Induction medium and method for promoting mammary epithelial cells to secrete milk-like exosomes

Abstract

The present disclosure relates to the technical field of biomedicine. Provided in the present disclosure are an induction medium and method for promoting mammary epithelial cells to secrete milk-like exosomes. Regarding the induction medium, estrogen and glucose are added to an MEpiCM medium, wherein the molar concentration of the estrogen is 50 to 200 μM, and the molar concentration of the glucose is 10 to 30 mM; and the pH of the induction medium is 6.4 to 7.1. An acidic microenvironment is formed by means of adjusting the pH, which increases the permeability of cell membranes and activates intracellular signaling pathways. In addition, 50 to 200 μM of estrogen and 10 to 30 mM of glucose are added, which interact with the acidic microenvironment formed by means of pH adjustment, thereby making the estrogen and glucose to regulate mammary epithelial cells and inducing the mammary epithelial cells to secrete exosomes.

Description

Inducing medium and method for promoting mammary epithelial cells to secrete milk-derived exosome-like

[0001] Cross-reference to Related Applications

[0002] The present disclosure claims priority to the Chinese patent application No. 202410613074.6, filed on May 17, 2024, entitled "Inducing medium and method for promoting mammary epithelial cells to secrete milk-derived exosome-like", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biomedical technology, and in particular to an inducing medium and method for promoting mammary epithelial cells to secrete milk-derived exosome-like. BACKGROUND

[0004] Exosomes are double-membrane vesicles with a diameter of 30-150 nm secreted by cells, containing specific proteins, lipids and nucleic acids, and can act as signal molecules for cell communication, playing an important role in disease diagnosis, treatment and infant growth and development. Milk-derived exosomes can prevent necrotizing enterocolitis, affect infant mental development, preschool language and psychological development. In addition, double animal experiments and clinical experiments have confirmed that the consumption of milk lacking exosomes can lead to an increase in purine metabolites in the body, causing health problems such as high uric acid. Although milk-derived exosomes have important functions, the difficulty in obtaining milk-derived exosomes limits the scientific research and clinical application of milk-derived exosomes.

[0005] In view of the above, the present disclosure is proposed. SUMMARY

[0006] One of the purposes of the present disclosure is to provide an inducing medium for promoting mammary epithelial cells to secrete milk-derived exosome-like, to solve the technical problems of low cell production of milk-derived exosome-like and difficulty in obtaining milk-derived exosome-like in the prior art.

[0007] The second purpose of the present disclosure is to provide a method for promoting mammary epithelial cells to secrete milk-derived exosome-like.

[0008] In order to achieve the above-mentioned purposes of the present disclosure, the following technical solutions are adopted:

[0009] In a first aspect, the present disclosure provides an inducing medium for promoting mammary epithelial cells to secrete milk-derived exosome-like, wherein estrogen and glucose are added to the MEpiCM medium;

[0010] The molar concentration of the estrogen is 50-200 μM, and the molar concentration of the glucose is 10-30 mM.

[0011] The pH of the induction medium is 6.4-7.1.

[0012] Further, the pH is 6.7, the concentration of glucose is 20 mM, and the concentration of estrogen is 50 mM.

[0013] Further, the pH is 6.4, the concentration of glucose is 30 mM, and the concentration of estrogen is 100 mM.

[0014] Further, the pH is 6.7, the concentration of glucose is 10 mM, and the concentration of estrogen is 100 mM.

[0015] Further, the pH is 6.7, the concentration of glucose is 10 mM, and the concentration of estrogen is 50 mM.

[0016] Further, the estrogen is estrone.

[0017] Further, it also includes a growth factor with a mass concentration of 0.5-1.0%, and serum with a mass concentration of ≤10%.

[0018] Further, the growth factor is MEpiCGS, EGF, or FGF.

[0019] The serum is fetal bovine serum, horse serum, or human umbilical cord serum.

[0020] In a second aspect, the present disclosure provides a method for promoting mammary epithelial cells to secrete milk-like exosomes, comprising culturing mammary epithelial cells using the above-mentioned induction medium.

[0021] Further, the culturing of mammary epithelial cells in the induction medium comprises inoculating mammary epithelial cells into the induction medium and culturing until the cell adhesion coverage is 70-80%.

[0022] The conditions for culturing the mammary epithelial cells include culturing in an environment with a temperature of 35-40°C and 4-6% CO2.

[0023] The present disclosure provides an induction medium for promoting mammary epithelial cells to secrete milk-like exosomes. By adjusting the pH to form an acidic microenvironment, the permeability of the cell membrane is improved, the intracellular signaling pathway is activated, and 50-200 mM of estrogen and 10-30 mM of glucose are added. The estrogen and glucose interact with the acidic microenvironment formed by the pH to regulate the secretion of exosomes by mammary epithelial cells, achieve the effect of inducing mammary epithelial cells to secrete exosomes, improve the protein concentration of exosomes in the supernatant, facilitate the extraction of exosomes, and solve the technical problems of low production of milk-like exosomes by cells and difficulty in obtaining milk-like exosomes in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the specific embodiments of the present disclosure or the prior art, the accompanying drawings required by the specific embodiments or prior art description will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present disclosure, and other drawings can also be obtained by those skilled in the art without any creative effort based on these drawings.

[0025] Figure 1 is a statistical diagram of MCF-10A cell growth activity under different concentrations of serum in Example 1 of the present disclosure.

[0026] Figure 2 is a statistical diagram of MCF-10A cell growth activity under different concentrations of growth factors in Example 1 of the present disclosure.

[0027] Figure 3 is a statistical diagram of the total protein concentration of MCF-10A cell supernatant exosomes under different pH interventions in Example 2 of the present disclosure.

[0028] Figure 4 is a statistical diagram of the total protein concentration of MCF-10A cell supernatant exosomes under different concentrations of glucose intervention in Example 2 of the present disclosure.

[0029] Figure 5 is a statistical diagram of the total protein concentration of MCF-10A cell supernatant exosomes under different concentrations of estrogen intervention in Example 2 of the present disclosure.

[0030] Figure 6 is a transmission electron microscope morphology diagram of MCF-10A cell exosomes in Example 3 of the present disclosure.

[0031] Figure 7 is an NTA exosome particle size diagram of MCF-10A cell exosomes in Example 3 of the present disclosure.

[0032] Figure 8 is a biological analysis flowchart of the raw data of lipid compounds of exosomes from different sources in Example 3 of the present disclosure.

[0033] Figure 9 is a relative content diagram of different types of lipids of exosomes from different sources in Example 3 of the present disclosure.

[0034] Figure 10 is the relative expression amount of miRNA in breast milk exosomes and MCF-10A cell exosomes in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0035] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definition. In this application, unless indicated otherwise, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0036] The methods and techniques of the present disclosure are generally performed in accordance with conventional methods well known in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated.

[0037] In a first aspect, the present disclosure provides an induced culture medium for promoting mammary epithelial cells to secrete milk-like exosomes, wherein estrogen and glucose are added to MEpiCM culture medium;

[0038] The molar concentration of the estrogen is 50-200 μM, and the molar concentration of the glucose is 10-30 mM.

[0039] The pH of the induced culture medium is 6.4-7.1.

[0040] By adjusting the pH to form an acidic microenvironment, the permeability of the cell membrane is improved, and the intracellular signaling pathway is activated. At the same time, 50-200 μM of estrogen and 10-30 mM of glucose are added, which interact with the acidic microenvironment formed by the pH, so that the estrogen and glucose regulate the secretion of exosomes by mammary epithelial cells, achieve the effect of inducing mammary epithelial cells to secrete exosomes, improve the protein concentration of exosomes in the supernatant, facilitate the extraction of exosomes, and solve the technical problem that it is difficult to extract exosomes due to the small volume and low yield of exosomes in the prior art.

[0041] The molar concentration of the estrogen can be, but is not limited to, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, or 200 μM, or any value between 50-200 μM.

[0042] The molar concentration of the glucose can be, but is not limited to, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM, or 30 mM, or any value between 10-30 mM.

[0043] The pH of the induced culture medium can be, but is not limited to, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, or 7.1, or any value between 6.4-7.1.

[0044] In some specific embodiments, the pH is 6.7, the concentration of the glucose is 20 mM, and the concentration of the estrogen is 50 μM. The yield of exosomes is further improved

[0045] In some specific embodiments, the PH is 6.4, the concentration of glucose is 30 mM, and the concentration of estrogen is 100 μM. The exosome production is further improved

[0046] In some specific embodiments, the PH is 6.7, the concentration of glucose is 10 mM, and the concentration of estrogen is 100 μM. The exosome production is further improved

[0047] In some specific embodiments, the PH is 6.7, the concentration of glucose is 10 mM, and the concentration of estrogen is 50 μM. The three factors have the greatest synergistic effect on improving the exosome production, and the exosome production is further improved.

[0048] In some specific embodiments, the estrogen is estrone.

[0049] In some specific embodiments, the induction medium further comprises a growth factor with a mass concentration of 0.5-1.0%, and serum with a mass concentration of ≤10%.

[0050] By limiting the concentrations of the growth factor and the serum, the culture environment is more conducive to the growth of mammary epithelial cells, and the cell growth rate is accelerated.

[0051] In some specific embodiments, the growth factor is MEpiCGS, EGF, or FGF.

[0052] The serum is fetal bovine serum, horse serum, or human umbilical cord serum.

[0053] In some preferred embodiments, the mammary epithelial cell growth factor is MEpiCGS, and the serum is fetal bovine serum.

[0054] In a second aspect, the present disclosure also provides a method for promoting the secretion of milk source-like exosomes by mammary epithelial cells, comprising culturing mammary epithelial cells using the above-mentioned induction medium.

[0055] In some specific embodiments, the culturing of mammary epithelial cells in the induction medium comprises inoculating mammary epithelial cells into the induction medium and culturing until the cell adhesion coverage is 70-80%.

[0056] The culture conditions for the mammary epithelial cells comprise culturing in an environment with a temperature of 35-40°C and 4-6% CO2.

[0057] In some preferred embodiments, the culture conditions comprise culturing in an environment with a temperature of 37°C and 5% CO2.

[0058] In a third aspect, the present disclosure also provides exosomes obtained by the method as indicated in any one of the above-mentioned embodiments.

[0059] In a fourth aspect, the present disclosure also provides a kit containing the induction medium as indicated in any one of the above, which can facilitate mammary epithelial cells to secrete milk- derived exosomes.

[0060] In a fifth aspect, the present disclosure also provides the use of the induction medium or the kit as indicated in any one of the above in facilitating mammary epithelial cells to secrete milk- derived exosomes.

[0061] In a sixth aspect, the present disclosure also provides the use of the induction medium or the kit as indicated in any one of the above in increasing the lipid content of exosomes, wherein the lipid comprises fatty acid (FA) and sphingomyelin (SM).

[0062] In a seventh aspect, the present disclosure also provides the use of the induction medium or the kit as indicated in any one of the above in increasing the expression amount of miRNA in exosomes, wherein the miRNA comprises let-7a-5p, miR-33b-3p, miR-1-5p, miR-6715b-3p and miR-208b-3p.

[0063] The technical solutions of the present disclosure will be described clearly and completely in connection with the embodiments below. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all the other embodiments obtained by those skilled in the art without creative labor shall fall within the scope of protection of the present disclosure.

[0064] Unless otherwise specified, the materials in the embodiments are prepared according to the existing methods or directly purchased from the market.

[0065] The main reagents used in the following embodiments are shown in Table 1.

[0066] Table 1 Main reagents

[0067] Mammary epithelial cell resuscitation: Take the frozen mammary epithelial cells out of the liquid nitrogen, quickly put them into a 37℃ water bath, slowly rub the frozen tube, dissolve it within 1 min, when there are small ice fragments in the tube, use the gun head to blow and suck and transfer to the centrifuge tube with pre-added medium, centrifuge at 1000xg for 5 min, discard the supernatant, resuspend the cell pellet with MEpiCM medium, inoculate into the culture bottle and put it into the 37℃, 5% CO2 incubator.

[0068] Mammary epithelial cell suspension: Take the resuscitated mammary epithelial cells, centrifuge to remove the supernatant, resuspend the cells with MEpiCM medium to a concentration of 10 4 mL.

[0069] Non-inducing medium: MEpiCM medium + 10% Exosome-depleted FBS + 1% Mammary epithelial cell growth supplement MEpiCGS + 1% Penicillin / Streptomycin solution P / S.

[0070] Inducing medium: Based on the non-inducing medium, add estrogen at a final concentration of 50-200 μΜ and 10-30 mM glucose, adjust the pH to 6.4-7.1.

[0071] Example 1 Optimization of mammary epithelial cell culture medium

[0072] (1) Effect of serum concentration on the growth activity of MCF-10A cells

[0073] Take 10 4 mL of MCF-10A cell suspension at 10 cells / mL, inoculate into a 12-well plate, set the serum concentration of the non-inducing medium at 0%, 5%, 10%, and 15% respectively, set 3 parallels for each concentration, sample once every 24 h, a total of 6 times, use cell number as the detection index, use a cell counter (Shenzhen Ruivode Life Science and Technology Co., Ltd., C100-SE) to measure the cell number, and after 6 days of experiment, make a cell growth curve under different serum concentration conditions, as shown in FIG. 1.

[0074] The results show that when 10% serum is added, the cells grow fastest; when no serum is added, the cells grow slowest. Within the serum concentration range of 0-10%, as the serum concentration increases, the cell growth accelerates, and the cell growth rate is fastest in 3-4 days. However, when the serum concentration is 15%, the cell growth rate is lower than that of 10% serum concentration.

[0075] (2) Effect of growth factor MEpiCGS on the growth activity of MCF-10A cells

[0076] Take 10 4 mL of MCF-10A cell suspension at 10 cells / mL, inoculate into a 12-well plate, set the growth factor addition amount of the non-inducing medium at 0%, 0.5%, 1%, and 2% respectively, set 3 parallels for each concentration, sample once every 24 h, a total of 6 times, use a cell counter to measure the cell number, and after 6 days of experiment, make a growth curve, as shown in FIG. 2 for the determination of the effect of growth factor MEpiCGS on mammary epithelial cells.

[0077] The results show that when the growth factor concentration is 1%, the cell growth curve is closer to the "S" type, which is more conducive to cell growth.

[0078] Therefore, the serum concentration in the non-inducing medium used in the following examples is 10%, and the growth factor concentration is 1%.

[0079] Example 2 Inducing mammary epithelial cells to produce exosomes

[0080] (1) Effect of pH intervention on MCF-10A cells producing exosomes

[0081] MCF-10A cells were cultured in non-inducing medium with pH = 7.1 (10 6 / ml), and then the pH of the non-inducing medium was adjusted to 6.7 with 1M hydrochloric acid. 10 6 / ml cells were seeded on the culture dish, and when the cells grew to 70%-90% confluence, the cells were passaged and sampled. After the cells were stable, the pH of the non-inducing medium was adjusted to 6.4 with 1M hydrochloric acid. 10 6 / ml cells (pH = 6.7) were seeded on a 6cm diameter culture dish, and when the cells grew to 70%-90% confluence, the cells were passaged and sampled. After the cells were stable, the pH of the non-inducing medium was adjusted to 6.0 with 1M hydrochloric acid. 10 6 / ml cells (pH = 6.4) were seeded on a 6cm diameter culture dish, and samples were taken every 24h. The specific results are shown in Figure 3.

[0082] The results showed that when the pH was 6.0, the average concentration of total exosome protein was 1.30g / L; when the pH was 6.4, the average concentration of total exosome protein was 1.47g / L; when the pH was 6.7, the average concentration of total exosome protein was 1.44g / L; and when the pH was 7.1, the average concentration of total exosome protein was 1.45g / L. The concentration of total exosome protein increased first and then decreased with increasing pH. The concentration of total exosome protein was the lowest at pH = 6.0, and the highest at pH = 6.4. This may be due to the fact that high concentration of acid destroys the permeability of the cell membrane, reducing cell viability and thus reducing the release of exosomes.

[0083] (2) Effect of glucose intervention on MCF-10A cells producing exosomes

[0084] Based on the non-inducing medium, 10mM, 20mM, 30mM and 40mM of glucose were added respectively to prepare several culture media. MCF-10A cells were cultured in these culture media respectively, and after 24h of culture, the cell supernatant was collected and exosomes were extracted. The specific results are shown in Figure 4.

[0085] The results show that when the glucose concentration is 10 mM, the total protein concentration of the exosome is 1.35 g / L; when the glucose concentration is 20 mM, the total protein concentration of the exosome is 1.37 g / L; when the glucose concentration is 30 mM, the total protein concentration of the exosome is 1.37 g / L; and when the glucose concentration is 40 mM, the total protein concentration of the exosome is 1.31 g / L. With the increase of the glucose concentration, the total protein concentration of the exosome first increases and then decreases. When the glucose concentration is 30 mM, the total protein concentration of the exosome is the highest, and then the total protein concentration of the exosome decreases.

[0086] (3) Effect of estrogen intervention on exosome production of MCF-10A cells

[0087] Based on the non-inducing medium, estrogen with a final concentration of 50 μM, 100 μM, 200 μM and 400 μM is added respectively, and MCF-10A cells are cultured with these prepared media. After 24 hours of culture, the cell supernatant is collected, and the exosome is extracted. The specific results are shown in FIG. 5.

[0088] The results show that with the increase of the estrogen concentration, the total protein concentration of the exosome first increases and then decreases. When the estrogen concentration is 50 μM, the average total protein concentration of the exosome is 1.36 g / L; when the estrogen concentration is 100 μM, the average total protein concentration of the exosome is 1.47 g / L; when the estrogen concentration is 200 μM, the average total protein concentration of the exosome is 1.37 g / L; and when the estrogen concentration is 400 μM, the average total protein concentration of the exosome is 1.32 g / L. When the estrogen concentration is 100 μM, the total protein concentration of the exosome is the highest. When the hormone concentration is higher than 200 μM, the total protein concentration of the exosome gradually decreases.

[0089] (4) Multi-factor combination experiment

[0090] According to the experiments of steps (1) to (3), pH (A), estrogen (B) and glucose (C) are selected as the multi-factor combination experiment factors, and the levels of each factor are shown in Table 2. The experiment is carried out according to the following induction culture method:

[0091] Induction culture: centrifuge the MCF-10A cells recovered in culture, discard the supernatant, wash the bottom of the cell culture bottle with 3 mL of PBS, add 8 mL of induction medium, gently shake the culture bottle to make it uniform, and continue to culture in a 37°C, 5% CO2 incubator until the cell adhesion coverage rate is 70% to 80%. Centrifuge to obtain the supernatant, and use Total Exosome Isolation (from cell culture media) to extract the exosome. The experimental results are shown in Table 3.

[0092] Table 2 Factor level table The results show that with the increase of the estrogen concentration, the total protein concentration of the exosome first increases and then decreases. When the estrogen concentration is 50 μM, the average total protein concentration of the exosome is 1.36 g / L; when the estrogen concentration is 100 μM, the average total protein concentration of the exosome is 1.47 g / L; when the estrogen concentration is 200 μM, the average total protein concentration of the exosome is 1.37 g / L; and when the estrogen concentration is 400 μM, the average total protein concentration of the exosome is 1.32 g / L. When the estrogen concentration is 100 μM, the total protein concentration of the exosome is the highest. When the hormone concentration is higher than 200 μM, the total protein concentration of the exosome gradually decreases.

[0089] (4) Multi-factor combination experiment

[0090] According to the experiments of steps (1) to (3), pH (A), estrogen (B) and glucose (C) are selected as the multi-factor combination experiment factors, and the levels of each factor are shown in Table 2. The experiment is carried out according to the following induction culture method:

[0091] Induction culture: centrifuge the MCF-10A cells recovered in culture, discard the supernatant, wash the bottom of the cell culture bottle with 3 mL of PBS, add 8 mL of induction medium, gently shake the culture bottle to make it uniform, and continue to culture in a 37°C, 5% CO2 incubator until the cell adhesion coverage rate is 70% to 80%. Centrifuge to obtain the supernatant, and use Total Exosome Isolation (from cell culture media) to extract the exosome. The experimental results are shown in Table 3.

[0092] Table 2 Factor level table

[0093] Table 3 Results of multi-factor combination experiments

[0094] As shown in Table 3, the supernatant exosome protein amount of the 27 multi-factor combination experiments was significantly higher than that of the supernatant exosome protein amount of the single-factor induced strips in experiments (1) to (3). It can be seen that the combination of estrogen, glucose and pH significantly improves the induction effect, and the exosome protein amount in the supernatant is significantly increased. Among them, the exosome total protein content corresponding to experiment number 10 is the highest, followed by experiment numbers 11, 8 and 13, i.e. A2B1C1 is the best combination formula for inducing cell exosomes.

[0095] Comparative Example 1

[0096] The recovered MCF-10A cells were centrifuged, the supernatant was discarded, and the cell culture bottle bottom was washed with 3 mL of PBS. Non-inducing medium 8 mL was added, the culture bottle was gently shaken to make it uniform, and it was continued to be cultured in a 37°C, 5% CO2 incubator until the cell adhesion coverage was 70% to 80%. The supernatant was taken by centrifugation at 1000 x g for 5 min, and the exosomes were extracted using Total Exosome Isolation (from cell culture media). The total protein concentration of the exosomes was 1.45 g / L.

[0097] The results show that compared with Comparative Example 1, the supernatant exosome protein amount of the single-factor induced strips in experiments (1) to (3) did not change significantly, but the supernatant exosome protein amount of the 27 multi-factor combination experiments was significantly improved. Therefore, in order to induce mammary epithelial cells to secrete milk-like exosomes, there is an interaction relationship between estrogen, glucose and pH.

[0098] Example 3 Comparison of the morphology, lipids and miRNA of mammary epithelial cell exosomes and breast milk exosomes

[0099] In this example, the exosomes obtained from non-inducing medium were selected for morphological identification. The mammary epithelial cell exosomes of the optimal combination (A2B1C1) in the multi-factor combination experiment were selected as the experimental group MCF-10A-exo, and the breast milk exosomes of colostrum were selected as the control group breast milk-exo. The lipid composition and miRNA relative expression were detected, respectively.

[0100] (1) Mammary epithelial cell exosome morphology

[0101] MCF-10A cells were cultured in non-inducing medium, and exosomes were extracted from the supernatant for morphological identification under transmission electron microscopy. Nanoparticle tracking analysis (NTA) technology was used to determine the particle size of the exosomes (Figures 6 and 7). It was found that the shape of the breast epithelial cell exosomes was intermediate, with a concave inward edge, like a "cup", and the background was relatively clean. The exosomes obtained by differential centrifugation had a relatively clean background, and the shape was similar to that of breast milk exosomes. The particle size was about 60-100 nm, and the mean distribution of the particle size of the cell-derived exosomes was slightly smaller than that of the breast milk-derived exosomes.

[0102] (2) Lipid composition of breast epithelial cell exosomes

[0103] LC-MS / MS technology was used to obtain the original data of the lipid composition of the optimal combination of breast epithelial cell exosomes and colostrum exosomes. Bioinformatics analysis was performed as shown in Figure 8. The original data was searched in the CD software library, and the retention time and mass-to-charge ratio parameters were screened. The peaks were aligned with a retention time deviation of 0.2 min and a mass deviation of 5 ppm to improve the identification accuracy. Then, the peaks were extracted according to the mass deviation, signal intensity, and signal-to-noise ratio, and the peak area was quantified. The molecular formula was predicted based on the molecular ion peak and fragment ion, and compared with the Lipidmaps and Lipidblast databases. The background ions were removed using blank samples, and the quantitative results were normalized. Finally, the lipid data was analyzed qualitatively and quantitatively. The results showed that the relative content of FA and SM in the optimal combination of cell exosomes (MCF-10A-exo) was higher than that in breast milk exosomes (breast milk-exo) (Figure 9). The equipment models used in the above experiments are shown in Table 4.

[0104] Table 4 Equipment models and manufacturers

[0105] (3) miRNA of breast epithelial cell exosomes

[0106] qRT-PCR was used to detect the miRNA composition of the optimal combination of breast epithelial cell exosomes and colostrum exosomes in the multi-factor combination experiment. The results showed that MCF-10A-exo and breast milk-exo had the same miRNA. The specific results are shown in Figure 10. The relative expression levels of let-7a-5p, miR-33b-3p, miR-1-5p, miR-6715b-3p, and miR-208b-3p of the optimal combination of breast epithelial cell exosomes were significantly higher than those of breast milk exosomes.

[0107] The specific detection steps are as follows:

[0108] Because the mature body of miRNA is very short, usually only about 21 nt, it cannot be directly subjected to conventional reverse transcription, and for the detection of the expression of specific miRNAs at different stages, qRT-PCR based on stem-loop reverse transcription is used. The primers are shown in Table 5.

[0109] Table 5 qRT-PCR primer sequences

[0110] 1) Reverse transcription reaction

[0111] ① Reverse transcription system

[0112] ② After gently mixing, centrifuge for 3-5 s, incubate the reaction mixture at 16°C for 30 min, then incubate at 37°C for 30 min, heat at 85°C for 5 min to inactivate the enzyme, and store at 4°C.

[0113] 2) Fluorescence quantitative detection

[0114] The qPCR experiment is performed with reference to the SYBR Green I intercalating fluorescence method kit of Novozyme Biological Company.

[0115] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present disclosure, and not to limit them; although the present disclosure has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present disclosure. Industrial applicability

[0116] The present disclosure provides an induction medium for promoting mammary epithelial cells to secrete milk-like exosomes, which can effectively induce mammary epithelial cells to secrete exosomes and improve the problem that the production of milk-like exosomes by cells is low and milk exosomes are difficult to obtain, thus having a wide application prospect and high market value.

Claims

1. An induction medium for promoting mammary epithelial cells to secrete milk-like exosomes, characterized in that, adding estrogen and glucose to MEpiCM medium; the molar concentration of the estrogen is 50-200 μM, and the molar concentration of the glucose is 10-30 mM; the PH of the induction medium is 6.4-7.

1.

2. The induction medium of claim 1, wherein, the PH is 6.7, the concentration of the glucose is 20 mM, and the concentration of the estrogen is 50 μM.

3. The induction medium of claim 1, wherein, the PH is 6.4, the concentration of the glucose is 30 mM, and the concentration of the estrogen is 100 μM.

4. The induction medium of claim 1, wherein, the PH is 6.7, the concentration of the glucose is 10 mM, and the concentration of the estrogen is 100 μM.

5. The induction medium of claim 1, wherein, the PH is 6.7, the concentration of the glucose is 10 mM, and the concentration of the estrogen is 50 μM.

6. The induction medium of claim 1, wherein, the estrogen is estrone.

7. The induction medium according to any one of claims 1 to 6, characterized in that, further comprising a growth factor with a mass concentration of 0.5-1.0%, and serum with a mass concentration of ≤10%.

8. The induction medium of claim 7, wherein, the growth factor is MEpiCGS, EGF or FGF; the serum is fetal bovine serum, horse serum or human umbilical cord serum.

9. A method of promoting mammary epithelial cell secretion of milk-like exosomes, characterized by, culturing mammary epithelial cells using the induction medium of any one of claims 1-8.

10. The method of claim 9, wherein, culturing mammary epithelial cells in the induction medium includes inoculating mammary epithelial cells into the induction medium and culturing until the cell adhesion coverage is 70-80%; the conditions for culturing the mammary epithelial cells include culturing at a temperature of 35-40°C in an environment with 4-6% CO2.

11. The exosome obtained by the method of claim 9 or 10.

12. A kit characterized in that, which contains the induction medium of any one of claims 1-8.

13. Use of the induction medium of any one of claims 1-8 or the kit of claim 12 in promoting mammary epithelial cells to secrete milk-derived exosomes.

14. Use of the induction medium according to any one of claims 1 to 8 or the kit according to claim 12 for increasing the lipid content of exosomes, characterized in that, the lipids include fatty acids and sphingomyelin.

15. Use of the induction medium according to any one of claims 1 to 8 or the kit according to claim 12 for increasing the amount of miRNA expressed in exosomes, characterized in that, the miRNAs include let-7a-5p, miR-33b-3p, miR-1-5p, miR-6715b-3p and miR-208b-3p.

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