Antioxidant synergistic exosome and preparation method thereof

Abstract

The present application relates to the technical field of biological medicine, and in particular to an antioxidant synergistic exosome and a preparation method thereof. The present application promotes the secretion of exosomes with stronger antioxidant efficacy by stimulating adipose mesenchymal stem cells with rutin and combining with a hypoxic culture mode, thereby improving the antioxidant efficacy of the exosomes. In addition, the present application can efficiently obtain antioxidant synergistic exosomes with high purity and stable and uniform quality in a short time by combining the methods of fractional filtration, ultrafiltration concentration and ultrahigh-speed centrifugation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an antioxidant synergistic exosome and a preparation method thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are a kind of multipotent cells, which are derived from the mesoderm in the earliest stage of embryonic development. MSCs secrete various cytokines such as growth factors and inflammatory factors through secretion modes such as autocrine and paracrine, and have significant effects in regulating immune response, inhibiting skin aging, promoting tissue repair and many other aspects. In recent years, with the in-depth research and application of MSCs, the exosomes secreted by MSCs have gradually become a new research hotspot.

[0003] Exosomes are a kind of double-layer disc-shaped vesicles with a diameter of about 40-150 nm, which are secreted by cells and contain important signal molecules such as proteins, mRNA, miRNA and lipids. Through binding with receptor cells, the signal pathways in the receptor cells are activated, and the information exchange between cells is involved. At present, many studies have shown that human mesenchymal stem cell-derived exosomes have good immune regulation and tissue repair functions, and have very high clinical value in the treatment of burns, aging-related diseases and respiratory system damage and many other diseases. However, the exosomes obtained under the traditional culture mode lack targeting, and their antioxidant effect is not significant. Moreover, the current exosome preparation method has the disadvantages of low extraction efficiency, too low purity of the obtained exosomes, easy aggregation of the exosomes into groups, and damage to the structure of the exosomes during the extraction process, which seriously affects the clinical application and industrial development of the exosomes. In addition, there is an urgent need to prepare antioxidant synergistic exosomes. SUMMARY

[0004] The present application aims at the problems existing in the prior art, and provides an antioxidant synergistic exosome and a preparation method thereof.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The present application first provides a preparation method of an antioxidant synergistic exosome, which comprises stimulating the culture of mesenchymal stem cells with rutin.

[0007] Further, the method comprises:

[0008] (1) stimulating mesenchymal stem cells with rutin:

[0009] When the mesenchymal stem cells are cultured to a confluence degree of 50-60%, the culture is stimulated with rutin for a certain period of time, and then the culture medium is replaced for continuous culture, to obtain supernatant containing exosomes.

[0010] Further, the mesenchymal stem cells are preferably P5-P8 generation adipose-derived mesenchymal stem cells, and the culture condition is that the cells are inoculated in DMEM / F12 culture solution containing 3-5% serum substitute.

[0011] Further, the rutin stimulation method is that the mesenchymal stem cells are transferred into DMEM / F12 culture solution containing 3-5% serum substitute containing rutin, and cultured for 24-48 h.

[0012] Further preferably, the concentration of rutin in the culture solution is 5-20 μmol / L.

[0013] Further, the replacement culture medium is a serum-free culture medium, and the continued culture condition is continued culture in an O2 environment of 1-3% for 45-50 h.

[0014] (2) Preparation of antioxidant synergistic exosomes:

[0015] The exosome-containing supernatant is centrifuged to remove impurities and collect the supernatant. The obtained supernatant is fractionally filtered to collect the filtered supernatant. The filtered supernatant is ultrafiltrated and concentrated to obtain crude antioxidant synergistic exosomes. The crude antioxidant synergistic exosomes are finely separated to obtain antioxidant synergistic exosomes.

[0016] Further, the centrifugation of the exosome-containing supernatant is performed at 1-5°C, and the centrifugation method comprises the following steps: the exosome-containing supernatant is centrifuged at 300-500 g for 10-20 min to collect the supernatant, then the collected supernatant is centrifuged at 2000-3000 g for 25-35 min to collect the supernatant, and then the collected supernatant is centrifuged at 9000-11000 g for 30-60 min to collect the supernatant.

[0017] Further, the membrane pore size used in the fractional filtration is 0.8 μm, 0.45 μm, and 0.22 μm in sequence.

[0018] Further, the ultrafiltration and concentration are performed using a 100 KD ultrafiltration tube, and the centrifugation parameters are 1500-3000 g and 30-50 min.

[0019] Further, the fine separation is performed using a high-speed centrifugation method, and the method comprises the following steps: the exosome precipitate is collected by centrifugation at 100000-120000 g for 90-150 min, the exosome precipitate is resuspended with 3-5°C pre-cooled phosphate buffer, the high-purity exosome precipitate is obtained by centrifugation at 100000-120000 g for 90-150 min, the high-purity exosome precipitate is resuspended with 0.2-0.5 mL 3-5°C pre-cooled phosphate buffer, and high-purity antioxidant synergistic exosomes with stable and uniform quality are obtained, which are stored after being divided into small portions.

[0020] The application also provides application of the exosome or the exosome prepared by the method in preparation of an antioxidant product.

[0021] Advantages of the application:

[0022] The application provides a method for efficiently preparing antioxidant synergistic exosomes, which improves the oxidative stress state of adipose mesenchymal stem cells by using rutin to stimulate the adipose mesenchymal stem cells, thereby promoting the adipose mesenchymal stem cells to secrete exosomes with antioxidant effects, so that the exosomes secreted by the adipose mesenchymal stem cells have higher antioxidant capacity, and the method combining fractional filtration, ultrafiltration centrifugation and ultrahigh-speed centrifugation efficiently obtains antioxidant synergistic exosomes with higher purity, better antioxidant effect and stable and uniform quality. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A schematic diagram of a process for treating mesenchymal stem cells with rutin.

[0024] Figure 2 A schematic diagram of a process for preparing antioxidant synergistic exosomes.

[0025] Figure 3 A nanoparticle tracking analysis (NTA) diagram of antioxidant synergistic exosomes.

[0026] Figure 4 A Western Blot analysis of the expression of specific membrane protein CD9 of antioxidant synergistic exosomes.

[0027] Figure 5 A transmission electron microscope (TEM) diagram of antioxidant synergistic exosomes.

[0028] Figure 6 An operation flowchart of an exosome antioxidant experiment.

[0029] Figure 7 Comparison results of antioxidant effects of different exosomes. DETAILED DESCRIPTION

[0030] The specific embodiments of the application will be further described in detail below with reference to the accompanying drawings and examples. The following examples are used to illustrate the application, but are not used to limit the scope of the application.

[0031] Example 1: Preparation of mesenchymal stem cell exosome supernatant

[0032] P5-P8 generation of adipose-derived mesenchymal stem cells (donated by Jiangsu Regional Cell Preparation Center, as a public cell) are inoculated in DMEM / F12 culture solution containing 3-5% serum substitute for culture. When the confluence reaches 50-60%, the original culture medium is replaced with DMEM / F12 culture solution containing 5-20 μmol / L rutin and 3-5% serum substitute, and placed in a cell culture box for rutin stimulation culture for 24-48 h. Then, the culture medium is replaced with serum-free culture medium (purchased from Huageng Biology), and the cells are placed in a 1-3% O2 environment for further culture for 45-50 h to collect the cell supernatant to obtain the exosome-containing supernatant.

[0033] Further, in the above step, the adipose-derived mesenchymal stem cells are cultured by inoculating P5-P8 generation of adipose-derived mesenchymal stem cells in DMEM / F12 culture solution containing 3-5% serum substitute, shaking, and then placing in a cell culture box for culture. When the confluence reaches 50-60%, the adipose-derived mesenchymal stem cells are transferred into DMEM / F12 culture solution containing rutin and 3-5% serum substitute, and placed in a cell culture box for culture for 24-48 h.

[0034] Preferably, the concentration of rutin in the culture solution in the above step is 5-20 μmol / L.

[0035] Figure 1 A schematic diagram of the process of treating mesenchymal stem cells with rutin in this embodiment, wherein the mesenchymal stem cells are human adipose-derived mesenchymal stem cells.

[0036] Example 2: Preparation of stem cell exosomes

[0037] (1) The exosome-containing supernatant obtained in Example 1 is centrifuged at 300-500 g for 10-20 min at 1-5°C to remove dead cells and apoptotic debris, and the supernatant ① is collected. The collected supernatant ① is centrifuged at 2000-3000 g for 25-35 min at 1-5°C to remove organelles and apoptotic bodies, and the supernatant ② is collected. Then, the second collected supernatant ② is centrifuged at 9000-11000 g for 30-60 min at 1-5°C to eliminate larger vesicles, and the supernatant ③ is collected.

[0038] (2) The final collected supernatant ③ in step (1) is subjected to fractional filtration with a vacuum filter to further remove impurities, and the filtered supernatant is collected. The membrane pore size of the vacuum filter is 0.8 μm, 0.45 μm, and 0.22 μm, respectively.

[0039] (3) The filtered supernatant is subjected to ultrafiltration concentration with a 100 KD ultrafiltration tube, and the crude anti-oxidation synergistic exosomes are obtained. The centrifugation parameters are 1500-3000 g for 30-50 min.

[0040] (4) The crude antioxidant synergistic exosomes are finely separated by using ultra-high-speed centrifugation to obtain high-purity antioxidant synergistic exosomes, and the specific steps are as follows:

[0041] a) Collect the exosome precipitate by centrifuging at 100000-120000 g for 90-150 min;

[0042] b) Resuspend the exosome precipitate with 3-5 ℃ pre-cooled phosphate buffer;

[0043] c) Obtain the high-purity exosome precipitate by centrifuging at 100000-120000 g for 90-150 min;

[0044] d) Resuspend the high-purity exosome precipitate with 0.2-0.5 mL of 3-5 ℃ pre-cooled phosphate buffer to obtain high-purity antioxidant synergistic exosomes with stable and uniform quality, and store them in a -80 ℃ refrigerator.

[0045] Figure 2 It is a schematic diagram of the preparation method of antioxidant synergistic exosomes.

[0046] Figure 3 It is a nanoparticle tracking analysis (NTA) diagram of antioxidant synergistic exosomes. As can be seen from the diagram, the main particle size of antioxidant synergistic exosomes is about 130 nm, which is consistent with the particle size distribution of exosomes, and the exosome particle size is mainly distributed in 100-200 nm. It can be seen that the extracted exosomes are relatively uniform, contain less impurities, and have high purity.

[0047] Figure 4 It is a Western Blot analysis of the expression of specific membrane protein CD9 of antioxidant synergistic exosomes. As can be seen from the diagram, antioxidant synergistic exosomes positively express specific membrane protein CD9, while the culture supernatant does not express CD9.

[0048] Figure 5 It is an electron microscope diagram of antioxidant synergistic exosomes. As can be seen from the diagram, the clear "tea tray-like" exosome structure and the clear background can be seen, and the extracted exosomes have high purity and contain less impurities.

[0049] Figure 3 、 4 , 5 The particle size, specific membrane protein and tea tray-like structure characteristics of exosomes are identified, which proves that the extracted substances meet the characteristics of exosomes, i.e. the extracted substances are exosomes.

[0050] Example 3: Antioxidant experiment

[0051] P2-P5 generation human dermal fibroblasts (HDF-a) (purchased from Ningbo Mingzhou Biotechnology Co., Ltd.) were inoculated in 1640 culture medium containing 10% fetal bovine serum overnight, and the exosome control group and the experimental group were added with 10-20 μg / mL of corresponding exosomes for 45-50 h of pretreatment, and the blank control group was added with an equal amount of phosphate buffer as a control. Then the culture medium was replaced with 1640 culture medium containing 200-400 μmol / L hydrogen peroxide solution for 2-4 h, and then the cells in each group were collected by digestion, 2-5 μmol / L DCFH-DA was added to the 1640 culture medium for incubation for 20-30 min, and the ROS level in each group of cells was detected by flow cytometry within 1 h.

[0052] Among them, the experimental group, that is, the antioxidant synergistic exosome group, uses the exosomes prepared in Example 2, the exosome control group uses the adipose mesenchymal stem cell exosomes extracted by the super-speed centrifugation method without rutin stimulation, and the blank control group is added with an equal amount of phosphate buffer as a control.

[0053] Figure 6 The flow chart for the exosome antioxidant experiment is shown in the figure, in which 10-20 μg / mL of the exosomes prepared in Example 2 are added to the antioxidant synergistic exosome group, 10-20 μg / mL of the adipose mesenchymal stem cell exosomes extracted by the super-speed centrifugation method without rutin stimulation are added to the exosome control group, and an equal amount of phosphate buffer is added to the blank control group.

[0054] Figure 7 The results of the antioxidant effect comparison of different exosomes are shown in the figure, and it can be seen that the antioxidant synergistic exosomes prepared by the method have the best antioxidant effect, and the ROS level is much lower than that of the blank control group and the exosome control group.

Claims

1. A method for preparing antioxidant synergistic exosomes, the method comprising stimulating mesenchymal stem cells with rutin. The method comprises: (1) stimulating mesenchymal stem cells with rutin: When the mesenchymal stem cells are cultured to a confluence of 50-60%, the cells are stimulated with rutin for a certain period of time, and then the medium is replaced with serum-free medium for further culture, to obtain an exosome-containing supernatant; The mesenchymal stem cells comprise P5-P8 generation adipose-derived mesenchymal stem cells; The stimulation with rutin comprises transferring the mesenchymal stem cells into a DMEM / F12 culture solution containing 3-5% serum replacement with rutin, and culturing for 24-48 h; the concentration of rutin in the culture solution is 5-20 μmol / L; (2) preparation of antioxidant exosomes: The exosome-containing supernatant is centrifuged to remove impurities and collect the supernatant; the obtained supernatant is fractionally filtered to collect the filtered supernatant; the filtered supernatant is ultrafiltrated and concentrated to obtain crude antioxidant synergistic exosomes; The crude antioxidant synergistic exosomes are finely separated to obtain antioxidant synergistic exosomes.

2. The method of claim 1, wherein, The centrifugation to remove impurities in the exosome-containing supernatant in step (2) is performed at 1-5℃, and the method comprises: centrifuging the exosome-containing supernatant at 300-500g for 10-20 min to collect the supernatant, centrifuging the collected supernatant at 2000-3000g for 25-35 min to collect the supernatant, and then centrifuging the collected supernatant at 9000-11000g for 30-60 min to collect the supernatant.

3. The method of claim 1, wherein, The membrane pore size used in the fractional filtration in step (2) is 0.8 μm, 0.45 μm, and 0.22 μm in sequence.

4. The method of claim 1, wherein, The ultrafiltration and concentration in step (2) are performed using a 100KD ultrafiltration tube, and the centrifugation parameters are 1500-3000g for 30-50 min.

5. The method of claim 1, wherein, The fine separation in step (2) is performed using a high-speed centrifugation method, which comprises: centrifuging at 100000-120000g for 90-150 min to collect the exosome precipitate, resuspending the exosome precipitate with 3-5℃ pre-cooled phosphate buffer, centrifuging at 100000-120000g for 90-150 min to obtain a high-purity exosome precipitate, resuspending the high-purity exosome precipitate with 0.2-0.5 mL of 3-5℃ pre-cooled phosphate buffer, to obtain the antioxidant synergistic exosomes. 6.The antioxidant synergistic exosomes prepared by the method of any one of claims 1-5. 7.Use of the antioxidant synergistic exosomes prepared by the method of any one of claims 1-5 or the exosomes of claim 6 in the preparation of antioxidant products.

Images