs serum-free medium. Continue culturing at 37°C and 5% CO2 saturated humidity. Observe the cell adhesion growth every day and change the medium every other day.

[0038] (2) Observe morphological characteristics: Observe the morphology, size and growth characteristics of cells under an inverted microscope, including whether they are spindle-shaped, whether they grow adherently to the wall, whether they grow in a whirlpool shape, and whether there are many filopodia around the cells.

[0039] ② Cytotoxicity experiments of X-ELNs and C-ELNs on MSCs: MSCs were plated at 1x104 The cells were inoculated at a density of [cells / well] in a 96-well plate and cultured overnight at 37 °C under 5% CO₂ and saturated humidity conditions. Then, the culture wells were divided into three groups, and serum-free medium for MSCs, PBS buffer with a volume ratio of 4:1, X-ELNs suspensions and C-ELNs suspensions with different concentrations (protein concentrations: 0.5, 1, 2, 4, 8 μg / mL) were added respectively. After continuous culture for 48 h, the medium was removed, and after washing 3 times with PBS buffer: ① MTT and DMSO were added successively for incubation, and then the OD value at 570 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader. The cells with PBS added were used as negative controls, and the cells treated with Triton X-100 (0.5%, v / v) were used as positive controls to detect the cell viability; ② The cells were digested with trypsin and collected by centrifugation at 1000 g for 5 min, and then the cells were suspended in Annexin V binding buffer, stained with Annexin V-FITC / propidium iodide (PI), and the apoptosis of the cells was detected using a flow cytometer.

[0040] ③ Experiment on the effects of X-ELNs and C-ELNs on the proliferation and migration abilities of MSCs: (1)Proliferation ability of MSCs: Adjust the cell concentration to 5×10 6 ⁵ cells / mL, inoculate on a 24-well plate, randomly divide the culture wells into three groups, and add PBS buffer with a volume ratio of 4:1, X-ELNs (protein concentration: 2 μg / mL) and C-ELNs (protein concentration: 2 μg / mL) to the serum-free medium for MSCs in each group respectively, and culture under the conditions of 37 °C, 5% CO₂ and saturated humidity. Digest and collect the cells in 3 wells every day, count 3 times, and finally count the average value. Continuously culture for 8 d according to this method, record the daily data. Use the culture time as the abscissa and the number of cells as the ordinate to draw a cell growth curve, and compare and observe the changes in the proliferation ability of MSCs.

[0041] (2)Migration ability of MSCs (cell scratch assay): MSCs were inoculated into a 6-well plate. After the cells adhered and grew confluently, a sterile pipette tip was used to draw a vertical line at the bottom of the six-well plate. After washing with PBS buffer, add the serum-free medium for MSCs, PBS buffer with a volume ratio of 4:1, medium containing X-ELNs (protein concentration: 2 μg / mL) and C-ELNs (protein concentration: 2 μg / mL), and continue to culture under the conditions of 37 °C, 5% CO₂ and saturated humidity. Take pictures at 0, 12, and 24 h respectively to observe the healing of the scratch.

[0042] ④ Experiment on the effects of X-ELNs and C-ELNs on the phenotypic characteristics of MSCs: The serum-free medium for MSCs was divided into three groups, and the serum-free medium for MSCs, PBS buffer with a volume ratio of 4:1, X-ELNs (protein concentration: 2 μg / mL), and C-ELNs (protein concentration: 2 μg / mL) were added respectively to prepare media containing PBS, X-ELNs, and C-ELNs. MSCs were inoculated into three groups of 6-well plates. When the cells adhered and grew to about 70%, the media were replaced with those containing PBS, X-ELNs, and C-ELNs respectively, and they were continuously cultured for 48 hours under the conditions of 37 °C and 5% CO₂ saturated humidity. The cells were collected and counted. Each group of cells was equally divided into five test tubes, and monoclonal antibodies against human CD73, CD105, CD34, CD45, and CD90 labeled with fluorescein were added respectively, and flow cytometry was used for phenotype detection.

[0043] ⑤ Experiment on the effects of X-ELNs and C-ELNs on the multi-differentiation ability of MSCs: The serum-free medium for MSCs was divided into three groups, and 1 / 4 volume of PBS, X-ELNs (protein concentration: 2 μg / mL), and C-ELNs (protein concentration: 2 μg / mL) were added respectively to prepare media containing PBS, X-ELNs, and C-ELNs. MSCs were inoculated into three groups of 6-well plates. When the cells adhered and grew to about 70%, the media were replaced with those containing PBS, X-ELNs, and C-ELNs respectively, and after continuous culture for 48 hours at 37 °C and 5% CO₂ saturated humidity, the following experiments were carried out: (1) Adipogenic induction differentiation: The culture media of the three groups of 6-well plates were replaced, and 10% FBS, 1 μmol / L dexamethasone, 10 mg / L insulin, 0.5 mmol / L 3-isobutyl-1-methylxanthine (IBM), and 0.2 mmol / L indomethacin were added to the DMEM / F12 culture medium for adipogenic induction. Morphological observation was continuously carried out for 2 - 3 weeks. After lipid droplets were formed, oil red O staining and hematoxylin staining were carried out, and observation and photography were carried out under an inverted microscope.

[0044] (2) Osteogenic induction differentiation: The culture media of the three groups of 6-well plates were replaced, and 10% FBS, 0.1 μmol / L dexamethasone, 50 μmol / L ascorbic acid, and 10 mmol / L β-glycerophosphate were added to the high-glucose DMEM culture medium for osteogenic induction. Morphological observation was continuously carried out for 1 - 2 weeks. After mineralized nodules were formed, alizarin red staining was carried out, and observation and photography were carried out under an inverted microscope.

[0045] ⑥ Experiment on the effects of cross-border transport of X-ELNs and C-ELNs on MSCs-derived exosomes (MSCs-exo): (1) Transmission electron microscopy was used to detect the morphology of three kinds of MSCs-derived exosomes; (2) The three types of MSC exosomes were isolated and purified, diluted with PBS, and then dripped onto a sample-loaded copper grid. The cells were negatively stained with 3% (wlv) sodium phosphotungstate solution (pH 6.8) at room temperature and allowed to dry. The cells were then observed and photographed using a transmission electron microscope. Twenty exosome vesicles were randomly selected and their diameters were measured.

[0046] ⑦ Experiments on the therapeutic effects of X-ELNs and C-ELNs cross-conversion into MSCs-exo (X-ELNs-MSCs-exo and C-ELNs-MSCs-exo) on allergic rhinitis included the following steps: (1) Female Balb / c mice aged 6-8 weeks were raised in an SPF animal room. OVA-AL(OH)3 sensitizer was injected into the peritoneal cavity of the mice in the first and third weeks, and saline was injected into the control group. From the fourth week onwards, 5% OVA solution was used for nasal stimulation every day for one week. The control group was given saline for nasal drops. The scratching and sneezing behaviors of the mice were observed and counted, and scored according to the behavioral scoring standard of the rhinitis model. A score of 5 or above indicated that the model was successfully established. (2) Model mice with allergic rhinitis successfully induced by OVA were randomly divided into model group, MSCs-exo group, X-ELNs-MSCs-exo group, and C-ELNs-MSCs-exo group, with 8 mice in each group; (3) Administration method and result detection: Normal saline, MSCs-exo, X-ELNs-MSCs-exo and C-ELNs-MSCs-exo were injected through the tail vein, once every three days, for 5 consecutive times; 5% OVA solution was administered intranasally every day for stimulation. On the 13th to 15th day, the mice were observed and counted for three consecutive days for behavioral analysis. On the 16th day, the mice were killed, nasal lavage was performed, blood was collected from the heart, and spleen, lung and head specimens were fixed in 4% paraformaldehyde for subsequent experiments. In the control group, 8 normal mice were injected with normal saline and administered intranasal saline every day; (4) Count the number of times the mice scratched their noses and sneezed after nasal drop stimulation; (5) Detect mucosal congestion and edema, mucosal thickness, immune cell infiltration, and changes in tissue structure; (6) Detection of cell exudation; (7) Detect the mRNA levels of IL-4, IL-10, Tbet, Gata3, and FoxP3; (8) Mice were anesthetized with 6% chloral hydrate, and blood was collected from the peritoneal vein. The immunoglobulin enzyme-linked immunosorbent assay kit was used according to the instructions to complete the experimental steps, and the absorbance at a wavelength of 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0047] It should be understood that the embodiments disclosed in the present invention are not limited to the specific processing steps or materials disclosed herein, but should extend to equivalent alternatives of such features understood by those of ordinary skill in the relevant art. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and do not imply limitation.

[0048] As used herein, the term "embodiment" means that a particular feature or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the phrase "an embodiment" or "embodiments" that appear throughout the specification are not necessarily all referring to the same embodiment.

[0049] In addition, the described features or characteristics may be combined in any other suitable manner in one or more embodiments. In the above description, some specific details, such as thickness, quantity, etc., are provided to provide a comprehensive understanding of the embodiments of the present invention. However, those skilled in the relevant art will understand that the present invention can be implemented without one or more of the above specific details or can also be implemented using other methods, components, materials, etc.

Claims

1. Preparation method of cross-border modified mesenchymal stem cell exosomes with tea nano vesicles for rhinitis treatment, characterized in that, Including the following steps: (A) Extraction of tea exosome-like nanovesicles (ELNs): The tea is divided into fresh tea leaves (X) and finished tea (C), and two ELNs samples (X-ELNs and C-ELNs) are obtained from fresh tea leaves (X) and finished tea (C) respectively; (B) Preparation and quantification of tea exosome-like nanovesicle cross-border modified mesenchymal stem cell exosomes (ELNs-MSCs-exo): The tea nanovesicles X-ELNs and C-ELNs are co-incubated with human mesenchymal stem cells (MSCs) respectively to cross-border modify the exosomes of mesenchymal stem cells (MSCs-exo). The mesenchymal stem cell exosomes X-ELNs-MSCs-exo and C-ELNs-MSCs-exo cross-border modified by tea exosome-like nanovesicles X-ELNs and C-ELNs are obtained respectively, and the two exosomes are quantified by protein content; (C) Measuring the particle size, zeta potential and morphological characteristics of the obtained ELNs samples; (D) Lipidomics analysis of the obtained ELNs samples; (E) Protein extraction and proteomic determination of the obtained ELNs samples.

2. The preparation method of the cross-border transformed mesenchymal stem cell exosomes using tea leaf nanovesicles for rhinitis treatment according to claim 1, wherein Before obtaining the ELNs sample (X-ELNs) in step (A), the fresh tea leaves (X) are processed. After weighing the fresh tea leaves (X), they are first washed with clean water, then put into 2 times the weight of cold PBS buffer at 4°C and homogenized with a tissue homogenizer at 20000 r / min for 2 min to make a tea slurry. After passing through a 300-mesh sieve, the tea juice is collected. The supernatant is taken by centrifugation at 1000 g for 10 min, 5000 g for 20 min, and 10000 g for 30 min respectively. Then, it is ultrafiltered through organic membranes with pore sizes of 30 nm and 250 nm using a vertical flow ultrafiltration machine to separate out a suspension of fresh tea leaf nanoparticles with a particle size range of 30 - 250 nm. Then, it is centrifuged at 100000 g for 1 h using a ultra-high speed and low temperature centrifuge, and the supernatant is discarded. The precipitate is suspended in cold PBS buffer at 4°C, and then purified by sucrose density gradient centrifugation. The gradient band between 45% and 60% sucrose is taken. Finally, it is washed with cold PBS at 4°C and centrifuged at 100000 g for 1 h using a ultra-high speed and low temperature centrifuge to remove the sucrose and impurities, and the fresh leaf ELNs sample (X-ELNs) is obtained.

3. The preparation method of cross-border modified mesenchymal stem cell exosomes with tea leaf nanovesicles for rhinitis treatment according to claim 1, wherein, Before obtaining the ELNs sample (C-ELNs) in step (A), the finished tea (C) was processed. The finished tea (C) was weighed and then frozen in a -80 °C low-temperature refrigerator for 2 hours, and then pulverized with a tissue grinder at 20,000 r / min for 2 min. After passing through a 60-mesh sieve, the tea powder was stirred and extracted with 12 times its weight of PBS buffer at 4 °C for 24 hours. After the extraction solution was filtered through a 300-mesh filter, the filtrate was centrifuged at 1000 g for 10 min to obtain the supernatant, centrifuged at 5000 g for 20 min to obtain the supernatant, and centrifuged at 10,000 g for 30 min to obtain the supernatant. Then, it was ultrafiltered through organic membranes with pore sizes of 30 nm and 250 nm using a vertical flow ultrafiltration machine to separate out a suspension of tea finished tea nanoparticles with a particle size range of 30 - 250 nm. Then, it was centrifuged at 100,000 g for 1 h using an ultra-high-speed refrigerated centrifuge, and the supernatant was discarded. The precipitate was suspended in cold PBS buffer at 4 °C, and then purified by sucrose density gradient centrifugation. The gradient band between 45% and 60% sucrose was taken, and finally, it was washed with cold PBS at 4 °C and centrifuged at 100,000 g for 1 h using an ultra-high-speed low-temperature centrifuge to remove the sucrose and impurities therein, thus obtaining the finished tea ELNs sample (C-ELNs).

4. The preparation method of the tea leaf nanovesicle cross-border modified mesenchymal stem cell exosomes for rhinitis treatment according to claim 1, characterized in that, In step (B), mesenchymal stem cells (MSCs) were prepared with a serum-free medium at a cell density of 2×10 5 cells / ml and inoculated into two batches of culture flasks, and cultured at 37°C and 5% CO2 saturated humidity for 24 hours. After discarding all the culture medium and washing the cells 3 times with PBS buffer, equal volumes of exosome-specific media containing 2 μg / mL X-ELNs and C-ELNs were added respectively, and continuously cultured at 37°C and 5% CO2 saturated humidity for 48 hours. All the cell culture media were collected, and the supernatants were taken after centrifugation at 1000g for 10 min, 5000g for 20 min, and 10000g for 30 min respectively. Then, using a vertical flow ultrafiltration machine, ultrafiltration was performed through organic membranes with pore sizes of 30 nm and 200 nm to separate mesenchymal stem cell exosome fluids with a particle size range of 30 - 200 nm. Then, using an ultra-high-speed low-temperature centrifuge, centrifugation was carried out at 100000g for 1 h, the supernatant was discarded, and the precipitate was suspended in cold PBS buffer at 4°C. Then, sucrose density gradient centrifugation was performed for purification, and the gradient band between 45% and 60% sucrose was taken. Finally, washing with cold PBS at 4°C and centrifugation at 100000g for 1 h using an ultra-high-speed low-temperature centrifuge were used to remove the sucrose and impurities therein, and mesenchymal stem cell exosomes X-ELNs-MSCs-exo and C-ELNs-MSCs-exo cross-transformed by tea exosome-like nanovesicles X-ELNs and C-ELNs were prepared. The BCA kit method was used to detect X-ELNs-MSCs-exo and C-ELNs-MSCs-exo, and an enzyme-linked immunosorbent assay (ELISA) reader was used to perform protein quantification at 405 nm.

5. The preparation method of the cross-border modified mesenchymal stem cell exosomes using tea nano vesicles for rhinitis treatment according to claim 1, wherein In step (C), the measurement method was as follows: 20 μg of the ELNs sample was dissolved in 1 mL of PBS (pH 7.4), and the average hydrodynamic diameter, polydispersity index, and Zeta potential of X-ELNs and C-ELNs were measured using a dynamic light scattering instrument (DLS) at 37 °C. The diluted X-ELNs and C-ELNs suspensions were dropped onto a ultra-thin carbon film copper grid and a mica sheet, and placed in a 40 °C oven. After the water was completely dried, the morphologies of the two kinds of ELNs were observed under a high-resolution transmission electron microscope respectively.

6. The preparation method of the cross-border transformed mesenchymal stem cell exosomes with tea leaf nanovesicles for rhinitis treatment according to claim 1, wherein, In step (D), the analysis method was as follows: 200 μL of the X-ELNs and C-ELNs samples were respectively added to 800 μL of methanol and 600 μL of ddH2O, centrifuged for 10 min to obtain the supernatant and evaporated to dryness. 400 μL of methanol / isopropanol 1:1 (v / v) was added to extract the supernatant, and the lipid composition was analyzed using a triple quadrupole mass spectrometer.

7. The preparation method of the cross-border transformed mesenchymal stem cell exosomes of the tea leaf nanovesicles for rhinitis treatment according to claim 1, characterized in that, In step (E), the analysis method was as follows: The protein bands in X-ELNs and C-ELNs were separated by SDS-PAGE electrophoresis, and the proteins were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

8. Experimental application of mesenchymal stem cell exosomes prepared by the method according to any one of claims 1-7, characterized in that Including: Experiments on the culture, passage, morphological and growth characteristics observation of mesenchymal stem cells (MSCs), cytotoxicity experiments of X-ELNs and C-ELNs on MSCs, experiments on the effects of X-ELNs and C-ELNs on the proliferation and migration abilities of MSCs, experiments on the effects of X-ELNs and C-ELNs on the phenotypic characteristics of MSCs, experiments on the effects of X-ELNs and C-ELNs on the multi-differentiation ability of MSCs, experiments on the effects of cross-border transport of X-ELNs and C-ELNs on MSCs exosomes (MSCs-exo), experiments on the therapeutic effects of cross-border modified MSCs-exo (X-ELNs-MSCs-exo and C-ELNs-MSCs-exo) of X-ELNs and C-ELNs on allergic rhinitis.

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