High VEGF protein / miR-210 extracellular vesicles induced by serum-free combined culture medium and application thereof

Abstract

The invention relates to the technical field of biological medicines, and provides a high VEGF protein / miR-210 extracellular vesicle induced by a serum-free combined culture medium and application of the high VEGF protein / miR-210 extracellular vesicle. According to the invention, high VEGF protein / miR-210 extracellular vesicles are obtained based on induction of a serum-free culture medium; the serum-free culture medium comprises a basic culture medium component combination: sugar-free RPMI1640 and MCDB131 in a volume ratio of 4: 1; the metabolic regulation additive is prepared by synergistically adding sodium pyruvate, cysteine and a proline hydroxylase inhibitor and taking alanine glutamine as an energy substitute; the growth factor is a combination of polyvinyl alcohol, 4-hydroxyethylpiperazine ethanesulfonic acid, ITS-X, epidermal growth factors and basic fibroblast growth factors, wherein the ITS-X, the epidermal growth factors and the basic fibroblast growth factors are added before use. CD63 and CD81 are specifically expressed on the surface of the prepared high VEGF protein / miR-210 extracellular vesicle, the high VEGF protein / miR-210 extracellular vesicle is rich in VEGF protein and miR-210, and the high VEGF protein / miR-210 extracellular vesicle is applied to targeted therapy of allergic rhinitis by inhibiting Th2 type immunoreaction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to extracellular vesicles with high VEGF protein / miR-210 induced by serum-free combined culture medium and their applications. Background Technology

[0002] Allergic rhinitis is a prevalent chronic inflammatory disease of the upper respiratory tract worldwide, clinically manifested as paroxysmal sneezing, runny nose, nasal congestion, and nasal itching. Allergic rhinitis not only significantly reduces patients' quality of life and work efficiency but can also trigger complications such as asthma, resulting in a heavy socioeconomic burden. Current treatment options primarily focus on avoiding allergen exposure and symptomatic drug therapy; however, long-term use of corticosteroids and antihistamines has significant side effects, and allergen-specific immunotherapy is limited by the complexity of individual allergens, restricting its clinical application. Therefore, developing safe and effective new treatment strategies has significant clinical value.

[0003] Mesenchymal stem cells (MSCs) have become a research hotspot in the treatment of allergic rhinitis due to their significant immunomodulatory functions. Their mechanism of action mainly involves the secretion of soluble factors (such as TGF-β1, IL-10, and PGE2) and extracellular vesicles (EVs), which can inhibit the activity of T / B lymphocytes, dendritic cells, and natural killer cells, thus regulating the balance between innate and adaptive immunity. Studies have shown that extracellular secretions such as exosomes derived from MSCs have exhibited therapeutic effects comparable to those of MSCs in various animal models. However, long-term culture of MSCs in traditional two-dimensional expansion systems is prone to problems such as decreased differentiation potential and altered immunomodulatory properties, limiting their clinical translation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides extracellular vesicles with high VEGF protein / miR-210 induced by serum-free combined culture medium and their applications, with the aim of solving the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides a method for preparing extracellular vesicles with high VEGF protein / miR-210 induced by serum-free combined culture medium, comprising the following steps: Step 1: Under aseptic conditions, Wharton's jelly tissue from the umbilical cord of a full-term fetus is obtained, cut into tissue blocks, placed in a culture flask, and then cultured in α-MEM complete medium containing penicillin, streptomycin and fetal bovine serum. When the cells reach 80%-90% confluence, human umbilical cord mesenchymal stem cells (primary) are obtained. Step 2: When the human umbilical cord mesenchymal stem cells (HMCs) have expanded to 80%-90%, discard the tissue block and culture medium, wash the HMCs, add 0.25% trypsin solution for digestion, and when the HMCs can detach, add digestion stop solution containing 1% fetal bovine serum to terminate digestion; resuspend the HMCs to obtain a cell suspension, centrifuge and discard the supernatant, add serum-containing culture medium for resuspending, and culture to obtain HMCs highly expressing VEGF / miR-210; change the culture flask and continue passage culture in the same way to continuously expand the cells until the HMCs have undergone the 5th passage culture; Step 3: When the confluence of human umbilical cord mesenchymal stem cells with high expression of VEGF / miR-210 reaches 80%-90%, discard the serum-containing culture medium, wash with phosphate buffer, add serum-free combined culture medium, culture, collect the supernatant, and extract extracellular vesicles with high VEGF protein / miR-210 by high-speed centrifugation. The serum-free combined culture medium included: 500 mL of mixed culture medium, which was a mixture of sugar-free RPMI 1640 medium and MCDB131 medium at a volume ratio of 4:1; 1 g of polyvinyl alcohol; 2.383 g of 4-hydroxyethylpiperazine ethanesulfonic acid; 25 μM roxadustat; 1 mM sodium pyruvate; 0.1 mM cysteine; 2 mM alanine glutamine; 1% ITS-X; 20 ng / mL epidermal growth factor; and 2 ng / mL basic fibroblast growth factor.

[0006] Furthermore, the serum-containing culture medium includes: 500 mL of mixed culture medium; 1 g of polyvinyl alcohol; 2.383 g of 4-hydroxyethylpiperazine ethanesulfonic acid; 25 μM roxadustat; 1 mM sodium pyruvate; 0.1 mM cysteine; 2 mM alanine glutamine; 10% fetal bovine serum; 20 ng / mL epidermal growth factor; and 2 ng / mL basic fibroblast growth factor.

[0007] Furthermore, the specific steps are as follows: Step S1: Under aseptic conditions, residual blood cells in the umbilical cord of a full-term fetus were removed using phosphate-buffered saline. The umbilical cord was then cut into fragments, blood vessels were removed, Wharton's jelly tissue was extracted, and the fragments were cut into tissue blocks. These blocks were then placed in culture flasks and α-MEM complete medium containing 100 U / mL penicillin, 100 mg / L streptomycin, and 10% fetal bovine serum were added. The flasks were then incubated at 37°C in a 5% CO2 incubator. The α-MEM complete medium included α-MEM liquid medium, L-glutamine, sodium pyruvate, β-mercaptoethanol, and epidermal growth factor. On day 3, half of the medium was replaced, and the medium was replaced again after one week. Thereafter, the medium was replaced every 3 days. The cell growth around the adhesion of the tissue blocks was observed. When the cells reached 80%-90% confluence, human umbilical cord mesenchymal stem cells (primary) were obtained. Step S2: When the human umbilical cord mesenchymal stem cells (HMCs) have expanded to 80%-90%, gently tap the side wall of the culture flask to detach the adherent tissue block. Discard the tissue block and α-MEM complete culture medium. Wash the HMCs with phosphate-buffered saline (PBS) and add 0.25% trypsin solution. Place the flask in a culture dish and incubate at 37°C for digestion. Once the HMCs can be gently shaken off the bottom of the culture dish, add a digestion stop solution containing 1% fetal bovine serum (FBS) to terminate the digestion. Slowly pipette and resuspend the HMCs to obtain a cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in serum-containing culture medium. Seed the cell suspension in culture dishes at a 1:3 ratio. Incubate the culture dishes at 37°C with 5% FBS. Human umbilical cord mesenchymal stem cells (HMCs) highly expressing VEGF / miR-210 were obtained by culturing in a CO2 incubator. The culture flasks were then replaced, and the cells were passaged in the same manner to continuously expand the cell count until the HMCs underwent their fifth passage. The digested HMCs were cultured at a density of 3000-6000 cells / cm³. 2 To carry out generational breeding; Step S3: When the confluence of human umbilical cord mesenchymal stem cells with high expression of VEGF / miR-210 reaches 80%-90%, discard the serum-containing culture medium supernatant, wash with phosphate buffer, add serum-free combined culture medium, and culture at 37℃ and 5% CO2 for 24-48h. Collect the supernatant and extract extracellular vesicles with high VEGF protein / miR-210 by high-speed centrifugation.

[0008] Furthermore, the method for preparing serum-containing culture medium includes the following steps: Step S201: Sugar-free RPMI 1640 medium and MCDB131 medium are mixed at a volume ratio of 4:1 to obtain a mixed medium; Step S202: Weigh 1g of polyvinyl alcohol and 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid and add them to 500mL of mixed culture medium to dissolve. After the polyvinyl alcohol and 4-hydroxyethylpiperazine ethanesulfonic acid are completely dissolved, add 25μM roxadustat, 1mM sodium pyruvate, 0.1mM cysteine ​​and 2mM alanine glutamine in sequence. Step S203: After filtration and sterilization, store at 4°C; before use, add 10% fetal bovine serum, 20 ng / mL epidermal growth factor, and 2 ng / mL basic fibroblast growth factor.

[0009] Furthermore, the method for preparing the serum-free combined culture medium includes the following steps: Step S301: Sugar-free RPMI 1640 medium and MCDB131 medium are mixed at a volume ratio of 4:1 to obtain a mixed medium; Step S302: Weigh 1g of polyvinyl alcohol and 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid and add them to 500mL of mixed culture medium to dissolve. After the polyvinyl alcohol and 4-hydroxyethylpiperazine ethanesulfonic acid are completely dissolved, add 25μM roxadustat, 1mM sodium pyruvate, 0.1mM cysteine ​​and 2mM alanine glutamine in sequence. Step S303: After filtration and sterilization, store at 4°C; before use, add 1% ITS-X, 20 ng / mL epidermal growth factor and 2 ng / mL basic fibroblast growth factor by volume.

[0010] Further, in step three, extracellular vesicles with high VEGF protein / miR-210 were extracted by high-speed centrifugation. The specific steps are as follows: the collected supernatant was centrifuged at 1000g for 10 min to obtain supernatant I; then supernatant I was centrifuged at 12000g for 20 min at 4℃ to obtain supernatant II; then supernatant II was centrifuged at 12000g for 40 min at 4℃ to obtain supernatant III; supernatant III was filtered through a 0.22μm filter to obtain supernatant IV; finally, supernatant IV was centrifuged at 100000g for 1 h at 4℃ to obtain extracellular vesicles with high VEGF protein / miR-210.

[0011] Secondly, the present invention provides extracellular vesicles with high VEGF protein / miR-210 induced by serum-free combined culture medium, which are prepared by a method for preparing extracellular vesicles with high VEGF protein / miR-210 induced by serum-free combined culture medium.

[0012] Furthermore, extracellular vesicles with high VEGF protein / miR-210 expression specifically express CD63 and CD81 on their surface and are rich in VEGF protein and miR-210.

[0013] Thirdly, the present invention provides the application of serum-free combined culture medium-induced high VEGF protein / miR-210 extracellular vesicles in the preparation of drugs for treating allergic rhinitis.

[0014] Furthermore, the drug treats allergic rhinitis by suppressing the Th2 immune response; The preferred dosage form of the drug is an injection, nasal drops, or nebulized inhaler.

[0015] The present invention has the following beneficial effects: (1) High VEGF protein / miR-210 extracellular vesicles were obtained by induction using serum-free culture medium. The serum-free culture medium included the following components: a 4:1 volume ratio of sugar-free RPMI1640 and MCDB131; metabolic regulators: sodium pyruvate, cysteine, and proline hydroxylase inhibitor (roxadustat) were added synergistically, with alanine glutamine as an energy substitute; stabilizers and growth factors: a combination of polyvinyl alcohol, 4-hydroxyethylpiperazine ethanesulfonic acid, and ITS-X, epidermal growth factor, and basic fibroblast growth factor added before use. This serum-free culture medium significantly reduced ROS accumulation by precisely regulating cellular metabolic pathways, maintaining the stemness and immunomodulatory function of human umbilical cord mesenchymal stem cells, and is a specific serum-free system.

[0016] (2) The high VEGF protein / miR-210 extracellular vesicles prepared specifically express CD63 and CD81 on their surface and are rich in VEGF protein and miR-210. By inhibiting the Th2 immune response, they can be applied to the targeted treatment of allergic rhinitis. Attached Figure Description

[0017] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 This is a comparison diagram of the cell morphology of HE-hUCMSCs in Example 2 of the present invention and Con-hUCMSCs in Comparative Example 1.

[0018] Figure 2 This is a comparison diagram of the cell proliferation capacity of HE-hUCMSCs from Example 2 of the present invention and Con-hUCMSCs from Comparative Example 1, wherein: Figure 2 In this context, A represents a comparison of the total cell count from generation P1 to generation P5; Figure 2 In the figure, B represents the total number of cells in generation P5, and *** indicates P < 0.001.

[0019] Figure 3This is a comparison of the expression levels of stemness marker genes (SOX2 and Nanog) of HE-hUCMSC in Example 2 of the present invention and Con-hUCMSC in Comparative Example 1. *** indicates P < 0.001.

[0020] Figure 4 These are typical images obtained by Western blotting of extracellular vesicle marker proteins (CD9, CD63, CD81) of HE-EVs in Example 2 and Con-EVs in Comparative Example 1.

[0021] Figure 5 This is a comparison of electron microscopy identification, extracellular vesicle yield, extracellular vesicle protein concentration, VEGF protein expression level, and miR-210 expression level between HE-EVs of Example 2 and Con-EVs of Comparative Example 1. *** indicates P < 0.001. Figure 5 In this context, A represents the electron microscopy result. Figure 5 In this context, B represents the statistical results of extracellular vesicle production. Figure 5 In this context, C represents the statistical result of extracellular vesicle protein concentration. Figure 5 D in the image is a typical picture obtained by Western blotting of VEGF protein. Figure 5 In this context, E represents the statistical result of VEGF protein expression level; Figure 5 F in the figure represents the expression level of miR-210.

[0022] Figure 6 This is a statistical result graph showing the effects of HE-EVs (Example 2) and Con-EVs (Comparative Example 1) on behavioral scores and serum IgE levels in mice with allergic rhinitis. Figure 6 In this context, A represents the behavioral score, and *** indicates P < 0.001; Figure 6 In the table, B represents the statistical value of serum IgE level, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0023] Figure 7 The images show pathological images (HE staining) of the nasal mucosa of mice with allergic rhinitis using HE-EVs from Example 2 and Con-EVs from Comparative Example 1. Scale bar: 20 μm.

[0024] Figure 8 This is a statistical graph showing the flow cytometry results of the inhibition of dendritic cell maturation by HE-EVs in Example 2 of the present invention and Con-EVs in Comparative Example 1. Detailed Implementation

[0025] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0027] In a first aspect, the present invention provides a method for preparing extracellular vesicles with high VEGF protein / miR-210 induced by serum-free combined culture medium, comprising the following steps: Step 1: Under aseptic conditions, Wharton's jelly tissue from the umbilical cord of a full-term fetus is obtained, cut into tissue blocks, placed in a culture flask, and then cultured in α-MEM complete medium containing penicillin, streptomycin and fetal bovine serum. When the cells reach 80%-90% confluence, human umbilical cord mesenchymal stem cells (primary) are obtained. Step 2: When the human umbilical cord mesenchymal stem cells (HMCs) have expanded to 80%-90%, discard the tissue block and culture medium, wash the HMCs, add 0.25% trypsin solution for digestion, and when the HMCs can detach, add digestion stop solution containing 1% fetal bovine serum to terminate digestion; resuspend the HMCs to obtain a cell suspension, centrifuge and discard the supernatant, add serum-containing culture medium for resuspending, and culture to obtain HMCs highly expressing VEGF / miR-210; change the culture flask and continue passage culture in the same way to continuously expand the cells until the HMCs have undergone the 5th passage culture; Step 3: When the confluence of human umbilical cord mesenchymal stem cells with high expression of VEGF / miR-210 reaches 80%-90%, discard the serum-containing culture medium, wash with phosphate buffer, add serum-free combined culture medium, culture, collect the supernatant, and extract extracellular vesicles with high VEGF protein / miR-210 by high-speed centrifugation. The serum-free combined culture medium included: 500 mL of mixed culture medium, which was a mixture of sugar-free RPMI 1640 medium and MCDB131 medium at a volume ratio of 4:1; 1 g of polyvinyl alcohol; 2.383 g of 4-hydroxyethylpiperazine ethanesulfonic acid; 25 μM roxadustat; 1 mM sodium pyruvate; 0.1 mM cysteine; 2 mM alanine glutamine; 1% ITS-X; 20 ng / mL epidermal growth factor; and 2 ng / mL basic fibroblast growth factor.

[0028] In some embodiments, the serum-containing culture medium comprises: 500 mL of mixed culture medium; 1 g of polyvinyl alcohol; 2.383 g of 4-hydroxyethylpiperazine ethanesulfonic acid; 25 μM roxadustat; 1 mM sodium pyruvate; 0.1 mM cysteine; 2 mM alanine glutamine; 10% fetal bovine serum; 20 ng / mL epidermal growth factor; and 2 ng / mL basic fibroblast growth factor.

[0029] In some embodiments, the specific steps are as follows: Step S1: Under aseptic conditions, residual blood cells in the umbilical cord of a full-term fetus were removed using phosphate-buffered saline. The umbilical cord was then cut into fragments, blood vessels were removed, Wharton's jelly tissue was extracted, and the fragments were cut into tissue blocks. These blocks were then placed in culture flasks and α-MEM complete medium containing 100 U / mL penicillin, 100 mg / L streptomycin, and 10% fetal bovine serum were added. The flasks were then incubated at 37°C in a 5% CO2 incubator. The α-MEM complete medium included α-MEM liquid medium, L-glutamine, sodium pyruvate, β-mercaptoethanol, and epidermal growth factor. On day 3, half of the medium was replaced, and the medium was replaced again after one week. Thereafter, the medium was replaced every 3 days. The cell growth around the adhesion of the tissue blocks was observed. When the cells reached 80%-90% confluence, human umbilical cord mesenchymal stem cells (primary) were obtained. Step S2: When the human umbilical cord mesenchymal stem cells (HMCs) have expanded to 80%-90%, gently tap the side wall of the culture flask to detach the adherent tissue block. Discard the tissue block and α-MEM complete culture medium. Wash the HMCs with phosphate-buffered saline (PBS) and add 0.25% trypsin solution. Place the flask in a culture dish and incubate at 37°C for digestion. Once the HMCs can be gently shaken off the bottom of the culture dish, add a digestion stop solution containing 1% fetal bovine serum (FBS) to terminate the digestion. Slowly pipette and resuspend the HMCs to obtain a cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in serum-containing culture medium. Seed the cell suspension in culture dishes at a 1:3 ratio. Incubate the culture dishes at 37°C with 5% FBS. Human umbilical cord mesenchymal stem cells (HMCs) highly expressing VEGF / miR-210 were obtained by culturing in a CO2 incubator. The culture flasks were then replaced, and the cells were passaged in the same manner to continuously expand the cell count until the HMCs underwent their fifth passage. The digested HMCs were cultured at a density of 3000-6000 cells / cm³. 2 To carry out generational breeding; Step S3: When the confluence of human umbilical cord mesenchymal stem cells with high expression of VEGF / miR-210 reaches 80%-90%, discard the serum-containing culture medium supernatant, wash with phosphate buffer, add serum-free combined culture medium, and culture at 37℃ and 5% CO2 for 24-48h. Collect the supernatant and extract extracellular vesicles with high VEGF protein / miR-210 by high-speed centrifugation.

[0030] In some embodiments, the method for preparing a serum-containing culture medium includes the following steps: Step S201: Sugar-free RPMI 1640 medium and MCDB131 medium are mixed at a volume ratio of 4:1 to obtain a mixed medium; Step S202: Weigh 1g of polyvinyl alcohol and 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid and add them to 500mL of mixed culture medium to dissolve. After the polyvinyl alcohol and 4-hydroxyethylpiperazine ethanesulfonic acid are completely dissolved, add 25μM roxadustat, 1mM sodium pyruvate, 0.1mM cysteine ​​and 2mM alanine glutamine in sequence. Step S203: After filtration and sterilization, store at 4°C; before use, add 10% fetal bovine serum, 20 ng / mL epidermal growth factor, and 2 ng / mL basic fibroblast growth factor.

[0031] In some embodiments, the preparation method of serum-free combined culture medium includes the following steps: Step S301: Sugar-free RPMI 1640 medium and MCDB131 medium are mixed at a volume ratio of 4:1 to obtain a mixed medium; Step S302: Weigh 1g of polyvinyl alcohol and 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid and add them to 500mL of mixed culture medium to dissolve. After the polyvinyl alcohol and 4-hydroxyethylpiperazine ethanesulfonic acid are completely dissolved, add 25μM roxadustat, 1mM sodium pyruvate, 0.1mM cysteine ​​and 2mM alanine glutamine in sequence. Step S303: After filtration and sterilization, store at 4°C; before use, add 1% ITS-X, 20 ng / mL epidermal growth factor and 2 ng / mL basic fibroblast growth factor by volume.

[0032] In some embodiments, in step three, extracellular vesicles with high VEGF protein / miR-210 are extracted by high-speed centrifugation. The specific steps are as follows: the collected supernatant is centrifuged at 1000g for 10 min to obtain supernatant I; then supernatant I is centrifuged at 12000g for 20 min at 4℃ to obtain supernatant II; then supernatant II is centrifuged at 12000g for 40 min at 4℃ to obtain supernatant III; supernatant III is filtered through a 0.22μm filter to obtain supernatant IV; finally, supernatant IV is centrifuged at 100000g for 1 h at 4℃ to obtain extracellular vesicles with high VEGF protein / miR-210.

[0033] In some embodiments, the present invention provides serum-free combined culture medium-induced high VEGF protein / miR-210 extracellular vesicles, which are prepared by a method for preparing serum-free combined culture medium-induced high VEGF protein / miR-210 extracellular vesicles.

[0034] In some embodiments, extracellular vesicles with high VEGF protein / miR-210 expression specifically express CD63 and CD81 on their surface and are rich in VEGF protein and miR-210.

[0035] In some embodiments, the present invention provides the use of serum-free combined culture medium-induced high VEGF protein / miR-210 extracellular vesicles in the preparation of drugs for treating allergic rhinitis.

[0036] In some embodiments, the drug treats allergic rhinitis by inhibiting the Th2 immune response.

[0037] In some embodiments, the dosage form of the drug is preferably an injection, nasal drops, or nebulized inhaler.

[0038] Example 1: Preparation of serum-free and serum-containing culture media 1. The specific preparation method for serum-free combined culture medium is as follows: (1) Measure 400 mL of sugar-free RPMI 1640 medium and 100 mL of MCDB131 medium, mix them evenly, and obtain 500 mL of mixed medium; (2) Weigh 1g of polyvinyl alcohol (PVA) and 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and add them to the mixed culture medium. Stir at 25-30℃ until completely dissolved. (3) After complete dissolution, add 25 μM roxadustat (final concentration), 1 mM sodium pyruvate (final concentration), 0.1 mM cysteine ​​(final concentration) and 2 mM alanine glutamine (final concentration) in sequence, mix well; adjust the pH value to 7.2-7.4; (4) Sterilize by filtration using a 0.22 μm filter membrane, dispense into portions and store at 4 °C to obtain basal culture medium A solution; (5) Before use, add 1 mL of ITS-X (volume ratio of 1%), 20 ng / mL of epidermal growth factor (EGF) and 2 ng / mL of basic fibroblast growth factor (bFGF) to every 100 mL of basic culture medium A, mix well, and the serum-free culture medium is obtained.

[0039] 2. The specific preparation method for serum-containing culture medium is as follows: (1) Measure 400 mL of sugar-free RPMI 1640 medium and 100 mL of MCDB131 medium, mix them evenly, and obtain 500 mL of mixed medium; (2) Weigh 1g of polyvinyl alcohol (PVA) and 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and add them to the mixed culture medium. Stir at 25-30℃ until completely dissolved. (3) After complete dissolution, add 25 μM roxadustat (final concentration), 1 mM sodium pyruvate (final concentration), 0.1 mM cysteine ​​(final concentration) and 2 mM alanine glutamine (final concentration) in sequence, mix well; adjust the pH value to 7.2-7.4; (4) Sterilize by filtration using a 0.22 μm filter membrane, dispense into portions and store at 4 °C to obtain basal culture medium A solution; (5) Before use, add 10% fetal bovine serum, 20 ng / mL epidermal growth factor (EGF) and 2 ng / mL basic fibroblast growth factor (bFGF) to each 100 mL of basic culture medium A solution, mix well, and the serum-containing culture medium is obtained.

[0040] Example 2: Preparation of extracellular vesicles with high VEGF protein / miR-210 induced by serum-free combined culture medium 1. Cell source and primary culture After obtaining informed consent from the mother and ruling out carriers of infectious diseases (EB, CMV, HIV, HBV, HCV), the umbilical cord of the full-term fetus is aseptically obtained.

[0041] (1) Wash the umbilical cord three times with phosphate-buffered saline (PBS) to remove residual blood cells.

[0042] (2) Cut the umbilical cord into 2.0cm segments, remove the blood vessels (1 umbilical vein and 2 umbilical arteries), peel off the Wharton's jelly tissue, and cut it into approximately 1mm segments. 3 The organizational blocks.

[0043] (3) Place the tissue block in a T75 culture flask and add α-MEM complete culture medium (containing L-glutamine, sodium pyruvate, β-mercaptoethanol and epidermal growth factor) containing 100 U / mL penicillin, 100 mg / L streptomycin and 10% fetal bovine serum.

[0044] (4) Cultured in a 37℃, 5% CO2 incubator. Half of the medium was changed on the 3rd day, and the whole medium was changed on the 7th day. Thereafter, the medium was changed every 3 days. When the cells around the tissue block grew to 80%-90% confluence, they were digested and passaged with 0.25% trypsin containing EDTA to obtain P1 generation (primary) human umbilical cord mesenchymal stem cells (hUCMSCs).

[0045] 2. Induction and expansion of cells with high expression (1) When the P1 generation human umbilical cord mesenchymal stem cells have expanded to 80%-90% fusion, gently tap the side wall of the culture flask to discard the tissue block, wash with PBS and add 0.25% trypsin for digestion.

[0046] (2) Add digestion termination solution containing 1% fetal bovine serum to terminate digestion, and collect cells by centrifugation (1000 rpm, 5 min).

[0047] (3) Resuspend the cells in a serum-containing culture medium.

[0048] (4) Passage the cells at a ratio of 1:3 and culture them in a 37°C, 5% CO2 incubator. Continue passage and expansion until the cells are passaged to the 5th generation (P5) human umbilical cord mesenchymal stem cells. The passage density is controlled at 3000-6000 cells / cm².

[0049] 3. Collection and extraction of extracellular vesicles (1) Take P5 generation HE-hUCMSCs (human umbilical cord mesenchymal stem cells with high expression of VEGF protein / miR-210 cultured in serum-free combined medium), and when the cell confluence reaches 80%-90%, discard the old culture medium and wash with PBS.

[0050] (2) Add serum-free combined culture medium. To ensure that enough supernatant is collected (not less than 50 mL), use 5 T175 large square bottles for culture. Add 15-20 mL of serum-free combined culture medium to each bottle, with a total volume of about 75-100 mL.

[0051] (3) Incubate at 37℃ and 5% CO2 for 24 hours.

[0052] (4) Collect the supernatant and extract vesicles using the following differential centrifugation method: centrifuge at 1000g for 10 min to remove cells; take supernatant I and centrifuge at 3000g for 20 min at 4℃ to remove cells and debris; take supernatant II and centrifuge at 12000g for 40 min at 4℃ to further remove large vesicles; take supernatant III and filter it through a 0.22μm filter to obtain supernatant IV; place supernatant IV in an ultracentrifugation tube and centrifuge at 100000g for 1 h at 4℃, then discard the supernatant; resuspend the precipitate with an appropriate amount of PBS to obtain a suspension of serum-free combined culture medium-induced high VEGF protein / miR-210 extracellular vesicles (HE-EVs).

[0053] Extracellular vesicle characterization analysis: (1) Particle size analysis (NTA): The results showed that the vesicle particle size was mainly concentrated in 30-150nm, which is typical of exosome distribution.

[0054] (2) Biomarker detection (Western Blot): The results showed that vesicles highly expressed CD63, CD81 and CD9, but did not express Calnexin.

[0055] (3) Electron microscopy (TEM): A typical saucer-like membrane structure can be seen under transmission electron microscopy.

[0056] Comparative Example 1: Preparation of extracellular vesicles cultured in conventional culture medium 1. Cell source and primary culture After obtaining informed consent from the mother and ruling out carriers of infectious diseases (EB, CMV, HIV, HBV, HCV), the umbilical cord of the full-term fetus is aseptically obtained.

[0057] (1) Wash the umbilical cord three times with phosphate-buffered saline (PBS) to remove residual blood cells.

[0058] (2) Cut the umbilical cord into 2.0cm segments, remove the blood vessels (1 umbilical vein and 2 umbilical arteries), peel off the Wharton's jelly tissue, and cut it into approximately 1mm segments. 3 The organizational blocks.

[0059] (3) Place the tissue block in a T75 culture flask and add conventional serum-containing culture medium (α-MEM + 10% FBS + 1% double antibiotics + 2 ng / mL bFGF + 10 ng / mL EGF).

[0060] (4) Cultured in a 37℃, 5% CO2 incubator. Half of the medium was changed on the 3rd day, and the whole medium was changed on the 7th day. Thereafter, the medium was changed every 3 days. When the cells around the tissue block grew to 80%-90% confluence, they were digested and passaged with 0.25% trypsin containing EDTA to obtain P1 generation (primary) human umbilical cord mesenchymal stem cells (hUCMSCs).

[0061] 2. Induction and expansion of cells with high expression (1) When the P1 generation human umbilical cord mesenchymal stem cells have expanded to 80%-90% fusion, gently tap the side wall of the culture flask to discard the tissue block, wash with PBS and add 0.25% trypsin for digestion.

[0062] (2) Add digestion termination solution containing 1% fetal bovine serum to terminate digestion, and collect cells by centrifugation (1000 rpm, 5 min).

[0063] (3) Resuspend cells in conventional serum-containing culture medium.

[0064] (4) Passage the cells at a ratio of 1:3 and culture them in a 37°C, 5% CO2 incubator. Continue passage and expansion until the cells are passaged to the 5th generation (P5) human umbilical cord mesenchymal stem cells. The passage density is controlled at 3000-6000 cells / cm².

[0065] 3. Collection and extraction of extracellular vesicles (1) Take P5 generation Con-hUCMSC (human umbilical cord mesenchymal stem cells cultured in conventional serum-containing medium), and when the cell confluence reaches 80%-90%, discard the old medium and wash with PBS.

[0066] (2) Change the culture medium (without specific components). The culture medium is α-MEM + 1% ITS-X + 1% double antibiotics + 2ng / mL bFGF + 10ng / mL EGF. It does not contain specific components such as roxadustat, sugar-free RPMI 1640, MCDB131 and polyvinyl alcohol. To ensure that enough supernatant is collected (not less than 50mL), five T175 large square bottles are used for culture. 15-20mL of culture medium (without specific components) is added to each bottle, with a total volume of about 75-100mL.

[0067] (3) Incubate at 37℃ and 5% CO2 for 24 hours.

[0068] (4) Collect the supernatant and extract vesicles using the following differential centrifugation method: centrifuge at 1000g for 10 min to remove cells; take supernatant I and centrifuge at 3000g for 20 min at 4℃ to remove cells and debris; take supernatant II and centrifuge at 12000g for 40 min at 4℃ to further remove large vesicles; take supernatant III and filter it through a 0.22μm filter to obtain supernatant IV; place supernatant IV in an ultrafiltration tube and centrifuge at 100000g for 1 h at 4℃, then discard the supernatant; resuspend the precipitate in an appropriate amount of PBS to obtain a suspension of extracellular vesicles (Con-EVs) cultured in conventional serum-containing medium.

[0069] Comparative analysis of results: (1) Comparison of cell morphology between HE-hUCMSCs in Example 2 and Con-hUCMSCs in Comparative Example 1 Figure 1 As shown; the cell proliferation capacity of HE-hUCMSCs in Example 2 and Con-hUCMSCs in Comparative Example 1 is compared. Figure 2 As shown; the expression levels of stemness marker genes (SOX2 and Nanog) of HE-hUCMSCs in Example 2 and Con-hUCMSCs in Comparative Example 1 are compared as follows: Figure 3 As shown; The results showed that both HE-hUCMSCs and Con-hUCMSCs had a spindle-shaped morphology and were firmly attached to the wall. Figure 1 Among them, HE-hUCMSCs have stronger proliferation capacity ( ); Figure 2 The expression of stemness marker genes SOX2 and Nanog is higher. Figure 3 Maintaining good stem cell characteristics is beneficial to their differentiation potential and survival ability.

[0070] (2) The expression of extracellular vesicle marker proteins (CD9, CD63, CD81) in HE-EVs of Example 2 and Con-EVs of Comparative Example 1 is as follows: Figure 4 As shown; The results showed that both HE-EVs and Con-EVs expressed extracellular vesicle marker proteins.

[0071] (3) Electron microscopy identification, extracellular vesicle yield, extracellular vesicle protein concentration, VEGF protein expression level, and miR-210 expression level comparison between HE-EVs of Example 2 and Con-EVs of Comparative Example 1. Figure 5 As shown; The results showed that the yield of HE-EVs in Example 2 was: particles / mL (in terms of particles / mL) (Cell count), significantly higher than the Con-EVs yield in Comparative Example 1. particles / mL ( Figure 5 (B in the text); BCA assay showed that the protein concentration of HE-EVs was 2.3 times that of Con-EVs (in the text). Figure 5 Both expressed VEGF / miR-210, but the expression level of VEGF / miR-210 was higher in HE-EVs (C). Figure 5 (DF in the image). This indicates that HE-EVs are extracellular vesicles that highly express VEGF / miR-210, suggesting their association with inhibiting dendritic cell maturation and regulating immunity.

[0072] Example 3: Validation of the therapeutic effect of serum-free combined culture medium-induced high VEGF protein / miR-210 extracellular vesicles (HE-EVs) on allergic rhinitis in mice. 1. Experimental grouping and modeling: Forty female Balb / c mice aged 6-8 weeks were randomly divided into 4 groups of 10 mice each. The control group was not modeled and was given saline. The model group was modeled and injected with PBS via the tail vein. The Con-EVs group was modeled and injected with Con-EVs via the tail vein (Comparative Example 1). The HE-EVs group was modeled and injected with HE-EVs via the tail vein (Example 2).

[0073] Modeling method: OVA-AL(OH)3 was injected intraperitoneally on days 1 and 14 to sensitize the animals; from days 21 to 27, 5% OVA solution was administered via nasal drops daily to challenge the animals; Administration method: Starting from the challenge period (day 21), the animals were injected via tail vein every other day for a total of 4 times, with a dose of 200 μg / animal (dissolved in 200 μL of PBS).

[0074] 2. Detection Indicators and Results (1) Behavioral scoring: Within 10 minutes after the last administration, observe the number of times the mice scratch their nose and sneeze; scoring criteria: 1 point for scratching the nose once, 2 points for sneezing once; Scoring results (Mean ± SD): Control group: 2.4 ± 0.6 points; Model group: 51.6 ± 7.4 points; Con-EVs group: 27.7 ± 8.3 points; HE-EVs group: 11.4 ± 3.6 points.

[0075] (2) Serum IgE level: detected by ELISA; ELISA test results: Control group: 201.4±17.5 pg / mL; Model group: 813.7±83.3 pg / mL; Con-EVs group: 433.5±72.7 pg / mL; HE-EVs group: 289.9±37.8 pg / mL.

[0076] The statistical results of the effects of HE-EVs in Example 2 and Con-EVs in Comparative Example 1 on behavioral scores and serum IgE levels in mice with allergic rhinitis are as follows: Figure 6 As shown, the results indicated that the HE-EVs group score was significantly lower than that of the model group and the Con-EVs group, meaning that the symptoms of allergic rhinitis mice improved more significantly after HE-EVs treatment. Figure 6 In the A section); allergic rhinitis mice treated with HE-EVs showed a more effective reduction in allergen-specific IgE levels ( ). Figure 6 (B in the middle).

[0077] (3) The pathological changes (HE staining) of the nasal mucosa of mice with allergic rhinitis were as follows: HE-EVs of Example 2 and Con-EVs of Comparative Example 1 Figure 7 As shown.

[0078] The results showed that the nasal mucosal epithelium in the model group was thickened and inflammatory cells were extensively infiltrated; the mucosal structure in the HE-EVs group was close to normal and the number of infiltrated inflammatory cells was significantly reduced (by about 75%), which was better than that in the Con-EVs group (by about 45%).

[0079] Example 4: Study on the mechanism by which serum-free combined culture medium-induced high VEGF protein / miR-210 extracellular vesicles (HE-EVs) inhibit dendritic cell (DC) maturation 1. Experimental methods: Human peripheral blood mononuclear cells were isolated and induced into immature dendritic cells (DCs) with IL-4 and GM-CSF, and divided into 4 groups; control group: containing only culture medium; model group: induced to mature with 1 μg / mL LPS; Con-EVs group: induced with LPS and then added with 50 μg / mL Con-EVs (Comparative Example 1); HE-EVs group: induced with LPS and then added with 50 μg / mL HE-EVs (Example 2).

[0080] 2. Flow cytometry detection: After 48 hours of culture, cells were collected and the expression positivity rates of surface markers CD83, CD86, and HLA-DR were detected.

[0081] 3. The statistical results of the percentage (%) of positive cells in each group are shown in Table 1.

[0082] Table 1. Statistical results of the percentage of positive cells in each group

[0083] The flow cytometry results of the inhibition of dendritic cell maturation by HE-EVs in Example 2 and Con-EVs in Comparative Example 1 are statistically shown below. Figure 8 As shown, the results indicated that LPS significantly promoted the maturation of dendritic cells (increased expression of markers); Con-EVs had a certain inhibitory effect, but HE-EVs had a significantly stronger inhibitory effect (P < 0.01), which could more effectively maintain dendritic cells in an immature state, thereby inhibiting the excessive activation of T cells (especially Th2 cells), which can explain the immunological mechanism of HE-EVs in treating allergic rhinitis.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for the preparation of serum-free combi-media induced high VEGF protein / miR-210 extracellular vesicles, characterized by: The method comprises the following steps: Step 1: under sterile conditions, obtain the Wharton's jelly tissue of the umbilical cord of a full-term pregnant delivery fetus, cut the tissue into tissue blocks, then place the tissue blocks in a culture bottle, and then add an alpha-MEM complete culture medium containing penicillin, streptomycin and fetal bovine serum to culture the tissue blocks, and when the cells reach 80%-90% confluence, human umbilical cord mesenchymal stem cells are obtained; Step 2: when the human umbilical cord mesenchymal stem cells are expanded to 80%-90%, discard the tissue blocks and the culture medium, wash the human umbilical cord mesenchymal stem cells, add a 0.25% trypsin solution to digest the human umbilical cord mesenchymal stem cells, and when the human umbilical cord mesenchymal stem cells can be detached, add a digestion termination liquid containing 1% fetal bovine serum to terminate the digestion; resuspend the human umbilical cord mesenchymal stem cells to obtain a cell suspension, centrifuge and discard the supernatant, resuspend the cells in a serum-containing culture medium, culture, and obtain human umbilical cord mesenchymal stem cells with high expression of VEGF / miR-210; continue to subculture and culture the cells in the same way, continuously expand the cells, and until the human umbilical cord mesenchymal stem cells are subcultured for the fifth time; Step 3: when the human umbilical cord mesenchymal stem cells with high expression of VEGF / miR-210 reach 80%-90% confluence, discard the serum-containing culture medium, wash the cells with a phosphate buffer, add a serum-free combination culture medium, culture, collect the supernatant, and extract the high-VEGF protein / miR-210 extracellular vesicles by high-speed centrifugation; The serum-free combination culture medium comprises: 500 mL of a mixed culture medium, the mixed culture medium being mixed by a volume ratio of 4:1 of a sugar-free RPMI 1640 culture medium and a MCDB131 culture medium; 1 g of polyvinyl alcohol; 2.383 g of 4-hydroxyethylpiperazine ethanesulfonic acid; 25 μM of Roxadustat; 1 mM of sodium pyruvate; 0.1 mM of cysteine; 2 mM of alanine glutamine; 1% of ITS-X; 20 ng / mL of epidermal growth factor; and 2 ng / mL of basic fibroblast growth factor.

2. The production method according to claim 1, characterized by: The serum-containing culture medium comprises: 500 mL of a mixed culture medium; 1 g of polyvinyl alcohol; 2.383 g of 4-hydroxyethylpiperazine ethanesulfonic acid; 25 μM of Roxadustat; 1 mM of sodium pyruvate; 0.1 mM of cysteine; 2 mM of alanine glutamine; 10% of fetal bovine serum; 20 ng / mL of epidermal growth factor; and 2 ng / mL of basic fibroblast growth factor.

3. The production method according to claim 1, wherein: Specifically, the following steps are included: Step S1: under sterile conditions, residual blood cells of the umbilical cord of the full-term pregnancy delivery fetus are removed using a phosphate buffer solution, the umbilical cord of the full-term pregnancy delivery fetus is then cut into fragments and blood vessels are removed, Wharton's jelly tissue is taken out and cut into tissue blocks, and then placed in a culture bottle, complete α-MEM culture medium containing 100 U / mL penicillin, 100 mg / L streptomycin and 10% fetal bovine serum is added, and placed in a 37°C incubator containing 5% CO2 for culture, wherein the complete α-MEM culture medium comprises a-MEM liquid medium, L-glutamine, sodium pyruvate, β-mercaptoethanol and epidermal cell growth factor; after 3 days, half of the liquid is changed, and the liquid is changed again after 1 week, and then the liquid is changed every 3 days, and the cell growth around the first tissue block is observed, and when the cells reach 80%-90% confluence, human umbilical cord mesenchymal stem cells are obtained; Step S2: when the human umbilical cord mesenchymal stem cells are expanded to 80%-90%, tap the sidewall of the culture bottle to make the adherent tissue block fall off, discard the tissue block and the α-MEM complete culture medium; wash the human umbilical cord mesenchymal stem cells with phosphate buffer solution, add 0.25% trypsin solution, place in a culture dish, and place in a 37°C cell incubator for digestion; when the human umbilical cord mesenchymal stem cells can be shaken off from the bottom of the culture dish, add a digestion termination solution containing 1% fetal bovine serum to terminate the digestion; resuspend the human umbilical cord mesenchymal stem cells by slow blowing, obtain a cell suspension, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend with a serum-containing culture medium, inoculate the cell suspension in a culture dish at a ratio of 1:3, place the culture dish in a 37°C incubator containing 5% CO2 for culture, and obtain human umbilical cord mesenchymal stem cells with high expression of VEGF / miR-210; continue to subculture in the same way, continuously expand the cells, and until the human umbilical cord mesenchymal stem cells are subcultured for the 5th time, wherein the human umbilical cord mesenchymal stem cells after digestion are inoculated in a culture dish at a density of 3000-6000 cells / cm 2 for subculture. Step S3: when the human umbilical cord mesenchymal stem cells are expanded to 80%-90%, tap the sidewall of the culture bottle to make the adherent tissue block fall off, discard the tissue block and the α-MEM complete culture medium; wash the human umbilical cord mesenchymal stem cells with phosphate buffer solution, add 0.25% trypsin solution, place in a culture dish, and place in a 37°C cell incubator for digestion; when the human umbilical cord mesenchymal stem cells can be shaken off from the bottom of the culture dish, add a digestion termination solution containing 1% fetal bovine serum to terminate the digestion; resuspend the human umbilical cord mesenchymal stem cells by slow blowing, obtain a cell suspension, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend with a serum-containing culture medium, inoculate the cell suspension in a culture dish at a ratio of 1:3, place the culture dish in a 37°C incubator containing 5% CO2 for culture, and obtain human umbilical cord mesenchymal stem cells with high expression of VEGF / miR-210; continue to subculture in the same way, continuously expand the cells, and until the human umbilical cord mesenchymal stem cells are subcultured for the 5th time, wherein the human umbilical cord mesenchymal stem cells after digestion are inoculated in a culture dish at a density of 3000-6000 cells / cm Step S3: when the confluence of the high-VEGF / miR-210 human umbilical cord mesenchymal stem cells reaches 80%-90%, the serum-containing culture medium supernatant is discarded, washed with a phosphate buffer solution, and a serum-free combination culture medium is added, and cultured at 37°C and 5% CO2 for 24-48 hours, and the supernatant is collected, and high-VEGF protein / miR-210 extracellular vesicles are obtained by high-speed centrifugation.

4. The method of claim 2, wherein: The preparation method of the serum-containing culture medium comprises the following steps: Step S201: mixing sugar-free RPMI 1640 medium and MCDB131 medium at a volume ratio of 4:1 to obtain a mixed culture medium; Step S202: 1g of polyvinyl alcohol and 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid are weighed and added to 500mL of the mixed culture medium for dissolution, and after the polyvinyl alcohol and 4-hydroxyethylpiperazine ethanesulfonic acid are completely dissolved, 25μM roxadustat, 1mM sodium pyruvate, 0.1mM cysteine and 2mM alanine glutamine are sequentially added; Step S203: store at 4°C after sterilization by filtration; before use, add fetal bovine serum at a volume ratio of 10%, epidermal growth factor at 20ng / mL and basic fibroblast growth factor at 2ng / mL.

5. The method of claim 1, wherein: The preparation method of the serum-free combination culture medium comprises the following steps: Step S301: mixing sugar-free RPMI 1640 medium and MCDB131 medium at a volume ratio of 4:1 to obtain a mixed culture medium; Step S302: 1g of polyvinyl alcohol and 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid are weighed and added to 500mL of the mixed culture medium for dissolution, and after the polyvinyl alcohol and 4-hydroxyethylpiperazine ethanesulfonic acid are completely dissolved, 25μM roxadustat, 1mM sodium pyruvate, 0.1mM cysteine and 2mM alanine glutamine are sequentially added; Step S303: store at 4°C after sterilization by filtration; before use, add ITS-X at a volume ratio of 1%, epidermal growth factor at 20ng / mL and basic fibroblast growth factor at 2ng / mL.

6. The method of claim 1, wherein: In step three, the high VEGF protein / miR-210 extracellular vesicles were obtained by high-speed centrifugation. The specific steps were as follows: the collected supernatant was centrifuged at 1000g for 10 min to obtain supernatant I; then the supernatant I was centrifuged at 12000g for 20 min at 4°C to obtain supernatant II; and then the supernatant II was centrifuged at 12000g for 40 min at 4°C to obtain supernatant III; The supernatant III was filtered through a 0.22μm filter to obtain supernatant IV; finally, the supernatant IV was centrifuged at 100000g for 1 h at 4°C to obtain the high VEGF protein / miR-210 extracellular vesicles.

7. Serum-free combination medium-induced high VEGF protein / miR-210 extracellular vesicles, characterized in that: The high VEGF protein / miR-210 extracellular vesicles were prepared by the method of claim 7.

8. The serum-free medium-induced high VEGF protein / miR-210 extracellular vesicles of claim 7, characterized in that: The high VEGF protein / miR-210 extracellular vesicles specifically expressed CD63 and CD81 on the surface, and were rich in VEGF protein and miR-210.

9. The high VEGF protein / miR-210 extracellular vesicles induced by the serum-free combined culture medium of any one of claims 1-6 for use in the preparation of a medicament for treating allergic rhinitis.

10. Use according to claim 9, wherein: The medicament treats allergic rhinitis by inhibiting Th2-type immune response.

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