원문 : Dexamethasone 및 전기 자극 처치 시 근세포로부터 유발된 exosome의 항 근위축 및 근비대 관련 microRNA 및 단백질의 변화에 미치는 효과

김정석 ( Jeong Suk Kim ),박소영 ( So Young Park ),윤정수 ( Chung Su Yoon ),박대령 ( Dae Ryoung Park ) 한국운동생리학회(구 한국운동과학회) 2014 운동과학 Vol.23 No.4

본 연구의 목적은 골격근 세포에 dexamethasone (Dex) 및 전기 자극(ES) 처치 시 발생하는 microRNA(miR)가 각각의 자극에 따라 어떠한 profile을 가지게 되며, 이러한 exosome을 골격근 primary cell에 처치 시 근위축 및 근비대 관련 인자의 인산화가 어떻게 변하는지를 규명하기 위한 것이었다. C2C12 세포주에 Dex 및 ES를 처치하여 골격근 세포로부터 유리된 exosome의 miR의 profile을 분석하였으며, 이러한 과정에서 얻어진 exosome을 골격근 primary cell에 재처리하여 골격근 세포에서의 근위축 및 근비대 관련 인자의 인산화 변화를 관찰하였다. C2C12 세포주에 Dex 혹은 ES를 처치한 결과, 근위축 관련 miR인 miR-1, miR-206, miR-322와 근비대 관련 miR-23a의 발현 양상이 유의하게 차이나는 것을 확인하였다. 또한 이러한 세포에서 얻어진 exosome을 골격근 primary cell에 처치한 결과, Dex를 처치한 세포에서 얻어진exosome은 근위축 관련 인자인 FOXO와 MuRF의 인산화를 증가시키는 것에 비해 ES를 처치한 세포에서 얻어진 exosome은 근비대관련인자인 mTOR와 p70S6K의 인산화를 증가시키는 것으로 나타났다. 이러한 결과는 각각의 신호체계에 의해 발생되는 골격근내 exosome이 세포의 신호전달 체계에 중요한 역할을 하고 있음을 제시하고 있으며, exosome에 의한 miR의 전달이 근위축 및 근비대와 관련된 신호단백질의 인산화를 조절할 수 있음을 제시하고 있다. The purpose of this study was to examine the effect of dexamethasone (Dex) treatment and electrical stimulation (ES) on skeletal muscle cell derived exosome`s microRNA (miR) profile and its effect on skeletal muscle signal protein expression. Exosome obtained from two different C2C12 cell were treated with Dex or ES. Then we profiled exosome`s miR expression and atrophy related miR-1, miR-206, miR-322 were significantly increased with DEX however, hypertrophy related miR-23a was significantly increased only in the ES. Further we treated two different types of exosome to Dex treated skeletal muscle primary cell and two types of exosome differently regulated atrophy related protein (FOXO/MurF) and hypertrophy related protein (mTOR/p70S6K) phosphorylaton. In conclusion, the results indicate that atrophy or hypertrophy signal induces different exosome including different profile of miR and it plays important role in skeletal muscle atrophy and hypertrophy.

Comparative Analysis of MSC-Derived Exosomes Depending on Cell Culture Media for Regenerative Bioactivity

Kim Jun Yong,임원규,Seo Hyo Jeong,Lee Joo Youn,박천권,Han Dong Keun 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.3

Background: In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. Methods: To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor™ CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. Results: The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. Conclusion: CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serum-derived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM. Background: In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. Methods: To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor™ CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. Results: The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. Conclusion: CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serum-derived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM.

Exosome isolation from hemolymph of white-spotted flower chafer, Protaetia brevitarsis (Kolbe) (Coleoptera: Scarabaeidae).

( Seokhyun Lee ),( Kisang Kwon ),( Myung-ha Song ),( Kwan-ho Park ),( O-yu Kwon ),( Ji-young Choi ) 한국잠사학회 2016 International Journal of Industrial Entomology Vol.33 No.2

Exosomes are homogenous vesicles of 40-100 nm diameter produced endogenously. Exosomes are generated by inward budding into multi-vesicular bodies (MVB) and then released to extracellular space. Exosomes contain various nucleic acid and protein cargoes from their cells of origin and this endosomal cellular molecules are used for intracellular communication and for both promotion and suppression of immune responses. Recently, they are also considered as delivery vehicle for therapeutic proteins due to their characteristics of stability in body fluids and ability for target uptake. Also, they show less immune reactivity because the isolated exosome harboring therapeutic proteins can be from the same host. White-spotted flower chafer, Protaetia brevitarsis is one of the major insect commercially reared in Korea. There are bacterial and fungal pathogens causing diseases in the beetle, and these diseases incur economic loss to the larva-rearing farms. Due to their endosomal cargoes, exosomes are good candidates in use of disease diagnosis. In this study, we isolated insect exosome from the hemolymph of P. brevitarsis, and verified it by analysis of the exosome-specific surface proteins and RNA.

Exosomes: Nomenclature, Isolation, and Biological Roles in Liver Diseases

Park Seol Hee,Lee Eun Kyeong,Yim Joowon,Lee Min Hoo,Lee Eojin,Lee Young-Sun,Seo Wonhyo 한국응용약물학회 2023 Biomolecules & Therapeutics(구 응용약물학회지) Vol.31 No.3

The biogenesis and biological roles of extracellular vesicles (EVs) in the progression of liver diseases have attracted considerable attention in recent years. EVs are membrane-bound nanosized vesicles found in different types of body fluids and contain various bioactive materials, including proteins, lipids, nucleic acids, and mitochondrial DNA. Based on their origin and biogenesis, EVs can be classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are the smallest EVs (30-150 nm in diameter), which play a significant role in cell-to-cell communication and epigenetic regulation. Moreover, exosomal content analysis can reveal the functional state of the parental cell. Therefore, exosomes can be applied to various purposes, including disease diagnosis and treatment, drug delivery, cell-free vaccines, and regenerative medicine. However, exosome-related research faces two major limitations: isolation of exosomes with high yield and purity and distinction of exosomes from other EVs (especially microvesicles). No standardized exosome isolation method has been established to date; however, various exosome isolation strategies have been proposed to investigate their biological roles. Exosome-mediated intercellular communications are known to be involved in alcoholic liver disease and nonalcoholic fatty liver disease development. Damaged hepatocytes or nonparenchymal cells release large numbers of exosomes that promote the progression of inflammation and fibrogenesis through interactions with neighboring cells. Exosomes are expected to provide insight on the progression of liver disease. Here, we review the biogenesis of exosomes, exosome isolation techniques, and biological roles of exosomes in alcoholic liver disease and nonalcoholic fatty liver disease.

Exosomal microRNA Derived from Hepatocellular Carcinoma Cells Regulates Pathways Associated with Cancer Progression

( Hyo Jung Cho ),( Gil Ho Lee ),( So Young Yoon ),( Jin Young Nam ),( Jeong Mi Kim ),( Do Wan Kim ),( Jae Youn Cheong ),( Sung Won Cho ),( Soon Sun Kim ) 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: Exosome plays important role in cell-to-cell communication. MicroRNAs in tumor-derived exosome appear to play an important role in cancer progression. The present study explored the differentially expressed microRNA profiles between hepatocellular carcinoma (HCC)-derived exosome (HEX) and normal hepatocyte-derived exosome (NEX) and investigated the target genes for microRNAs in HEX by using network analysis. Methods: Exosomes were isolated from human HCC cell lines (Hep3B and Huh-7) and immortalized normal hepatocyte cell line (THLE-2). Exosomal small RNAs were extracted and differentially expressed exosomal microRNAs between HEX and NEX were screened by using Ilumina Hiseq2000 deep sequencing. Ingenuity Pathway Analysis (IPA) software was used to identify the microRNA regulatory network analysis. Results: A total of 1,830 differentially expressed microRNAs in HEX were identified by deep sequencing. Among them, top 30 up-regulated and top 30 down-regulated exosomal microRNAs were identified in each Huh-7 and Hep3B. By using the differentially expressed microRNA profiles, network analyses were performed. Finally, SMAD3, IFNGR1, and WWP1 were identified as over-expressed hub gene in target network in Hep3B derived exosomal microRNA, while IGF1R, RAC1, and TNF were predicted as over-expressed hub gene in Huh-7 derived exosomal microRNA network. Conclusions: In conclusion, HCC derived exosomal microRNAs could have a central role in HCC progression by acting on the oncogenic pathways. Several key genes for HCC progression were successfully identified by regulatory network analysis of microRNA in HCC derived exosome.

Exosome isolation from hemolymph of white-spotted flower chafer, Protaetia brevitarsis (Kolbe) (Coleoptera: Scarabaeidae).

Lee, Seokhyun,Kwon, Kisang,Song, Myung-Ha,Park, Kwan-ho,Kwon, O-Yu,Choi, Ji-young Korean Society of Sericultural Science 2016 International Journal of Industrial Entomology Vol.33 No.2

Exosomes are homogenous vesicles of 40-100 nm diameter produced endogenously. Exosomes are generated by inward budding into multi-vesicular bodies (MVB) and then released to extracellular space. Exosomes contain various nucleic acid and protein cargoes from their cells of origin and this endosomal cellular molecules are used for intracellular communication and for both promotion and suppression of immune responses. Recently, they are also considered as delivery vehicle for therapeutic proteins due to their characteristics of stability in body fluids and ability for target uptake. Also, they show less immune reactivity because the isolated exosome harboring therapeutic proteins can be from the same host. White-spotted flower chafer, Protaetia brevitarsis is one of the major insect commercially reared in Korea. There are bacterial and fungal pathogens causing diseases in the beetle, and these diseases incur economic loss to the larva-rearing farms. Due to their endosomal cargoes, exosomes are good candidates in use of disease diagnosis. In this study, we isolated insect exosome from the hemolymph of P. brevitarsis, and verified it by analysis of the exosome-specific surface proteins and RNA.

Cardiac-specific delivery by cardiac tissue-targeting peptide-expressing exosomes

Kim, Hyoeun,Yun, Nuri,Mun, Dasom,Kang, Ji-Young,Lee, Seung-Hyun,Park, Hyelim,Park, Hyewon,Joung, Boyoung Elsevier 2018 Biochemical and biophysical research communication Vol.499 No.4

<P><B>Abstract</B></P> <P>Naturally occurring RNA carriers such as exosomes might be an untapped source of effective delivery vehicles. However, if exosomes are to be exploited for therapeutic applications, they must target specific tissues or cell types to avoid off-target effects. This study evaluated whether genetic modification of exosomes could enhance exosome delivery to heart cells and heart tissue without toxicity. Exosomes expressing cardiac-targeting peptide (CTP)-Lamp2b on the exosomal membrane (CTP-Exo) were generated by introducing vectors encoding CTP-Lamp2b into HEK 293 cells. The expression of CTP-Lamp2b peptide on exosomes was stabilized by attaching glycosylation sequences. Exosomes expressing only Lamp2b on exosomal membranes (CTL-Exo) were generated as a control. The <I>in vitro</I> and <I>in vivo</I> uptake of CTL-Exo and CTP-Exo was evaluated in cell lines and mice. Both exosomes were delivered to HEK 293 and H9C2 cells. The delivery of the exosome was not different between CTP-Exo and CTL-Exo in HEK 293 cells, whereas the delivery of CTP-Exo was 16% greater than that of CTL-Exo in H9C2 cells (P = 0.047). Cell viability was maintained at almost 100% with different dosages of both CTL-Exo and CTP-Exo. Moreover, compared with CTL-Exo, the <I>in vivo</I> delivery of exosomes to the hearts of mice was increased by 15% with CTP-Exo (P = 0.035). The delivery to livers and spleens was not different between the two exosomes. Genetic modification of exosomes by expressing CTP-Lamp2b on the exosomal membrane enhanced exosome delivery to heart cells and the heart tissue. These results suggested that CTP-Exo might be used as a therapeutic tool for heart disease.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Genetic modification of exosomes could enhance exosome delivery to heart. </LI> <LI> CTP-Exo did not show toxic effects <I>in vitro</I> or <I>in vivo.</I> </LI> <LI> CTP-Exo might be used as a therapeutic tool for heart disease. </LI> </UL> </P>

Influence of Storage Condition on Exosome Recovery

이미정,반재준,임우석,김만호 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.2

Most of mammalian cells release extracellular vesicles including exosomes which mediate intercellular communication by delivering a variety of molecules. Despite of their importance in normal physiology and disease progression, the standard criteria of storage condition is indefinite and controversial. Therefore, we investigated exosome’s recovery yield and stability by various storage conditions. To investigate the effect of short-term storage temperature on exosome stability, exosomes were incubated at temperatures ranging from -70 to 90°C for 30 min. Immunoblot results showed that all exosome-associated proteins incubated at 90°C were mostly degraded for a short period of time. To examine the effect of long-term storage, isolated exosomes were incubated for 10 days at from -70°C to room temperature (RT), and exosomal protein, RNA and exosome markers were examined. Protein and RNA amounts were most reduced at RT compared with -70 and 4°C. Incubation at 4°C and RT resulted in major loss of CD63, and decreasing level of HSP70 was shown at only RT. In addition, flow cytometry result showed that exosome population became more dispersed after RT incubation for 10 days compared with -70°C incubated or freshly isolated exosomes. In summary, our results indicate that different storage temperature and period influences recovery yield and morphology of exosome, and storage at below -70°C is the favorable condition for preservation of fresh exosomes for clinical application and basic researches.

Photodynamic Therapy for the Treatment of Pancreatic Cancer Using Chlorin e6 loaded Exosome

정재훈,장용호,오경연,김민균,김현철 한국공업화학회 2020 한국공업화학회 연구논문 초록집 Vol.2020 No.-

Exosome, extracellular vesicles for cell-to-cell communications, has been utilized as drug delivery system. In this study, tumor-derived exosome was sonicated and reassembled for a drug delivery carrier. Chlorin e6 (Ce6), a representative photosensitizer, was loaded into tumor-derived exosome during re-assembly of exosomes. The reassembled exosomes was found to have membrane proteins like the exosomes. By removing the internal substances, cancer formation from tumor-derived exosomes could be resolved. In vitro experimental data showed that Ce6-loaded re-assembled exosomes were selectively targeted to original-derived cells. Due to the high specific targeting ability of exosome, the Ce6 could be effectively delivered. Furthermore, after effective intracellular delivery, sufficient reactive oxygen species (ROS) were generated with very small amounts of Ce6 in the target cells under the laser irradiation. In conclusion, photodynamic therapy could be obtained with Ce6- loaded exosome.

HDAC5-mediated exosomal Maspin and miR-151a-3p as biomarkers for enhancing radiation treatment sensitivity in hepatocellular carcinoma

이승민,조정인,최수진,김동하,류재원,김인기,우동철,성영훈,정진용,백인정,백찬기,노진경,이상욱,하창훈 한국생체재료학회 2023 생체재료학회지 Vol.27 No.00

Background Tumor-derived exosomes are critical elements of the cell–cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes. Methods We observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components. Results We found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size. Conclusions Our present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity.