A culture medium and its application, and a method for in vitro isolation, culture and adipogenic differentiation of goat intramuscular precursor adipocytes

Abstract

The application discloses a culture medium and application thereof, and a method for in-vitro separation and culture of goat intramuscular precursor fat cells and adipogenic differentiation of the cells, and relates to the technical field of cell culture. The culture medium comprises the following components: 8-12 mu g / mL insulin, 8-12% fetal bovine serum in volume fraction, 95-105 U / mL penicillin, 95-105 mu g / mL streptomycin, and the like. On the basis of the prior art, the application mainly adds appropriate glycyrrhizin and pilocarpine in the culture medium, and finds that the two components are in cooperation, and the adipogenic differentiation ability of cells such as mesenchymal stem cells and precursor fat cells is significantly improved, and the application range is wide. On the basis, the application further provides a complete method for in-vitro separation and culture of goat intramuscular precursor fat cells and adipogenic differentiation of the cells, and provides a method and a way for a series of researches on goat primary intramuscular precursor fat cells in the future.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a culture medium and its application, and a method for the in vitro isolation, culture and adipogenic differentiation of goat intramuscular preadipocytes. Background Technology

[0002] Among the many research directions in cell culture, the study of adipogenic differentiation is of paramount importance. Adipose tissue is not only an important energy storage organ in the body, but also plays a crucial role in endocrine regulation, immune response, and mechanical protection. Abnormalities in the adipogenic differentiation process are closely related to the occurrence and development of various metabolic diseases such as obesity, diabetes, and fatty liver. Therefore, in-depth research and exploration of effective methods for regulating adipogenic differentiation have significant theoretical value and clinical application prospects for the prevention and treatment of these metabolic diseases.

[0003] Currently, while various culture media for adipogenic differentiation have been developed in the field of cell culture, these media still have many limitations. Traditional adipogenic differentiation media typically contain basic components such as insulin, fetal bovine serum, and penicillin, as well as some inducers that can promote adipogenic differentiation, such as 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone (Dex). However, the adipogenic differentiation induction effects of existing media vary significantly for different cell types, and the induction efficiency is often not ideal. Taking mesenchymal stem cells as an example, these cells have the potential to differentiate into adipocytes in vivo, but their adipogenic differentiation ability is constrained by various factors during in vitro culture. The component combinations and concentration settings in existing media are insufficient to fully activate the relevant intracellular adipogenic differentiation signaling pathways, resulting in low degrees and efficiency of adipogenic differentiation, which cannot meet the growing research needs. Summary of the Invention

[0004] To address the above problems, this invention provides a culture medium and its application, and a method for the in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes.

[0005] In a first aspect, the present invention provides a culture medium comprising the following components:

[0006] 8-12 μg / mL insulin, 8-12% fetal bovine serum (FBS), 95-105 U / mL penicillin, 95-105 μg / mL streptomycin, 0.3-0.7 mmol / L 3-isobutyl-1-methylxanthine, 0.7-1.2 μmol / L dexamethasone, 5-10 μmol / L glycyrrhizin, 15-20 μmol / L pilocarpine, with the remainder being the first basal culture medium.

[0007] Furthermore, the ratio of the concentration C1 of pilocarpine to the concentration C2 of glycyrrhizin is 3.

[0008] Furthermore, the concentration C1 of the pilocarpine is 18 μmol / L;

[0009] And / or, the concentration C2 of the glycyrrhizin is 6 μmol / L.

[0010] Furthermore, the first basal culture medium includes DMEM / F12 medium.

[0011] Furthermore, the culture medium comprises the following components:

[0012] 10 μg / mL insulin, 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.5 mmol / L 3-isobutyl-1-methylxanthine, 1.0 μmol / L dexamethasone, 6 μmol / L glycyrrhizin, 18 μmol / L pilocarpine, with the remainder being the basal culture medium.

[0013] Secondly, based on the same inventive concept, the present invention provides the application of the culture medium described in any one of the first aspects in adipogenic differentiation of cells, wherein the cells include at least one of mesenchymal stem cells and preadipocytes.

[0014] Thirdly, based on the same inventive concept, this invention provides a method for the in vitro isolation and culture of goat intramuscular preadipocytes and their adipogenic differentiation, such as... Figure 1 As shown, the method for in vitro isolation and culture of goat intramuscular preadipocytes and their adipogenic differentiation includes the following steps:

[0015] Primary goat intramuscular preadipocytes were obtained;

[0016] When the primary goat intramuscular preadipocytes have grown to complete fusion, they are subjected to adipogenic differentiation treatment using the culture medium described in the first aspect, and then adipogenic maintenance treatment is performed using adipogenic maintenance culture medium to obtain mature goat intramuscular adipocytes.

[0017] The lipid-maintaining culture medium comprises the following components: 8-12 μg / mL insulin, 8-12% fetal bovine serum (FBS), 95-105 U / mL penicillin, 95-105 μg / mL streptomycin, and the remainder being a second basal culture medium.

[0018] Furthermore, the method of obtaining the primary goat intramuscular preadipocytes includes isolating and culturing intramuscular preadipocytes from the longissimus dorsi muscle tissue of goats using collagenase digestion.

[0019] Furthermore, the adipogenic differentiation treatment lasts for 1 to 3 days, and the adipogenic retention treatment lasts for 4 to 5 days.

[0020] Furthermore, the second basal culture medium includes DMEM / F12 medium.

[0021] The technical solutions provided in the embodiments of the present invention have at least the following advantages compared with the prior art:

[0022] This invention provides a culture medium and its application, as well as a method for the in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes. Building upon existing technologies, this invention primarily improves the adipogenic differentiation capacity of cells such as mesenchymal stem cells and preadipocytes by adding appropriate amounts of glycyrrhizin and pilocarpine to the culture medium, demonstrating their synergistic effect. Furthermore, this invention provides a complete set of methods for the in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes, offering a framework and pathway for future research using primary goat intramuscular preadipocytes. Attached Figure Description

[0023] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic flowchart illustrating a method for the in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes, as provided in an embodiment of the present invention.

[0026] Figure 2 This is a cell diagram of primary goat intramuscular preadipocytes obtained in Test Example 2 of the present invention using the culture medium of Example 1 for in vitro isolation and culture of goat intramuscular preadipocytes and their adipogenic differentiation.

[0027] Figure 3 This is an image showing the Oil Red O staining results of mature goat intramuscular adipocytes obtained in Test Example 2 of the present invention, using the culture medium of Example 1 for in vitro isolation and culture of goat intramuscular preadipocytes and their adipogenic differentiation.

[0028] Figure 4This is an image showing the Oil Red O staining results of mature goat intramuscular adipocytes obtained in Test Example 2 of the present invention, using a control culture medium for the in vitro isolation and culture of goat intramuscular preadipocytes and their adipogenic differentiation. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. For example, DMEM / F12 culture medium (or DMEM / F-12 culture medium), pilocarpine (CAS No.: 92-13-7), glycyrrhizin, and other raw materials can be directly used as commercially available products. Furthermore, unless otherwise specified or defined, the steps and parameters involved, such as obtaining primary goat intramuscular preadipocytes, can be performed according to the procedures for goat... The preparation process steps and parameters disclosed in existing technologies, such as the in vitro culture experiment of preadipocytes (DOI: 10.3969 / J.ISSN.1674-148X.2013.01.001) and the study on the expression patterns of genes related to the isolation, culture and differentiation of preadipocytes in Tan sheep muscle (DOI: 10.3969 / j.issn.1673-1182.2020.11.007), will be used or existing equipment will be used directly according to the instruction manual. These will not be repeated in this document.

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed according to national standards. If no corresponding national standard exists, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0032] Example 1

[0033] This example provides a culture medium comprising the following components: 10 μg / mL insulin, 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin, 0.5 mmol / L 3-isobutyl-1-methylxanthine, 1.0 μmol / L dexamethasone, 6 μmol / L glycyrrhizin, 18 μmol / L pilocarpine, with the remainder being basal culture medium (specifically DMEM / F12 medium).

[0034] Example 2

[0035] This example provides a culture medium comprising the following components: 12 μg / mL insulin, 12% fetal bovine serum (FBS), 105 U / mL penicillin, 105 μg / mL streptomycin, 0.7 mmol / L 3-isobutyl-1-methylxanthine, 1.2 μmol / L dexamethasone, 5 μmol / L glycyrrhizin, 15 μmol / L pilocarpine, with the remainder being basal medium (specifically DMEM / F12 medium).

[0036] Example 3

[0037] This example provides a culture medium comprising the following components: 8 μg / mL insulin, 8% fetal bovine serum (FBS), 95 U / mL penicillin, 95 μg / mL streptomycin, 0.3 mmol / L 3-isobutyl-1-methylxanthine, 0.7 μmol / L dexamethasone, 6 μmol / L glycyrrhizin, 18 μmol / L pilocarpine, with the remainder being basal medium (specifically DMEM / F12 medium).

[0038] Example 4

[0039] This example provides a culture medium, which differs from Example 1 only in that:

[0040] (1) The concentration of pilocarpine C1 is 20 μmol / L, and the concentration of glycyrrhizin C2 is 10 μmol / L.

[0041] Comparative Example 1

[0042] This example provides a culture medium that differs from Example 1 only in that pilocarpine is not added (i.e., pilocarpine concentration is 0 μmol / L); all other steps and parameters are the same.

[0043] Comparative Example 2

[0044] This example provides a culture medium that differs from Example 1 only in that glycyrrhizin is not added (i.e., glycyrrhizin concentration is 0 μmol / L); all other steps and parameters are the same.

[0045] Test Example 1

[0046] This example examines the effect of the culture media provided in the above-described embodiments and comparative examples on the adipogenic differentiation of mesenchymal stem cells. The test method includes the following steps: P6 generation AD-MSCs (adipose-derived mesenchymal stem cells) were passaged and cultured at a cell density of 20,000 / cm³. 2 Cells were reseeded into 12-well plates and cultured for 24 hours before differentiation was induced. The culture media provided in Examples 1-4 and Comparative Examples 1-2 were added to each well for induction. Fresh culture medium was added every two days, and differentiation was induced for 10 days. After differentiation, the differentiation medium was aspirated from the 12-well cell culture plates, and 1 mL of DPBS (Dubor's phosphate buffer) was added to gently rinse the bottom of the plates. The DPBS was then aspirated. In a fume hood, 1 mL of 4% formaldehyde solution was added, and the plates were gently shaken to ensure even wetting of the bottom surface. The plates were incubated at room temperature for 30 minutes. The fixative was discarded, and 1 mL of DPBS was added to rinse the cells twice. 500 μL of 0.3% Oil Red O staining solution was added, and the cells were stained at room temperature for 30 minutes. The staining solution was aspirated, and the cells were rinsed three times with 1 mL of DPBS. The lipid droplet abundance (%) was calculated using a CSI (chemical shift imaging) device.

[0047] The test results are shown in Table 1.

[0048] Table 1

[0049]

[0050] As can be seen from Table 1,

[0051] 1) Compared with comparative examples 1-2, the culture medium provided by examples 1-3 of the present invention can significantly increase the abundance value of lipid droplets, indicating that the present invention significantly enhances the adipogenic differentiation ability of mesenchymal stem cells through the combined action of glycyrrhizin and pilocarpine added to the culture medium.

[0052] 2) By comparing the test results of Example 1 and Example 4, it can be seen that by optimizing the dosage relationship between glycyrrhizin and pilocarpine in the culture medium and limiting the concentration ratio of the two to 3, the present invention can further enhance the adipogenic differentiation ability of the obtained culture medium for mesenchymal stem cells.

[0053] Test Example 2

[0054] This example further applies the culture medium provided in Example 1 to the in vitro isolation and culture of goat intramuscular preadipocytes and their adipogenic differentiation, with a culture medium without glycyrrhizin and pilocarpine (i.e., the concentrations of glycyrrhizin and pilocarpine in the culture medium provided in Example 1 are both 0 μmol / L) used as a control culture medium. Specific tests include the following procedures:

[0055] (1) In vitro isolation and culture of primary goat intramuscular preadipocytes

[0056] Two-day-old goat longissimus dorsi muscle tissue was selected, sterilized, separated, and its connective tissue was removed and minced to obtain goat longissimus dorsi muscle tissue blocks. These blocks were digested using type II collagenase. After digestion, the digestion was terminated with complete culture medium (10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and the remainder 90% DMEM / F12 basal medium). Larger, undigested tissue was removed by filtration, and the mixture was passed through a cell sieve to obtain a cell filtrate. The filtrate was centrifuged, and the cells were resuspended in erythrocyte lysate, with excess erythrocytes removed. The resuspended cells were then centrifuged again and seeded into cell culture dishes for adherent culture. After 2 hours, the culture medium was removed, and fresh complete culture medium was added to obtain purified primary goat intramuscular preadipocytes.

[0057] (2) Adipogenic differentiation

[0058] When the primary goat intramuscular preadipocytes reached complete fusion, they were subjected to adipogenic differentiation treatment for 1.5 days using the culture medium provided in Example 1 or the control culture medium, followed by adipogenic maintenance treatment for 4.5 days using adipogenic maintenance culture medium to obtain mature goat intramuscular adipocytes. The adipogenic maintenance culture medium included the following components: 10 μg / mL insulin, 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin, and the remainder was a second basal culture medium (specifically DMEM / F12 medium).

[0059] like Figure 2 As shown, the primary goat intramuscular preadipocytes obtained in this example, observed under an inverted fluorescence microscope, exhibited a spindle-shaped or polygonal shape, similar to fibroblasts. The cytoplasm was uniformly translucent and highly refractive. Furthermore, during in vitro culture, the preadipocytes maintained a highly undifferentiated state, preserving their significant differentiation potential. In addition, the Oil Red O staining results were as follows... Figure 3 and Figure 4 As shown, the test results indicate that, compared with the control culture medium, the culture medium of Example 1 with added glycyrrhizin and pilocarpine significantly improved the oil red staining intensity of intracellular lipid droplets, indicating a significant increase in lipid droplet content.

[0060] In summary, the embodiments of this invention provide a culture medium and its application, as well as a method for the in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes. Based on existing technologies, this invention primarily improves the adipogenic differentiation capacity of cells such as mesenchymal stem cells and preadipocytes by adding appropriate amounts of glycyrrhizin and pilocarpine to the culture medium, demonstrating their synergistic effect. Furthermore, this invention provides a complete set of methods for the in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes, offering a framework and approach for future research using primary goat intramuscular preadipocytes.

[0061] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A culture medium, characterized in that, The culture medium comprises the following components: 8-12 μg / mL insulin, 8-12% fetal bovine serum (FBS), 95-105 U / mL penicillin, 95-105 μg / mL streptomycin, 0.3-0.7 mmol / L 3-isobutyl-1-methylxanthine, 0.7-1.2 μmol / L dexamethasone, 5-10 μmol / L glycyrrhizin, 15-20 μmol / L pilocarpine, with the remainder being the first basal culture medium; The first basal culture medium is DMEM / F12 medium; The ratio of the concentration C1 of pilocarpine to the concentration C2 of glycyrrhizin is 3.

2. The culture medium according to claim 1, characterized in that, The concentration C1 of the pilocarpine is 18 μmol / L; And / or, the concentration C2 of the glycyrrhizin is 6 μmol / L.

3. The culture medium according to claim 1, characterized in that, The culture medium comprises the following components: 10 μg / mL insulin, 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.5 mmol / L 3-isobutyl-1-methylxanthine, 1.0 μmol / L dexamethasone, 6 μmol / L glycyrrhizin, 18 μmol / L pilocarpine, with the remainder being the first basal culture medium.

4. The application of the culture medium according to any one of claims 1 to 2 in adipogenic differentiation of cells, characterized in that, The cells include at least one of mesenchymal stem cells and preadipocytes.

5. A method for the in vitro isolation and culture of goat intramuscular preadipocytes and their adipogenic differentiation, characterized in that, The method for in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes includes the following steps: Primary goat intramuscular preadipocytes were obtained; When the primary goat intramuscular preadipocytes have grown to complete fusion, they are subjected to adipogenic differentiation treatment using the culture medium according to any one of claims 1 to 3, and then adipogenic maintenance treatment is performed using adipogenic maintenance culture medium to obtain mature goat intramuscular adipocytes. The lipid-maintaining culture medium comprises the following components: 8-12 μg / mL insulin, 8-12% fetal bovine serum (FBS), 95-105 U / mL penicillin, 95-105 μg / mL streptomycin, and the remainder being a second basal culture medium.

6. The method for in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes according to claim 5, characterized in that, The method for obtaining the primary goat intramuscular preadipocytes includes isolating and culturing intramuscular preadipocytes from the longissimus dorsi muscle tissue of goats using collagenase digestion.

7. The method for in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes according to claim 5, characterized in that, The duration of the adipogenic differentiation treatment is 1 to 3 days, and the duration of the adipogenic maintenance treatment is 4 to 5 days.

8. The method for in vitro isolation, culture, and adipogenic differentiation of goat intramuscular preadipocytes according to claim 5, characterized in that, The second basal culture medium includes DMEM / F12 medium.

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