Synthetic method and application of mammary epithelial cell casein regulated and controlled by gene and signal channels

Abstract

The invention provides a synthetic method of mammary epithelial cell casein regulated and controlled by gene and signal channels, which comprises the following steps: 1, cell culture and proliferation: selecting mammary epithelial cells, adding 10% of fetal calf serum (FBS) and 1% of antibiotics into a basic culture medium, and culturing to 80% of confluence degree; the second step, specific allogenic substances and valine are added, specifically, the specific allogenic substances and valine are further added into the culture medium, culture continues to be conducted for 48 hours, it is ensured that the gene is fully expressed, and the specific allogenic substances specifically comprise a CSN1S1 interference agent, an mTOR signal channel accelerant and diallyl disulfide; and step 3, cell treatment and sample collection. According to the method, the casein synthesis rate of the mammary epithelial cells can be increased; and the total amount of casein synthesized by mammary epithelial cells is increased.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for synthesizing mammary epithelial cell casein regulated by genes and signal pathways. Background Art

[0002] Casein, as one of the important proteins secreted by mammary epithelial cells, has important application value in many fields such as food, medicine and cosmetics. However, during the in vitro culture of mammary epithelial cells, the synthesis rate of casein is generally low, resulting in its synthesis amount often only reaching the level of micrograms per milliliter (μg / mL) or nanograms per milliliter (ng / mL), even lower than the sensitivity threshold of conventional detection technology. This low yield and low rate limitation seriously affects the accuracy of experimental analysis and restricts subsequent applications.

[0003] At present, in order to improve the casein synthesis level of mammary epithelial cells, the following methods are mainly used:

[0004] Prolong the culture cycle: Increase the total amount of mammary epithelial cells synthesized by extending the culture time. Although this method is simple, it is inefficient, and the extension of the culture cycle will significantly increase the economic and time costs. At the same time, long-term cell culture may bring uncontrollable experimental variables, increase the uncertainty of experimental results, and affect the reliability of data.

[0005] Scaling up cell culture: Increasing the total casein production by increasing the volume of culture medium and the number of cells. However, this approach requires larger culture equipment and more resources, and is difficult to scale up efficiently under laboratory conditions. In addition, due to the metabolic characteristics of mammary epithelial cells, the expansion of culture scale is often limited by the balance between cell density and nutrient supply.

[0006] Adding promoters: Adding certain nutrients or growth factors, such as amino acids, vitamins or growth factors (such as prolactin), to the culture medium to increase the metabolic activity of cells. Although these additives can promote casein synthesis to a certain extent, the single effect is limited and the concentration and addition time need to be precisely controlled.

[0007] At the genetic level, casein synthesis in mammary epithelial cells is regulated by multiple genes. Among them, the CSN1S1 gene is a key gene encoding αS1-casein and is directly involved in the synthesis and secretion of casein. Studies have shown that the expression level of the CSN1S1 gene is negatively correlated with the amount of casein synthesis. However, the high expression of the CSN1S1 gene is often restricted by transcriptional and post-translational regulation. Therefore, regulating the expression of the CSN1S1 gene may become an important means to increase casein production.

[0008] Existing studies have found that RNA interference (RNAi) technology has important applications in gene regulation. By targeting the CSN1S1 gene, its expression level can be significantly affected. Adding a specific concentration of amino acids to the culture medium of mammary epithelial cells can produce a negative correlation effect with the expression of the CSN1S1 gene, thereby indirectly increasing the synthesis of casein. Further combining the RNA interference technology of the CSN1S1 gene can significantly improve the efficiency of casein synthesis in mammary epithelial cells through synergistic effects.

[0009] In addition, genes with similar functions to CSN1S1, such as CSN1S2 (αS2-casein gene), CSN2 (β-casein gene) and CSN3 (κ-casein gene), are also important genes for regulating casein secretion by mammary epithelial cells. By regulating the expression of these genes, the synthesis rate and total amount of casein can also be affected to varying degrees. However, the current research on the regulation of CSN1S1 and its similar genes is still in its initial stage, and no efficient and operational technical solutions have yet been formed.

[0010] In summary, the efficiency of mammary epithelial cells in casein synthesis is still a current technical difficulty. By optimizing culture conditions, combining gene regulation technology, especially the regulation of CSN1S1 and its related genes, and designing synergistic culture methods, it is expected to significantly increase the synthesis rate and total amount of casein, thereby providing reliable technical support for industrial production. Summary of the invention

[0011] The purpose of the present invention is to provide a method for synthesizing casein in mammary epithelial cells by jointly regulating genes and signal pathways. By optimizing culture conditions and combining gene and signal pathway regulation technology, especially regulating CSN1S1 and its related genes, the rate of casein synthesis in mammary epithelial cells can be increased; and the total amount of casein synthesized by mammary epithelial cells can be increased.

[0012] In order to solve the above technical problems, the present invention provides a method for synthesizing casein in mammary epithelial cells by combined regulation of genes and signal pathways, which comprises the following steps:

[0013] Step 1: Cell culture proliferation: mammary epithelial cells were selected, 10% fetal bovine serum (FBS) and 1% antibiotics were added to the basal culture medium, and cultured to 80% confluence;

[0014] Step 2: Adding specific exogenous substances and valine: adding specific exogenous substances and valine that increase the expression of casein to the culture medium, and continuing to culture for 48 hours to ensure that the gene is fully expressed. The specific exogenous substances specifically include CSN1S1 disruptors, mTOR signaling pathway promoters, and diallyl disulfide.

[0015] The third step is cell processing and sample collection.

[0016] The added amount of the specific exogenous substances is 25-50 μg / mL for CSN1S1 interfering agent, 5-10 μg / mL for mTOR signaling pathway promoter, and 5-10 μg / mL for diallyl disulfide.

[0017] Wherein, the added amount of valine is 6.4mmol / L-12.8mmol / L.

[0018] In the first step, the oxygen volume concentration in the culture environment is set to 21%, and in the second step, the oxygen volume concentration is adjusted to 18%.

[0019] The volume concentration of carbon dioxide is maintained at 5% during the first and second step cultivation processes.

[0020] Wherein, the C / N ratio of the culture medium in the first step is 20:1-30:1, and the C / N ratio of the culture medium in the second step is 15:1 to 20:1.

[0021] Beneficial Effects of the Invention

[0022] The present invention significantly increases the expression of casein by adding specific exogenous substances (CSN1S1 disruptors, mTOR signaling pathway promoters, diallyl disulfide) and valine to the culture medium to regulate the metabolic pathway of mammary epithelial cells. Through the optimized addition strategy, these exogenous factors can enhance the protein synthesis ability of cells and improve the synthesis efficiency and secretion level of casein. DETAILED DESCRIPTION

[0023] Although mammary epithelial cells can secrete a large amount of casein, they may not be able to naturally maintain efficient casein synthesis when cultured in vitro. The reason is the lack of activation of the necessary signal pathways or the lack of exogenous incentives. The present invention can effectively activate the key pathways in these cells by exogenously adding CSN1S1 disruptors, mTOR signaling pathway promoters and diallyl disulfide, thereby enhancing casein synthesis and overcoming the disadvantages of mammary epithelial cells synthesizing casein in vitro culture.

[0024] CSN1S1 is one of the genes mainly responsible for synthesizing casein in mammary epithelial cells. Studies have shown that CSN1S1 is highly expressed in mammary tissue and is a direct control factor for casein synthesis in mammary cells. Under the condition of adding a specific concentration of valine, the synthesis of casein can be directly enhanced by exogenously adding a CSN1S1 interfering agent. Preferably, the CSN1S1 interfering agent is CSN1S1 siRNA.

[0025] The mTOR signaling pathway regulates cell growth, proliferation, survival, and protein synthesis. In mammary epithelial cells, the mTOR signaling pathway regulates multiple links in the translation process by activating downstream key factors such as S6K1 (ribosomal protein S6 kinase 1) and 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1), thereby promoting protein synthesis. Casein, as an important secretory protein of mammary epithelial cells, is significantly dependent on the activity of the mTOR signaling pathway. Exogenous addition of mTOR signaling pathway promoters, such as rapamycin derivatives or mTOR signaling pathway activators, can indirectly enhance the protein synthesis capacity of mammary epithelial cells by increasing the activity of the mTOR signaling pathway. This chemical activation mechanism can more efficiently promote the phosphorylation of translation-related factors, enhance the cell's ability to regulate casein synthesis, and improve the metabolic activity and functional state of mammary epithelial cells.

[0026] Diallyl disulfide is a natural compound found in garlic. Studies have shown that it may affect cell function and gene expression through a variety of mechanisms. Among them, promoting the expression of C / EBPα is one of its mechanisms of action. C / EBPα is a transcription factor that is involved in regulating a variety of cellular processes, including cell differentiation, proliferation, and metabolism. It plays an important role in mammary gland development and has also been found to promote the expression of milk proteins such as casein by regulating the transcriptional activity of mammary epithelial cells. As a transcription factor, C / EBPα can directly regulate the expression of casein genes. The addition of diallyl disulfide can increase the transcription level of casein genes in mammary epithelial cells by increasing the expression level of C / EBPα, thereby enhancing casein synthesis.

[0027] Valine is a branched-chain amino acid (BCAA) that is known to promote protein synthesis. Studies have shown that BCAA can promote protein synthesis by activating the mTOR pathway, especially during cell division and growth. In mammary epithelial cells, valine can increase the expression of casein through the following mechanism.

[0028] The present invention can effectively change the disadvantage of casein expression of mammary epithelial cells in in vitro culture by adding specific exogenous substances (such as CSN1S1 interfering agents, mTOR signaling pathway promoters, diallyl disulfide) and amino acids (such as valine): under the condition of adding a specific valine concentration, the exogenous substance CSN1S1 interfering agent is added to interfere with CSN1S1 directly regulating the synthesis of casein, while mTOR and C / EBPα further enhance the expression of casein by activating protein synthesis and transcription mechanisms. Valine, as a branched chain amino acid, can promote protein synthesis, especially when the mTOR pathway is activated, further enhancing the synthesis of casein. Through this combined strategy, the expression of casein in mammary epithelial cells is significantly improved, overcoming their natural disadvantages in in vitro culture, enabling them to synthesize and secrete casein more effectively.

[0029] Based on this, the present invention provides a method for synthesizing casein in mammary epithelial cells regulated by genes and signal pathways, which comprises the following steps: a first step, cell culture proliferation: selecting mammary epithelial cells, adding 10% fetal bovine serum (FBS) and 1% antibiotics to the basal culture medium, and culturing to 80% confluence; a second step, adding specific exogenous substances and valine: further adding specific exogenous substances and valine that increase the expression of casein to the culture medium, and continuing to culture for 48 hours to ensure sufficient gene expression, the specific exogenous substances specifically include CSN1S1 disruptors, mTOR signaling pathway promoters, and diallyl disulfide; a third step, cell processing and sample collection. The added amount of the specific exogenous substance is 25-50 μg / mL of CSN1S1 interfering agent, 5-10 μg / mL of mTOR signaling pathway promoter, and 5-10 μg / mL of diallyl disulfide. It is further preferred that the CSN1S1 interfering agent concentration is 50 μg / mL, the mTOR signaling pathway promoter concentration is 10 μg / mL, the diallyl disulfide concentration is 10 μg / mL, and the valine added concentration is preferably 6.4 mmol / L-12.8 mmol / L.

[0030] Mammary epithelial cells require adequate oxygen supply during proliferation and differentiation to maintain their normal metabolic activities and protein synthesis. By adjusting the oxygen concentration, the respiration and ATP production of cells can be improved, and the synthesis of casein can be promoted. Too high oxygen concentration may cause oxidative stress, while too low oxygen concentration may slow down cell metabolism and affect protein synthesis. During the initial culture (cell proliferation stage), the oxygen concentration in the culture environment is set to 21%, which is the oxygen concentration in regular air. This concentration can usually support rapid cell growth. After entering the casein expression stage, the oxygen concentration can be gradually adjusted to 18% to reduce the negative effects of excess oxygen, improve the metabolic efficiency of cells, and promote the synthesis of intracellular proteins.

[0031] Cells produce carbon dioxide during metabolism. Excessive carbon dioxide concentrations will cause the pH of the culture medium to drop, affecting cell growth and protein synthesis. A moderate carbon dioxide concentration can help adjust the pH of the culture medium and maintain a stable physiological environment for the cells. During the cell proliferation stage, the carbon dioxide concentration is set to 5% to keep the pH of the culture medium within the physiological range (usually 7.2-7.4) and prevent the culture medium from becoming too acidic. After the cells enter the casein synthesis stage, a 5% CO2 concentration can continue to be maintained, because a higher carbon dioxide concentration helps maintain the acid-base balance in the cells and avoids affecting the synthesis of cells due to excessive pH fluctuations.

[0032] The basal medium provided by the present invention can be a commercially available medium. In the cell proliferation stage, the C / N ratio of the preferred medium is 20:1-30:1. At this time, the carbon source (glucose) and the nitrogen source (amino acid, glutamine, etc.) are maintained at a relatively high concentration to promote the rapid growth of the cells. After the cells reach 80% confluence, the casein expression stage is entered. At this time, by adjusting the C / N ratio in the medium, preferably adjusting to 15:1 to 20:1, the metabolism of the cells is directed to casein synthesis rather than cell proliferation.

[0033] In addition to adding valine, other amino acids such as glutamine, arginine, leucine, aspartic acid, glycine, etc. may also be further added.

[0034] The following embodiments are used to explain the implementation methods of the present invention in detail, so that the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0035] Effects of different exogenous substances on casein synthesis in mammary epithelial cells

[0036] Cell culture: HC11 mammary epithelial cells (or other suitable mammary cell lines) were selected for culture.

[0037] Culture medium: DMEM / F-12 medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin / streptomycin).

[0038] Reagents: RIPA buffer, valine, gene transfection reagent, ELISA kit, Western Blot reagent, CSN1S1 interfering agent, mTOR signaling pathway promoter and diallyl disulfide.

[0039] Equipment: cell culture incubator, centrifuge, ultrasonic disruptor, immunoblotter, ELISA reader.

[0040] The cells were seeded in 6-well plates and cultured at 37°C, 5% CO2, and 21% O2 until 80% confluence. Different exogenous substances were added, and the oxygen concentration was adjusted to 18%. The cells were cultured for 48 h to ensure that the genes were fully expressed and acted. The cell supernatant and cell lysate were collected for subsequent analysis.

[0041] Collection and treatment of supernatant:

[0042] The cell supernatant in the culture medium was collected, and after low-speed centrifugation (2000 rpm, 10 minutes) to remove cell debris, the supernatant was saved for casein concentration determination.

[0043] Treatment of broken cells:

[0044] The collected cells were lysed with a cell lysis buffer (such as RIPA buffer), the lysed cell solution was centrifuged at high speed (12000 rpm, 15 minutes), and the supernatant (i.e., the broken cell group) was collected for subsequent analysis, such as Western Blot and ELISA determination.

[0045] The experiments were divided into the following groups:

[0046] Comparative Example 1: No exogenous substances were added, and the natural casein synthesis of cells was observed.

[0047] Example 1: Commercially available CSN1S1 disruptor, mTOR signaling pathway promoter, and diallyl disulfide were added to the culture medium to study their effects on casein synthesis. The concentration of the CSN1S1 disruptor was 50 μg / mL, the concentration of the mTOR signaling pathway promoter was 10 μg / mL, and the concentration of diallyl disulfide was 10 μg / mL.

[0048] Example 2: The same as Example 1, except that the concentration of CSN1S1 interfering agent is 25 μg / mL, the concentration of mTOR signaling pathway promoter is 10 μg / mL, and the concentration of diallyl disulfide is 10 μg / mL.

[0049] Comparative Example 2: The same as Example 1, except that the concentration of the CSN1S1 interfering agent is 10 μg / mL, the concentration of the mTOR signaling pathway promoter is 10 μg / mL, and the concentration of diallyl disulfide is 10 μg / mL.

[0050] Comparative Example 3: The same as Example 1, except that the concentration of CSN1S1 interfering agent is 100 μg / mL, the concentration of mTOR signaling pathway promoter is 10 μg / mL, and the concentration of diallyl disulfide is 10 μg / mL.

[0051] Comparative Example 4: The same as Example 1, except that the concentration of the CSN1S1 interfering agent is 50 μg / mL, the concentration of the mTOR signaling pathway promoter is 5 μg / mL, and the concentration of diallyl disulfide is 10 μg / mL.

[0052] Comparative Example 5: The same as Example 1, except that the concentration of the CSN1S1 interfering agent is 50 μg / mL, the concentration of the mTOR signaling pathway promoter is 20 μg / mL, and the concentration of diallyl disulfide is 10 μg / mL.

[0053] Comparative Example 6: The same as Example 1, except that the concentration of the CSN1S1 interfering agent is 50 μg / mL, the concentration of the mTOR signaling pathway promoter is 10 μg / mL, and the concentration of diallyl disulfide is 5 μg / mL.

[0054] Comparative Example 7: The same as Example 1, except that the concentration of the CSN1S1 interfering agent is 50 μg / mL, the concentration of the mTOR signaling pathway promoter is 10 μg / mL, and the concentration of diallyl disulfide is 20 μg / mL.

[0055] Gene addition method:

[0056] Gene vector: Select commercially available recombinant plasmids or synthetic gene fragments to ensure that they can stably exist in the culture medium and can be effectively absorbed by cells.

[0057] Treatment time: After gene addition, cells were cultured for 48 hours to ensure gene expression and effect on casein synthesis.

[0058] Method for adding specific exogenous substances

[0059] CSN1S1 siRNA, product number: SI03208602, was prepared in the form of lipid nanoparticles (LNPs) and added directly to the culture medium.

[0060] mTOR signaling pathway promoter MHY1485, product model: SML0810, purity ≥ 95%, dissolved in an appropriate amount of DMSO and directly added to the cell culture medium.

[0061] Diallyl disulfide, product brand and product number: Sigma, SMB00378, ≥98% (HPLC), dissolved in an appropriate amount of DMSO and directly added to the cell culture medium.

[0062] Timing of addition: Specific exogenous substances were added when the cells were 80% confluent, and the experimental analysis was continued after 48 hours of culture.

[0063] Experimental data collection and analysis

[0064] Gene expression analysis: The expression levels of gene mRNA and protein were analyzed by qRT-PCR and Western Blot.

[0065] Analysis of casein synthesis: The concentration of casein in cell culture medium was determined using the ELISA method.

[0066] Cell survival analysis: MTT assay was used to evaluate the survival rate of cells under different treatment conditions.

[0067] Each experiment in the embodiments and comparative examples was repeated 3 times and the average value was taken.

[0068] Results presentation and data analysis

[0069] Gene expression (mRNA) analysis: The mRNA expression levels of CSN1S1, mTOR, and C / EBPα genes were analyzed by real-time quantitative PCR (qRT-PCR). The results are shown in Table 1.

[0070] Table 1

[0071] Group CSN1S1 (mRNA expression) mTOR (mRNA expression) C / EBPα (mRNA expression) Example 1 1.12 1.05 2.15 Example 2 1.05 2.03 1.05 Comparative Example 1 1.00 1.00 1.00 Comparative Example 2 2.08 1.02 1.12 Comparative Example 3 2.11 2.10 1.05 Comparative Example 4 1.05 1.10 1.02 Comparative Example 5 1.02 2.03 1.05 Comparative Example 6 1.17 1.05 0.95 Comparative Example 7 1.06 1.02 2.08

[0072] CSN1S1 is one of the main genes for casein synthesis, which directly affects the casein synthesis ability of mammary epithelial cells. In theory, excessive or low expression of CSN1S1 will affect casein synthesis. Excessive CSN1S1 concentration may lead to excessive casein accumulation, causing stress response in cells, and thus affecting the synthesis and secretion process. As a regulator of cell growth and protein synthesis, mTOR usually promotes casein synthesis by regulating translation mechanisms. If its concentration is too high, it may activate negative feedback mechanisms, inhibit other related signaling pathways, and thus affect casein synthesis; while if the concentration is too low, it may not be able to effectively activate protein synthesis. C / EBPα is a transcription factor that can regulate cell differentiation and thus affect casein synthesis. Excessive C / EBPα concentration may lead to excessive cell metabolic load and inhibit casein synthesis; while if the concentration is too low, it may not be able to effectively activate the differentiation process, thus affecting casein production.

[0073] CSN1S1 is a core gene for casein synthesis, and its expression level directly affects the efficiency of casein synthesis. CSN1S1 is a gene specifically expressed in mammary epithelial cells and is the main component of casein. It provides the basic framework for casein synthesis by participating in the formation and secretion of mammary alveoli. Therefore, the concentration of CSN1S1 needs to be maintained in an appropriate range. Excessive CSN1S1 expression (such as in Comparative Examples 2 and 3) may cause endoplasmic reticulum stress, making it impossible for cells to effectively process excessive casein, thereby inhibiting the correct folding, secretion and accumulation of casein, resulting in decreased synthesis efficiency. In Example 1 (CSN1S1: 1.12), the expression level of CSN1S1 is moderate, which can fully activate casein synthesis without causing cell stress, thereby ensuring the effective synthesis of casein.

[0074] The role of mTOR is mainly through regulating the protein synthesis of cells. As a key signaling pathway, mTOR activates the protein synthesis process and promotes the synthesis of casein by sensing environmental factors such as nutrients, oxygen, and energy. By regulating the mTOR signaling pathway activator MHY1485, the activity of the mTOR signaling pathway is enhanced, thereby promoting cell growth, proliferation, and protein synthesis. When the expression level of mTOR (such as 1.05 in Example 1) is at a moderate level, it can effectively coordinate CSN1S1 to enhance the protein synthesis ability of cells. However, the excessive expression of mTOR (such as in Comparative Example 3) may trigger an unnecessary negative feedback mechanism, thereby inhibiting the synthesis of casein.

[0075] C / EBPα, as a transcription factor, mainly functions to regulate the differentiation process of cells. In mammary epithelial cells, C / EBPα promotes the differentiation of mammary cells and casein synthesis. By adding diallyl disulfide, the expression of C / EBPα may be indirectly promoted by regulating related signaling pathways, thereby enhancing the synthesis of casein in mammary epithelial cells. In this experiment, high expression of C / EBPα (such as 2.15 in Example 1) enhanced cell differentiation and further improved the synthesis of casein. On the contrary, too low expression of C / EBPα may lead to limited cell differentiation and poor casein synthesis.

[0076] Protein expression analysis: The expression levels of CSN1S1, mTOR and C / EBPα proteins were detected by Western Blot technology. The results are shown in Table 2.

[0077] Table 2

[0078]

[0079]

[0080] As a core gene, CSN1S1 plays a decisive role in casein synthesis. Moderate CSN1S1 expression (such as 1.82 in Example 1) can effectively promote casein synthesis, while excessive expression (such as 2.12 in Comparative Example 3) may lead to endoplasmic reticulum stress or excessive accumulation of casein, thereby inhibiting its synthesis efficiency. Too low CSN1S1 expression will also limit casein synthesis, showing the key role of CSN1S1 in casein synthesis.

[0081] mTOR also plays an auxiliary role in regulating casein synthesis. In some groups (such as 1.83 in Comparative Example 5), high expression of mTOR can enhance protein synthesis, but excessive mTOR expression may cause cellular metabolic burden and inhibit normal synthesis process. Therefore, the concentration of mTOR should be moderate to avoid over-activation.

[0082] As a transcription factor, C / EBPα can further promote casein synthesis by regulating cell differentiation. Higher C / EBPα expression (such as 1.80 in Comparative Example 7) can enhance synthesis, but too high expression may also bring negative effects and affect the metabolic balance of cells.

[0083] In general, a balance needs to be maintained between the protein expression levels of CSN1S1, mTOR, and C / EBPα. Too high or too low expression will have an adverse effect on casein synthesis. The best protein synthesis effect occurs when the expression levels of these genes are in a moderate range, especially the expression of CSN1S1 needs to be at an optimized level to maximize casein synthesis.

[0084] Casein synthesis analysis: ELISA was used to determine the casein concentration in the supernatant and disrupted cell fluid. The results are shown in Table 3.

[0085] Table 3

[0086] Group Supernatant group (μg / mL) Broken cell group (μg / mL) Example 1 79.23 89.47 Example 2 69.58 84.32 Comparative Example 1 55.35 70.10 Comparative Example 2 60.41 74.97 Comparative Example 3 73.67 84.56 Comparative Example 4 74.82 85.21 Comparative Example 5 68.44 79.34 Comparative Example 6 64.52 79.98 Comparative Example 7 69.87 84.62

[0087] Too high or too low gene concentrations will have a negative impact on casein synthesis. Too high gene concentrations may cause cell stress, inhibit the correct folding and secretion of casein, and even induce cell death or autophagy. Too low gene concentrations cannot effectively activate the casein synthesis mechanism, resulting in insufficient synthesis. CSN1S1 is a key gene for casein synthesis.

[0088] In Comparative Example 3 (CSN1S1 100 μg / mL, mTOR 10 μg / mL, C / EBPα 10 μg / mL), although the concentration of CSN1S1 is very high, casein synthesis is reduced relative to Example 1. This shows that excessive gene concentration may cause metabolic burden on cells, leading to cell stress response. Specifically, excessively high CSN1S1 concentration may cause endoplasmic reticulum stress, thereby affecting the correct folding, secretion and accumulation of casein. At the same time, mTOR and C / EBPα may promote excessive cell proliferation or differentiation at excessively high concentrations, further increase the metabolic burden of cells, and inhibit the normal synthesis of casein. That is, excessively high gene expression concentration not only hinders the synthesis of casein, but may also induce cell autophagy or death, inhibiting the normal biosynthesis process.

[0089] In Comparative Example 2, although casein synthesis increased, the effect was relatively weak. This phenomenon shows that when the gene concentration is insufficient, the translation mechanism of the cell is not fully activated and the synthesis of casein fails to reach the optimal level. The low concentrations of mTOR and C / EBPα limit the activation of protein synthesis and fail to significantly promote casein synthesis. Therefore, the concentration of the gene needs to be maintained within an appropriate range to ensure its effective promotion of casein synthesis.

[0090] mTOR is an important signaling protein that promotes protein synthesis, especially during cell growth and division. It can promote casein synthesis by activating protein synthesis-related pathways, such as the PI3K / Akt / mTOR pathway. However, when the concentration of mTOR is too high, it may also trigger negative feedback regulation, leading to adverse metabolic reactions, such as excessive cell proliferation and even cell death. C / EBPα is a key transcription factor that regulates cell differentiation and metabolic processes. C / EBPα has a synergistic effect on casein synthesis, especially when it works with mTOR and CSN1S1. Its high concentration may cause excessive cell differentiation or increased metabolic load, thereby inhibiting casein synthesis.

[0091] The effect is best in Example 1, which can give full play to the synergistic effect of the three, and the casein synthesis is the highest. In Comparative Example 5, when mTOR is too high, and in Comparative Example 7, when C / EBPα is too high, casein synthesis does not increase significantly further, but is slightly lower than the moderate concentration group, which indicates that too high a concentration may cause cell stress or metabolic burden, thereby affecting casein synthesis. In Comparative Example 4, when mTOR is too low, and in Comparative Example 6, when C / EBPα is too low, casein synthesis decreases significantly, especially in the supernatant group and the broken cell group, indicating that low concentrations of mTOR or C / EBPα are not enough to activate an effective protein synthesis mechanism.

[0092] Cell viability analysis results are shown in Table 4.

[0093] Table 4

[0094] Group Cell survival rate (%) Example 1 92.35 Example 2 91.60 Comparative Example 1 90.00 Comparative Example 2 91.00 Comparative Example 3 90.50 Comparative Example 4 91.50 Comparative Example 5 90.90 Comparative Example 6 92.00 Comparative Example 7 89.80

[0095] The cell survival rates in Examples 1 and 2 are relatively high, which may be related to the reasonable deployment of the three gene concentrations. Example 1 (CSN1S1 50 μg / mL, mTOR 10 μg / mL, C / EBPα 10 μg / mL) provides the best gene concentration combination, supports casein synthesis without causing obvious cell stress, and the cell survival rate is 92.35%. In Example 2, the concentration of CSN1S1 is slightly reduced, but the survival rate is still relatively high (91.60%), indicating that CSN1S1 can still effectively promote casein synthesis at a lower concentration without significantly affecting cell survival. In Comparative Example 3, the concentration of CSN1S1 is increased to 100 μg / mL, which may lead to the occurrence of cell stress response, so the cell survival rate drops to 90.50%. Excessively high CSN1S1 concentrations may induce endoplasmic reticulum stress, inhibit the normal folding and secretion of casein, and thus affect the health of cells. In comparative example 5, the concentration of mTOR increased to 20 μg / mL, which may increase the metabolic burden of cells. Although mTOR can promote protein synthesis, overexpression may cause adverse reactions, thereby slightly reducing cell survival rate (90.90%). In comparative example 7, the concentration of C / EBPα was too high to 20 μg / mL, which may interfere with the normal metabolism and differentiation process of cells, causing the cell survival rate to drop to 89.80%.

[0096] In summary, CSN1S1 is a key gene that promotes casein synthesis: the expression and protein synthesis of CSN1S1 increased significantly, reflecting its core role in casein synthesis in mammary cells. mTOR and C / EBPα have auxiliary effects: although mTOR and C / EBPα have weak effects on casein synthesis when added alone, their combined effect with CSN1S1 can significantly increase casein synthesis, especially in the three-target combined addition group, where the concentration and protein expression of casein reached the maximum. The synergistic effect of the three targets enhances casein synthesis: the combined addition of the three target genes (CSN1S1, mTOR, and C / EBPα) significantly enhanced casein synthesis, not only increasing the mRNA and protein expression levels, but also improving cell survival rate. Different effects of gene expression: different genes have different effects on mRNA expression and protein expression levels. CSN1S1 has the most significant effect on casein synthesis, while the effects of mTOR and C / EBPα are amplified under synergistic effects.

[0097] Effect of valine concentration on casein expression

[0098] Experimental group design:

[0099] Control group (untreated): no specific exogenous substances and valine were added

[0100] Valine concentration group: different concentrations of valine were added, namely: 1.6mmol / L (Example 3), 3.2mmol / L (Example 4), 6.4mmol / L (Example 5), 12.8mmol / L (Example 6), 25.6mmol / L (Example 7), 51.2mmol / L (Example 8), and three target genes (CSN1S1, mTOR and C / EBPα) were added together, and the concentration of each gene was 50μg / mL. After valine was dissolved, it was added to the culture medium, and the treatment time was 48h.

[0101] The cell viability percentages are shown in Table 5.

[0102] Table 5

[0103]

[0104]

[0105] Low concentrations of valine (1.6mmol / L and 3.2mmol / L) can slightly promote cell activity in the initial stage (2 hours to 12 hours), especially between 4 hours and 12 hours. As the culture time increases, cell activity gradually stabilizes, probably because the cells adapt to the supply of valine. Low concentrations of valine have a certain stimulating effect on cell activity, but do not produce excessive stress, so there are fewer negative effects on cells.

[0106] Moderate concentrations of valine (6.4mmol / L and 12.8mmol / L) were effective in promoting cell proliferation and increasing cell activity, especially at 12 hours when cell activity reached the highest value (123.27% and 123.51%, respectively). These concentrations provided sufficient nitrogen sources for cells, promoting cell metabolism and proliferation. However, these concentrations fell back to a certain extent at 48 hours (95.58%). This may be because excessive addition of valine caused some burden on cell metabolism, or that cells showed a certain decline in adaptability after a longer period of culture.

[0107] Treatment with high concentrations of valine (25.6mmol / L and 51.2mmol / L) can promote cell activity in the early stage (2 hours to 12 hours), but it shows a strong negative effect, especially at 48 hours, when cell activity decreased significantly. This suggests that excessive valine concentrations are toxic or inhibitory to cells. Excessive valine concentrations may increase the metabolic stress of cells, thereby inhibiting the normal proliferation and metabolic activity of cells.

[0108] The results of casein synthesis are compared in Table 6.

[0109] Table 6

[0110] Group Supernatant group (μg / mL) Broken cell group (μg / mL) Comparative Example 1 55.35 70.10 Example 3 88.53 94.29 Example 4 91.37 97.82 Example 5 95.03 102.39 Example 6 97.16 105.50 Example 7 98.59 106.83 Example 8 95.31 103.12

[0111] According to the experimental results and data analysis, the addition of valine had a significant positive effect on casein synthesis and cell activity under the combined action of three target genes (CSN1S1, mTOR, C / EBPα). Low to moderate concentrations of valine (1.6mmol / L to 12.8mmol / L) promoted casein synthesis, which was manifested as a steady increase in casein concentration in the supernatant group and the broken cell group, and cell activity was also significantly improved. Within this concentration range, valine enhances the protein synthesis capacity and metabolic activity of cells by activating the mTOR signaling pathway and providing essential amino acids, thereby optimizing the synergistic effect of the three target genes. Especially in the range of 6.4mmol / L to 12.8mmol / L, casein synthesis and cell activity reached the highest values.

[0112] However, when the valine concentration is too high (for example, 25.6 mmol / L and above), the synthesis of casein tends to stabilize or slightly decline, and cell activity begins to decline. This suggests that excessive valine concentration may lead to a burden on intracellular amino acid metabolism, produce a negative feedback effect, inhibit protein synthesis and reduce cell health. Therefore, reasonable control of valine concentration within a certain range is the key to optimizing casein synthesis, while excessive valine concentration may not bring more benefits, but may have adverse effects.

[0113] In summary, the addition of valine and the synergistic effect of the three target genes significantly improved the efficiency of casein synthesis, and the medium concentration of valine showed the best effect. Reasonable adjustment of the concentration of valine can not only promote casein synthesis, but also maintain normal metabolic activity of cells, which is an effective strategy to optimize breast cell protein synthesis.

[0114] All of the above are primary implementations of this intellectual property and do not set limitations on other forms of implementation of this new product and / or new method. Those skilled in the art will use this important information and modify the above content to achieve similar implementations. However, all modifications or transformations based on the new product of this invention belong to the reserved rights.

[0115] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for synthesizing casein in mammary epithelial cells regulated by genes and signal pathways, characterized in that: The following steps are involved: Step 1: Cell culture proliferation: mammary epithelial cells were selected, 10% fetal bovine serum (FBS) and 1% antibiotics were added to the basal culture medium, and cultured to 80% confluence; Step 2: Adding genes and valine: adding specific exogenous substances that increase casein expression and valine to the culture medium, and continuing to culture for 48 hours to ensure that the gene is fully expressed. The specific exogenous substances specifically include CSN1S1 disruptors, mTOR signaling pathway promoters, and diallyl disulfide. The third step is cell processing and sample collection.

2. The method for synthesizing casein in mammary epithelial cells regulated by genes and signal pathways as claimed in claim 1, characterized in that: The added amounts of the specific exogenous substances are as follows: the concentration of CSN1S1 interfering agent is 25-50 μg / mL, the concentration of mTOR signaling pathway promoter is 5-10 μg / mL, and the concentration of diallyl disulfide is 5-10 μg / mL.

3. The method for synthesizing casein in mammary epithelial cells regulated by genes and signal pathways as claimed in claim 1, characterized in that: The added amounts of the specific exogenous substances are as follows: the concentration of CSN1S1 interfering agent is 50 μg / mL, the concentration of mTOR signaling pathway promoter is 10 μg / mL, and the concentration of diallyl disulfide is 10 μg / mL.

4. The method for synthesizing casein in mammary epithelial cells regulated by genes and signal pathways as claimed in claim 1, characterized in that: The added amount of valine is 6.4mmol / L-12.8mmol / L.

5. The method for increasing casein expression in mammary epithelial cells according to claim 1, characterized in that: In the first step, the oxygen volume concentration in the culture environment was set to 21%, and in the second step, the oxygen volume concentration was adjusted to 18%.

6. The method for synthesizing casein in mammary epithelial cells regulated by genes and signal pathways as claimed in claim 1, characterized in that: The volume concentration of carbon dioxide was maintained at 5% during the first and second step cultivations.

7. The method for synthesizing casein in mammary epithelial cells regulated by genes and signal pathways as claimed in claim 1, characterized in that: The C / N ratio of the culture medium in the first step is 20:1-30:1, and the C / N ratio of the culture medium in the second step is 15:1 to 20:

1.

8. A composite additive for use in the method for synthesizing casein in mammary epithelial cells regulated by genes and signal pathways as claimed in any one of claims 1 to 7, characterized in that: include: Specific exogenous substances and valine, the specific exogenous substances specifically include CSN1S1 interfering agent, mTOR signaling pathway promoter and diallyl disulfide, the added amount is CSN1S1 interfering agent concentration of 25-50μg / mL, mTOR signaling pathway promoter concentration of 5-10μg / mL, diallyl disulfide concentration of 5-10μg / mL, and the added amount of valine is 6.4mmol / L-12.8mmol / L.

9. Application of the method for mammary epithelial cell casein synthesis regulated by the gene and signal pathway according to claims 1 to 7 in food processing, dairy product production, biopharmaceuticals, and animal feed production.

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