Culture Method and Regulation Mechanisms of Stages of C2C12 Cell Line Myogenesis
- 作者: Isayeva M.O.1, Gadzhiyeva F.Т.1, Abalenikhina Y.V.1, Shchul’kin A.V.1, Yakusheva E.N.1
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隶属关系:
- Ryazan State Medical University
- 期: 卷 31, 编号 4 (2023)
- 页面: 525-534
- 栏目: Original study
- ##submission.dateSubmitted##: 04.05.2023
- ##submission.dateAccepted##: 10.10.2023
- ##submission.datePublished##: 22.11.2023
- URL: https://journals.eco-vector.com/pavlovj/article/view/375362
- DOI: https://doi.org/10.17816/PAVLOVJ375362
- ID: 375362
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INTRODUCTION: C2C12 is an immortalized mouse myoblast cell line actively used as in vitro experimental models to study skeletal muscle metabolism in biomedical research. C2C12 cells can differentiate into myocytes under appropriate culture conditions. It is important to understand the specifics of each stage of myogenesis and its regulatory mechanisms for the targeted exposure and development of medical drugs, taking into account the mechanisms of pathogenesis of various diseases.
AIM: To describe the method of culturing С2С12 cell line and to study regulatory mechanisms of С2С12 cell line myogenesis.
MATERIALS AND METHODS: The study was conducted on C2C12 cell line. Differentiation of myoblasts was induced in the nutrient medium containing 2% horse serum. The differentiation stages were evaluated by studying the cells on the 1st, 4th and 7th days. Cells before differentiation were used as a comparison group. At each differentiation stage, the myoblast fusion index (IM) was evaluated with preliminary staining of the cells by Romanowsky–Giemsa method. The cell differentiation mechanism was evaluated by the level of myosin, a-actin, myogenic differentiation protein (MyoD), myogenin (MyoG) using Western blot method.
RESULTS: On the 1st day of C2C12 cells culturing, formation of myotubes was observed (IM = 0.15 ± 0.05), on the 4th day — fusion of myoblasts with the formation of binucleated cells accompanied by an increase in the amount of MyoD and α-actin protein (IM = 0.44 ± 0.14). By the 7th day of differentiation, the fusion of cells increased with the formation of myotubes containing more than two nuclei; the content of MyoD and α-actin did not differ from the control, and the amount of MyoG and myosin increased (IM = 0.77 ± 0.04).
CONCLUSION: The described method of culturing C2C12 cell line in Dulbecco’s Modified Eagle’s Medium with a high glucose content (4500 mg/l), containing 2% horse serum, L-glutamine (4 mМ), 100 Un/ml and 100 µg/ml of penicillin and streptomycin, is suitable for the formation of muscle cells, where MyoD and MyoG participate in the regulation of the increase in the amount of specific muscle proteins — α-actin and myosin.
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作者简介
Mariya Isayeva
Ryazan State Medical University
编辑信件的主要联系方式.
Email: mia.poroshina@yandex.ru
ORCID iD: 0000-0002-7237-1789
SPIN 代码: 7833-7030
Assistant of the Department of Biological Chemistry
俄罗斯联邦, RyazanFidan Gadzhiyeva
Ryazan State Medical University
Email: fidagadzhiieva2003@gmail.com
ORCID iD: 0009-0001-0676-0487
SPIN 代码: 9074-2340
Student
俄罗斯联邦, RyazanYuliya Abalenikhina
Ryazan State Medical University
Email: abalenihina88@mail.ru
ORCID iD: 0000-0003-0427-0967
SPIN 代码: 4496-9027
MD, Dr. Sci. (Med.), Associate Professor
俄罗斯联邦, RyazanAleksey Shchul’kin
Ryazan State Medical University
Email: alekseyshulkin@rambler.ru
ORCID iD: 0000-0003-1688-0017
SPIN 代码: 2754-1702
MD, Dr. Sci. (Med.), Associate Professor
俄罗斯联邦, RyazanElena Yakusheva
Ryazan State Medical University
Email: e.yakusheva@rzgmu.ru
ORCID iD: 0000-0001-6887-4888
SPIN 代码: 2865-3080
MD, Dr. Sci. (Med.), Professor
俄罗斯联邦, Ryazan参考
- Wonga CY, Al-Salamia H, Dassa CR. C2C12 cell model: its role in understanding of insulin resistance at the molecular level and pharmaceuticalт development at the preclinical stage. J Pharm Pharmacol. 2020;72(12):1667–93. doi: 10.1111/jphp.13359
- Mangnall D, Bruce C, Fraser RB. Insulin-stimulated glucose uptake in C2C12 myoblasts. Biochem Soc Trans. 1993;21(4):438s. doi: 10.1042/bst021438s
- Nedachi T, Kanzaki M. Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes. Am J Physiol Endocrinol Metab. 2006;291(4):E817–28. doi: 10.1152/ajpendo.00194.2006
- Ludolph DC, Konieczny SF. Transcription factor families: muscling in on the myogenic program. FASEB J. 1995;9(15):1595–604. doi: 10.1096/fasebj.9.15.8529839
- Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987;51(6):987–1000. doi: 10.1016/0092-8674(87)90585-x
- Kopantseva EE, Belyavsky AV. Key regulators of skeletal myogenesis. Molecular Biology. 2016;50(2):195–222. (In Russ). doi: 10.7868/S0026898416010079
- Shishkin SS. Miostatin i nekotoryye drugiye biokhimicheskiye faktory, reguliruyushchiye rost myshechnykh tkaney u cheloveka i ryada vysshikh pozvonochnykh. Uspekhi Biologicheskoy Khimii. 2004;44:209–62. (In Russ).
- Sin J, Andres AM, Taylor DJR, et al. Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts. Autophagy. 2016;12(2):369–80. doi: 10.1080/15548627.2015.1115172
- Ferri P, Barbieri E, Burattini S, et al. Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. J Cell Biochem. 2009;108(6):1302–17. doi: 10.1002/jcb.22360
- Emelin AM, Buev DO, Slabikova AA, et al. Kolichestvennaya otsenka miogennoy differentsirovki kletochnoy linii C2C12 s ispol'zovaniem polietilenglikolya i induktsionnykh sred in vitro. Genes & Cells. 2019;14(S):87-87. (In Russ). doi: 10.23868/gc122609
- Sestili P, Barbieri E, Martinelli C, et al. Creatine supplementation prevents the inhibition of myogenic differentiation in oxidatively injured C2C12 murine myoblasts. Mol Nutr Food Res. 2009;53(9):1187–204. doi: 10.1002/mnfr.200800504
- Asfour HA, Allouh MZ, Said RS. Myogenic regulatory factors: The orchestrators of myogenesis after 30 years of discovery. Exp Biol Med (Maywood). 2018;243(2):118–28. doi: 10.1177/1535370217749494
- Lipton B, Schultz E. Developmental fate of skeletal musclesatellite cells. Science. 1979;205(4412):1292–4. doi: 10.1126/science.472747
- Partridge TA, Grounds M, Sloper JC. Evidence of fusionbetween host and donor myoblasts in skeletal muscle grafts. Nature. 1978;273(5660):306–8. doi: 10.1038/273306a0
- Mendell JR, Kissel JT, Amato AA, et al. Myoblast transfer in the treatment ofDuchenneʼs muscular dystrophy. N Engl J Med. 1995;333(13):832–8. doi: 10.1056/NEJM199509283331303
- Giordani L, Parisi A, Le Grand F. Satellite Cell Self‐Renewal. Curr Top Dev Biol. 2018;126:177–203. doi: 10.1016/bs.ctdb.2017.08.001
- Bouchentouf M, Benabdallah BF, Dumont M, et al. Real‐time imaging of myoblast transplantation using the human sodium iodide symporter. Biotechniques. 2005;38(6):937–42. doi: 10.2144/05386IT01
- Incitti T, Magli A, Darabi R, et al. Pluripotent stem cell‐derived myogenic progenitors remodel their molecular signature upon in vivo engraftment. Proc Natl Acad Sci USA. 2019;116(10):4346–51. doi: 10.1073/pnas.1808303116
- Patz TM, Doraiswamy A, Narayan RJ, et al. Two‐dimensional differential adherence and alignment ofC2C12 myoblasts. Materials Science and Engineering: B. 2005;123(3):242–7. doi: 10.1016/j.mseb.2005.08.088
- Calzia D, Ottaggio L, Cora A, et al. Characterization of C2C12 cells in simulated microgravity: Possible use for myoblast regeneration. J Cell Physiol. 2020;235(4):3508–18. doi: 10.1002/jcp.29239
- Kachesova AA, Shchurova EN, Sayfutdinov MS, et al. Functional Capaсities of Limb Muscles in Patients with Partial Damage to the Cervical Spinal Cord. I. P. Pavlov Russian Medical Biological Herald. 2022;30(2):203–12. (In Russ). doi: 10.17816/PAVLOVJ96752