식이성 폴리페놀 (-)-epigallocatechin-3-gallate가 mouse C2C12 myoblast 분화에 미치는 영향

김혜진,이원준,Kim, Hye-Jin,Lee, Won-Jun 한국생명과학회 2007 생명과학회지 Vol.17 No.3

본 연구에서는 유전자 발현에 중요한 조절 역할을 하는 DNA 메틸화를 식이성 폴리페놀의 하나인 녹차의 대표적인 추출물 EGCG로 억제하였을 때 C2C12 myoblast 세포에 일어나는 현상을 살펴보았다. 그 결과 smooth muscle의 지표인 transgelin, smooth muscle ${\alpha}-actin$ mRNA와 단백질이 현저히 증가함을 보였고, 형태학적으로도 smooth muscle의 전형적인 모습을 보였다. 식이에 포함된 DNA 메틸화 억제제인 EGCG가 C2C12 myoblast를 smooth muscle로 분화를 유도하였으며, 암 예방 차원에서의 EGCG의 역할 외에 혈관질환과 같은 smooth muscle에 관련된 예방과 치료차원에서 EGCG의 역할이 있을 것으로 사료된다. 본 연구는 C2C12 myoblast를 smooth muscle로 유도하는 결정적인 신호전달 역할을 하는 유전자에 대한 연구는 수행하지 못하였다. 따라서 EGCG에 의해 변화되는 유전자에 대한 기전연구가 필요하다고 하겠다. In the present investigation, we studied the modulating effects of (-)-epigallocatechin-3-gallate(EGCG) on the differentiation of mouse C2C12 myoblasts. We found that the strong inhibitory effect of EGCG on DNA methyltransferase-mediated DNA methylation induced transdifferentiation of C2C12 myoblasts into smooth muscle cells demonstrated by both morphological changes and immunofluorescent staining. C2C12 myoblasts treated with EGCG for 4 days expressed smooth muscle ${\alpha}-actin$ protein. Real-time PCR data revealed that smooth muscle ${\alpha}-actin$ mRNA was induced by EGCG treated C2C12 myoblasts in a concentration-dependent manner. Smooth muscle ${\alpha}-actin$ mRNA concentration increased 330% and 490% after 2 and 3 days of 50 ${\mu}M$ of EGCG treatment. The expression of another smooth muscle marker, transgelin, mRNA was also increased up to 9-fold by 4 days of EGCG treatment compared with control in a concentration-dependent manner. These results suggested that C2C12 enables to transdifferentiate into smooth muscle when gene expression patterns are changed by the inhibition of DNA methylation induced by EGCG. In conclusion, transdifferentiation of C2C12 myoblasts into smooth muscle is resulted from the modulating effects of EGCG on DNA methylation which subsequently results in changing the expression pattern of several genes playing a critical role in the differentiation of C2C12 myoblasts.

설치류 식도에서 가로무늬근육-민무늬근육 이행부의 미세구조에 관한 연구

김영기,남광일,박성식 대한해부학회 1998 Anatomy & Cell Biology Vol.31 No.1

Muscle-derived Stem Cells Differentiate into Functional Smooth Muscle Cells for Ureter Tissue Engineering: An Experimental Study

Zhan-Kui Zhao,Hong-Lian Yu,Fei Xiao,Shi-Wen Li,Wen-Biao Liao,Kai-Liang Zhao 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.3

We assessed the ability of muscle-derived stem cells (MDSC) to differentiate into smooth muscle cells (SMC) and their potential to promote the regeneration of smooth muscle with a vessel extracellular matrix (VECM)for tissue engineering of the ureter. MDSC were isolated,proliferated, and identified by flow cytometry. SMC phenotype differentiation was induced with a smooth muscle induction medium. Gene expression was evaluated by real-time quantitative polymerase chain reaction (PCR)and Western blot studies. The VECM was obtained by a decellularization process, and cytotoxic effects were evaluated by exposing the induced cells to a VECM extract. The induced cells were seeded onto VECM in vitro for 1 week, and then the compound grafts were used for ureter reconstitution in vivo. The grafts were obtained for histological studies at 2, 4, 8, and 16 weeks post-operation. Intravenous urography was used to evaluate renal function and ureteral patency. Flow cytometry demonstrated that the MDSC expressed Sca-1 and desmin, but did not express CD45. After induction, SMC phenotype gene expression was confirmed in the induced cells by real-time quantitative PCR and Western blot studies. VECM exhibited a nontoxic effect on the induced cells in vitro. At 16 weeks postoperation,a histological evaluation showed that multilayered urothelium and organized muscle fiber bundles had formed in the grafts. Intravenous urography demonstrated no evidence of ureteral stricture or hydroureteronephrosis. These results demonstrate that MDSC can be induced into SMC and that this was useful for promoting regeneration of smooth muscles for ureter tissue engineering. We assessed the ability of muscle-derived stem cells (MDSC) to differentiate into smooth muscle cells (SMC) and their potential to promote the regeneration of smooth muscle with a vessel extracellular matrix (VECM)for tissue engineering of the ureter. MDSC were isolated,proliferated, and identified by flow cytometry. SMC phenotype differentiation was induced with a smooth muscle induction medium. Gene expression was evaluated by real-time quantitative polymerase chain reaction (PCR)and Western blot studies. The VECM was obtained by a decellularization process, and cytotoxic effects were evaluated by exposing the induced cells to a VECM extract. The induced cells were seeded onto VECM in vitro for 1 week, and then the compound grafts were used for ureter reconstitution in vivo. The grafts were obtained for histological studies at 2, 4, 8, and 16 weeks post-operation. Intravenous urography was used to evaluate renal function and ureteral patency. Flow cytometry demonstrated that the MDSC expressed Sca-1 and desmin, but did not express CD45. After induction, SMC phenotype gene expression was confirmed in the induced cells by real-time quantitative PCR and Western blot studies. VECM exhibited a nontoxic effect on the induced cells in vitro. At 16 weeks postoperation,a histological evaluation showed that multilayered urothelium and organized muscle fiber bundles had formed in the grafts. Intravenous urography demonstrated no evidence of ureteral stricture or hydroureteronephrosis. These results demonstrate that MDSC can be induced into SMC and that this was useful for promoting regeneration of smooth muscles for ureter tissue engineering.

Altered Esophageal Smooth Muscle Phenotype in Achalasia

( David M Rodrigues ),( Sandra R Lourenssen ),( Jay Kataria ),( William G Paterson ),( Michael G Blennerhassett ),( Robert Bechara ) 대한소화기기능성질환·운동학회 2024 Journal of Neurogastroenterology and Motility (JNM Vol.30 No.2

Background/Aims Achalasia is a disorder characterized by impairment in lower esophageal sphincter relaxation and esophageal aperistalsis, caused primarily by loss of inhibitory innervation. However, little is known about associated changes in esophageal smooth muscle. We examined the contractile phenotype and innervation of the circular smooth muscle, as well as inflammatory status, and correlated these with patient-specific parameters. Methods Circular smooth muscle biopsies were obtained in consecutive patients with achalasia undergoing peroral endoscopic myotomy. Axonal innervation and neurotransmitter subtypes were determined with immunocytochemistry, and this was used with quantitative Polymerase Chain Reaction (qPCR) to characterize smooth muscle proliferation and cellular phenotype, as well as collagen expression. These were compared to control tissue obtained at esophagectomy and correlated with patient demographic factors including age, onset of symptoms, and Eckhardt score. Results Biopsies of smooth muscle were obtained from 25 patients with achalasia. Overall, there was increased mast cell number and collagen deposition but increased smooth muscle cell proliferation vs control. There was a striking drop in axon density over controls, with no differences among subtypes of achalasia. Immunocytochemical analysis showed increased expression of the contractile marker α-smooth muscle actin, principally in Type 1 achalasia, that increased with disease duration, while qPCR identified increased mRNA for smoothelin with decreased myosin heavy chain and collagen 3a1, but not collagen 1a1. Conclusions The thickened circular smooth muscle layer in achalasia is largely denervated, with an altered contractile phenotype and fibrosis. Biopsies obtained during peroral endoscopic myotomy provide a means to further study the pathophysiology of achalasia. (J Neurogastroenterol Motil 2024;30:166-176)

가토 대동맥 평활근에서 인삼 알콜 추출물에 의한 Calcium 동원에 관한 연구

김용배,이영호,강복순,강두희,Kim, Yong-Bae,Lee, Young-Ho,Kang, Bok-Soon,Kang, Doo-Hee 대한생리학회 1990 대한생리학회지 Vol.24 No.1

There have been conflicting reports concerning the effect of Panax ginseng on the contractility of vascular smooth muscle, i.e., Panax ginseng extract has been reported to cause relaxation, contraction or to have no effect on the tension of vascular smooth muscle. A further investigation of $Ca^{++}$ stores which supply $Ca^{++}$ for contraction of vascular smooth muscle is needed to understand the underlying mechanisms of this conflicting effect of ginseng alcohol extract (GAE). The present study was intended to examine the sources of calcium mobilized for contraction of vascular smooth muscle by GAE. Aortic ring preparations were made from the rabbit thoracic aorta and endothelial cells were removed from the ring. The contractility of the aortic ring was measured under various experimental conditions and $Ca^{++}$ flux across the membrane of aortic ring and the sarcoplasmic reticulum and mitochondria were measured with a calcium selective electrode. The result were summarized as follows; 1) At low concentration of extracellular $Ca^{++}$, GAE increased the contractility of vascular smooth muscle in dose-dependent fashion except high concentration $Ca^{++}$ (1 mM). 2) In the presence of ryanodine, GAE still increased contractility of vascular smooth muscle as much as control group, but in the presence of caffeine, GAE increased it significantly. i.e. Their effects seemed to be additive. 3) In the presence of verapamil+lanthanum, and verapamil+lanthanum+ryanodine, the contractility of the vascular smooth muscle was decreased, but a dose dependent increase in vascular tension was still demonstrated by GAE although total tension was low. 4) GAE increased $Ca^{++}$ efflux from vascular smooth muscle cells, but have no effect on $Ca^{++}$ influx. 5) GAE increased $Ca^{++}$ efflux from sarcoplasmic reticulum and mitochondria vesicles. From the above results, it may be concluded that GAE increased the release of $Ca^{++}$ from sarcoplasmic reticulum, mitochondria or other intracellular $Ca^{++}$ stores of vascular smooth muscle, but it does not increase $Ca^{++}$ influx across the plasma membrane.

수뇨관 평활근에서의 퓨린 수용체 특성에 관한 연구

공인덕,전상준,박규상,이중우 연세대학교 원주의과대학 1993 원주의대논문집 Vol.6 No.1

탈분극과 근장그물 내 $Ca^{2+}$ 고갈-유도 평활근의 수축 및 세포 내 $Ca^{2+}$ 변동에 관여하는 L-형 $Ca^{2+}$ 통로의 상관성

김중환,Kim, Jung-Hwan 대한물리치료학회 2007 대한물리치료학회지 Vol.19 No.5

Purpose: It is generally accepted that smooth muscle contraction is triggered by intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) released from intracellular $Ca^{2+}$ stores such as sarcoplasmic teticulum (SR) and from the extracellular space. The increased $[Ca^{2+}]^i$ can phosphorylate the 20,000 dalton myosin light chain $(MLC_{20})$ by activating MLC kinase (MLCK), and this initiates smooth muscle contraction. In addition to the $[Ca^{2+}]_i$MACK-tension pathway, a number of intracellular signal molecules, including mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and others, play important roles in the regulation of smooth muscle contraction. However, the mechanisms regulating contraction of depletion of SR $Ca^{2+}$ in mouse gastric smooth muscle strips is not still clear. Methods: To investigate the rotes of $Ca^{2+}$ influx and SR $Ca^{2+}$ release channel on gastric motility, isometric contraction and $[Ca^{2+}]_i$ were examined in mouse gastric smooth muscle strips. Results: High KCl, ryanodine, an activator of $Ca^{2+-}$induced $Ca^{2+}$ release channel, and cyclopiazonic acid (CPA), an inhibitor of SR $Ca^{2+-}$ATPase evoked a sustained increase in muscle contraction and $[Ca^{2+}]_i$. These increases induced by high KCl, ryanodine, and CPA were partially blocked by application of verapamil ($10{\mu}M$), a L-type $Ca^{2+}$ channel inhibitor. Additionally, in $Ca^{2+-}$free solution (1 mM EGTA), ryanodine and CPA had no effect contraction and $[Ca^{2+}]_i$ in fundic muscle strips. Conclusion: These results that extracellular $Ca^{2+}$ influx and depletion of SR trigger $Ca^{2+}$ influx through verapamil-sensitive $Ca^{2+}$ channel, and extracellular and SR $Ca^{2+}$ store may functionally involve in the subcellular $Ca^{2+}$ mobilization in mouse gastric muscle.

탈분극과 근장그물 내 Ca²⁺ 고갈-유도 평활근의 수축 및 세포 내 Ca²⁺ 변동에 관여하는 L-형 Ca²⁺통로의 상관성

김중환 ( Jung-hwan Kim ) 대한물리치료학회 2007 대한물리치료학회지 Vol.19 No.5

Purpose: It is generally accepted that smooth muscle contraction is triggered by intracellular Ca²⁺ ([Ca²⁺]_i) released from intracellular Ca²⁺ stores such as sarcoplasmic reticulum (SR) and from the extracellular space. The increased [Ca²⁺]ⁱ can phosphorylate the 20,000 dalton myosin light chain (MLC₂₀) by activating MLC kinase (MLCK), and this initiates smooth muscle contraction. In addition to the [Ca²⁺]_i-MLCK-tension pathway, a number of intracellular signal molecules, including mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and others, play important roles in the regulation of smooth muscle contraction. However, the mechanisms regulating contraction of depletion of SR Ca²⁺ in mouse gastric smooth muscle strips is not still clear. Methods: To investigate the roles of Ca²⁺ influx and SR Ca²⁺ release channel on gastric motility, isometric contraction and [Ca²⁺]ⁱ were examined in mouse gastric smooth muscle strips. Results: High KC1, ryanodine, an activator of Ca^2+- induced Ca²⁺ release channel, and cyclopiazonic acid (CPA), an inhibitor of SR Ca^2+- ATPase evoked a sustained increase in muscle contraction and [Ca²⁺]ⁱ. These increases induced by high KC1, ryanodine, and CPA were partially blocked by application of verapamil (10 μM), a L-type Ca²⁺ channel inhibitor. Additionally, in Ca^2+-free solution (1 mM EGTA), ryanodine and CPA had no effect on contraction and [Ca²⁺]ⁱ, in fundic muscle strips. Conclusion: These results suggest that extracellular Ca²⁺ influx and depletion of SR trigger Ca²⁺ influx through verapamil-sensitive Ca²⁺ channel, and extracellular and SR Ca²⁺ store may functionally involve in the subcellular Ca²⁺ mobilization in mouse gastric muscle. (J Kor Soc Phys Ther 2007; 19(5):65-76)

[P106] A case of linear atrophic smooth muscle hamartoma

( Myeongheon Chae ),( Suhyun Park ),( Jiyeoun Lee ),( Taeyoung Yoon ) 대한피부과학회 2017 대한피부과학회 학술발표대회집 Vol.69 No.1

Smooth muscle hamartoma is a benign proliferation of mature smooth muscle. It is usually found on the trunk or extremity, and it can manifests as several forms: a single patch or plaque that can be accompanied with follicular prominence, or grouped solitary lesions (rarely with a linear distribution). The lesions can exhibit variable hyperpigmentation and hypertrichosis. A 50-year-old woman presented with linear brownish atrophic patches on her scalp for 1 week. The lesion did not cause any discomfort to the patient. No hypertrichosis or follicular prominence were noted. The clinical appearance suggested a diagnosis of linear morphea. An incisional biopsy showed a marked increase of well-defined bundles of smooth muscle fibers scattered throughout the dermis and basal layer hyperpigmention. Based on these findings, a diagnosis of smooth muscle hamartoma was made. Herein, we report a rare case of smooth muscle hamartoma that showed linear, atrophic features resembling a linear morphea.

Airway Smooth Muscle에 미치는 자울(紫울)의 효과

나경상 ( Kyung Sang Na ),권의광 ( Eui Kwang Kwon ),소응향 ( Yeung Hyung Soo ),서은미 ( Eun Mi Suh ),한종현 ( Jong Hyun Han ) 대한경락경혈학회 2001 Korean Journal of Acupuncture Vol.18 No.1

Radix Asteris has been used in Korea for many centuries as a treatment for respiratory disease. The effect of Radix Asteris on tracheal smooth muscle is not known. The purpose of the present study is to determine the effect of Radix Asteris on histamine induced tracheal smooth muscle contraction in rats and guinea pigs. Guinea pig(500g, male) and Sprague Dawley rats (250g, male) were killed by CO2 exposure and a segment (8-10mm) of the thoracic trachea from each rat and guinea pig was cut into equal segments and mounted `in pairs` in a tissue bath. Contractile force was measured with force displacement transducers under 0.5g loading tension. The dose of histamine (His) which evoked 50% of maximal response (ED50) was obtained from cumulative dose response curves for histamine (10(-7)~10(-4)M). Contractions evoked by His (ED50) were inhibited significantly by Radix Asteris. In guinea pig tracheal smooth muscle, the mean percent inhibition of histamine induced contraction was 120.5% (p<0.01) after 100㎕/㎖Radix Asteris. In rat tracheal smooth muscle, the mean percent inhibition of histamine induced contraction was 135.4% (p<0.01) after 100㎕/㎖Radix Asteris. Propranolol (10(-7)M) slightly but significantly attenuated the inhibitory effects of Radix Asteris. Following treatment with propranolol, the mean percent inhibition caused by 100㎕/㎖Radix Asteris fell to 44.6% in guinea pig induced by histamine contraction and by 100㎕/㎖Radix Asteris fell to 18.7% (p<0.05) in rat induced by histamine contraction. Indomethacin and methylene blue(10(-7)M) did not significantly alter the inhibitory effect of Radix Asteris. These results indicate that Radix Asteris can relax histamine induced contraction of guinea pig and rat tracheal smooth muscle, and that this inhibition involves sympathetic effects.