CN111363718A - By exogenously adding a differentiation promoting reagent YYQ1 capable of obviously promoting the myoblast differentiation of mouse C2C12 - Google Patents
By exogenously adding a differentiation promoting reagent YYQ1 capable of obviously promoting the myoblast differentiation of mouse C2C12 Download PDFInfo
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Abstract
A differentiation promoting reagent YYQ1 capable of obviously promoting myoblast differentiation of a mouse C2C12 is added through an external source, belongs to the technical field of cell biology, and is characterized in that: the differentiation promoting reagent YYQ1 has the component of 10 mu M retinol, the differentiation promoting reagent YYQ1 is added into the C2C12 cells cultured in vitro, better differentiation efficiency can be obtained, the differentiation of the C2C12 cells is promoted, the aim of remarkably promoting the differentiation of the mouse C2C12 myoblasts after external source addition is finally achieved, the differentiation promoting reagent YQ1 provides a basis for promoting the muscle differentiation of animals, the retinol is used for promoting the muscle differentiation, and the practical application value is larger. The invention has the advantages of simple manufacture, strong operability, low cost and obvious effect.
Description
Technical Field
The invention relates to a differentiation promoting reagent YYQ1 capable of remarkably promoting myoblast differentiation of a mouse C2C12 by exogenous addition, and belongs to the technical field of cell biology.
Background
Retinol (called ROL for short) is a fat-soluble alcohol substance, and plays an important role in maintaining normal visual function, maintaining normal growth and development of bones, and promoting cell proliferation and growth. Vitamin A has various molecular forms, is an unsaturated monohydric alcohol with alicyclic ring, comprises two kinds of vitamin A1 and vitamin A2 from animal food sources, vitamin A1 is mostly present in the liver of mammals and saltwater fish, while vitamin A2 is often present in the liver of freshwater fish, and the vitamin A is commonly referred to as vitamin A1 due to the low activity of vitamin A2. Vitamin a as used in this application is also vitamin a 1. The previous research on retinol mainly focuses on the aspect of dietary deficiency of retinol, and when the retinol is deficient, the retinol can affect the growth of bones, lead to the differentiation impairment of leydig cells, thus leading to the decline of reproductive function and dry and rough skin, and particularly has great influence on eyes, such as dry ophthalmia, corneal softening, nyctalopia and the like. Cell differentiation refers to the process by which cells of the same origin gradually produce populations of cells with different morphological, structural and functional characteristics, resulting in spatial differences between cells and temporal differences between the same cell and its previous state. The essence of cell differentiation is the selective expression of the genome in time and space, with the expression of different genes turned on or off, ultimately producing marker proteins. In general, the process of cell differentiation is irreversible, however, under certain conditions, the differentiated cell is unstable and its gene expression pattern may change reversibly, returning to its undifferentiated state, which is called dedifferentiation. At present, there is no study on the influence of retinol on the differentiation of myoblasts cultured in vitro. The fibroblast is the main cell component of loose connective tissue, the cell is flat, has multiple bulges, is in a star shape, has rich cytoplasm, is alkalescent, has larger nucleus, is flat oval, has loose chromatin, is lightly colored, has obvious nucleolus, and has a rough endoplasmic reticulum, a free ribosome and a developed Golgi complex in the cytoplasm under an electron microscope, which indicates that the function of the cell for synthesizing protein is vigorous. Fibroblasts synthesize and secrete both collagen and elastin, producing collagen fibers, reticular fibers and elastic fibers, and also matrix components such as glycosaminoglycans and glycoproteins. When fibroblasts are in a quiescent state, they are called fibroblasts, and they become small, long fusiform, small nuclei, deep coloration, few rough endoplasmic reticulum in cytoplasm, undeveloped Golgi complex, and eosinophilic. Under certain conditions, such as wound repair and connective tissue regeneration, fibroblasts can be transformed into fibroblasts again, and the fibroblasts can divide and proliferate. Fibroblasts are usually attached to collagen fibers by the mediation of matrix glycoproteins, and are slowly moved in a certain direction by the attraction of chemokines such as lymphokines, complement, etc. In 2018, scientists at the institute of Barcelona biomedical research institute, etc., explained how skin fibroblasts are aged by research and published the results on the International journal Cell (Marion Claudia Salzer, Atefh Lafzi, Antoni Berenger Llergo, et al. identity Noise and didegenic peptides Characterize Dermal fiberlast. Cell, 2018; DOI:10.1016/j. cell.2018.10.012): skin fibroblasts are very critical for the production of collagen and other proteins constituting the dermis, and are also important for the function of protecting the barrier of the skin and repairing skin damage. With age, these skin fibroblasts will cause their cell identity to be as if they had forgotten what they are, and thus the activity of the cells will be altered, the dermis will lose its ability to produce collagen, and its ability to repair wounds will be significantly impaired. Elderly people often face these problems because the skin of their body does not heal properly and barrier function is reduced, which increases the risk of skin infections and systemic infections of the body, thereby affecting the health of the body's tissues. The mouse fibroblast cell line C2C12 is established by Yaffe and other clones, and the C2C12 cells cultured in vitro can be differentiated into multinucleated myotubes under the condition of low serum induction and express various marker proteins of mature skeletal muscles such as myostatin and myogenin and the like, so the C2C12 cells generally become a first-choice model for researching proliferation and differentiation of the fibroblast muscle cells in vitro. In the actual experiment process of culturing the C2C12 myoblasts in mice, the cells are not in good state, are only in strong and excessive growth, are not differentiated in induction, cause great influence on the smooth development of the experiment and seriously influence the progress of the experiment. Therefore, how to find a reagent which can remarkably promote the differentiation of the mouse C2C12 myoblasts after being added by external sources so as to rapidly develop related experiments of the fibroblast becomes a great problem to be solved urgently, so that the invention of the differentiation promoting reagent YYQ1 which can remarkably promote the differentiation of the mouse C2C12 myoblasts by being added by external sources is necessary.
Disclosure of Invention
In order to overcome the problems that the cell state is not good, the cell is only a strong overgrowth and is not differentiated in the process of a mouse C2C12 myoblast culture experiment, the differentiation promoting reagent YYYQ 1 capable of remarkably promoting the differentiation of the mouse C2C12 myoblast is added externally, the differentiation promoting reagent YQ1 capable of remarkably promoting the differentiation of the mouse C2C12 myoblast is added externally, the differentiation promoting reagent YQ1 prepared from retinol with the concentration of 10 mu M is added into C2C12 cells cultured in vitro, better differentiation efficiency can be obtained, the differentiation of the C2C12 cells is promoted, the aim of remarkably promoting the differentiation of the mouse C2C12 myoblast after the exogenous addition is finally achieved, the differentiation promoting reagent YQ1 provides a basis for promoting the differentiation of animal muscle, the retinol is used for promoting the muscle, and the practical application value is higher.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention adds a differentiation promoting reagent YYQ1 which can obviously promote the differentiation of mouse C2C12 myoblasts through exogenous addition, and is characterized in that: the component of the differentiation promoting reagent YYQ1 is 10 mu M Retinol (Retinol, ROL for short).
The C2C12 cell culture and retinol addition experiments: mouse C2C12 cell line was purchased from Shanghai, China, and the cells were passaged and seeded into six-well plates for culture, C2C12 cells were cultured in growth Medium of Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and penicillin-streptomycin; when the cell density reaches 80-90%, changing a differentiation culture solution containing 2% of horse serum and penicillin-streptomycin to induce differentiation, simultaneously adding ROL with final concentration of 0, 5, 10 and 15 mu M into each hole respectively, and screening the optimal final concentration of exogenously added ROL; place the cells in 5% CO2Cultured in an incubator at 37 ℃.
The western blot analysis method: the cells were induced to differentiate in C2C12 cells and 10. mu.M ROL was added thereto at the same time1、D3、 D7Collecting protein, washing with PBS for 3 times, lysing cells with protein lysate RIPA, adding buffer, boiling for 10min, and centrifuging at 4000rpm/min for 10 min; egg separation by 10% SDS-PAGE gel electrophoresisTransferring white matter to polyvinylidene fluoride (PVDF) membrane, incubating the membrane with MyoG and GAPDH primary antibody, and adding goat anti-rabbit immunoglobulin G [ IgG ]]The secondary antibody of (1); protein visualization using Super ECL Plus detection kit; the membrane was exposed in a Mini chemiluminescence imaging and analysis system to obtain an image.
The experiment for detecting the fusion rate of myotubes in the differentiation process of the mouse C2C12 by desmin immunofluorescence comprises the following steps: C2C12 cells are subcultured and inoculated into a six-well plate, when the cell density reaches 80% -90%, 10 mu M ROL is added into each well while 2% of differentiation culture solution is replaced, and the cells are respectively cultured at D1、D3、D7Washing with PBS for 3 times, fixing in cold methanol for 20min, and placing in a refrigerator at-20 deg.C; washing with PBS 3 times, blocking with PBST containing 5% Bovine Serum Albumin (BSA) (PBS containing 0.5% Triton X-100) at 37 deg.C for 2h, diluting with PBST containing 5% BSA to obtain specific Desmin (1:50), and shaking overnight at 4 deg.C; washing with PBST for 3 times, and incubating the cells with a fluorescently-labeled goat anti-rabbit-FITC (1:20) secondary antibody at 37 ℃ for 2 h; washing 3 times with PBST, staining nuclei for 3min in 4, 6-diamino-2-phenylindole (DAPI), washing cells three times with PBST, mounting with an anti-fluorescence quencher, and observing and obtaining images using a fluorescence microscope (Olympus, BeiJing, China); by counting nuclei in differentiated myotubes: (>2) The rate of myoblast fusion was evaluated as a percentage of the total number of nuclei (mononuclear and polynuclear).
The invention has the beneficial effects that the differentiation promoting reagent YYQ1 capable of obviously promoting the differentiation of the mouse C2C12 myoblasts is added through an external source, the differentiation promoting reagent YYQ1 prepared from retinol with the concentration of 10 mu M is added to the C2C12 cells cultured in vitro, better differentiation efficiency can be obtained, the differentiation of the C2C12 cells is promoted, the aim of obviously promoting the differentiation of the mouse C2C12 myoblasts after the external source is added is finally achieved, the differentiation promoting reagent YQ1 provides a basis for promoting the animal muscle differentiation, the retinol is used for promoting the muscle differentiation, and the practical application value is higher. The invention has the advantages of simple manufacture, strong operability, low cost and obvious effect.
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The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a graph showing the effect of exogenously added ROL with a final concentration of 10. mu.M, which is an differentiation promoting agent YYQ1 capable of significantly promoting the differentiation of mouse C2C12 myoblasts, on mouse C2C12 cell differentiation genes at different stages of differentiation.
In the figure: (A) inducing differentiation and adding ROL to fix cell at D1, D3 and D7, respectively, and then detecting desmin (green) by immunofluorescence. Nuclei were stained with DAPI (blue). DMSO was added as a control. Scale bar, 100 μm. (B) Quantitative analysis of myotube fusion index for D1, D3, D7 was counted against panel a.
FIG. 2 is a graph showing the optimal final concentration of exogenously added ROL screened by exogenously adding a differentiation-promoting agent YYQ1 capable of significantly promoting the differentiation of mouse C2C12 myoblasts
In the figure: (A) and (3) detecting the expression quantity change of MyoG after exogenous ROL with different concentrations is added by western blotting. (B) Gray-scale scans of MyoG protein after addition of different concentrations of ROL are shown in Panel A. The cross section structure of the air-jet heating pipe is schematic.
FIG. 3 is a graph showing the effect of exogenously added ROL with a final concentration of 10. mu.M, which is an differentiation promoting agent YYQ1 capable of significantly promoting the differentiation of mouse C2C12 myoblasts, on mouse C2C12 cell differentiation genes at different stages of differentiation.
In the figure: (A) and (3) detecting the expression quantity change of MyoG with different differentiation degrees after adding the ROL from an external source by western blotting. (B) Gray-scale scanning of MyoG protein in Panel A.
Detailed Description
The first embodiment is as follows:
as shown in the figure, the differentiation promoting reagent YYQ1 which can obviously promote the myoblast differentiation of the mouse C2C12 is added by external sources and comprises the following components: the experimental process related to the activity of 10 mu M Retinol (ROTinol, ROL for short) and the differentiation promoting reagent YYQ1 for promoting the differentiation of mouse C2C12 myoblasts is as follows:
C2C12 cell culture and retinol addition experiments:
the mouse C2C12 cell line was purchased from ShangHai (ShangHai, China). To investigate the effect of ROL on mouse C2C12 cell differentiation, cells were passaged and plated into six-well plates, and C2C12 cells were cultured in medium containing10% fetal bovine serum and penicillin-streptomycin in Dulbecco's Modified Eagle's Medium (DMEM). When the cell density reaches 80% -90%, the differentiation culture solution containing 2% horse serum and penicillin-streptomycin is replaced to induce differentiation, meanwhile, 0, 5, 10 and 15 mu M ROL (Sigma-Aldrich, R7632) are respectively added into each hole, and the optimal final concentration of exogenously added ROL is screened. Place the cells in 5% CO2Cultured in an incubator at 37 ℃.
2. Western blot analysis
Adding 10 μ M ROL to collect protein at D1, D3 and D7 respectively while inducing differentiation of C2C12 cells, washing with PBS 3 times, lysing cells with protein lysate RIPA, adding buffer boiled water for 10min, and centrifuging at 4000rpm/min for 10 min. Proteins were separated by electrophoresis on a 10% SDS-PAGE gel, transferred to a polyvinylidene fluoride (PVDF) membrane, incubated with primary MyoG, GAPDH antibodies, and then with secondary antibodies to which goat anti-rabbit immunoglobulin G [ IgG ] was added. Protein was visualized using the SuperECL Plus detection kit. The membrane was exposed in a Mini chemiluminescence imaging and analysis system to obtain an image.
3. Immunofluorescence detection
The rate of myotube fusion during differentiation of mouse C2C12 was detected by desmin immunofluorescence. C2C12 cells were passaged and inoculated into six-well plates for culture, when the cell density reached 80% -90%, 10. mu.M ROL was added to each well while replacing 2% differentiation medium, washed 3 times with PBS at D1, D3, D7, respectively, fixed in cold methanol for 20min and placed in a refrigerator at-20 ℃. Washed 3 times with PBS and treated with PBST containing 5% Bovine Serum Albumin (BSA) (PBS containing 0.5% Triton X-100) at 37 ℃.
Claims (4)
1. A differentiation promoting reagent YYQ1 capable of obviously promoting the differentiation of mouse C2C12 myoblasts is added by external sources, and the method is characterized in that: the component of the differentiation promoting reagent YYQ1 is 10 mu M retinol.
2. The differentiating agent YYQ1 capable of significantly promoting the differentiation of mouse C2C12 myoblasts by exogenous addition according to claim 1, characterized in that: the above-mentionedC2C12 cell culture and retinol addition experiments: mouse C2C12 cell line was purchased from Shanghai, passaged and inoculated into six-well plates for culture, C2C12 cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and penicillin-streptomycin; when the cell density reaches 80-90%, changing a differentiation culture solution containing 2% of horse serum and penicillin-streptomycin to induce differentiation, simultaneously adding retinol with final concentrations of 0, 5, 10 and 15 mu M into each hole respectively, and screening the optimal final concentration of externally added retinol; place the cells in 5% CO2Cultured in an incubator at 37 ℃.
3. The differentiating agent YYQ1 capable of significantly promoting the differentiation of mouse C2C12 myoblasts by exogenous addition according to claim 1, characterized in that: the western blot analysis method: adding 10 μ M retinol while inducing differentiation of C2C12 cells, collecting proteins at D1, D3 and D7 respectively, washing with PBS for 3 times, lysing cells with protein lysate RIPA, adding buffer, boiling for 10min, and centrifuging at 4000rpm/min for 10 min; separating proteins by adopting 10% SDS-PAGE gel electrophoresis, transferring the proteins to a polyvinylidene fluoride membrane, incubating the membrane with primary antibodies of MyoG and GAPDH, and then adding a secondary antibody of goat anti-rabbit immunoglobulin G; protein visualization using Super ECL Plus detection kit; the membrane was exposed in a Mini chemiluminescence imaging and analysis system to obtain an image.
4. The differentiating agent YYQ1 capable of significantly promoting the differentiation of mouse C2C12 myoblasts by exogenous addition according to claim 1, characterized in that: the experiment for detecting the fusion rate of myotubes in the differentiation process of the mouse C2C12 by desmin immunofluorescence comprises the following steps: C2C12 cells are subcultured and inoculated into a six-well plate for culture, when the cell density reaches 80% -90%, 2% of differentiation culture solution is replaced, meanwhile, 10 mu M retinol is added into each well, the cells are washed for 3 times by PBS (phosphate buffer solution) when being respectively in D1, D3 and D7, and the cells are fixed in cold methanol for 20min and then are placed in a refrigerator at the temperature of-20 ℃; washing with PBS 3 times, blocking with PBST containing 5% bovine serum albumin (0.5% Triton X-100) in PBS at 37 deg.C for 2h, diluting with PBST containing 5% BSA (1:50), and shaking overnight at 4 deg.C; washing with PBST for 3 times, and incubating the cells with a fluorescently-labeled goat anti-rabbit-FITC (1:20) secondary antibody at 37 ℃ for 2 h; washing with PBST for 3 times, staining nuclei with 4, 6-diamino-2-phenylindole for 3min, washing cells with PBST for three times, adding an anti-fluorescence quencher seal, and observing and obtaining images by using a fluorescence microscope; the rate of myoblast fusion was assessed by counting the percentage of nuclei in differentiated myotubes to the total number of nuclei including mononuclear and polynuclear nuclei.
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| CN113528432A (en) * | 2021-07-19 | 2021-10-22 | 东北林业大学 | Method for promoting bovine myoblast differentiation by using miR-18inhibitor |
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| CN113528432A (en) * | 2021-07-19 | 2021-10-22 | 东北林业大学 | Method for promoting bovine myoblast differentiation by using miR-18inhibitor |
| CN113528432B (en) * | 2021-07-19 | 2024-04-19 | 东北林业大学 | Method for promoting bovine myoblast differentiation by using miR-18 inhibitor |
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