Culture medium for vascular endothelial progenitor cells, culture method and application

Abstract

The invention relates to the technical field of biological medicine, in particular to a culture medium for vascular endothelial progenitor cells, a culture method and application, the culture medium for the vascular endothelial progenitor cells is improved, and the prepared vascular endothelial progenitor cells have higher proliferative activity and better angiogenesis ability and have the strongest ability of promoting secretion of active factors. Factors such as IL-10 and TGF-beta secreted by the EPCs have potential functions of inhibiting mast cell degranulation and reducing release of histamine and inflammatory mediators so as to relieve wind mass and pruritus. In addition, the EPCs promote endothelial cell proliferation by secreting VEGF, Ang-1 and other factors, repair damaged vascular endothelium and reduce vascular leakage and edema. In urticaria vasculitis, the EPCs can inhibit hyperplasia of vascular endothelium and relieve chronic inflammation. The culture medium disclosed by the invention can be used for culturing the vascular endothelial progenitor cells, is beneficial to screening potential medicines for treating urticaria, and has a huge application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a culture medium for vascular endothelial progenitor cells, a culture method and an application thereof. Background Art

[0002] Vascular endothelial progenitor cells (EPCs) are a type of precursor cells with the potential to differentiate into mature endothelial cells and play an important role in angiogenesis, endothelial repair and inflammation regulation. Research shows that EPCs can inhibit the release of inflammatory factors and promote the repair of vascular endothelial barrier function through paracrine action, providing new ideas for the treatment of diseases with abnormal vascular permeability such as urticaria. However, there are the following problems in the culture of EPCs in the prior art: 1. The components of the culture medium are complex: Traditional culture media (such as M199, DMEM) need to add fetal bovine serum (FBS) and various growth factors (such as VEGF, EGF), but the serum batch differences are large, the cost is high and there is a risk of exogenous contamination.

[0003] 2. The cell proliferation efficiency is low: The purity of EPCs isolated by the existing methods is insufficient (the proportion of CD34+ / CD133+ cells < 30%), and the number of passages is limited (usually ≤ 3 generations), which is difficult to meet the needs of large-scale treatment.

[0004] 3. The functional verification is not sufficient: The existing research mostly focuses on the angiogenesis ability of EPCs, lacking systematic verification of their anti-inflammatory and immunomodulatory effects, especially the pharmacodynamic evaluation for the treatment of urticaria.

[0005] Therefore, there is an urgent need to develop a culture medium with clear components, low cost and capable of efficiently expanding high-purity EPCs. Summary of the Invention

[0006] In order to achieve the above object, the present invention first provides a culture medium for vascular endothelial progenitor cells, which comprises serum-free DMEM / F12 as a basal medium, and olea europaea leaf exosome extract, beta vulgaris root exosome extract, VEGF, bFGF, EGF, IGF-1, TGF-β, PDGF, vitamin C, heparin, streptomycin.

[0007] 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.

[0008] Preferably, the preparation method of the olive leaf exosome extract comprises the following steps: (1) After drying and pulverizing the olive leaves, sieving them, adding a buffer solution, and stirring to obtain a material; (2) Subjecting the material obtained in step (1) to low-speed centrifugation, retaining the supernatant 1; subjecting the supernatant 1 to medium-speed centrifugation, retaining the supernatant 2; subjecting the supernatant 2 to high-speed centrifugation to obtain a precipitate; (3) Ultrafiltration concentration: Resuspending the precipitate obtained in 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: Adding a freeze-drying protectant to the concentrate, followed by freeze-drying to obtain a freeze-dried powder, which is the olive leaf exosome extract.

[0009] Preferably, the buffer solution contains Na 2 HPO 4 , KH 2 PO 4 , CaCl 2 , NaCl and water, with a pH of 6 - 7.

[0010] Preferably, in step (3), a 30 kDa ultrafiltration membrane is used to concentrate the precipitate resuspension.

[0011] Preferably, the preparation method of the olive leaf exosome extract comprises the following steps: (1) After drying and pulverizing 50 g of olive ( Olea europaea. ) leaves and sieving them through a 100-mesh sieve, adding 500 mL of separation buffer solution (containing 10 mmol / L Na 2 HPO 4 , 10 mmol / L KH 2 PO 4 , 1 mmol / L CaCl 2 , 0.5 mmol / L NaCl, pH 6.5), and stirring for 30 minutes to obtain a material; (2) Gradient centrifugation purification: Processing the material obtained in step (1) as follows: First, perform low-speed centrifugation: 1000 g, for 20 minutes, and retain the supernatant 1; Perform medium-speed centrifugation on supernatant 1: 5000 g, for 30 minutes, and retain the supernatant 2; Perform high-speed centrifugation on supernatant 2: 12,000 g, for 1 hour, to obtain a precipitate; (3) Ultrafiltration concentration: Resuspend the precipitate with ddH 2 O to obtain a resuspended precipitate solution, and then concentrate the resuspended precipitate solution using a 30 kDa ultrafiltration membrane with a concentration factor of 20 times to obtain a concentrate; (4) Freeze-drying process: Add 5% (w / v, unit: g / mL) sucrose to the concentrate as a freeze-drying protectant, and perform freeze-drying to obtain a freeze-dried powder, which is the olive leaf exosome extract.

[0012] Preferably, the preparation method of the beetroot exosome extract includes the following steps: Cut the beetroot into pieces, add PBS, quickly freeze and grind into powder in liquid nitrogen, add cellulase, shake and enzymolyze, inactivate the enzyme, centrifuge, take the first supernatant, then centrifuge to obtain the second supernatant, centrifuge the second supernatant to obtain the third supernatant, add a freeze-drying protectant, and perform freeze-drying to obtain a freeze-dried powder, which is the beetroot exosome extract.

[0013] Preferably, add the cellulase solution according to a solid-liquid ratio of 1:10 (w / v, unit: g / mL).

[0014] Preferably, the freeze-drying protectant is sucrose or a sucrose solution.

[0015] Preferably, the preparation method of the beetroot exosome extract includes the following steps: Cut the beetroot into pieces (5×5 mm), add pre-cooled PBS (containing 1% PVP), quickly freeze and grind into powder in liquid nitrogen, add the cellulase solution according to a solid-liquid ratio of 1:10 (w / v, unit: g / mL), enzymolyze by shaking (200 rpm) at 37°C for 2 hours, inactivate the enzyme by boiling at 100°C for 5 minutes, centrifuge at 12,000×g for 15 minutes, take the first supernatant, then centrifuge the supernatant at 4000 g for 15 min to obtain the second supernatant, and finally centrifuge the second supernatant at 10,000 g for 40 minutes to remove impurities to obtain the third supernatant, add 5% (w / v) sucrose as a freeze-drying protectant, and perform freeze-drying to obtain a freeze-dried powder, which is the beetroot exosome extract.

[0016] The present invention also provides a method for culturing vascular endothelial progenitor cells, and the method includes the step of culturing the above-mentioned vascular endothelial progenitor cells using a medium.

[0017] The present invention also provides an application of the above-mentioned medium for vascular endothelial progenitor cells, and the application is to prepare vascular endothelial progenitor cells.

[0018] Compared with the prior art, the beneficial effects of the above technical solution of the present invention are as follows: The present invention improves the culture medium of vascular endothelial progenitor cells. The vascular endothelial progenitor cells prepared therefrom have higher proliferation activity, better angiogenesis ability, and the strongest ability to promote the secretion of active factors. 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 relieving wheals and itching. In addition, EPCs promote endothelial cell proliferation, repair damaged vascular endothelium, and reduce vascular leakage and edema by secreting factors such as VEGF and Ang-1. In urticarial vasculitis, EPCs can inhibit excessive proliferation of vascular endothelium and relieve chronic inflammation. Therefore, the culture medium of the present invention can be used for culturing vascular endothelial progenitor cells, which helps to screen potential drugs for treating urticaria and has great application value. Detailed implementation manners

[0019] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to specific embodiments.

[0020] Example 1. Preparation of olea europaea leaf exosome extract (1) 50 g of olea europaea ( Olea europaea. ) leaves are dried and pulverized through a 100-mesh sieve, and 500 mL of separation buffer (containing 10 mmol / L Na 2 HPO 4 , 10 mmol / L KH 2 PO 4 , 1 mmol / L CaCl 2 , 0.5 mmol / L NaCl, pH 6.5) is added, and stirred for 30 minutes to obtain a material; (2) Gradient centrifugation for purification: The material obtained in step (1) is processed as follows: First, use low-speed centrifugation: 1000 g, 20 minutes, and retain supernatant 1 (to remove cell debris).

[0021] Perform medium-speed centrifugation on supernatant 1: 5000 g, 30 minutes, and retain supernatant 2 (to remove large vesicles); Perform high-speed centrifugation on supernatant 2: 12,000 g, 1 hour, to obtain a precipitate; (3) Ultrafiltration concentration: Resuspend the precipitate with ddH 2 O to obtain a precipitate resuspension, and then concentrate the precipitate resuspension using a 30 kDa ultrafiltration membrane with a concentration factor of 20 times to obtain a concentrate; (4) Freeze-drying process: Add 5% (w / v, unit: g / mL) sucrose to the concentrate as a freeze-drying protectant, and then perform freeze-drying to obtain the freeze-dried powder, which is the extract of olive leaf exosomes.

[0022] Example 2. Preparation of beetroot exosome extract The steps include: Beet ( Beta vulgaris L. ) Cut the root into pieces (5×5 mm), add pre-cooled PBS (containing 1% PVP), quickly freeze with liquid nitrogen and grind into powder. Add cellulase solution according to the solid-liquid ratio of 1:10 (w / v, unit: g / mL), enzymolyze at 37°C with shaking (200 rpm) for 2 hours, inactivate the enzyme by boiling at 100°C for 5 minutes, centrifuge at 12,000×g for 15 minutes, take the first supernatant, then centrifuge the supernatant at 4000g for 15 min to obtain the second supernatant, and finally centrifuge the second supernatant at 10000g for 40 minutes to remove impurities and obtain the third supernatant. Add 5% (w / v, unit: g / mL) sucrose to the third supernatant as a freeze-drying protectant, and perform freeze-drying to obtain the freeze-dried powder, which is the extract of beetroot exosomes.

[0023] Example 3. Components and ratios of the culture medium Culture medium 1: Serum-free DMEM / F12 as the basal medium Add the following components: The extract of olive leaf exosomes prepared in Example 1 (20 ng / mL), the extract of beetroot exosomes 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), streptomycin (100 U / mL); Culture medium 2: On the basis of culture medium 1, omit the extract of olive leaf exosomes, change the concentration of the extract of beetroot exosomes to 40 ng / mL, and the other conditions are the same as those of culture medium 1.

[0024] Culture medium 3: On the basis of culture medium 1, omit the extract of beetroot exosomes, change the concentration of the extract of olive leaf exosomes to 40 ng / mL, and the other conditions are the same as those of culture medium 1.

[0025] Culture medium 4: On the basis of culture medium 1, omit the extract of olive leaf exosomes and the extract of beetroot exosomes, and the other conditions are the same as those of culture medium 1.

[0026] Example 4. Culture of vascular endothelial progenitor cells 1. Cell isolation: Peripheral blood or umbilical cord blood was collected, and mononuclear cells (MNCs) were obtained by Ficoll gradient centrifugation (density 1.077 g / mL, 2000 rpm, 20 min).

[0027] After washing the MNCs with PBS, they were seeded in culture dishes coated with gelatin (0.1% gelatin, incubated at 37°C for 1 h).

[0028] 2. Primary culture: The above-mentioned Medium 1 - Medium 4 were added respectively, and static culture was carried out in an incubator at 37°C and 5% CO 2 2.

[0029] Medium change strategy: Half of the medium was changed on the first day, and then the whole medium was changed every 2 days.

[0030] 3. Subculture: After the primary cells reached 80% density, they were digested with 0.25% trypsin and subcultured at a ratio of 1:3.

[0031] Purification by differential adhesion method: The first adherent cells (24 h) were EPCs, and the non - adherent cells were discarded.

[0032] 4. Amplification to P5 - P8: The culture cycle for each generation was 7 - 10 days. The cell morphology was like paving stones, and flow cytometry detection showed that CD34+ / CD133+ / VEGFR - 2+≥85%.

[0033] Example 5. Testing the effect of the medium 1. Cell proliferation: The in vitro proliferation ability of EPCs obtained with Medium 1 - Medium 4 was determined by cell counting.

[0034] Calculation formula: Population doubling number (PD) = log2 (number of cells at harvest - number of cells at seeding), and population doubling time (PDTs) = time interval between seeding and harvest / PD. The results are shown in Table 1.

[0035] Table 1. Doubling time Grouping PDT (h) Culture medium 1 28.2±1.3 Culture medium 2 38.2±3.1 Culture medium 3 41.2±3.3 Culture medium 4 52.4±5.2 The results in Table 1 show that the in vitro proliferation ability of EPCs obtained by culturing with Medium 1 was the strongest, and it was significantly better than that of Medium 4 (p < 0.01).

[0036] 2. Function verification: Angiogenic ability: The Matrigel lumen formation assay showed that the results were as shown in Table 2.

[0037] Table 2. Angiogenic ability Grouping Number of lumens per field of view (200x) Culture medium 1 110±5 Culture medium 2 90±7 Culture medium 3 75±6 Culture medium 4 62±3 The results in Table 2 show that the EPCs obtained by culturing with Medium 1 have the strongest ability to promote angiogenesis, and are significantly more effective than Medium 4 (p < 0.01).

[0038] 3. Detection of active factors For the detection of active factors of EPCs obtained by culturing with Medium 1 - Medium 4, ELISA was mainly used to detect IL-10, TGF-β, VEGF and Ang-1, and based on the expression levels of EPCs obtained by culturing with Medium 4, the relative expression levels of each group were calculated. The results are shown in Table 3.

[0039] Table 3. Relative expression levels of each active factor Experimental group IL-10 TGF-β VEGF Ang-1 Culture medium 1 3.3±0.1 2.9±0.2 1.8±0.2 2.3±0.1 Culture medium 2 2.5±0.3 2.3±0.2 1.4±0.1 1.6±0.2 Culture medium 3 2.3±0.2 1.8±0.1 1.3±0.3 1.4±0.1 The results in Table 3 show that the EPCs obtained by culturing with Medium 1 have the strongest ability to promote the secretion of active factors, and are significantly more effective than Medium 4 (p < 0.01).

[0040] Moreover, 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 itching. In addition, EPCs promote the proliferation of endothelial cells, repair damaged vascular endothelium, and reduce vascular leakage and edema by secreting factors such as VEGF and Ang-1. In urticarial vasculitis, EPCs can inhibit excessive proliferation of vascular endothelium and relieve chronic inflammation. Therefore, the medium of the present invention can be used for culturing endothelial progenitor cells, which helps to screen potential drugs for treating urticaria.

[0041] It should be understood that the above are only some embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the creative concept of the present invention, other deformations and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A culture medium for endothelial progenitor cells, characterized in that The culture medium contains 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.

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 1-10ng / mL, the concentration of bFGF is 5-20ng / 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 100-300μg / mL, the concentration of heparin is 1-10U / mL, and the concentration of streptomycin is 50-200 U / mL.

3. The culture medium according to claim 1, characterized in that 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) centrifuging the material obtained in step (1) at a low speed to retain supernatant 1; centrifuging supernatant 1 at a medium speed to retain supernatant 2; and centrifuging supernatant 2 at a high speed 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: 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.

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

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

6. The culture medium according to claim 1, characterized in that 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 lyophilizing protective agent, and freeze-drying to obtain a lyophilized powder, which is the beetroot exosome extract.

7. The culture medium according to claim 6, characterized in that Add cellulose enzymatic hydrolysate at a material-liquid ratio of 1:10 (w / v).

8. The culture medium according to claim 3 or 6, characterized in that The freeze-drying protective agent is sucrose or sucrose solution.

9. 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 8.

10. A use of the culture medium for endothelial progenitor cells according to any one of claims 1 to 8, characterized in that: The application is to prepare endothelial progenitor cells.