Name: purification of extracellular trypanosomes, including african, from blood by anion-exchangers (diethylaminoethyl-cellulose columns)
Uploaded: 2019-04-06T13:00:00
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我们感谢 UMR 177 INTERTRYP IRD CIRAD 波尔多大学的所有成员。这项研究得到了波尔多大学内部资助的支持, 来自 ANR、LABEX ParaFrap ANR-11-LAB-0024 以及寄生虫和热带医学发展协会的支持。班吉 (Centrafrique)

根据《实验室动物护理和使用准则》 (NIH 第85±23号出版物, 1996年修订本) 进行的调查。协议得到了我们当地道德委员会的批准。
1. 动物
<ol><li>在每次实验前 15天, 将8至10周大的瑞士雌性老鼠, 20-25g 的雌性瑞士老鼠放在动物住房设施中。将它们放在通风的盒子里, 存放在受保护的温度 (22°c) 和湿度 (50%) 中控制室, 12小时关闭光循环。</li>
	<li>让动物免费获得食物和水。尽量...

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	<li>B&#252;scher, P., Cecchi, G., Jamonneau, V., Priotto, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis.">Human African trypanosomiasis.</a> Lancet. 390 (10110), 2397-2409 (2017).</li><li>Lejon, V., Bentivoglio, M., Franco, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis.">Human African trypanosomiasis.</a> Handbook of Clinical Neurology. 114, 169-181 (2013).</li><li>Giordani, F., Morrison, L. J., Rowan, T. G., De Koning, H. P., Barrett, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+animal+trypanosomiases+and+their+chemotherapy:+a+review.">The animal trypanosomiases and their chemotherapy: a review.</a> Parasitology. 143 (14), 1862-1889 (2016).</li><li>Aksoy, S., Buscher, P., Lehane, M., Solano, P., Van Den Abbeele, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis+control:+Achievements+and+challenges.">Human African trypanosomiasis control: Achievements and challenges.</a> PLoS Neglected Tropical Diseases. 11 (4), e0005454 (2017).</li><li>B&#252;scher, P., Deborggraeve, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+molecular+diagnostics+contribute+to+the+elimination+of+human+African+trypanosomiasis?.">How can molecular diagnostics contribute to the elimination of human African trypanosomiasis?.</a> Expert Review of Molecular Diagnostics. 15 (5), 607-615 (2015).</li><li>Truc, P., 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=Atypical+human+infections+by+animal+trypanosomes.">Atypical human infections by animal trypanosomes.</a> PLoS Neglected Tropical Diseases. 7 (9), e2256 (2013).</li><li>Lumsden, W. H., Kimber, C. D., Evans, D. A., Doig, S. 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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=Trypanosoma+brucei+CYP51:+Essentiality+and+Targeting+Therapy+in+an+Experimental+Model.">Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.</a> PLoS Neglected Tropical Diseases. 10 (11), e0005125 (2016).</li><li>Raz, B., Iten, M., Grether-B&#252;hler, Y., Kaminsky, R., Brun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Alamar+Blue+assay+to+determine+drug+sensitivity+of+African+trypanosomes+(T.+b.+rhodesiense+and+T.+b.+gambiense)+in+vitro.">The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro.</a> Acta Tropica. 68 (2), 139-147 (1997).</li><li>Albright, J. W., Albright, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+growth+of+Trypanosoma+musculi+in+cell-free+medium+conditioned+by+rodent+macrophages+and+mercaptoethanol.">In vitro growth of Trypanosoma musculi in cell-free medium conditioned by rodent macrophages and mercaptoethanol.</a> International Journal for Parasitology. 10 (2), 137-142 (1980).</li><li>Gobert, A. P., 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=L-Arginine+availability+modulates+local+nitric+oxide+production+and+parasite+killing+in+experimental+trypanosomiasis.">L-Arginine availability modulates local nitric oxide production and parasite killing in experimental trypanosomiasis.</a> Infection and Immunity. 68 (8), 4653-4657 (2000).</li><li>De Muylder, G., 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=A+Trypanosoma+brucei+kinesin+heavy+chain+promotes+parasite+growth+by+triggering+host+arginase+activity.">A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity.</a> PLoS Pathogens. 9 (10), e1003731 (2013).</li><li>Nzoumbou-Boko, R., 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=Trypanosoma+musculi+Infection+in+Mice+Critically+Relies+on+Mannose+Receptor-Mediated+Arginase+Induction+by+a+TbKHC1+Kinesin+H+Chain+Homolog.">Trypanosoma musculi Infection in Mice Critically Relies on Mannose Receptor-Mediated Arginase Induction by a TbKHC1 Kinesin H Chain Homolog.</a> Journal of Immunology. 199 (5), 1762-1771 (2017).</li><li>Bonhivers, M., Nowacki, S., Landrein, N., Robinson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+the+trypanosome+endo-exocytotic+organelle+is+cytoskeleton+mediated.">Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated.</a> PLoS Biology. 6 (5), e105 (2008).</li><li>Albisetti, 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=Interaction+between+the+flagellar+pocket+collar+and+the+hook+complex+via+a+novel+microtubule-binding+protein+in+Trypanosoma+brucei.">Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei.</a> PLoS Pathogens. 13 (11), e1006710 (2017).</li><li>Cross, G. A. M., Klein, R. A., Linstead, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+amino+acids+by+Trypanosoma+brucei+in+culture:+L-threonine+as+a+precursor+for+acetate.">Utilization of amino acids by Trypanosoma brucei in culture: L-threonine as a precursor for acetate.</a> Parasitology. 71 (2), 311-326 (1975).</li><li>Bringaud, F., Rivi&#232;re, L., Coustou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+trypanosomatids:+adaptation+to+available+carbon+sources.">Energy metabolism of trypanosomatids: adaptation to available carbon sources.</a> Molecular and Biochemical Parasitology. 149 (1), 1-9 (2006).</li><li>Mazet, M., 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=Revisiting+the+central+metabolism+of+the+bloodstream+forms+of+Trypanosoma+brucei:+production+of+acetate+in+the+mitochondrion+is+essential+for+the+parasite+viability.">Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for the parasite viability.</a> PLoS Neglected Tropical Diseases. 7 (12), e2587 (2013).</li><li>Coutton, 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=Mutations+in+CFAP43+and+CFAP44+cause+male+infertility+and+flagellum+defects+in+Trypanosoma+and+human.">Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.</a> Nature Communications. 9 (1), 686 (2018).</li><li>Cnops, J., Magez, S., De Trez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+mechanisms+of+African+trypanosomes:+Why+trypanosomosis+is+keeping+us+awake.">Escape mechanisms of African trypanosomes: Why trypanosomosis is keeping us awake.</a> Parasitology. 142 (3), 417-427 (2015).</li><li>Barrett, M. 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E., Cover, B., Gaborak, M. <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+blood+and+intracellular+forms+of+Trypanosoma+cruzi+from+rats+and+other+rodents+and+preliminary+studies+of+their+metabolism.">Isolation of blood and intracellular forms of Trypanosoma cruzi from rats and other rodents and preliminary studies of their metabolism.</a> Parasitology. 76 (2), 159-176 (1978).</li><li>Cruz-Saavedra, 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=Purification+of+Trypanosoma+cruzi+metacyclic+trypomastigotes+by+ion+exchange+chromatography+in+sepharose-DEAE,+a+novel+methodology+for+host-pathogen+interaction+studies.">Purification of Trypanosoma cruzi metacyclic trypomastigotes by ion exchange chromatography in sepharose-DEAE, a novel methodology for host-pathogen interaction studies.</a> Journal of Microbiological Methods. 142, 27-32 (2017).</li><li>Lemesre, J. 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纯化的色氨酸酶是研究免疫学、生物化学、细胞和分子生物学的有力手段。从锥虫体中获得了大量的数据和结果, 这有助于从其他真核细胞30 中获得信息。锥虫体也是重要和有趣的研究的主题, 因为它们设计了许多机制, 使它们能够在两种截然不同的环境中生存和生长: 采采蝇飞行载体和哺乳动物宿主 23, 31. 据报告, 分离锥虫的各种技术,...

这里介绍的方法允许从血液中分离锥虫体、负责动物和人类非洲锥虫病 (HAT) 的寄生虫。这是诊断第一阶段 HAT 的最佳方法, 而且这种寄生虫纯化方法可以进行可靠的血清学和研究研究。
HAT 是由采采蝇传播的冈比亚锥虫和罗得西亚酮 1.引起的。这些原生动物寄生虫在疾病的第一阶段 (血液淋巴阶段) 在血液、淋巴和间质液体中外源性繁殖。第二阶段 (脑膜-脑期) 从寄生虫越过血脑屏障开始;神经体征, 包括睡眠障碍, 将其命名为 "昏睡病", 是第二阶段2的典型。相关的锥虫体 (t. evansi, t. congolense, t. vivax, t. b. brucei) 是非洲动物锥虫病 (aat) 3 的致病因子.
世界卫生组织 (世卫组织) 的目标是到2020年消除 HAT 这一公共卫生问题, 到2030年停止传播.最近引入的快速诊断测试改善了血清学诊断 1,4,5。已经开发了一些分子诊断测试, 但它们在现场诊断中的作用尚未确立5。它们被用来鉴定布鲁谢群和非典型锥虫病引起的由负责动物锥虫病的寄生虫6引起的亚种。
寄生虫的检测对诊断、治疗和随访至关重要, 因为血清学可以给出假阳性, 不幸的是假阴性结果1。在由冈比亚 t. b. b.引起的 hat 情况下, (95% 以上的病例) 通常是低寄生虫, 而对于导致引起的 hat, 则很困难。血液中经常存在的寄生虫数量。采用了各种浓缩技术, 如厚滴和毛细管离心 (CTC), 但通过一列阴离子交换器 (DEAE 纤维素) 从血液中分离寄生虫, 然后对其进行离心和微观观察。颗粒, 是最敏感的方法 (约50种血液寄生虫)1,7。因此, 采用这种阴离子交换器 (DEAE 纤维素) 方法纯化锥虫体是最好的, 到目前为止, 也是将血液中的寄生虫可视化和分离的参考方法, 用于 HAT 的诊断。在野外条件下, 成功地使用了 deae 纤维素的小型柱, 并通过了一些改进, 促进了显微镜观察 7,8。
从血液中分离锥虫的方法, 描述如下, 取决于寄生虫表面电荷, 这是较少阴性比哺乳动物血细胞9。有趣的是, 这种方法是50年前由希拉·兰姆博士在1968年开发的, 仍然是检测和制备血液锥虫的黄金标准。它是快速和可重复的唾液锥虫体从广泛的哺乳动物, 允许诊断动物和人类锥虫病10。
为了获得活的、纯化的寄生虫, 被感染的血液被添加到阴离子交换器柱上。色谱条件 (主要是 pH 值, 液比物介质的离子强度) 必须适应每一种锥虫属, 更普遍的是, 适合哺乳动物血细胞和色样体 10的每一混合物。对于大多数非洲色胺体 10, 洗脱缓冲液精确地调整到 ph 值8。这种方法有利于患者血液中发现的寄生虫浓度, 因为寄生虫可能太低, 仅通过显微镜观察就无法检测到, 而且还能进行实验室调查。使用这种技术处理新鲜分离的锥虫体和受感染动物的血液, 比在实验室内无限期培养的针对在无动条件下培养的寄生虫的研究更相关。
寄主寄生虫的关系最好与感染其自然寄主的寄生虫研究, 因此, 肌肉 t ,一种天然的小鼠寄生虫, 它是细胞外锥虫体的代表, 有许多优点, 因为小鼠感染涉及在小型实验动物, 不需要生物危害安全水平 (BSL) 条件。与其他许多锥虫类, 包括人类病原体不同, 肌肉 t 不会杀死具有免疫能力的小鼠。在 t 细胞缺乏的小鼠中没有消除肌肉 t ,在受感染的小鼠中, 通过改变食物和营养摄入量11, 可以增加寄生虫。这种寄生虫调节免疫反应, 共同感染与其他病原体12。受感染小鼠的肌肉 t 与培养的肌肉组织有差异 ,例如, 与从受感染小鼠身上纯化的寄生虫相比, 肌肉组织的膜 fc 受体在抗酸菌培养中失去了表达,14. 在轴突状培养物中, 分泌物分泌的因子 (esf) 在质量和数量上也较少表达, 在流行地区分离的菌株之间存在差异15。ESF 是第一个向宿主免疫系统显示的抗原, 因此在最初的宿主免疫反应 16中发挥着重要作用。
在用于实验室调查的实验感染动物中, 该协议有助于对更多的寄生虫进行实验, 最大限度地减少所需的小鼠数量, 特别是在使用免疫抑制动物时。在大规模筛选的锥虫病卡凝集试验中使用的可变表面糖蛋白 (Vsg) 仍然从在大鼠体内繁殖的锥虫体中纯化。目前可在现场使用的两项快速诊断测试 (单独包装的盒式磁带) 仍在使用本地 vsg 的感染模型源, 而不是体外培养的锥虫 1,4, 5. 由于这些 deae 纤维素纯化的寄生虫可以很容易地从自然或实验感染的宿主, 特别是啮齿类动物那里大量获得, 因此促进了锥虫学和生物学研究的进展。

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			<td style="width:184px;">352,070</td>
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			<td height="21" style="height:21px;width:299px;">Centrifuge</td>
			<td style="width:381px;">Sigma Aldrich</td>
			<td style="width:184px;">4K15</td>
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			<td style="width:184px;">s/c- 211213</td>
			<td>100 G</td>
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			<td style="width:184px;">100 28026;260</td>
			<td>500 G</td>
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			<td height="21" style="height:21px;width:299px;">Nalgene Plastic Media Bottles size 500 mL</td>
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			<td style="width:381px;">EUROBIO</td>
			<td style="width:184px;">CABPES01 OU</td>
			<td>100 mL</td>
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			<td height="21" style="height:21px;width:299px;">Phenol red</td>
			<td style="width:381px;">Sigma Aldrich</td>
			<td style="width:184px;">P0290</td>
			<td>100 mL</td>
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			<td height="21" style="height:21px;width:299px;">Syringue&#160;</td>
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			<td>ANTAT 1.1</td>
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			<td>ITMAP 1893</td>
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			<td style="width:184px;"></td>
			<td>Partinico II</td>
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purification of extracellular trypanosomes, including african, from blood by anion-exchangers (diethylaminoethyl-cellulose columns)

这种方法允许分离锥虫体, 寄生虫负责动物和人类非洲锥虫病 (HAT), 从受感染的血液。这是诊断第一阶段 HAT 的最佳方法, 而且这种寄生虫纯化方法允许血清学和研究调查。
HAT 是由采采蝇传播的冈比亚锥虫和罗得西亚 t. b.引起的。相关的锥虫体是动物锥虫病的病原体。锥虫体检测对于 HAT 的诊断、治疗和随访至关重要。这里描述的技术是最敏感的寄生虫检测技术, 适用于现场条件的诊断冈比亚 hat , 可以在一个小时内完成。血液被分层到以前调整为 pH 值8的阴离子交换器柱 (DEAE 纤维素) 上, 并添加洗脱缓冲液。高负电荷的血细胞被吸附在色谱柱上, 而负电荷的锥虫体通过的程度较低。采集的色氨酸体是通过离心造粒和显微镜观察的。此外, 寄生虫的制备没有细胞损伤, 同时保持其传染性。
免疫检测需要纯化的色氨酸体;它们被用于色氨酸溶解试验, 这是 HAT 血清学中的金标准。染色寄生虫用于卡片凝集试验 (CATT) 的现场血清学。纯化的锥细胞的抗原, 如不同的表面糖蛋白, 外抗原, 也用于各种免疫检测。这里描述的程序是为非洲锥虫设计的;因此, 色谱条件必须适应每一个锥虫株, 更广泛地说, 适应每种寄主哺乳动物的血液。
这些迷人的病原体很容易被纯化, 可用于生化、分子和细胞生物学研究, 包括与宿主细胞共培养, 以调查在膜受体、信号和基因水平上的宿主-寄生虫关系表达;体外药物检测;研究代谢过程、细胞骨架生物发生和寄生虫存活的基因缺失、突变或过度表达。

纯化的色氨酸体已被用于药物测试。寄生虫被转移到含有特定药物连续稀释的培养井, 单独或混合19。微观观察, 评估运动是一个标志的活力, 可以执行时, 只有少数的哑弹正在进行测试, 而 AlamarBlue 细胞活力检测是一个很好的方法, 大的动力检测在药物筛选20。在 hat 治疗中使用的参考药物----戊胺素的作用如图 1所示?...

Watch this Scientific Journal Video about Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns at JoVE.com

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

这种从血液中分离锥虫的方法取决于它们的表面电荷比哺乳动物的血细胞更少阴性。受感染的血液被放置在阴离子交换器柱上并进行治疗。这种方法是非洲锥虫病最适合的诊断方法, 为免疫、生物、生化、药物和分子生物学研究提供纯化寄生虫。

阴离子交换剂 (二乙基氨基乙基纤维素柱) 纯化包括非洲在内的细胞外锥虫体

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

非洲锥虫体是造成人类非洲锥虫病，或昏睡病和非洲动物锥虫病的责任人。引起昏睡病的锥虫体是采采蝇，这些寄生虫在撒哈拉以南非洲的许多地理病区存在。寄生虫的检测对于诊断、治疗和随从至关重要。血清学可以给出假阳性和不幸的阴性结果。为了帮助血液的检测，必须集中锥体。各种寄生虫浓缩技术已被使用。最敏感的方法是通过一列心安的血管交换器、DEAE纤维素分离出血液中的寄生虫，然后进行离心和微观观察。因此，DEAE纤维素法是最好的，并且至今是一种将寄生虫从血液中可视化和隔离的参考方法，用于人类非洲锥虫病的诊断，特别是在野外条件下。这种方法是非洲锥虫病的最合适诊断方法，也为免疫学、生物、生化、制药和分子生物学研究提供了纯化寄生虫。所有调查均符合实验室动物护理和使用指南，并已获得法国当局的同意，使用的所有协议均已获得我们当地道德委员会的批准。根据书面协议称出每种物质。加入蒸馏水，用磷酸二氢钾精确调节至pH 8，制成以下缓冲溶液，浓缩磷酸盐缓冲液盐水2X。加入蒸馏水和葡萄糖，用于磷酸盐缓冲盐水葡萄糖。对于100毫升的洗脱缓冲液，每毫升青霉素加入100个单位，每毫升链霉素100微克，向磷酸盐缓冲盐水葡萄糖添加5微克毫克的酚红色。100克DEAE纤维素首先用三升蒸馏水洗净，然后离开沉淀。细颗粒被丢弃。洗涤重复，直到上清液是明确的。添加浓缩磷酸盐缓冲盐水2X，搅拌DEAE纤维素。pH值调整为8，含磷酸钾。然后用蒸馏水洗两次纤维素，用磷酸盐缓冲盐水葡萄糖洗两次。DEAE纤维素和磷酸盐缓冲盐水葡萄糖以相等的体积混合。合金在室温或四摄氏度或零下 20 摄氏度下四分之一分，以进行长时间储存。锥体感染的血液储存在液氮中。一毫升的合金夸脱在室温下解冻。使用针头和一毫升注射器从冷冻管中仔细收集寄生虫。解冻寄生虫的可行性由感染前光显微镜观察所可视化的活力评估。寄生虫被注射到两只小鼠的内侧腔中，每只小鼠500微升。感染后的每一天，寄生虫病都通过用针头从尾巴上收集一滴血，然后通过显微镜检查进行评估。当寄生虫病处于适当水平时，会采集受感染的血液。夹子或支架中支持 10 毫升注射器，并添加预切的滤纸。准备好的DEAE纤维素倒入注射器，高达8或9毫升的水平。用洗脱缓冲器清洗列。两毫升受感染的血液被小心地放置在柱上，在50毫升离心管中收集在柱状水液中。根据锥体体传递情况定期添加洗脱缓冲液。通过定期服用一滴列水化，并使用光显微镜观察锥形体，检查锥形体通过柱的传递。当不再在伊卢酸盐中观察到锥形体时，锥形管在1，800次G下离心，在4摄氏度下10分钟。上清液被丢弃，托盘中的锥体用血细胞计计数。收集的锥体随后被稀释在计划调查所需的介质中。这种方法是非洲锥虫病最合适的诊断方法，为免疫学、生物、生化、制药和分子生物学研究提供了纯化寄生虫。这些研究之所以得到发展，是因为DEAE纤维素纯化寄生虫很容易从自然或实验感染的宿主，特别是啮齿动物获得大量。通过微观观察评估药物制剂存在的锥度体生存能力。寄生虫的生存能力与运动性相关，因此，可以在光学显微镜下以 40 倍或更高的放大倍率进行眼睛检查。寄生虫的生存能力通过测量动体形式的百分比进行评估。在模拟处理控制井时，将测试化合物的稀释添加到 96 孔组织培养板的每个孔中。每个浓度以三钙评估。每次稀释的可行寄生虫数量与控制井中的寄生虫数量进行比较。本图显示了在非洲人类锥虫病治疗中使用的参考药物五溴二胺的影响。锥体巨噬细胞共培养允许在细胞水平上研究宿主寄生虫的相互作用。在巨噬细胞寄生虫共培养中，额外的细胞锥体会诱导产生巨噬细胞精氨酸酶，水解精氨酸到食肉素。苯胺是多胺和苯丙酮的前体，对寄生虫的存活和生长至关重要。此外，精氨酸的耗竭减少了一氧化氮的产生。随后，加入一种阿金酶抑制剂S-L-半胱氨酸，以联合培养，显著减少巨噬细胞诱导寄生虫的生长。阿吉娜斯诱导由锥体排泄和/或分泌因子进行调解。这种精氨酸诱导因子已被确定为奥辛基宁。激酶结合到C型莱金曼尼诺斯结合受体。精氨酸酶被诱导并产生有利于寄生虫生长的食肉素。在巨噬细胞中，用12.5毫摩尔人或巨噬细胞培养的金氨酸不会诱发阿吉娜斯，而巨噬细胞的人类受体已被删除。使用DAEA纤维素纯化锥体细胞生物学和生化实验的进一步实例在已识别出新的细胞骨骼蛋白（如BILBO1）的研究中得到证明。BILBO1 是重要细胞骨骼结构和寄生虫生存的生物发生所需的。因此，BILBO1 是致病性锥体干预的重要目标。图中显示了一个显示血液培养形式的免疫浮游标记，用抗BILBO1抗体探测的锥形瘤布鲁西细胞。此外，葡萄糖和曲氨酸代谢已描述使用DEAE纤维素纯化锥体和参与这些过程的酶已经通过实验验证。图中显示了血液形成锥形体中葡萄糖和三元代谢的示意图。通过心安的核酸交换从血液中纯化非洲锥虫体，DEAE纤维素柱最近有所改进，仍然是锥体检测的黄金标准，不仅在流行地区寄生虫病含量低的自然宿主，而且大量需要寄生虫进行实验研究。研究已经能够识别锥体分子在寄生虫的生存和生长中起着至关重要的作用。已确定的目标可能是未来疫苗的基础，这种疫苗具有对人类和动物的潜在抗原，采用一种健康方法。

Research

JoVE Journal

Immunology and Infection

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

Depletion of Specific Cell Populations by Complement Depletion

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Isolation of Mouse Peritoneal Cavity Cells

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...