• 한국진공학회:학술대회논문집
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• 한국진공학회 2015년도 제49회 하계 정기학술대회 초록집
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• pp.158-158
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• 2015

Myoblast are myogenic precursors that proliferate, activate, and differentiate on muscle injury to sustain the regenerative capacity of skeletal muscle; The neuronal isoform of nitric oxide synthase (nNOS, termed also NOS-I) is expressed in normal adult skeletal muscle, suggesting important functions for Nitric oxide (NO) in muscle biology1,2,3. However, the expression and subcellular localization of NO in muscle development and myoblast differentiation are largely unknown. In this study, we examined effects of the nitric oxide generated by a microwave plasma torch, on proliferation/differentiation of rat myoblastic L6 cells. Experimental data pertaining to nitric oxide production are presented in terms of the oxygen input in units of cubic centimetres per minute. The various levels of nitric oxide are observed depending on the flow rate of nitrogen gas, the ratio of oxygen gas, and the microwave power4. In order to evaluate the potential of nitric oxide as an activator of cell differentiation, we applied nitric oxide generated from the microwave plasma torch to L6 skeletal muscles. Differentiation of L6 cells into myotubes was significantly enhanced the differentiation after nitric oxide treatment. Nitric oxide treatment also increase the expression of myogenesis marker proteins and mRNA level, such as myogenin and myosin heavy chain (MHC), as well as cyclic guanosine monophosphate (cGMP), However during the myotube differentiation we found that NO activate oxidative stress signaling erks expression. Therefore, these results establish a role of NO and cGMP in regulating myoblast differentiation and elucidate their mechanism of action, providing a direct link with oxidative stress signalling, which is a key player in myogenesis. Based on these findings, nitric oxide generated by plasma can be used as a possible activator of cell differentiation and tissue regeneration.

• 동의생리병리학회지
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• 제28권4호
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• pp.411-416
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• 2014

Simple formulas (單方) of muscle section in Donguibogam (東醫寶鑑) have long been prescribed for strengthening muscle and/or prevention of age-related muscle loss. However, biological activity and mechanisms by which they influence myoblast differentiation have not been studied. Therefore, in this study, we evaluated the effects of 14 simple formulas on myoblast differentiation in C2C12 myoblast cells under non-cytotoxic ($0.5mg/m{\ell}$) conditions. C2C12 cells were treated with water extracts of simple formulas for 72 h, and RT-PCR was performed to determine the gene expression levels of myogenic regulatory factors (MRFs), including myoD, myogenin, MRF4, myf5, and insulin like growth factor-1 (IGF-1). Treatment with Colocasiae Rhizoma (CR), Pini Semen (PS), and Sesami Semen (SS) resulted in a significant increase in expression of myogenin in C2C12 cells. Treatment with Allii Macrostemi Bulbus (AM), Colocasiae Rhizoma (CR), and Pini Semen (PS) also resulted in increased expression of MRF4 in C2C12 cells. In addition, enhanced expression of IGF-1 was observed in treatment with Eucommiae cortex (EC), Dioscoreae Rhizoma (DR), Colocasiae Rhizoma (CR), Pini Semen (PS), and Sesami Semen (SS) in C2C12 cells. These results indicate that simple formulas of muscle section in Donguibogam could potentially enhance myoblast differentiation at least in part via increasing expression of myogenin, and/or MRF4 and/or IGF-1.

• BMB Reports
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• 제51권7호
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• pp.350-355
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• 2018

HnRNPK is a multifunctional protein that participates in chromatin remodeling, transcription, RNA splicing, mRNA stability and translation. Here, we uncovered the function of hnRNPK in regulating the proliferation and differentiation of myoblasts. hnRNPK was mutated in the C2C12 myoblast cell line using the CRISPR/Cas9 system. A decreased proliferation rate was observed in hnRNPK-mutated cells, suggesting an impaired proliferation phenotype. Furthermore, increased G2/M phase, decreased S phase and increased sub-G1 phase cells were detected in the hnRNPK-mutated cell lines. The expression analysis of key cell cycle regulators indicated mRNA of Cyclin A2 was significantly increased in the mutant myoblasts compared to the control cells, while Cyclin B1, Cdc25b and Cdc25c were decreased sharply. In addition to the myoblast proliferation defect, the mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was decreased sharply in hnRNPK-mutated cells compared to control myoblasts during differentiation. The deficiency in hnRNPK also resulted in the repression of Myog expression, a key myogenic regulator during differentiation. Together, our data demonstrate that hnRNPK is required for myoblast proliferation and differentiation and may be an essential regulator of myoblast function.

• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.479-486
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• 2016

A previous genome-wide association study (GWAS) exposed histone deacetylase 2 (HDAC2) as a possible candidate gene for breast muscle weight in chickens. The present research has examined the possible role of HDAC2 in skeletal muscle development in chickens. Gene expression was measured by quantitative polymerase chain reaction in breast and thigh muscles during both embryonic (four ages) and post-hatch (five ages) development and in cultures of primary myoblasts during both proliferation and differentiation. The expression of HDAC2 increased significantly across embryonic days (ED) in breast (ED 14, 16, 18, and 21) and thigh (ED 14 and 18, and ED 14 and 21) muscles suggesting that it possibly plays a role in myoblast hyperplasia in both breast and thigh muscles. Transcript abundance of HDAC2 identified significantly higher in fast growing muscle than slow growing in chickens at d 90 of age. Expression of HDAC2 during myoblast proliferation in vitro declined between 24 h and 48 h when expression of the marker gene paired box 7 (PAX7) increased and cell numbers increased throughout 72 h of culture. During induced differentiation of myoblasts to myotubes, the abundance of HDAC2 and the marker gene myogenic differentiation 1 (MYOD1), both increased significantly. Taken together, it is suggested that HDAC2 is most likely involved in a suppressive fashion in myoblast proliferation and may play a positive role in myoblast differentiation. The present results confirm the suggestion that HDAC2 is a functional gene for pre-hatch and post-hatch (fast growing muscle) development of chicken skeletal muscle.

• 한국동물학회지
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• 제31권3호
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• pp.207-217
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• 1988

배양 계배 근원세포의 분화에 미치는 계배추출물의 영향을 조사하고, 이 추출물로부터 근워세포의 분화에 필수적인 myotrophic protein(MP)를 순수분리하였다. 그리고 이 MP는 철 운반 단백질인 trsnaferrin과 동일하거나 또는 대단히 유사한 단백질임을 알 수 있었다. 이 단백질은 철을 근원세포에 공급하고 이 철근이 근원세포의 융합에 필수적인 역할을 하는 것으로 보인다. 또 계배추출물속에는 이 MP이외에 근원세포의 융합을 억제하는, 그리고 열에 비교적 안정한 단백질이 존재하다고 생각된다. 이 MP의 수용체(receptor)분석을 한 결과, 수용체의 수는 근원세포가 융합을 하고 나면 급속히 감소하는 것으로 나타났다. 그리고 근원세포의 내로의 철과 MP의 수송에는 약 10분이 소요되는 것으로 나타났다.

• 생명과학회지
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• 제31권2호
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• pp.137-143
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• 2021

근육 위축증은 근섬유의 손상으로 인한 근육세포의 감소에 의해 일어나며 심장질환, 당뇨, 각종 노인성 만성질환을 일으킨다고 알려져 있다. 본 연구는 미역 추출물 및 이의 성분인 알긴산의 근육세포보호, 근육감소억제 및 근육재생효과를 확인하고자 하였다. 미역 추출물 및 알긴산을 분화된 C2C12 myoblast 세포에 처리한 결과 TNF-α에 의한 근육섬유의 감소가 억제하였고murf1과 MaFbx의 발현량도 감소하였다. 또한, 마우스에 미역 추출물 및 알긴산을 10주간 급여한 결과 cardiotoxin에 의한 다리부종이 감소하였으며 근육단백질인 MyHC와 PGC-1α의 발현량이 증가 하였다. 따라서 미역 추출물 및 알긴산은 근감소 억제 및 근육재생 효과를 나타내어 기능성소재의 활용 가능성을 확인하였다.

• Animal cells and systems
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• 제1권1호
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• pp.63-69
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• 1997

We have previously reported that NF-kB is involved in the regulation of nitric oxide synthase gene expression during differentiation of chick embryonic myoblasts. However, how NF-kB is timely activated during myogenesis remains elusive. One of the most prominent events in myogenesis is myoblast membrane fusion, which is accompanied with massive cytoskeletal reorganization. Here we show that the activity of NF-kB markedly increases in L6 rat myogenic cells that have just initiated morphological changes by treating nocodazole, a microtubule-disrupting agent. Furthermore, the induction of NF-kB activation was closely correlated with the myoblast fusion. In addition, a variety of agents that disrupt microtubules stimulated the myoblast fusion as well as the induction of NF-kB activation. In contrast, taxol, a microtubule-stabilizing agent, suppressed the induction of NF-kB activation and inhibited spontaneous differentiation of L6 cells as well. In addition, we found that the NF-KB in the cells consists of p50/p65 heterodimers. These results support the idea that reorganization of microtubule at early stages of differentiation plays a role as a signal for NF-KB activation during myogenesis.

• 한국동물학회지
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• 제35권3호
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• pp.322-331
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• 1992

To investigate the svnthyesis of muscle proteins during differentiation of chicken myoblast, cvtosolic and membrane fractions were used for both sodium dodecvl sulfate polvcrylamide gel eBectrophoresis and two-dimensional gel electrophoresis. An extensive cell fusion was observed in 4 day culture. In the protein pattern of the cvtosolic fraction from SDS-PAGE. several protein bands including 250 kDa and 46 kDa showed remarkable changes during culture. the protein of 46 kDa was the most prominent one ann its optical density was the highest in 5 day culture (OD = 1.30). In the membrane fraction, band of 19.8 kDa showed the highest absorbance with 0.93 OD at 12 hr after initial plating and decreased gradually thereafter to 0.23 in 5 nay culture. From the results of two-dimensional gel electrophoresis of cytosolic fraction, the 46 kDa spot was observed as ko separated forms from culture 2 nary culture, and the sixte of this spot was the largest in 5 nay culture. In the pattern of membrane protein, the extensive appearance of newiv synthesized Proteins was found in a naut culture, but no Prominent spot was observed throughout culture. From the results of the present clay, we found that, during myoblast differentiation, the most prominent proteins were bands of 46 kDa and 19.8 kDa in cvtosolic and membrane fraction, respectively, and the appearance of new proteins was initiated at 48 hr after initial plating, and the 46 kDa protein was predominant in the cytoplasm of late culture in which extensive cell fusion was observed.

• Journal of Ginseng Research
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• 제42권1호
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• pp.116-121
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• 2018

Background: Black ginseng (BG) has greatly enhanced pharmacological activities relative to white or red ginseng. However, the effect and molecular mechanism of BG on muscle growth has not yet been examined. In this study, we investigated whether BG could regulate myoblast differentiation and myotube hypertrophy. Methods: BG-treated C2C12 myoblasts were differentiated, followed by immunoblotting for myogenic regulators, immunostaining for a muscle marker, myosin heavy chain or immunoprecipitation analysis for myogenic transcription factors. Results: BG treatment of C2C12 cells resulted in the activation of Akt, thereby enhancing hetero-dimerization of MyoD and E proteins, which in turn promoted muscle-specific gene expression and myoblast differentiation. BG-treated myoblasts formed larger multinucleated myotubes with increased diameter and thickness, accompanied by enhanced Akt/mTOR/p70S6K activation. Furthermore, the BG treatment of human rhabdomyosarcoma cells restored myogenic differentiation. Conclusion: BG enhances myoblast differentiation and myotube hypertrophy by activating Akt/mTOR/p70S6k axis. Thus, our study demonstrates that BG has promising potential to treat or prevent muscle loss related to aging or other pathological conditions, such as diabetes.

• 한국동물학회지
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• 제33권1호
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• pp.108-118
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• 1990

혈청을 비롯해서 extracellular matrix에 존재하는 당단백질인 fibronectin은 근세포의 융합과 밀접한 관계가 있는 것으로 알려져 있다. 본 연구실에서는 최근에 근세포가 분화하는 동안에 fibronectin의 수준이 감소되며, 이러한 감소는 fibronectin의 28 kDa fragment에 대한 수용체으 유용성이 감소하는 결과로 밝혀낸 바 있다. 본 연구에서는 근세포으 융합을 억제하는 물질로 알려진 EGTA를 이용하여 근세포의 융합과 28 kDa fragmen receptor의 관계를 검토하여 보았다.EGTA를 처리한 경우 EGTA를 처리하지 않은 근세포에 비해서 fibronectin의 수준과 28 kDa fragmen binding이 훨씬 적게 감소하였으며, 융합이 봉쇄된 근세포에서 EGTA를 제거하여 융합을 재개시키면 fibronectin의 수준과 28 kDa fragmen의 binding이 정상 근세포 수준으로 환원되었다. 이상의 실험 결과로 볼 때 28 kDa fragmen에 대한 수용체의 감소 또는 변화가 근세포의 분화과정에서 일어나는 fibronectin 수준의 감소와 연관성이 있음을 알 수 있다. 한편, 배양액 내에 trypsin을 처리한 경우에는 처리하지 않은 경우에 비해서 28 kDa fragmen의 binding이 현저하게 감소되었고, gangliosides를 처리한 상태에서는 gangliosides의 농도에 정비례해서 28 kDa fragmen의 binding이 감소되었다. 이 밖에 gel overlay technique을 이용하여 28 kDa fragmen가 SDS-PAGE gel에서 분자량이 약 43kDa인 단백질 및 gangliosides와 binding하는 사실을 알 수 있었다. 이러한 실험 결과를 종합하여 볼 때, 근세포의 융합은 28 kDa fragmen에 대한 receptor의 감소와 관계가 있으며, 그 수용체는 gangliosides와 비슷한 당을 가지고 있는 당단백질일 것으로 추정된다.