Intramuscular adipogenesis mechanism analysis method, regulatory factor and evaluation method

Abstract

The invention discloses an intramuscular adipogenesis mechanism analysis method, a regulatory factor and an evaluation method, and the method comprises the following steps: constructing a co-culture system of bovine adipocytes and skeletal muscle cells, and establishing a single culture control group; carrying out whole transcriptome sequencing on the co-cultured adipocytes and adipocytes of a control group, and enriching and analyzing a differential signal pathway by combining a KEGG pathway; analyzing that a lysosome signal channel is a key regulatory factor in adipocyte differentiation, respectively activating and inhibiting differential signal channels through drug intervention of an activator and an inhibitor, and observing the adipocyte differentiation condition; and the effect of the differential signal pathway in adipose differentiation is evaluated. The intramuscular adipogenesis mechanism is analyzed, a method is provided for exploring a differential signal channel which plays a key role in adipose differentiation, a new target spot is provided for treating obesity and related metabolic diseases, new therapeutic drugs are convenient to screen and develop, and a new method is provided for clinical treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of cell co-culture, and relates to an analysis method for the mechanism of intramuscular fat formation, a regulating factor and an evaluation method. Background Art

[0002] As consumer demand for high-quality beef continues to grow, optimizing fat deposition and improving meat quality have become core issues in animal husbandry research. Intramuscular fat (IMF) is considered a key quality indicator affecting meat quality. The formation of IMF involves a complex differentiation process of bovine adipocytes (BAds) and bovine skeletal muscle cells (BSMCs), both of which are derived from mesenchymal stem cells (MSCs) in the mesoderm. Traditional single-cell culture systems cannot accurately replicate the dynamic cell-to-cell interactions that occur in vivo, hindering a comprehensive understanding of the complex communication between BAds and BSMCs. To address this limitation, the current study used a cell culture insert device (Transwell) co-culture system to better simulate the physiological interactions between these cell types.

[0003] On the one hand, maintaining an ideal cell ratio and microenvironmental balance when establishing a co-culture model is a technical challenge. Differences in survival and growth rates of different cell types may lead to instability in the model. In addition, the medium requirements of the co-culture system are complex, and interactions between cells may affect the stability and repeatability of the experimental results. On the other hand, the mechanism of intramuscular fat formation is still in the research stage and needs to be further interpreted. Summary of the invention

[0004] In view of the above problems, the present invention proposes an analysis method, regulatory factors and evaluation method for the mechanism of intramuscular fat formation, which well solve the problems in the prior art.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for analyzing the mechanism of intramuscular fat formation, comprising:

[0007] S1. Construct a co-culture system of bovine adipocytes and skeletal muscle cells, simulate the in vivo microenvironment, co-culture bovine adipocytes and skeletal muscle cells, observe the interaction between cells, establish a control group, culture bovine adipocytes alone, and the culture process is the same as the co-culture system;

[0008] S2. Whole transcriptome sequencing was performed on the co-cultured adipocytes and the control group adipocytes, and differential signaling pathways were analyzed in combination with KEGG pathway enrichment analysis.

[0009] S3. Activate and inhibit different signaling pathways through intervention with activator and inhibitor drugs, and observe the differentiation of adipocytes;

[0010] S4. Evaluate the role of differential signaling pathways in adipogenic differentiation.

[0011] Optionally, the S1 includes:

[0012] S11, inoculating bovine adipocytes and skeletal muscle cells at a ratio of 1:2, placing the adipocytes in the upper chamber of the cell culture insert, and placing the skeletal muscle cells in the lower chamber, and using a high-glucose DMEM culture medium containing 10% FBS;

[0013] S12, after the cells adhere to the wall, assemble the upper and lower chambers of the cell culture insert device to start co-culture, and incubate at 37°C, 5% CO 2 Continue to maintain in the incubator;

[0014] S13, after the cells have grown to full size, collect the cells for total RNA and protein extraction;

[0015] S14, performing step S11 again, after the two types of cells are fully grown, inducing differentiation of the two types of cells respectively, inducing differentiation of bovine skeletal muscle cells using skeletal muscle differentiation medium, and replacing the differentiation induction medium every 2 days;

[0016] S15, when the bovine skeletal muscle cells are induced to differentiate on the 4th day, the bovine adipocytes are induced to differentiate using adipose differentiation medium, and after 2 days of induction of differentiation, the culture medium is replaced with maintenance medium for continued cultivation, and the maintenance medium is replaced every 2 days during the period;

[0017] S16, when the bovine skeletal muscle cells are differentiated on the 8th day and the bovine adipocytes are differentiated on the 4th day, assemble the upper and lower chambers of the cell culture insert device, and continue to co-culture for 4 days, using a high-glucose DMEM culture medium containing 5% FBS, and replace the culture medium every 2 days;

[0018] S17, after the co-culture period is over, the cells are collected for total RNA and protein extraction;

[0019] S18. A control group was set up. Adipocytes were seeded in the upper chamber of the cell culture insert under the same conditions and density as in S11. Only culture medium was set in the lower chamber. The culture process was the same as S11-S17.

[0020] Optionally, the S13 also includes detecting the proliferation effect of bovine adipocytes by EdU staining, flow cytometry, qPCR and WB after the cells are fully grown; the S17 also includes detecting the differentiation effect of co-cultured bovine adipocytes by using Oil Red 0, BODIPY, Nile Red staining, TG determination, qPCR and WB after the co-culture period ends.

[0021] A regulatory factor, wherein the regulatory factor is the above-mentioned regulatory factor of fat cells, and the regulatory factor is a lysosomal signaling pathway.

[0022] An evaluation method, which is the evaluation method of a regulatory factor mentioned above: comprising:

[0023] A. Determine the concentrations of the activator and inhibitor in step S2;

[0024] B. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and the expression of lysosomal signaling pathway and autophagy-related genes was detected by qPCR;

[0025] C. After 4 days of treatment with activators or inhibitors, the culture medium was discarded, the cells were fixed after washing with PBS, and the adipogenesis of cells was detected by staining with Oil Red O, BODIPY, and Nile Red;

[0026] D. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and RNA was extracted, and the expression of adipogenesis-related genes was detected by qPCR;

[0027] E. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and proteins were extracted, and the phosphorylation expression of adipogenesis-related proteins was detected by WB.

[0028] Optionally, step A comprises:

[0029] A1. Cultivate bovine adipocytes at 37°C and 5% CO2 until the confluence is 70-80%, discard the culture medium, wash the cells with PBA, and replace with new complete culture medium;

[0030] A2. Design the concentration gradient of the activator or inhibitor, and set up three replicate wells for each concentration group;

[0031] A3. Dilute the activator or inhibitor to the corresponding working concentration according to the designed concentration gradient, divide the bovine adipocytes into a control group and different concentration drug treatment groups, each group has about 1×10^5 cells / well, add the corresponding concentration of activator or inhibitor to each group, shake the culture plate to evenly distribute the drug, and after the cells are full, induce differentiation of the adipocytes, and replace the cell differentiation induction culture medium containing the drug every two days;

[0032] A4. After 4 days of induction of differentiation, cells from each group were collected, total RNA was extracted, cDNA synthesis and qPCR reaction were performed using a qPCR kit, and specific primers were used to quantitatively analyze genes related to the lysosomal signaling pathway. The optimal concentration of the activator or inhibitor was screened out by comparing the qPCR results of each concentration group and time point.

[0033] Compared with the prior art, the present invention has the following beneficial effects: the analysis of the mechanism of intramuscular fat formation by this method provides a method for exploring differential signaling pathways that play a key role in adipose differentiation. Furthermore, by exploring the key differential signaling pathways in adipose differentiation, new targets are provided for the treatment of obesity and related metabolic diseases, which facilitates the screening and development of new therapeutic drugs and provides new methods for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a model diagram of the co-culture system of bovine bone cells and adipocytes in a cell culture insert device;

[0035] Figure 2 are the EdU staining and quantification images of mono-cultured and co-cultured adipocytes;

[0036] Figure 3 It is the result graph and quantification graph of cell cycle detection and apoptosis detection of mono-cultured and co-cultured bovine adipocytes;

[0037] Figure 4 This is a graph showing the expression changes of marker genes and proteins of bovine adipocyte proliferation in single culture and co-culture;

[0038] Figure 5 It is the Oil Red O, Nile Red and BODIPY staining images of mono-cultured and co-cultured bovine adipocytes, as well as the Oil Red O quantification image and TG content determination result image;

[0039] Figure 6 It is the detection result and quantitative diagram of the expression of marker genes and marker proteins of adipogenic differentiation of single culture and co-cultured bovine adipocytes;

[0040] Figure 7 It is the KEGG bubble diagram and mRNA enrichment circle diagram;

[0041] Figure 8 It is the KEGG enrichment circle map of miRNA;

[0042] Fig. 9 It is the KEGG enrichment circle map of circRNA;

[0043] Fig.10 It is the KEGG enrichment circle map of cireRNA-mRNA interaction;

[0044] Fig.11 It is the KEGG enrichment circle diagram of lncRNA-mRNA interaction;

[0045] Fig.12It is the result graph of the optimal concentration of lysosomal signaling pathway activator EN6 and inhibitor CQ screened by qPCR, and the result graph of the expression changes of lysosomal and autophagy related genes detected by qPCR with activator EN6 and inhibitor CQ;

[0046] Fig.13 is a picture showing the effect of activator EN6 on bovine adipocyte differentiation detected by Oil Red 0, Nile Red and BODIPY staining;

[0047] Fig.14 This is the result graph and quantification graph of the expression changes of adipogenic differentiation marker genes and related proteins in bovine adipocytes by activator EN6 and inhibitor CQ;

[0048] Fig.15 It is a graph showing the effects of Oil Red 0 staining to detect the activator EN6 and Nile Red and BODIPY staining to detect the effects of the inhibitor CQ on bovine adipocyte differentiation. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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.

[0050] The embodiment of the present invention discloses a method for analyzing the mechanism of intramuscular fat formation, comprising the following steps:

[0051] S1. Construct a co-culture system of bovine adipocytes and skeletal muscle cells, simulate the in vivo microenvironment, co-culture bovine adipocytes and skeletal muscle cells, observe the interaction between cells, establish a control group, culture bovine adipocytes alone, and the culture process is the same as the co-culture system;

[0052] S2. Whole transcriptome sequencing was performed on the co-cultured adipocytes and the control group adipocytes, and differential signaling pathways were analyzed in combination with KEGG pathway enrichment analysis.

[0053] S3. Activate and inhibit different signaling pathways through intervention with activator and inhibitor drugs, and observe the differentiation of adipocytes;

[0054] S4. Evaluate the role of differential signaling pathways in adipogenic differentiation.

[0055] Specifically, by establishing a co-culture system of bovine adipocytes and skeletal muscle cells, the cell-cell interactions were observed, and compared with the adipocytes cultured alone in the control group. By sequencing the co-cultured adipocytes and the control group adipocytes, differential signaling pathways were screened, and combined with KEGG pathway enrichment analysis, the mechanism of action of the differential signaling pathways on adipose differentiation was further analyzed and verified, and drug intervention was performed by setting activators and inhibitors to further test the role of the differential signaling pathways.

[0056] In this way, the analysis of the mechanism of intramuscular fat formation by this method provides a method for exploring differential signaling pathways that play a key role in adipose differentiation. Furthermore, by exploring the key differential signaling pathways in adipose differentiation, it provides new targets for the treatment of obesity and related metabolic diseases, facilitates the screening and development of new therapeutic drugs, and provides new methods for clinical treatment.

[0057] As a specific implementation of the method for analyzing the mechanism of intramuscular fat formation provided in the application, please refer to Figure 1 The co-culture system of bovine adipocytes and skeletal muscle cells includes a proliferation system and a differentiation system, specifically including:

[0058] S11, inoculating bovine adipocytes and skeletal muscle cells at a ratio of 1:2, placing the adipocytes in the upper chamber of the cell culture insert, and placing the skeletal muscle cells in the lower chamber, and using a high-glucose DMEM culture medium containing 10% FBS;

[0059] S12, after the cells adhere to the wall, assemble the upper and lower chambers of the cell culture insert device and incubate at 37°C, 5% CO 2 Continue to maintain in the incubator;

[0060] S13, after the cells have grown to full size, collect the cells for total RNA and protein extraction;

[0061] S14, performing step S11 again, inducing differentiation of the two cell types respectively, inducing differentiation of bovine skeletal muscle cells using skeletal muscle differentiation medium, and replacing the differentiation induction medium every 2 days;

[0062] S15, when the bovine skeletal muscle cells are induced to differentiate on the 4th day, the bovine adipocytes are induced to differentiate using adipose differentiation medium, and after 2 days of induction of differentiation, the culture medium is replaced with maintenance medium for continued cultivation, and the maintenance medium is replaced every 2 days during the period;

[0063] S16, when the bovine skeletal muscle cells are differentiated on the 8th day and the bovine adipocytes are differentiated on the 4th day, assemble the upper and lower chambers of the cell culture insert device, and continue to co-culture for 4 days, using a high-glucose DMEM culture medium containing 5% FBS, and replace the culture medium every 2 days;

[0064] S17, after the co-culture period is over, the cells are collected for total RNA and protein extraction;

[0065] S18. A control group was set up. Adipocytes were seeded in the upper chamber of the cell culture insert under the same conditions and density as in S11. Only culture medium was set in the lower chamber. The culture process was the same as S11-S17.

[0066] It should be understood that in steps S11-S13, the proliferation system of the co-culture system is established, and in steps S14-S17, the differentiation system of the co-culture system is established. By establishing a stable co-culture system of bovine adipocytes and bovine skeletal muscle cells, it is convenient to analyze the interaction between cells. Similarly, in step S18, two control groups are set up, and control groups are established for the proliferation system and the differentiation system respectively.

[0067] In some feasible embodiments, the upper chamber of the cell culture insert device (Transwell) is a 0.4 μm pore size polycarbonate membrane, the skeletal muscle differentiation medium is a high-glucose DMEM medium with 2% HS, the adipose differentiation medium is a high-glucose DMEM medium containing 1 μmol / L dexamethasone, 1 μmol / L rosiglitazone, 0.5 mmol / L IBMX, and 10 μg / mL insulin, and the maintenance medium is a high-glucose DMEM medium containing 1 μmol / L rosiglitazone and 10 μg / mL insulin.

[0068] Furthermore, step S13 also includes detecting the proliferation effect of bovine adipocytes by EdU staining, flow cytometry, qPCR and WB after the cells are fully grown; step S17 also includes detecting the differentiation effect of co-cultured bovine adipocytes by using Oil Red O, BODIPY, Nile Red staining, TG determination, qPCR and WB after the co-culture period ends.

[0069] In some feasible methods, detecting the proliferation effect of bovine fat cells includes: after the cells are fully grown, discarding the old culture medium, washing the cells with PBS, adding 4% paraformaldehyde to fix the bovine fat cells, staining the cells with an EdU staining kit, and then taking pictures under an inverted fluorescence microscope to record the fluorescence signal; collecting bovine fat cells for flow cytometry, performing cell cycle detection and cell apoptosis detection respectively; collecting bovine fat cells for total RNA and protein extraction, and performing qPCR and WB to detect the expression of proliferation marker genes and proteins respectively.

[0070] In this example, see Figure 2-Figure 4, respectively, are the EdU staining and quantification of the control group (NC) and co-cultured (co-culture) adipocytes, the cells of the control group (NC) and co-cultured (co-culture) bovine adipocytes, and the expression changes of the proliferation marker genes and marker proteins of the control group (NC) and co-cultured (co-culture) bovine adipocytes. The results show that co-culture inhibits the proliferation of bovine adipocytes. Among them, ** represents P<0.01, that is, there is a very significant difference between the two groups in statistical analysis; * represents P<0.05, that is, there is a significant difference; ns represents P>0.05, that is, there is no significant difference.

[0071] In some feasible methods, detecting the differentiation effect of co-cultured bovine adipocytes includes: after the co-culture period is over, discarding the old culture medium, washing the cells with PBS, fixing the cells with 4% paraformaldehyde, staining with Oil Red O, BODIPY, and Nile Red to detect cell adipogenesis, and taking pictures and recording with a microscope; collecting cells, disrupting the cells, and then detecting them with a TG content determination kit; collecting cells and extracting total RNA and protein, and then detecting the expression of adipogenic marker genes and proteins with qPCR and WB.

[0072] In this example, see Figure 5-Figure 6 , respectively, Oil Red O staining and quantification of bovine adipocytes in the control group (NC) and co-culture, Nile Red staining and BODIPY staining of bovine adipocytes in the control group (NC) and co-culture, TG content determination results of bovine adipocytes in the control group (NC) and co-culture, and adipogenic differentiation marker gene and marker protein expression detection results and quantification of bovine adipocytes in the control group (NC) and co-culture. The results show that co-culture inhibits the differentiation of bovine adipocytes.

[0073] Based on the above method, the present invention also discloses a regulatory factor, which is a key differential signaling pathway in adipocyte differentiation, namely, a lysosomal signaling pathway.

[0074] See also Figure 7-Figure 11 ,By performing whole transcriptome sequencing of the control group and co-culture and combining with KEGG pathway enrichment analysis, the results showed that the lysosomal signaling pathway was a key regulatory factor in the regulation of adipogenesis under co-culture conditions. Figure 7 Bubble chart and Figure 8-Figure 11The circular histogram further confirmed the core role of the lysosomal pathway in adipocyte differentiation. The frequency distribution of the top 15 KEGG signaling pathways was quantitatively analyzed and showed that the lysosomal signaling pathway was highly enriched, which could inhibit lipid deposition under co-culture conditions and provide a new molecular target for improving beef quality.

[0075] Based on the above regulatory factors, the present invention also discloses an evaluation method, comprising:

[0076] A. Determine the concentrations of the activator and inhibitor in step S2;

[0077] B. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and the expression of lysosomal signaling pathway and autophagy-related genes was detected by qPCR;

[0078] C. After 4 days of treatment with activators or inhibitors, the culture medium was discarded, the cells were fixed after washing with PBS, and the adipogenesis of cells was detected by staining with Oil Red 0, BODIPY, and Nile Red;

[0079] D. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and RNA was extracted, and the expression of adipogenesis-related genes was detected by qPCR;

[0080] E. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and proteins were extracted, and the phosphorylation expression of adipogenesis-related proteins was detected by WB.

[0081] Further, step A comprises:

[0082] A1. Cultivate bovine adipocytes at 37°C and 5% CO2 until the confluence is 70-80%, discard the culture medium, wash the cells with PBA, and replace with new complete culture medium;

[0083] A2. Design the concentration gradient of the activator or inhibitor, and set up three replicate wells for each concentration group;

[0084] A3. Dilute the activator or inhibitor to the corresponding working concentration according to the designed concentration gradient, divide the bovine adipocytes into a control group and different concentration drug treatment groups, each group has about 1×10^5 cells / well, add the corresponding concentration of activator or inhibitor to each group, shake the culture plate to evenly distribute the drug, and after the cells are full, induce differentiation of the adipocytes, and replace the cell differentiation induction culture medium containing the drug every two days;

[0085] A4. After 4 days of induction of differentiation, cells from each group were collected, total RNA was extracted, cDNA synthesis and qPCR reaction were performed using a qPCR kit, and specific primers were used to quantitatively analyze genes related to the lysosomal signaling pathway. The optimal concentration of the activator or inhibitor was screened out by comparing the qPCR results of each concentration group and time point.

[0086] In some feasible methods, the activator is EN6 and the inhibitor is CQ. The concentration gradient of EN6 can be set to 1μM, 2.5μM, 5μM, 10μM; the concentration gradient of CQ can be set to 1μM, 2μM, 5μM, 10μM, 20μM, 30μM. Three replicate wells are set to set three independent biological replicates for each experimental condition to ensure the biological reliability and statistical significance of the data.

[0087] See also Fig.12 , is a result diagram of the optimal concentration of lysosomal signaling pathway activator EN6 and inhibitor CQ screened by qPCR, and a result diagram of the expression changes of lysosomal and autophagy related genes detected by qPCR with activator EN6 and inhibitor CQ. The results show that in this embodiment, the optimal concentration of EN6 for treating bovine adipocytes is 5 μM, and the optimal concentration of CQ for treating bovine adipocytes is 10 μM. After EN6 treatment, the activity of lysosomal signaling pathway in bovine adipocytes is effectively activated, and after CQ treatment, the activity of lysosomal signaling pathway in bovine adipocytes is effectively inhibited.

[0088] See also Figure 13-14 , respectively, the figure of the effect of activator EN6 on bovine adipocyte differentiation detected by Oil Red 0 staining, the figure of the effect of activator EN6 on bovine adipocyte differentiation detected by Nile Red and BODIPY staining, and the result figure and quantification figure of the expression change of adipogenic differentiation marker genes and related proteins in bovine adipocytes by activator EN6. The results show that the activation of lysosomal signaling pathway inhibits the differentiation of bovine adipocytes.

[0089] See also Fig.15 , respectively, Oil Red 0 staining to detect the effect of activator EN6 on bovine adipocyte differentiation, Nile Red staining and BODIPY staining to detect the effect of inhibitor CQ on bovine adipocyte differentiation, and inhibitor CQ on the expression changes of adipogenic differentiation marker genes and related proteins in bovine adipocytes and quantitative graphs. The results show that the inhibition of lysosomal signaling pathway activity promotes the differentiation of bovine adipocytes.

[0090] To facilitate the implementation of this embodiment, the sources of cells and reagents involved in this embodiment are also disclosed as follows:

[0091] Bovine adipocytes were obtained by collecting newborn calf adipose tissue and isolating it using the tissue block method; bovine skeletal muscle cells were obtained by collecting newborn calf longissimus dorsi muscle tissue and isolating it using the enzymatic digestion method; PCR amplification primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; PBS, DMEM / High-Glucose, and 0.25% trypsin were purchased from Hyclone; LipofectamineTM3000 transfection reagent and Annexin V-FITC / P cell apoptosis detection kit were purchased from Invigentech; BeyoClickTMEdU-555 cell proliferation detection kit, cell cycle and cell apoptosis detection kit, and 4% paraformaldehyde were purchased from Beyotime; IBMX, dexamethasone, insulin, rosiglitazone, EN6, and chloroquine (CQ) were purchased from Sigma; fetal bovine serum (FBS) was purchased from BI; reverse transcription kit and RNAiso Plus were purchased from Takara; 2×M5Hiper SYBR Premix EsTaq was purchased from Polymer; Oil Red 0 was purchased from Solarbio; BODIPY493 / 503 and Nile Red staining reagents were purchased from Anjieke; triglyceride (TG) determination kit (single reagent GPO-PAP method) (microplate reader and biochemical analyzer) was purchased from Nanjing Jiancheng Bioengineering Institute; anti-PCNA antibody (Shanghai Bioengineering), anti-CDK2 antibody (Abways), anti-FABP4 antibody (Abways), anti-PPARγ antibody (Biyuntian), anti-ERK1 / 2 antibody (Abways), anti-p-ERK1 / 2 antibody (Abways), anti-JNK1 / 2 / 3 antibody (Abways), anti-p-JNK1 / 2 / 3 antibody (Abways), anti-GAPDH (Abways), Goat Anti-Rabbit IgG secondary antibody (Affinity), and other WB-related reagents were purchased from Yamei.

[0092] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing the mechanism of intramuscular fat formation, characterized in that: include: S1. Construct a co-culture system of bovine adipocytes and skeletal muscle cells, simulate the in vivo microenvironment, co-culture bovine adipocytes and skeletal muscle cells, observe the interaction between cells, establish a control group, culture bovine adipocytes alone, and the culture process is the same as the co-culture system; S2. Whole transcriptome sequencing was performed on the co-cultured adipocytes and the control group adipocytes, and differential signaling pathways were analyzed in combination with KEGG pathway enrichment analysis. S3. Activate and inhibit different signaling pathways through intervention with activator and inhibitor drugs, and observe the differentiation of adipocytes; S4. Evaluate the role of differential signaling pathways in adipogenic differentiation.

2. The method for analyzing the mechanism of intramuscular fat formation according to claim 1, characterized in that: The S1 includes: S11, inoculating bovine adipocytes and skeletal muscle cells at a ratio of 1:2, placing the adipocytes in the upper chamber of the cell culture insert, and placing the skeletal muscle cells in the lower chamber, and using a high-glucose DMEM culture medium containing 10% FBS; S12, after the cells adhere to the wall, assemble the upper and lower chambers of the cell culture insert device to start co-culture, and continue to maintain in a 37° C., 5% CO2 incubator; S13, after the cells have grown to full size, collect the cells for total RNA and protein extraction; S14, performing step S11 again, after the two types of cells are fully grown, inducing differentiation of the two types of cells respectively, inducing differentiation of bovine skeletal muscle cells using skeletal muscle differentiation medium, and replacing the differentiation induction medium every 2 days; S15, when the bovine skeletal muscle cells are induced to differentiate on the 4th day, the bovine adipocytes are induced to differentiate using adipose differentiation medium, and after 2 days of induction of differentiation, the culture medium is replaced with maintenance medium for continued cultivation, and the maintenance medium is replaced every 2 days during the period; S16, when the bovine skeletal muscle cells are differentiated on the 8th day and the bovine adipocytes are differentiated on the 4th day, assemble the upper and lower chambers of the cell culture insert device, and continue to co-culture for 4 days, using a high-glucose DMEM culture medium containing 5% FBS, and replace the culture medium every 2 days; S17, after the co-culture period is over, the cells are collected for total RNA and protein extraction; S18. A control group was set up. Adipocytes were seeded in the upper chamber of the cell culture insert under the same conditions and density as in S11. Only culture medium was set in the lower chamber. The culture process was the same as S11-S17.

3. The experimental method of the mechanism of intramuscular fat formation according to claim 2, characterized in that: The S13 also includes detecting the proliferation effect of bovine adipocytes by EdU staining, flow cytometry, qPCR and WB after the cells are fully grown; the S17 also includes detecting the differentiation effect of co-cultured bovine adipocytes by using Oil Red O, BODIPY, Nile Red staining, TG determination, qPCR and WB after the co-culture period ends.

4. A regulatory factor, which is a regulatory factor for adipocytes in a method for analyzing the mechanism of intramuscular fat formation according to claim 1, characterized in that: The regulatory factor is the lysosomal signaling pathway.

5. An evaluation method, the evaluation method being the evaluation method of a regulatory factor according to claim 4: comprising A. Determine the concentrations of the activator and inhibitor in step S2; B. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and the expression of lysosomal signaling pathway and autophagy-related genes was detected by qPCR; C. After 4 days of treatment with activators or inhibitors, the culture medium was discarded, the cells were fixed after washing with PBS, and the adipogenesis of cells was detected by staining with Oil Red O, BODIPY, and Nile Red; D. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and RNA was extracted, and the expression of adipogenesis-related genes was detected by qPCR; E. After 4 days of treatment with activators or inhibitors, bovine adipocytes were collected and proteins were extracted, and the phosphorylation expression of adipogenesis-related proteins was detected by WB.

6. An evaluation method according to claim 5, characterized in that: The step A comprises: A1. Cultivate bovine adipocytes at 37°C and 5% CO2 until the confluence is 70-80%, discard the culture medium, wash the cells with PBA, and replace with new complete culture medium; A2. Design the concentration gradient of the activator or inhibitor, and set up three replicate wells for each concentration group; A3. Dilute the activator or inhibitor to the corresponding working concentration according to the designed concentration gradient, divide the bovine adipocytes into a control group and different concentration drug treatment groups, each group has about 1×10^5 cells / well, add the corresponding concentration of activator or inhibitor to each group, shake the culture plate to evenly distribute the drug, and after the cells are full, induce differentiation of the adipocytes, and replace the cell differentiation induction culture medium containing the drug every two days; A4. After 4 days of induction of differentiation, cells from each group were collected, total RNA was extracted, cDNA synthesis and qPCR reaction were performed using a qPCR kit, and specific primers were used to quantitatively analyze genes related to the lysosomal signaling pathway. The optimal concentration of the activator or inhibitor was screened out by comparing the qPCR results of each concentration group and time point.

Images