Culture medium, culture method and application of endothelial progenitor cells

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a culture medium, culture method and application for endothelial progenitor cells. The present invention improves the culture medium for endothelial progenitor cells, and the endothelial progenitor cells prepared therefrom have higher proliferation activity and better angiogenesis ability, and their ability to promote the secretion of active factors is the strongest. Factors such as IL-10 and TGF-β secreted by EPCs have the potential to inhibit mast cell degranulation, reduce the release of histamine and inflammatory mediators, and thus relieve wheals and itching. In addition, EPCs promote endothelial cell proliferation by secreting factors such as VEGF and Ang-1, repair damaged vascular endothelium, and reduce vascular leakage and edema. In urticarial vasculitis, EPCs can inhibit excessive proliferation of the vascular endothelium and relieve chronic inflammation. The culture medium of the present invention can be used for endothelial progenitor cell culture, which is helpful for screening potential drugs for treating urticaria and has great application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a culture medium, a culture method and an application of endothelial progenitor cells. Background Art

[0002] Endothelial progenitor cells (EPCs) are a type of precursor cell with the potential to differentiate into mature endothelial cells. They play a vital role in angiogenesis, endothelial repair, and inflammation regulation. Studies have shown that EPCs can inhibit the release of inflammatory factors and promote the repair of endothelial barrier function through paracrine effects, providing new insights into the treatment of vascular permeability disorders such as urticaria. However, existing EPC cultivation techniques present the following challenges:

[0003] 1. Complex culture medium composition: Traditional culture media (such as M199 and DMEM) require the addition of fetal bovine serum (FBS) and multiple growth factors (such as VEGF and EGF). However, serum batches vary greatly, the cost is high, and there is a risk of exogenous contamination.

[0004] 2. Low cell proliferation efficiency: The EPCs isolated by existing methods are of insufficient purity (CD34+ / CD133+ cell ratio <30%), and the number of passages is limited (usually ≤3 generations), making it difficult to meet the needs of large-scale treatment.

[0005] 3. Insufficient functional validation: Existing studies have mostly focused on the angiogenic ability of EPCs, lacking systematic validation of their anti-inflammatory and immune regulation, especially the pharmacodynamic evaluation for the treatment of urticaria.

[0006] Therefore, there is an urgent need to develop a culture medium with clear composition, low cost and the ability to efficiently expand high-purity EPCs. Summary of the Invention

[0007] In order to achieve the above-mentioned object, the present invention first provides a culture medium for endothelial progenitor cells, which comprises serum-free DMEM / F12 as a basal culture medium and olive leaf exosome extract, beetroot exosome extract, VEGF, bFGF, EGF, IGF-1, TGF-β, PDGF, vitamin C, heparin, and streptomycin.

[0008] Preferably, the concentration of the olive leaf exosome extract is 10-30 ng / mL, the concentration of the beetroot exosome extract is 10-30 ng / mL, the concentration of VEGF is 1-10 ng / mL, the concentration of bFGF is 5-20 ng / mL, the concentration of EGF is 5-20 ng / mL, the concentration of IGF-1 is 1-10 ng / mL, the concentration of TGF-β is 10-30 ng / mL, the concentration of PDGF is 10-30 ng / mL, the concentration of vitamin C is 100-300 μg / mL, the concentration of heparin is 1-10 U / mL, and the concentration of streptomycin is 50-200 U / mL.

[0009] Preferably, the method for preparing the olive leaf exosome extract comprises the following steps:

[0010] (1) After drying, the olive leaves are crushed and sieved, a buffer solution is added, and the mixture is stirred to obtain a material;

[0011] (2) The material obtained in step (1) is subjected to low-speed centrifugation to retain supernatant 1; supernatant 1 is subjected to medium-speed centrifugation to retain supernatant 2; supernatant 2 is subjected to high-speed centrifugation to obtain a precipitate;

[0012] (3) Ultrafiltration concentration: resuspending the precipitate from step (2) to obtain a precipitate resuspension, and then concentrating the precipitate resuspension using an ultrafiltration membrane to obtain a concentrate;

[0013] (4) Freeze-drying process: A freeze-drying protective agent is added to the concentrate, and freeze-dried to obtain freeze-dried powder, which is the olive leaf exosome extract.

[0014] Preferably, the buffer comprises Na2HPO4, KH2PO4, CaCl2, NaCl and water, pH 6-7.

[0015] Preferably, in step (3), a 30 kDa ultrafiltration membrane is used to concentrate the precipitate resuspension.

[0016] Preferably, the method for preparing the olive leaf exosome extract comprises the following steps:

[0017] (1) 50g of olive oil ( Olea europaea. ) The leaves were dried and crushed through a 100-mesh sieve, and 500 mL of separation buffer (containing 10 mmol / L Na2HPO4, 10 mmol / L KH2PO4, 1 mmol / L CaCl2, 0.5 mmol / L NaCl, pH 6.5) was added and stirred for 30 minutes to obtain the material;

[0018] (2) Gradient centrifugation purification: The material obtained in step (1) is treated as follows:

[0019] First, use low-speed centrifugation: 1000g, 20 minutes, and retain the supernatant 1;

[0020] Centrifuge supernatant 1 at medium speed: 5000 g for 30 minutes, and retain supernatant 2;

[0021] Supernatant 2 was centrifuged at high speed: 12,000 g for 1 hour to obtain a precipitate;

[0022] (3) Ultrafiltration concentration: resuspend the precipitate in ddH2O to obtain a precipitate resuspension, and then concentrate the precipitate resuspension using a 30 kDa ultrafiltration membrane at a concentration factor of 20 to obtain a concentrate;

[0023] (4) Freeze-drying process: 5% (w / v, g / mL) sucrose was added to the concentrate as a freeze-drying protective agent, and freeze-dried to obtain freeze-dried powder, which is the olive leaf exosome extract.

[0024] Preferably, the preparation method of the beetroot exosome extract comprises the following steps: cutting beetroot into pieces, adding PBS, quick-freezing with liquid nitrogen and grinding into powder, adding cellulase, oscillating enzymolysis, inactivating the enzyme, centrifuging, taking a first supernatant, and then centrifuging to obtain a second supernatant, centrifuging the second supernatant to obtain a third supernatant, adding a lyoprotectant, freeze-drying, and obtaining a lyophilized powder, which is the beetroot exosome extract.

[0025] Preferably, the cellulose hydrolysate is added at a material-liquid ratio of 1:10 (w / v, unit: g / mL).

[0026] Preferably, the lyoprotectant is sucrose or a sucrose solution.

[0027] Preferably, the preparation method of the beetroot exosome extract comprises the following steps: cutting beetroot into pieces (5×5 mm), adding pre-cooled PBS (containing 1% PVP), quick-freezing with liquid nitrogen and grinding into powder, adding cellulose hydrolysate at a material-liquid ratio of 1:10 (w / v, unit: g / mL), enzymolysis at 37°C with shaking (200 rpm) for 2 hours, boiling at 100°C for 5 minutes to inactivate the enzyme, centrifuging at 12,000×g for 15 minutes, taking the first supernatant, and then centrifuging the supernatant at 4000g for 15 minutes to obtain a second supernatant, and finally centrifuging the second supernatant at 10,000g for 40 minutes to remove impurities to obtain a third supernatant, adding 5% (w / v) sucrose as a lyoprotectant, and freeze-drying to obtain a lyophilized powder, which is the beetroot exosome extract.

[0028] The present invention also provides a method for culturing endothelial progenitor cells, comprising the step of culturing the endothelial progenitor cells using the above-mentioned culture medium.

[0029] The present invention also provides a use of the above-mentioned culture medium for endothelial progenitor cells, wherein the use is for preparing endothelial progenitor cells.

[0030] Compared with the prior art, the above technical solution of the present invention has the following beneficial effects:

[0031] The present invention improves the culture medium for endothelial progenitor cells (EPCs). The EPCs produced thereby exhibit higher proliferation activity and enhanced angiogenesis capacity, and possess the strongest ability to secrete active factors. Factors secreted by EPCs, such as IL-10 and TGF-β, have the potential to inhibit mast cell degranulation and reduce the release of histamine and inflammatory mediators (such as TNF-α and IL-6), thereby alleviating wheals and itching. Furthermore, EPCs secrete factors such as VEGF and Ang-1, promoting endothelial cell proliferation, repairing damaged vascular endothelium, and reducing vascular leakage and edema. In urticarial vasculitis, EPCs can inhibit excessive endothelial proliferation and alleviate chronic inflammation. Therefore, the culture medium of the present invention can be used for culturing endothelial progenitor cells, aiding in the screening of potential therapeutic drugs for urticaria, and possesses significant application value. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to specific embodiments.

[0033] Example 1. Preparation of olive leaf exosome extract

[0034] (1) 50g of olive oil ( Olea europaea. ) The leaves were dried and crushed through a 100-mesh sieve, and 500 mL of separation buffer (containing 10 mmol / L Na2HPO4, 10 mmol / L KH2PO4, 1 mmol / L CaCl2, 0.5 mmol / L NaCl, pH 6.5) was added and stirred for 30 minutes to obtain the material;

[0035] (2) Gradient centrifugation purification: The material obtained in step (1) is treated as follows:

[0036] First, perform low-speed centrifugation: 1000 g, 20 minutes, and retain the supernatant 1 (to remove cell debris).

[0037] Centrifuge supernatant 1 at 5000 g for 30 minutes and retain supernatant 2 (to remove large vesicles).

[0038] Supernatant 2 was centrifuged at high speed: 12,000 g for 1 hour to obtain a precipitate;

[0039] (3) Ultrafiltration concentration: resuspend the precipitate in ddH2O to obtain a precipitate resuspension, and then concentrate the precipitate resuspension using a 30 kDa ultrafiltration membrane at a concentration factor of 20 to obtain a concentrate;

[0040] (4) Freeze-drying process: Add 5% (w / v, g / mL) sucrose as a freeze-drying protectant to the concentrate according to the proportion, freeze-dry, and obtain freeze-dried powder, which is the olive leaf exosome extract.

[0041] Example 2. Preparation of Beetroot Exosome Extract

[0042] The steps include:

[0043] beet( Beta vulgaris L. ) roots were cut into pieces (5×5 mm), added with pre-cooled PBS (containing 1% PVP), quick-frozen in liquid nitrogen, and ground into powder. Cellulose hydrolysate was added at a solid-liquid ratio of 1:10 (w / v, g / mL), and enzymatic hydrolysis was carried out at 37°C with shaking (200 rpm) for 2 hours. The enzyme was boiled at 100°C for 5 minutes to inactivate the enzyme, and the mixture was centrifuged at 12,000×g for 15 minutes. The first supernatant was collected, and then the supernatant was centrifuged at 4000g for 15 minutes to obtain a second supernatant. Finally, the second supernatant was centrifuged at 10,000g for 40 minutes to remove impurities to obtain a third supernatant. 5% (w / v, g / mL) sucrose was added to the third supernatant as a lyoprotectant in proportion, and the mixture was freeze-dried to obtain lyophilized powder, which is the beetroot exosome extract.

[0044] Example 3: Culture medium components and proportions

[0045] Medium 1: serum-free DMEM / F12 as basal medium

[0046] Add the following ingredients:

[0047] Olive leaf exosome extract prepared in Example 1 (20 ng / mL), beetroot exosome extract prepared in Example 2 (20 ng / mL), VEGF (5 ng / mL), bFGF (10 ng / mL), EGF (10 ng / mL), IGF-1 (5 ng / mL), TGF-β (20 ng / mL), PDGF (20 ng / mL), vitamin C (200 μg / mL), heparin (2 U / mL), and streptomycin (100 U / mL);

[0048] Medium 2: Based on Medium 1, olive leaf exosome extract was omitted and the concentration of beetroot exosome extract was changed to 40 ng / mL. The other conditions were the same as Medium 1.

[0049] Medium 3: Based on Medium 1, the beetroot exosome extract was omitted and the concentration of olive leaf exosome extract was changed to 40 ng / mL. The other conditions were the same as Medium 1.

[0050] Medium 4: omitting the olive leaf exosome extract and beetroot exosome extract from Medium 1, and other conditions were the same as Medium 1.

[0051] Example 4: Culture of endothelial progenitor cells

[0052] 1. Cell separation:

[0053] Peripheral blood or cord blood was collected and mononuclear cells (MNCs) were obtained by Ficoll gradient centrifugation (density 1.077 g / mL, 2000 rpm, 20 min).

[0054] After washing with PBS, MNCs were seeded on gelatin-coated (0.1% gelatin, incubated at 37°C for 1 h) culture dishes.

[0055] 2. Primary Culture:

[0056] Add the above-mentioned culture medium 1 to culture medium 4 respectively, and culture statically in a 37°C, 5% CO2 incubator.

[0057] Medium exchange strategy: half-volume medium exchange on the first day, and full-volume medium exchange every 2 days thereafter.

[0058] 3. Subculture:

[0059] When the primary cells reached 80% density, they were digested with 0.25% trypsin and passaged at a ratio of 1:3.

[0060] Differential adhesion purification: The cells that adhered for the first time (24 h) were EPCs, and non-adherent cells were discarded.

[0061] 4. Amplification to P5-P8:

[0062] Each culture cycle is 7-10 days. The cell morphology is cobblestone-like. Flow cytometry detection shows CD34+ / CD133+ / VEGFR-2+≥85%.

[0063] Example 5, culture medium effect test

[0064] 1. Cell proliferation:

[0065] The in vitro proliferation capacity of EPCs obtained from medium 1 to medium 4 was determined by cell counting.

[0066] The calculation formula is: population doubling number (PD) = log2 (cell number at harvest - cell number at inoculation), and population doubling time (PDTs) = time interval between inoculation and harvest / PD. The results are shown in Table 1.

[0067] Table 1. Doubling time

[0068] Grouping PDT(h) Medium 1 28.2±1.3 Medium 2 38.2±3.1 Medium 3 41.2±3.3 Medium 4 52.4±5.2

[0069] The results in Table 1 show that the in vitro proliferation ability of EPCs obtained by culture in medium 1 is the strongest, and the effect is significant compared with that in medium 4 (p<0.01).

[0070] 2. Functional Verification:

[0071] Angiogenesis ability: Matrigel tube formation assay was performed, and the results are shown in Table 2.

[0072] Table 2. Angiogenesis ability

[0073] Grouping Number of lumens per field of view (200x) Medium 1 110±5 Medium 2 90±7 Medium 3 75±6 Medium 4 62±3

[0074] The results in Table 2 show that EPCs cultured in medium 1 have the strongest ability to promote angiogenesis, and the effect is significantly higher than that in medium 4 (p<0.01).

[0075] 3. Active factor detection

[0076] The active factors of EPCs obtained by culture in medium 1 to medium 4 were detected mainly by using Elisa to detect IL-10, TGF-β, VEGF and Ang-1. The relative expression levels of each group were calculated based on the expression levels of EPCs obtained by culture in medium 4. The results are shown in Table 3.

[0077] Table 3. Relative expression levels of each active factor

[0078] Experimental group IL-10 TGF-β VEGF Ang-1 Medium 1 3.3±0.1 2.9±0.2 1.8±0.2 2.3±0.1 Medium 2 2.5±0.3 2.3±0.2 1.4±0.1 1.6±0.2 Medium 3 2.3±0.2 1.8±0.1 1.3±0.3 1.4±0.1

[0079] The results in Table 3 show that the EPCs cultured using medium 1 have the strongest ability to promote the secretion of active factors, and the effect is significant compared with medium 4 (p < 0.01).

[0080] Factors such as IL-10 and TGF-β secreted by EPCs have the potential to inhibit mast cell degranulation and reduce the release of histamine and inflammatory mediators (such as TNF-α and IL-6), thereby alleviating wheals and pruritus. Furthermore, EPCs secrete factors such as VEGF and Ang-1, promoting endothelial cell proliferation, repairing damaged vascular endothelium, and reducing vascular leakage and edema. In urticarial vasculitis, EPCs can inhibit excessive endothelial proliferation and alleviate chronic inflammation. Therefore, the culture medium of the present invention can be used for culturing endothelial progenitor cells, facilitating the screening of potential therapeutic drugs for urticaria.

[0081] It should be understood that the above are only some embodiments of the present invention. It should be pointed out that for ordinary technicians in this field, other variations and improvements can be made without departing from the creative concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A culture medium for endothelial progenitor cells, characterized in that The culture medium comprises serum-free DMEM / F12 as a basal medium, as well as olive leaf exosome extract, beetroot exosome extract, VEGF, bFGF, EGF, IGF-1, TGF-β, PDGF, vitamin C, heparin, and streptomycin; The preparation method of the olive leaf exosome extract comprises the following steps: (1) After drying, the olive leaves are crushed and sieved, a buffer solution is added, and the mixture is stirred to obtain a material; (2) The material obtained in step (1) was centrifuged at 1000 g for 20 minutes, and supernatant 1 was retained; supernatant 1 was centrifuged at 5000 g for 30 minutes, and supernatant 2 was retained; supernatant 2 was centrifuged at 12,000 g for 1 hour to obtain a precipitate; (3) Ultrafiltration concentration: resuspending the precipitate from step (2) to obtain a precipitate resuspension, and then concentrating the precipitate resuspension using an ultrafiltration membrane to obtain a concentrate; (4) Freeze-drying process: The concentrate is added with a freeze-drying protective agent and freeze-dried to obtain a freeze-dried powder, which is the olive leaf exosome extract; The preparation method of the beetroot exosome extract comprises the following steps: cutting beetroot into pieces, adding PBS, quick-freezing with liquid nitrogen, grinding into powder, adding cellulase, performing enzymatic hydrolysis by oscillation, inactivating the enzyme, centrifuging, taking a first supernatant, and then centrifuging the supernatant at 4000g for 15 minutes to obtain a second supernatant, and finally centrifuging the second supernatant at 10000g for 40 minutes to remove impurities to obtain a third supernatant, adding a lyoprotectant, and freeze-drying to obtain a lyophilized powder, which is the beetroot exosome extract.

2. The culture medium according to claim 1, characterized in that The concentration of the olive leaf exosome extract is 10-30 ng / mL, the concentration of the beetroot exosome extract is 10-30 ng / mL, the concentration of VEGF is 1-10 ng / mL, the concentration of bFGF is 5-20 ng / mL, the concentration of EGF is 5-20 ng / mL, the concentration of IGF-1 is 1-10 ng / mL, the concentration of TGF-β is 10-30 ng / mL, the concentration of PDGF is 10-30 ng / mL, the concentration of vitamin C is 100-300 μg / mL, the concentration of heparin is 1-10 U / mL, and the concentration of streptomycin is 50-200 U / mL.

3. The culture medium according to claim 1, characterized in that The buffer contains Na2HPO4, KH2PO4, CaCl2, NaCl and water, pH 6-7.

4. The culture medium according to claim 1, characterized in that In step (3), the precipitate resuspension is concentrated using a 30 kDa ultrafiltration membrane.

5. The culture medium according to claim 1, characterized in that The freeze-drying protective agent is sucrose or sucrose solution.

6. A method for culturing endothelial progenitor cells, characterized in that: The method comprises the step of culturing the endothelial progenitor cells using the culture medium according to any one of claims 1 to 5.

7. A use of the culture medium for endothelial progenitor cells according to any one of claims 1 to 5, characterized in that: The application is to culture endothelial progenitor cells.