이 연구는, 일부 또는 전체, 국가 자연 과학 재단의 중국 (31472187 및 31672550) 및 농업 과학, 농업 과학의 중국 아카데미의 기술 혁신 프로그램 지원.

모든 동물 실험 상하이 수의학 연구소, 농업 과학의 중국 아카데미의 로컬 윤리 위원회에 의해 승인 (허가 번호: SHVRIAU-14-0101).
1. Schistosomes 의 생체 외에서 문화
참고: Schistosomes 는 생물을 나타냅니다. 노동자 때 생물 학적 재료 관련 웜, schistosomal 정지, 또는 다른 처리 시간 라텍스 장갑을 전혀 착용 해야 한다. 뉴질랜드 토끼 복 부 노출 통해 ~ 2000 cercariae 감염 되었습니다. 간 단계에서 schistosomes 토끼 간 및 mesenteric 혈관의 관류에 의해 감염 된 후 28 일에 미 나무 cercariae 감염에서 수집 했다. 웜 컬렉션의 절차 이전 간행물 쥐18연구 보고에서 참조 되어 있다.
<ol><li>500 mL 비 커에 기생충을 수집 합니다. 그들을 철저 하 게 세척 하 고 200 mL와 함께 부드럽게 3 x preheated (37 ° C) PBS (pH 7.4).</li>
	<li>각 90 mm 페 트리 접시 ~ 200 벌레 쌍을 구분 합니다. 다음 기생충을 철저 하 게 세척 하 고 50 mL와 함께 부드럽게 2 x preheated (37 ° C) 페니실린과 스의 100 mg/mL의 U 100을 포함 된 RPMI-1640 문화 매체.</li>
	<li>37 ° C 미만 5% CO2 ~ 5 웜 쌍 /mL 90 mm 페 트리 h 2의 조밀도에 접시에 항생제 없이 데워 RPMI 1640 문화 매체의 40 mL에 기생충을 유지 합니다. 노트: RPMI 1640 매체 혈 청을 포함 해서는 안, 그렇지 않으면 exogenous extracellular 소포를 소개 합니다.</li>
</ol>2. 사전 처리 상태 미디어
<ol><li>문화 매체를 수집 하 고 계란을 제거 하 고 웜 tegmental 파편 30 분 2000 × g에서 4 ° C에서 매체를 원심.</li>
	<li>신선한 튜브는 상쾌한 전송 하 고 0.22 μ m 주사기 필터를 사용 하 여 솔루션을 필터링 합니다.</li>
	<li>Dialyzed 가방에 필터링된 솔루션을 수집 (분자량 컷오프: 3.5kD)와 다음 하룻밤 4 ° C에서 8 %PEG8000 솔루션을 사용 하 여 솔루션을 dialyze. 노트: 이 단계는 SjEVs 절연의 수율을 높이기 위한 SjEVs을 풍부 하 게 하는 것이 중요.</li>
</ol>3입니다. SjEVs의 격리
<ol><li>새로운 튜브에 dialyzed 솔루션을 전송 하 고 총 exosome 격리 시 약의 0.5 볼륨을 추가 합니다.</li>
	<li>솔루션 vortexing 또는 솔루션의 동질성을 되도록 아래로 pipetting으로 잘 섞는다.</li>
	<li>2 ° C ~ 8 ° C에 혼합물을 밤새 품 어.</li>
	<li>2 ° C 8 ° c에서 1 시간을 위한 15000 × g에서 혼합물을 원심</li>
	<li>발음 하 고는 상쾌한 삭제. 노트: EVs (대부분의 경우에는 표시 되지 않음) 튜브의 하단에 펠 릿에 포함 됩니다.</li>
	<li>2 ml PBS의 EVs 펠 릿을 resuspend 하 고 추가 분석까지-80 ° C에서 EVs 솔루션을 저장 합니다.</li>
</ol>4입니다. 서 부 Blotting에 의해 EVs의 특성
<ol><li>SjEVs 샘플, 예, BCA 분석 결과 의해의 단백질 농도 결정 합니다.</li>
	<li>22.5 µ L RIPA 버퍼에 EVs의 10-20 µ g을 준비 합니다. 그런 다음 버퍼와 95 ° c.에 5 분에 대 한 열 로드 x 4의 7.5 µ L을 추가</li>
	<li>12 %SDS 페이지 젤에 의해 별도 SjEVs 단백질.</li>
	<li>PVDF 막에는 단백질을 전송 하 고 안티-CD63 항 체 (1:1, 000 희석), 뒤에 안티-토끼 IgG-HRP (1:5, 000 희석)와 프로브와 프로브.</li>
	<li>막 개발을 위한 ECL 검출 시 약을 사용 하 고 화학 이미징 시스템에 의해 신호를 감지.</li>
</ol>5. 전송 전자 현미경 검사 법에 의해 EVs의 특성
<ol><li>200 메쉬 formvar 코팅 격자를 고립 된 SjEVs의 2 µ L을 추가 하 고 실 온에서 건조를 허용.</li>
	<li>1 분에 대 한 Phosphotungstic 산 2 %stain 고 실 온에서 건조.</li>
	<li>전송 전자 현미경 샘플 홀더에 격자를 로드 하 고 이미지 캡처에 대 한 80 kV 전자 빔에 노출.</li>
</ol>6. SjEVs 레이블 PKH67와 셀 이해 분석 결과
노트: 달리 명시 하지 않는 한 모든 후속 단계 온도 (20-25 ° C)에서 수행 했다.
<ol><li>원뿔 바닥 폴 리 프로필 렌 튜브에 5 µ g SjEVs 솔루션을 전송 및 RPMI 1640 매체를 추가 하 여 12 mL을 볼륨을 조정.</li>
	<li>SjEVs 펠 렛을 4 ° C에서 90 분 동안 110000 × g에서 SjEVs 원심</li>
	<li>원심, 후 신중 하 게는 상쾌한 발음. 참고: PKH67 ethanolic 염료 이기종 얼룩 및 감소 된 세포 생존 능력에 발생할 것이 SjEV 펠 릿에 직접 추가할 수 없습니다.</li>
	<li>SjEV 펠 릿;에 희석제 C의 1 mL을 추가 하 여 2 x EV 정지 솔루션 준비 완전 한 분산 되도록 부드러운 pipetting으로 resuspend.</li>
	<li>희석제 C, 얼룩이 지기 전에 폴 리 프로필 렌 원심 분리기 튜브에서 희석제 C의 1 mL를 PKH67 ethanolic 염료 해결책의 2 µ L을 추가 하 고 분산을 잘 혼합 하 여 신선한 2x 염료 솔루션을 준비 합니다.</li>
	<li>빠르게 EV 정지 (단계 6.4) 즉시 염료 솔루션 (단계 6.5) x 2의 1 mL를 혼합 샘플 pipetting 2 x의 1 mL를 추가 합니다.</li>
	<li>4 분 동안 실내 온도에 혼합물을 품 어.</li>
	<li>FBS의 4 mL를 추가 하 여 얼룩을 중지 하 고 1 분 동안 품 어.</li>
	<li>12 mL을 볼륨을 조정 하 고 다음 90 분 동안 4 ° C에서 110000 × g에서 원심을 RPMI1640 매체를 추가 합니다.</li>
	<li>제거는 상쾌한 고 RPMI1640 매체 SjEV 펠 릿을 표시 하는 PKH67 resuspend를 추가 합니다.</li>
	<li>한번 세척 한 후 PBS resuspend SjEVs 펠 릿을 추가 90 분 동안 4 ° C에서 110000 × g에서 원심.</li>
</ol>7. SjEVs 셀 이해 분석 결과
<ol><li>NCTC 같은 종자 포유류 세포 1469 셀 또는 HEK293T 셀 6 잘 플레이트 (잘 당 2 × 105 셀)에 복제 하 고 하룻밤 셀 문화.</li>
	<li>5 µ g PKH67 표시 SjEVs를 추가 하 고 2-4 h 37 ° C에서 세포 문화.</li>
	<li>후에 부 화, 매체를 제거 하 고 PBS 가진 셀을 두 번 세척.</li>
	<li>15 분 동안 4% 포름알데히드 솔루션 셀을 수정 하 고 PBS를 가진 더 두 번 세척.</li>
	<li>4, 6-diamidino-2-phenylindole (DAPI)와 세포 핵 얼룩.</li>
	<li>형광 현미경 검사 법을 사용 하 여 셀을 관찰 합니다.</li>
</ol>

<ol>
	<li>Tetta, C., Ghigo, E., Silengo, L., Deregibus, M. C., Camussi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+an+emerging+mechanism+of+cell-to-cell+communication.">Extracellular vesicles as an emerging mechanism of cell-to-cell communication.</a> Endocrine. 44 (1), 11-19 (2013).</li><li>Abels, E. R., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+Extracellular+Vesicles:+Biogenesis,+RNA+Cargo+Selection,+Content,+Release,+and+Uptake.">Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake.</a> Cell Mol Neurobiol. 36 (3), 301-312 (2016).</li><li>Riaz, F., Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-like+vesicles+of+helminths:+implication+of+pathogenesis+and+vaccine+development.">Exosome-like vesicles of helminths: implication of pathogenesis and vaccine development.</a> Ann Transl Med. 5 (7), 175 (2017).</li><li>Zhang, H. G., Grizzle, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+cancer:+a+newly+described+pathway+of+immune+suppression.">Exosomes and cancer: a newly described pathway of immune suppression.</a> Clin Cancer Res. 17 (5), 959-964 (2011).</li><li>Colombo, M., Raposo, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annu Rev Cell Dev Biol. 30, 255-289 (2014).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNA+is+enriched+and+stable+in+exosomes:+a+promising+biomarker+for+cancer+diagnosis.">Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.</a> Cell Res. 25 (8), 981-984 (2015).</li><li>Marcilla, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+in+parasitic+diseases.">Extracellular vesicles in parasitic diseases.</a> J Extracell Vesicles. 3, 25040 (2014).</li><li>Cwiklinski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Extracellular+Vesicles+of+the+Helminth+Pathogen,+Fasciola+hepatica:+Biogenesis+Pathways+and+Cargo+Molecules+Involved+in+Parasite+Pathogenesis.">The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.</a> Mol Cell Proteomics. 14 (12), 3258-3273 (2015).</li><li>Buck, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+secreted+by+nematode+parasites+transfer+small+RNAs+to+mammalian+cells+and+modulate+innate+immunity.">Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity.</a> Nat Commun. 5, 5488 (2014).</li><li>Sotillo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+secreted+by+Schistosoma+mansoni+contain+protein+vaccine+candidates.">Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.</a> Int J Parasitol. 46 (1), 1-5 (2016).</li><li>Nowacki, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+and+small+non-coding+RNA-enriched+extracellular+vesicles+are+released+by+the+pathogenic+blood+fluke+Schistosoma+mansoni.">Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni.</a> J Extracell Vesicles. 4, 28665 (2015).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-like+vesicles+derived+by+Schistosoma+japonicum+adult+worms+mediates+M1+type+immune-+activity+of+macrophage.">Exosome-like vesicles derived by Schistosoma japonicum adult worms mediates M1 type immune- activity of macrophage.</a> Parasitol Res. 114 (5), 1865-1873 (2015).</li><li>Koh, Y. Q., Almughlliq, F. B., Vaswani, K., Peiris, H. N., Mitchell, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+enrichment+by+ultracentrifugation+and+size+exclusion+chromatography.">Exosome enrichment by ultracentrifugation and size exclusion chromatography.</a> Front Biosci (Landmark Ed). 23, 865-874 (2018).</li><li>Safdar, A., Saleem, A., Tarnopolsky, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+endurance+exercise-derived+exosomes+to+treat+metabolic+diseases.">The potential of endurance exercise-derived exosomes to treat metabolic diseases.</a> Nat Rev Endocrinol. 12 (9), 504-517 (2016).</li><li>Livshits, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+exosomes+by+differential+centrifugation:+Theoretical+analysis+of+a+commonly+used+protocol.">Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.</a> Sci Rep. 5, 17319 (2015).</li><li>Zhu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+extracellular+vesicles+containing+small+RNAs+from+the+eggs+of+Schistosoma+japonicum.">Release of extracellular vesicles containing small RNAs from the eggs of Schistosoma japonicum.</a> Parasit Vectors. 9 (1), 574 (2016).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+S.+japonicum+exosome-like+vesicles+reveals+their+regulatory+roles+in+parasite-host+interactions.">Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions.</a> Sci Rep. 6, 25885 (2016).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. , 11 (2001).</li><li>Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+miRNAs:+roles+in+cancer+diagnosis,+prognosis+and+therapy.">Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy.</a> Adv Drug Deliv Rev. 81, 75-93 (2015).</li></ol>

저자는 공개 없다.

EVs에 대 한 최근 연구는 schistosome EVs 호스트 병원 체 상호 작용3,9,,1216에 중요 한 역할을 설명 했다. 추가 주소에 그들의 규정 하는 기능 필수적 이다 schistosomes에서 EVs를 분리. 여기, 우리 SjEV 절연을 위한 다른 방법을 설명합니다. 이 메서드 생성, SjEVs의 넓은 범위 크기 100에서 400 nm nm, 성인 미 나무</...

Extracellular 소포 (EVs)는 작은 막 도약 소포와 다양 한 단백질, 지질, 핵 산 캡슐화의 인구. 최근 연구 보여준 EVs 셀 셀 통신에 중요 한 역할을 하 고 셀 개발, 면역 조절, 신생, 세포 마이그레이션2, 등 수많은 생리 적 프로세스의 규칙에 관련 된 3,,45. 증거를 축적 EVs, exosomes, 그리고 그들의 miRNA 화물 순환 특정 질병6의 잠재적인 생체 나타냅니다 나타냅니다.
여러 가지 원생 동물 Trichomonas vaginalis, Trypanosoma cruzi, 등 Leishmania spp., EVs;를 분 비 수 표시 되었습니다 helminths 발견 되었습니다 또한7호스트 생활에 EVs를 분 비. 기생 EVs pathogenicity8, 감염과 면역 규칙9의 유지 보수에 포함 될 표시 되었습니다. 최근 두 Schistosoma mansoni (S. mansoni) 10,11 schistosomes에서 공부와 미 나무12 성인 schistosomes 분 수 있는 exosome 같은 소포 비 표명 특정 생물 학적 과정의 기능 규정에 참여.
날짜 하려면, 여러 가지 방법은 extracellular 소포, ultracentrifugation, 외13, 폴리머 기반 시 약, 크기 배제 크로마토그래피, immunoaffinity 격리의 사용을 사용 되었습니다. 이러한 다른 방법을 그들의 자신의 이점 및 제한 사항14. 일반적으로, ultracentrifugation는 소포 격리에 대 한 표준 방법으로 간주 됩니다. 그러나,이 방법은 잠재적인 EV 집계14의 한계에서 고통.
Schistosomes에서 방법의 몇 가지 EV 격리에 대 한 보고 되었습니다: ultracentrifugation11,,1210 와 여러 단계 (달걀16, 상업 EV 격리 키트16 를 사용 하 여 포함 됩니다 schistosomula11, 성인 schistosomes10,,1217). Microvesicles 천 나노미터 몇 백 나노미터 크기의 넓은 범위는, 그 주어진 우리 벤치 원심 분리기, 한, 그리고 분리에서 EVs 상업 킷은 절연을 사용 하 여 결합 하는 대체 방법 개발 성인 Schistosoma 나무. 격리 된 EVs는 일반적으로 100-400 nm의 직경을 소유 하 고 성공적으로 받는 세포에 의해 내 면화 했다.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Total Exosome Isolation Reagent (from cell culture media)</td><td>ThermoFisher SCIENTIFIC</td><td>4478359</td><td>For isolating S. japonicum extracellular vesicles</td></tr><tr><td>PKH67</td><td>Sigma-aldrich</td><td>MINI67</td><td>For labeling Evs</td></tr><tr><td>RPMI 1640 Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11875119</td><td>For parasite culture</td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher SCIENTIFIC</td><td>15140122</td><td></td></tr><tr><td>0.22&amp;mu;m syringe filter</td><td>Merck</td><td>SLGP033RB</td><td></td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo SCIENTIFIC</td><td>68035</td><td></td></tr><tr><td>PEG8000</td><td>Sangon Biotech</td><td>A100159&amp;nbsp;</td><td></td></tr><tr><td>RIPA</td><td>Beyotime</td><td>P0013B</td><td></td></tr><tr><td>EMD Millipore&amp;trade; Immobilon&amp;trade; Western Chemiluminescent HRP Substrate (ECL)</td><td>Fisher scientific</td><td>WBKLS0100&amp;nbsp;</td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>4083</td><td></td></tr><tr><td>BCA kit</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>CD63 antibodies</td><td>Sangon Biotech</td><td>D160973-0025</td><td></td></tr><tr><td>PVDF</td><td>Bio-Rad</td><td>1704273</td><td></td></tr><tr><td>Formvar/Carbon 400 mesh</td><td>Ted Pella</td><td>01754-F</td><td></td></tr><tr><td>Phosphotungstic Acid</td><td>Ted Pella</td><td>19402</td><td></td></tr><tr><td>anti-mouse IgG-HRP</td><td>CWBIO</td><td>CW0102</td><td></td></tr><tr><td>NCTC clone 1469 cells</td><td>ATCC</td><td>ATCC&amp;reg; CCL-9.1&amp;trade;</td><td></td></tr><tr><td>FBS</td><td>HyClone</td><td>SV30087.02</td><td></td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE chemoluminescance imaging system</td><td>GE</td><td>ImageQuant LAS4000mini</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7600</td><td></td></tr><tr><td>Milli-Q water</td><td>Milli-Q</td><td>Milli-Q Elix</td><td></td></tr><tr><td>Eppendor Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5804R</td><td></td></tr><tr><td>Zetasizer Nano</td><td>Malvern</td><td>Zetasizer Nano ZS&amp;nbsp;</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima L-100 XP Ultracentrifuge</td><td></td></tr><tr><td>Incubator</td><td>ESCO</td><td>CCL-107B</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Zeiss Axin Observer Z1</td><td></td></tr></tbody></table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

Extracellular 소포 (EVs)는 막 소포 세포 외 microenvironment로 셀의 다양 한 발표. EVs는 누구의 크기 범위 40 1000 nm 사이 이기종 vesicles의 인구를 나타냅니다. 축적 된 증거는 EVs에 중요 한 역할 규정 호스트 병원 체 상호 작용에 표시. Schistosome EVs에 대 한 깊은 이해는 기본 schistosomiasis에 대 한 새로운 전략의 개발을 활성화 하는 schistosome-호스트 상호 작용 메커니즘에 대 한 통찰력을 제공 해야 합니다. 여기, 우리는 성인 Schistosoma 나무 (S. 머 위)에서 절연 및 EVs의 특성화에 대 한 프로토콜을 제시 하 여 EVs 함수 schistosomes에 진학 하고자 합니다. EVs 문화 매체 체 외에 상업 exosome 격리 키트와 결합 하는 원심 분리를 사용 하 여 격리 했다. 고립 된 미 나무 EVs (SjEVs) 일반적으로 100-400 nm의 직경을가지고 그리고 전송 전자 현미경 검사 법 및 서쪽 blotting에 의해 특징입니다. 염료-표시 된 SjEVs PKH67의 사용 SjEVs 받는 사람 세포에 의해 내 면화는 설명 했다. 전반적으로, 우리의 프로토콜 성인 schistosomes;에서 EVs를 격리 하기 위한 대체 방법을 제공 한다 격리 된 SjEVs 기능 분석에 적합 수 있습니다.

설명 된 프로토콜을 사용 하 여 격리 된 SjEVs의 수익률을 계량, 우리는 28 일 성인 schistosomes에서 고립 된 SjEVs의 단백질 농도 액세스할 수 BCA 단백질 분석 결과 사용. 표 1에서 같이, SjEV 단백질 농도의 매체 (표 1) 100 mL 당 250 µ g을 208 µ g에서 배열 했다. 입자 크기 Malvern 나노 분석에 의해 결정 된 대로 표시 된 격리 SjEVs 100에서 240 400 nm nm, 높은 인구 약...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

절연 및 Extracellular 소포 특성 성인 Schistosoma 나무 에서

여기, 선물이 성인 Schistosoma 나무에서 체 외에서 배양된 매체에 extracellular 소포 (EVs) 격리를 설명 하는 프로토콜.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

isolation-characterization-extracellular-vesicles-from-adult

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

7.8K 조회수.  Ministry of Agriculture. 여기, 선물이 성인 Schistosoma 나무에서 체 외에서 배양된 매체에 extracellular 소포 (EVs) 격리를 설명 하는 프로토콜.

비디오: Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Cercarial 변화와 체외에서</em의> 양성 Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

연구

면역학 및 감염병학

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

에서 계란을 사용하여<em&gt; Schistosoma mansoni</em&gt; 등<em&gt; 생체내에서</em기생충 유도된 폐 염증의&gt; 모델

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

대량 절연 및 인간의 혈액 우연 Schistosoma Mansoni 의 Intramolluscan 단계의 생체 외에서 재배

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

발생학

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

간암 조직에서 유래한 작은 세포외 소포체의 농축 방법

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

암 연구학

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

조절된 세포 배양 배지에서 세포외 소포를 분리하기 위한 크기 배제 크로마토그래피

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

신경과학

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus(Echinococcus granulosus에서 작은 세포외 소포의 분리 및 수지상 세포 흡수)

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

예쁜꼬마선충의 정화 및 분석 세포외 소포

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

생물학

<meta charset="utf8"></head><body>Murine Spleen-Derived Exosome 분리를 위한 정제 기법

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

<meta charset="utf8"></head><body>Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

절연 및 Extracellular 소포 특성 성인 Schistosoma 나무 에서

요약

더 많은 비디오 탐색

이 비디오의 챕터

Cercarial 변화와 체외에서 양성 Schistosoma mansoni* Schistosomules*

에서 계란을 사용하여

대량 절연 및 인간의 혈액 우연 Schistosoma Mansoni 의 Intramolluscan 단계의 생체 외에서 재배

간암 조직에서 유래한 작은 세포외 소포체의 농축 방법

조절된 세포 배양 배지에서 세포외 소포를 분리하기 위한 크기 배제 크로마토그래피

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus(Echinococcus granulosus에서 작은 세포외 소포의 분리 및 수지상 세포 흡수)

예쁜꼬마선충의 정화 및 분석 세포외 소포

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

절연 및 Extracellular 소포 특성 성인 Schistosoma 나무 에서

요약

더 많은 비디오 탐색

이 비디오의 챕터

Cercarial 변화와 체외에서 양성 Schistosoma mansoni Schistosomules

에서 계란을 사용하여

대량 절연 및 인간의 혈액 우연 Schistosoma Mansoni 의 Intramolluscan 단계의 생체 외에서 재배

간암 조직에서 유래한 작은 세포외 소포체의 농축 방법

조절된 세포 배양 배지에서 세포외 소포를 분리하기 위한 크기 배제 크로마토그래피

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus(Echinococcus granulosus에서 작은 세포외 소포의 분리 및 수지상 세포 흡수)

예쁜꼬마선충의 정화 및 분석 세포외 소포

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues(생쥐 비장 조직에서 유래한 엑소좀의 분리 및 특성 분석)

Cercarial 변화와 체외에서 양성 Schistosoma mansoni* Schistosomules*