[논문]성게 생식소 유래 세포외소포체 특성 분석 및 신경세포에 미치는 영향 연구

최병훈; 조성한; 박상혁

doi:10.9718/jber.2024.45.1.20

[국내논문] 성게 생식소 유래 세포외소포체 특성 분석 및 신경세포에 미치는 영향 연구
Characterization of Sea Urchin Gonad-derived Extracellular Vesicles and Study of Their Effects on Nerve Cells

Journal of biomedical engineering research : the official journal of the Korean Society of Medical & Biological Engineering, v.45 no.1, 2024년, pp.20 - 25

최병훈 (국립부경대학교 4차산업융합바이오닉스공학과) , 조성한 (국립부경대학교 4차산업융합바이오닉스공학과) , 박상혁 (국립부경대학교 4차산업융합바이오닉스공학과)

Abstract ▼ AI-Helper

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by cells. EVs act as messengers for cell-to-cell communication. Inside, it contains various substances that show biological activity, such as proteins, lipids, nucleic acids, and metabolites. The study of EVs extracted from terrestrial organisms and stem cells on inflammatory environments and tissue regeneration have been actively conducted. However, marine organisms-derived EVs are limited. Therefore, we have extracted EVs from sea urchins belonging to the Echinoderm group with their excellent regenerative ability. First, we extracted extracellular matrix (ECM) from sea urchin gonads treated with hypotonic buffer, followed by collagenase treatment, and filtration to collect ECM-bounded EVs. The size of sea urchin gonad-derived EVs (UGEVs) is about 20-100 nm and has a round shape. The protein content was higher after EVs burst than before, which is evidence that proteins are contained inside. In addition, proteins of various sizes are distributed inside. PKH-26 was combined with UGEVs, which means that UGEVs have a lipid membrane. PHK-26-labeled UGEVs were successfully uptaken by cells. UGEVs can be confirmed to have the same characteristics as traditional EVs. Finally, it was confirmed that Schwann cells were not toxic by increasing proliferation after treatment.

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표/그림 (5)

그림 그림 1. 성게 생식소로부터 세포외소포체 추출 과정 모식도 Fig. 1. Extracellular vesicles extraction process derived from sea urchin gonad
그림 그림 2. UGEVs의 물리적 특성 및 내부 단백질 함량(A) UGEVs의 크기 분포도는 DLS를 이용하여 측정(B) UGEVs의 형태는 SEM을 이 용하여 측정(Scale bar: 250 nm) (C) UGEVs 내부 단백질 함량 측정은 Bradford assay로 측정, ***p < 0.001는 Student's t-test로 계산 Fig. 2. Physical characterization and internal protein content of UGEVs (A) The size distribution of UGEVs was measured using dynamic light scattering (DLS) (B) Morphology of UGEVs identified using scanning electron microscope (SEM) (Scale bar: 250nm) (C) The internal protein content of UGEVs was measured using Bradford assay, Data are presented as the mean ± SD from three independent experiments (***p < 0.001 as calculated using Student's t-test)
그림 그림 3. S16 세포에 PKH-26이 결합된 UGEVs를 처리한 후 세포내이입을 확인, 대조염색은 DAPI를 사용 (Scale bar: 20 μm) Fig. 3. Endocytosis was confirmed after treating S16 cells with PKH-26-labeled UGEVs and DAPI was used for counterstaining (Scale bar: 20 μm)
그림 그림 4. S16 슈반 세포에 0,1.25, 2.5, 5, 10, 15, 20 ug/mL 농도의 UGEVs를 24시간 동안 처리한 후 세포 증식을 평가, 세포 증식 평 가는 water-soluble tetrazolium salt (WST)를 사용하여 진행, *p < 0.05, **p < 0.01, *** p < 0.001는 one-way ANOVA로 계산 Fig. 4. S16 Schwann cells were treated with UGEVs at concentrations of 0, 1.25, 2.5, 5, 10, 15, and 20 ug/mL for 24 hours, and then cell proliferation was evaluated. Cell proliferation evaluation was conducted using water-soluble tetrazolium salt (WST), Data are presented as the mean ± SD from independent experiments (n = 4) (*p < 0.05, **p < 0.01, *** p < 0.001 as calculated using one-way ANOVA)
그림 그림 5. S16세포에 UGEVs 20 mg/mL처리 24시간 후 세포 Live/Dead 분석을 진행 (Scale bar: 200 μm) Fig. 5. Live/dead cell viability assay was performed 24 hours after treating S16 cells with 20 μg/mL UGEVs suspension media (Scale bar: 200 μm)

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