Application of Forskolin in promoting sialic acid synthesis in mammary epithelial cells

Abstract

The present invention provides an application of Forskolin in promoting mammary epithelial cells to synthesize sialic acid, and relates to the field of biotechnology. The inventors have proved through experiments that the use of Forskolin to treat mammary epithelial cells can significantly promote the content of sialic acid in cell culture fluid. It has been proved that Forskolin can promote mammary epithelial cells to synthesize sialic acid. A new way to regulate sialic acid secretion is provided.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to the application of Forskolin in promoting mammary epithelial cells to synthesize sialic acid. Background Art

[0002] Sialic acid secreted by mammary epithelial cells plays an important role in mammary gland development and the formation of breast milk components. The level of sialic acid is closely related to the function of mammary epithelial cells and the nutritional components of breast milk. Therefore, regulating sialic acid secretion is of great significance for improving mammary gland function and breast milk quality.

[0003] Since the natural production of sialic acid is relatively low, the methods disclosed in the prior art are based on the synthesis pathway of N-acetylneuraminic acid in vivo, using relatively reasonably priced raw materials and using a two-step enzymatic method for biosynthesis. The first step is to convert glucosamine (GlcNAc) into N-acetylmannosamine (ManNAc) using N-acetylglucosamine-2-epimerase (AGE), and the second part is to connect ManNAc and pyruvate (Pyruvate) into N-acetylneuraminic acid (Neu5Ac) using N-acetylneuraminic acid aldolase (NAL). The reaction efficiency of sialic acid in this method still cannot meet the requirements of industrial production.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide the use of Forskolin in promoting the synthesis of sialic acid in mammary epithelial cells, so as to solve the technical problem of the lack of substances for regulating the secretion of sialic acid in the prior art.

[0006] The second object of the present invention is to provide the use of Forskolin in producing sialic acid.

[0007] The third object of the present invention is to provide the use of Forskolin in increasing the sialic acid content in milk.

[0008] A fourth object of the present invention is to provide a method for regulating sialic acid synthesis in mammary epithelial cells in vitro.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] In a first aspect, the present invention provides the use of Forskolin in promoting sialic acid synthesis in mammary epithelial cells.

[0011] Furthermore, the mammary epithelial cells are from mammals.

[0012] Furthermore, the mammal includes any one of humans, cattle, sheep, camels, horses, donkeys, reindeer, alpacas or llamas.

[0013] In a second aspect, the present invention provides the use of Forskolin in producing sialic acid.

[0014] In a third aspect, the present invention provides the use of Forskolin in increasing the sialic acid content in milk.

[0015] In a fourth aspect, the present invention provides a method for regulating sialic acid synthesis by mammary epithelial cells in vitro, wherein mammary epithelial cells are cultured using Forskolin, wherein the concentration of Forskolin is 0.3-30 μM.

[0016] Furthermore, when the mammary epithelial cells are non-human mammary epithelial cells, the concentration of Forskolin is 3-30 μM.

[0017] Furthermore, the concentration of Forskolin is 3 μM.

[0018] Furthermore, the culturing time is at least 24 hours.

[0019] Furthermore, the method of culturing mammary epithelial cells using Forskolin includes culturing mammary epithelial cells using a culture medium supplemented with Forskolin.

[0020] The invention provides an application of Forskolin in promoting mammary epithelial cells to synthesize sialic acid. The inventors have proved through experiments that the use of Forskolin to treat mammary epithelial cells can significantly promote the content of sialic acid in the cell culture medium. It has been proved that Forskolin can promote mammary epithelial cells to synthesize sialic acid. A new way to regulate sialic acid secretion is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The results of the detection of sialic acid content in bovine MAC-T cell culture fluid treated with different concentrations of Forskolin provided in Example 1 of the present invention;

[0023] Figure 2The results of the detection of sialic acid content in the culture medium of human MCF-10A cells treated with different concentrations of Forskolin provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings 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 external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0025] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0026] In one aspect, the present invention provides the use of Forskolin in promoting mammary epithelial cells to synthesize sialic acid.

[0027] The inventors have proved through experiments that using Forskolin to treat mammary epithelial cells can significantly promote the content of sialic acid in the cell culture medium. It has been proved that Forskolin can promote the synthesis of sialic acid in mammary epithelial cells, providing a new way to regulate the secretion of sialic acid.

[0028] In some specific embodiments, the mammary epithelial cells are from a mammal.

[0029] The mammals in the present invention include animals that can secrete milk to feed their young.

[0030] In mammary epithelial cells from different mammals, the synthesis pathway of sialic acid generally involves glucose metabolism, hexosamine pathway, synthesis of CMP-sialic acid, and regulation of mTOR signaling pathway, and its synthesis pathway has high similarity among different species. In some specific embodiments, the mammal includes any one of human, cattle, sheep, camel, horse, donkey, reindeer, alpaca or llama.

[0031] Forskolin treats mammary epithelial cells and successfully promotes the secretion of sialic acid in the mammary epithelial cells. According to another aspect of the present invention, the use of Forskolin in the production of sialic acid is also provided.

[0032] Forskolin regulates the secretion of sialic acid in mammary epithelial cells, thereby increasing the sialic acid content in breast milk, and has potential application value, especially in the field of breast milk quality optimization and breast health research. According to another aspect of the present invention, there is also provided the use of Forskolin in increasing the sialic acid content in breast milk.

[0033] According to another aspect of the present invention, a method for regulating sialic acid synthesis by mammary epithelial cells in vitro is provided, wherein the mammary epithelial cells are cultured using Forskolin, wherein the concentration of the Forskolin is 0.3-30 μM.

[0034] The concentration of Forskolin may be, but is not limited to, 0.3 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, 10 μM, 13 μM, 15 μM, 20 μM, 23 μM, 25 μM or 30 μM, and may be any concentration value between 0.3 and 30 μM.

[0035] In some specific embodiments, when the mammary epithelial cells are non-human mammary epithelial cells, the concentration of Forskolin is 3-30 μM.

[0036] In some specific embodiments, the culturing time is at least 24 hours.

[0037] In some specific embodiments, the concentration of Forskolin is 3 μM, which promotes the maximum amount of sialic acid synthesis in mammary epithelial cells.

[0038] In some specific embodiments, the culturing mammary epithelial cells using Forskolin comprises culturing mammary epithelial cells using a culture medium supplemented with Forskolin.

[0039] Among them, the culture medium with Forskolin added is a lactation culture medium, which includes serum-free culture medium, prolactin, ITS (cell culture additive composed of insulin, transferrin and selenium), hydrocortisone, EGF (epidermal growth factor), insulin and estrogen. Before culturing mammary epithelial cells, the mammary epithelial cells can also be starved for 12 hours when the cells are attached and fused to 80% using a basal culture medium (serum-free culture medium).

[0040] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Bovine MAC-T cells: from Shanghai Cell Bank;

[0042] Human MCF-10A cells: from Shanghai Jinyuan Biotechnology Co., Ltd.

[0043] Forskolin: purchased from MCE (MedChemExpress);

[0044] Basic culture medium: Huakan Bio serum-free basic culture medium (Cat. No. RMZ112-PYJ);

[0045] Lactation culture medium: Huakan Bio serum-free basal medium + prolactin, ITS, hydrocortisone, EGF, insulin and estrogen.

[0046] Example 1

[0047] 1. Cell culture: Bovine MAC-T cells and human MCF-10A cells were cultured with basal culture medium until the cells adhered to the wall and were fused to 80%. After starvation treatment for 12 hours, they were switched to lactation culture medium.

[0048] 2. Experimental methods: Bovine MAC-T cells and human MCF-10A cells were divided into 6 groups, 1 group was the control group, and cells were treated with 0.1% DMSO, and 5 groups were experimental groups, and cells were treated with lactation medium containing 0.3μM, 1μM, 3μM, 10μM and 30μM concentrations of Forskolin. The treatment time was 24 hours, and the cell culture fluid was collected by centrifugation. The supernatant was hydrolyzed with sialic acid (sigma, N8271), and the total sialic acid content in the cell culture fluid was detected by combining the sialic acid kit (abcam, ab83375).

[0049] The results are as follows Figure 1 and Figure 2 As shown. In human MCF-10A cells, there were significant differences in the concentrations of 0.3μM~30μM compared with the control group; in bovine MAC-T cells, there were significant differences in the concentrations of 3μM~30μM compared with the control group. Among them, the sialic acid content in the 3μM Forskolin treatment group was the highest, and the sialic acid level decreased compared with the 3μM group under the treatment of 10μM and 30μM. Through the treatment of Forskolin, the secretion of sialic acid in mammary epithelial cells was successfully promoted, especially at a concentration of 3μM.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Forskolin in promoting sialic acid synthesis in mammary epithelial cells.

2. The use according to claim 1, characterized in that: The mammary epithelial cells are from mammals.

3. The use according to claim 2, characterized in that: The mammal includes any one of humans, cattle, sheep, camels, horses, donkeys, reindeer, alpacas or llamas.

4. Application of Forskolin in the production of sialic acid.

5. Application of Forskolin in increasing the sialic acid content in breast milk.

6. A method for regulating sialic acid synthesis in mammary epithelial cells in vitro, characterized in that: Forskolin is used to culture mammary epithelial cells, wherein the concentration of Forskolin is 0.3-30 μM.

7. The method according to claim 6, characterized in that When the mammary epithelial cells are non-human mammary epithelial cells, the concentration of Forskolin is 3-30 μM.

8. The method according to claim 6, characterized in that The concentration of Forskolin was 3 μM.

9. The method according to claim 6, characterized in that The culturing time is at least 24 hours.

10. The method according to any one of claims 6 to 9, characterized in that: The method of culturing mammary epithelial cells using Forskolin includes culturing mammary epithelial cells using a culture medium supplemented with Forskolin.

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