• International Journal of Industrial Entomology and Biomaterials
• /
• 제33권2호
• /
• pp.85-91
• /
• 2016

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.

• Asian-Australasian Journal of Animal Sciences
• /
• 제33권11호
• /
• pp.1824-1836
• /
• 2020

Objective: On the hypothesis that grazing of cattle prompts organs to secrete or internalize circulating microRNAs (c-miRNAs) in parallel with changes in energy metabolism, we aimed to clarify biological events in adipose, skeletal muscle, and liver tissues in grazing Japanese Shorthorn (JSH) steers by a transcriptomic approach. Methods: The subcutaneous fat (SCF), biceps femoris muscle (BFM), and liver in JSH steers after three months of grazing or housing were analyzed using microarray and quantitative polymerase chain reaction (qPCR), followed by gene ontology (GO) and functional annotation analyses. Results: The results of transcriptomics indicated that SCF was highly responsive to grazing compared to BFM and liver tissues. The 'Exosome', 'Carbohydrate metabolism' and 'Lipid metabolism' were extracted as the relevant GO terms in SCF and BFM, and/or liver from the >1.5-fold-altered mRNAs in grazing steers. The qPCR analyses showed a trend of upregulated gene expression related to exosome secretion and internalization (charged multivesicular body protein 4A, vacuolar protein sorting-associated protein 4B, vesicle associated membrane protein 7, caveolin 1) in the BFM and SCF, as well as upregulation of lipolysis-associated mRNAs (carnitine palmitoyltransferase 1A, hormone-sensitive lipase, perilipin 1, adipose triglyceride lipase, fatty acid binding protein 4) and most of the microRNAs (miRNAs) in SCF. Moreover, gene expression related to fatty acid uptake and inter-organ signaling (solute carrier family 27 member 4 and angiopoietin-like 4) was upregulated in BFM, suggesting activation of SCF-BFM organ crosstalk for energy metabolism. Meanwhile, expression of plasma exosomal miR-16a, miR-19b, miR-21-5p, and miR-142-5p was reduced. According to bioinformatic analyses, the c-miRNA target genes are associated with the terms 'Endosome', 'Caveola', 'Endocytosis', 'Carbohydrate metabolism', and with pathways related to environmental information processing and the endocrine system. Conclusion: Exosome and fatty acid metabolism-related gene expression was altered in SCF of grazing cattle, which could be regulated by miRNA such as miR-142-5p. These changes occurred coordinately in both the SCF and BFM, suggesting involvement of exosome in the SCF-BFM organ crosstalk to modulate energy metabolism.

• Journal of Ginseng Research
• /
• 제48권2호
• /
• pp.211-219
• /
• 2024

Background: Recently, plant-derived exosome-like nanoparticles (PDENs) have been isolated, and active research was focusing on understanding their properties and functions. In this study, the characteristics and molecular properties of ginseng root-derived exosome-like nanoparticles (GrDENs) were examined in terms of skin protection. Methods: HPLC-MS protocols were used to analyze the ginsenoside contents in GrDENs. To investigate the beneficial effect of GrDENs on skin, HaCaT cells were pre-treated with GrDENs (0-2 × 10⁹ particles/mL), and followed by UVB irradiation or H₂O₂ exposure. In addition, the antioxidant activity of GrDENs was measured using a fluorescence microscope or flow cytometry. Finally, molecular mechanisms were examined with immunoblotting analysis. Results: GrDENs contained detectable levels of ginsenosides (Re, Rg1, Rb1, Rf, Rg2 (S), Gyp17, Rd, C-Mc1, C-O, and F2). In UVB-irradiated HaCaT cells, GrDENs protected cells from death and reduced ROS production. GrDENs downregulated the mRNA expression of proapoptotic genes, including BAX, caspase-1, -3, -6, -7, and -8 and the ratio of cleaved caspase-8, -9, and -3 in a dose-dependent manner. In addition, GrDENs reduced the mRNA levels of aging-related genes (MMP2 and 3), proinflammatory genes (COX-2 and IL-6), and cellular senescence biomarker p21, possibly by suppressing activator protein-1 signaling. Conclusions: This study demonstrates the protective effects of GrDENs against skin damage caused by UV and oxidative stress, providing new insights into beneficial uses of ginseng. In particular, our results suggest GrDENs as a potential active ingredient in cosmeceuticals to promote skin health.

• 대한화장품학회지
• /
• 제42권4호
• /
• pp.359-366
• /
• 2016

엑소좀은 세포에서 분비되는 작은 소낭체로서, 기원세포와 조직에 따라 다양한 기능을 수행하며, 세포생존 및 세포 간 커뮤니케이션에 중요한 역할을 한다. 최근 엑소좀을 활용하여 종양연구, 면역질환 개선, 질병진단 bio-marker 개발 등 다양한 분야에서 연구가 진행되고 있으나, 피부세포에서 분비기전 및 피부 생리적 기능에 대한 연구는 미흡한 실정이다. 따라서 본 연구에서 인체피부 유래 각질형성세포(HaCaT)로부터 분리된 엑소좀이 피부각질형성세포의 증식과 이주에 미치는 영향을 확인하고자 하였다. HaCaT으로부터 ExoQuick-TC를 활용하여 엑소좀을 분리하고, 열처리 엑소좀(boiled exosome)과 무처리 엑소좀(unboiled exosome)으로 구분하였다. HaCaT 유래 엑소좀은 농도 의존적으로($0.1{\sim}20{\mu}g/mL$) HaCaT의 증식을 유도하였으며, $20{\mu}g/mL$에서 대조군(control)에 비해 각각 $186.96{\pm}3.87%$(열처리) 또는 $193.48{\pm}10.48%$(무처리)의 증식 유도활성을 나타내었다. 또한 HaCaT 유래 무처리 엑소좀은 농도 의존적인($0.1{\sim}20{\mu}g/mL$) HaCaT의 이주활성을 나타내었고, $20{\mu}g/mL$에서 대조군에 비해 $179.39{\pm}4.89%$의 이주를 유도하였다. 그러나 열처리 엑소좀은 이주 유도활성을 나타내지 않았다. 뿐만 아니라 무처리 HaCaT 유래 엑소좀은 collagen sprout outgrowth를 농도 의존적으로 유도함을 확인하였다. 이러한 결과를 통해서 HaCaT 유래 엑소좀은 지질 및 열에 안정한 물질이 세포의 증식을 유도하고, 단백질 또는 열에 불안정한 물질이 세포의 이동 및 sprout out growth 활성에 관여하는 것으로 확인되었다. 따라서 피부각질형성세포 유래 엑소좀은 피부의 재상피화 및 상처치유 등의 활성을 나타낼 수 있으며, 향후 화장품소재로서 응용 가능성이 확인되었다.

• Animal Bioscience
• /
• 제36권6호
• /
• pp.851-860
• /
• 2023

Objective: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 ㎍/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative realtime polymerase chain reaction. Results: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion: The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

• The Korean Journal of Physiology and Pharmacology
• /
• 제27권1호
• /
• pp.61-73
• /
• 2023

Esophageal squamous cell carcinoma (ESCC) is a kind of malignant tumor with high incidence and mortality in the digestive system. The aim of this study is to explore the function of lnc-ABCA12-3 in the development of ESCC and its unique mechanisms. RT-PCR was applied to detect gene transcription levels in tissues or cell lines like TE-1, EC9706, and HEEC cells. Western blot was conducted to identify protein expression levels of mitochondrial apoptosis and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway. CCK-8 and EdU assays were carried out to measure cell proliferation, and cell apoptosis was examined by flow cytometry. ELISA was used for checking the changes in glycolysis-related indicators. Lnc-ABCA12-3 was highly expressed in ESCC tissues and cells, which preferred it to be a candidate target. The TE-1 and EC9706 cells proliferation and glycolysis were obviously inhibited with the downregulation of lnc-ABCA12-3, while apoptosis was promoted. TLR4 activator could largely reverse the apoptosis acceleration and relieved the proliferation and glycolysis suppression caused by lnc-ABCA12-3 downregulation. Moreover, the effect of lnc-ABCA12-3 on ESCC cells was actualized by activating the TLR4/NF-κB signaling pathway under the mediation of exosome. Taken together, the lnc-ABCA12-3 could promote the proliferation and glycolysis of ESCC, while repressing its apoptosis probably by regulating the TLR4/NF-κB signaling pathway under the mediation of exosome.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권8호
• /
• pp.3471-3476
• /
• 2014

Background: Aberrant expression of the microRNA-29 family is associated with tumorigenesis and cancer progression. As transport carriers, tumor-derived exosomes are released into the extracellular space and regulate multiple functions of target cells. Thus, we assessed the possibility that exosomes could transport microRNA-29c as a carrier and correlations between microRNA-29c and apoptosis of bladder cancer cells. Materials and Methods: A total of 28 cancer and adjacent tissues were examined by immunohistochemistry to detect BCL-2 and MCL-1 expression. Disease was Ta-T1 in 12 patients, T2-T4 in 16, grade 1 in 8, 2 in 8 and 3 in 12. The expression of microRNA-29c in cancer tissues was detected by quantitative reverse transcriptase PCR (QRT-PCR). An adenovirus containing microRNA-29c was used to infect the BIU-87 human bladder cancer cell line. MicroRNA-29c in exosomes was measured by QRT-PCR. After BIU-87 cells were induced by exosomes-derived microRNA-29c, QRT-PCR was used to detect the level of microRNA-29c. Apoptosis was examined by flow cytometry and BCL-2 and MCL-1 mRNA expressions were assessed by reverse transcription-polymerase chain reaction. Western blotting was used to determine the protein expression of BCL-2 and MCL-1. Results: The expressions of BCL-2 and MCL-1 protein were remarkably increased in bladder carcinoma (p<0.05), but was found mainly in the basal and suprabasal layers in adjacent tissues. The expression of microRNA-29c in cancer tissues was negatively correlated with the BCL-2 and MCL-1. The expression level of microRNA-29c in exosomes and BIU-87 cells from the experiment group was higher than that in control groups (p<0.05). Exosome-derived microRNA-29c induced apoptosis (p<0.01). Although only BCL-2 was reduced at the mRNA level, both BCL-2 and MCL-1 were reduced at the protein level. Conclusions: Human bladder cancer cells infected by microRNA-29c adenovirus can transport microRNA-29c via exosomes. Moreover, exosome-derived microRNA29c induces apoptosis in bladder cancer cells by down-regulating BCL-2 and MCL-1.

• BMB Reports
• /
• 제49권11호
• /
• pp.585-586
• /
• 2016

Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells.

• Journal of Animal Science and Technology
• /
• 제66권5호
• /
• pp.1021-1033
• /
• 2024

Exosomes have been extensively studied as disease biomarker in humans, given their role in transporting bioactive molecules. However, despite the great potential of exosomes as noninvasive diagnostic markers and therapeutic nanocarriers for bovine diseases, few studies have been conducted on bovine exosome. Thus, this study aimed to quantitatively and qualitatively compare three isolation methods to identify a suitable method for bovine serum. Exosomes were isolated using ultracentrifugation alone (UC), a combination of ultracentrifugation and size exclusion chromatography (US), or membrane affinity-based exoEasy kit (EE). Isolated particles were evaluated using a range of complementary techniques. Transmission electron microscopy showed that all three isolation methods resulted in particles with a cup-shaped morphology. The particle concentration measured by nanoparticle trafficking analyzer of US was lower compared to those of UC and EE method. As a result of immunoblotting, exosome markers including TSG101, CD81, and HSP70 were detected in US particles, while in UC and EE, only TSG101 expression was confirmed. Particles isolated from UC and EE showed a contamination with the blood protein albumin, whereas particles from US did not show albumin contamination. In addition, to evaluate the possibility of using exosomes as biomarkers, the profiles of the small RNA in the exosomes were compared using the bioanalyzer 2100. As a result, in the EE method, the band of small RNA (25-200 nt) was most prominent, and in the US methods, a distinct band was observed in the small RNA range. Collectively, the purity of exosomes without non-exosomal contamination was highest in the US method. However, for the detection of small RNA, the EE method was found to be the most suitable. Therefore, the results suggest that the optimal isolation method varies depending on the specific purpose of exosome isolation.

• IMMUNE NETWORK
• /
• 제5권2호
• /
• pp.105-109
• /
• 2005

Background: Dendritic cells (DCs) are the most potent APCs (antigen-presenting cells) and playa critical role in immune responses. Galectin-3 is a biological lectin with a beta-galactoside binding affinity. Recently, proteomic analysis revealed the presence of galectin-3 in the exosome of mature DCs. However, the expression and function of galectin-3 in DCs remains unclear yet. Methods: We used bone marrow-derived DCs of mouse and showed the expression of galectin-3 in DCs by using flow cytometry analysis and Western blot analysis. Results: Galectin-3 was determined as single band of 35 kDa in Western blot analysis. Flow cytometry analysis showed the major growth factor for DCs, granulocyte-macrophage colony stimulating factor (GM-CSF) and maturing agents, anti-CD40 monoclonal antibody (mAb) and lipopolysaccharide (LPS) consistently increased the intracellular expression of galectin-3 in DCs compared to medium alone. In addition, DCs treated with maturing agents did marginally express galectin-3 on their surface. Conclusion: This study suggests that galectin-3 in DCs may be regulated by critical factors for DC function.