Name: isolation and purification of fungal &#946;-glucan as an immunotherapy strategy for glioblastoma
Uploaded: 2023-06-02T14:20:00
Description: Watch this Scientific Journal Video about Isolation and Purification of Fungal Î²-Glucan as an Immunotherapy Strategy for Glioblastoma at JoVE.com

我们要感谢Vasiliki Economopoulos博士在ImageJ中测量富洛尔斯信号的内部脚本。我们还要感谢CITIUS（塞维利亚大学）及其所有人员在示威期间的支持。这项工作得到了塞维利亚大学的西班牙FEDER I + D + i-USE，US-1264152和科学，创新和大学部PID2021-126090OA-I00的支持。

本协议中描述的四种不同的蘑菇变体是从商业来源获得的（见 材料表）。
1. 真菌β-葡聚糖的分离
<ol><li>可溶性蘑菇多糖的提取和分离 注意：可溶性蘑菇多糖（SMP）是根据 图1所示的示意性程序获得的。<ol><li>在蒸馏水中轻轻冲洗新鲜的 P. ostreatus， P. djamor， H. erinaceus和 G. lucidum 子实体（约2，000克/...

<ol>
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C., Li, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages/microglia+in+the+glioblastoma+tumor+microenvironment.">Macrophages/microglia in the glioblastoma tumor microenvironment.</a> International Journal of Molecular Sciences. 22 (11), 5775 (2021).</li><li>Sevenich, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+&amp;#34;cold&amp;#34;+into+&amp;#34;hot&amp;#34;+tumors-opportunities+and+challenges+for+radio-immunotherapy+against+primary+and+metastatic+brain+cancers.">Turning &amp;#34;cold&amp;#34; into &amp;#34;hot&amp;#34; tumors-opportunities and challenges for radio-immunotherapy against primary and metastatic brain cancers.</a> Frontiers in Oncology. 9, 163 (2019).</li><li>Niesel, 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+immune+suppressive+microenvironment+affects+efficacy+of+radio-immunotherapy+in+brain+metastasis.">The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis.</a> EMBO Molecular Medicine. 13 (5), e13412 (2021).</li><li>McCann, F., Carmona, E., Puri, V., Pagano, R. E., Limper, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+internalization+of+fungal+beta-glucans+is+not+necessary+for+initiation+of+related+inflammatory+responses.">Macrophage internalization of fungal beta-glucans is not necessary for initiation of related inflammatory responses.</a> Infection and Immunity. 73 (10), 6340-6349 (2005).</li><li>Vetvicka, V., Teplyakova, T. V., Shintyapina, A. B., Korolenko, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+medicinal+fungi-derived+&#946;-glucan+on+tumor+progression.">Effects of medicinal fungi-derived &#946;-glucan on tumor progression.</a> Journal of Fungi. 7 (4), 250 (2021).</li><li>Chan, G. C. F., Chan, W. K., Sze, D. M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+beta-glucan+on+human+immune+and+cancer+cells.">The effects of beta-glucan on human immune and cancer cells.</a> Journal of Hematology &amp; Oncology. 2, 25 (2009).</li><li>Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> The Journal of Biological Chemistry. 193 (1), 265-275 (1951).</li><li>Klaus, 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=Antioxidative+activities+and+chemical+characterization+of+polysaccharides+extracted+from+the+basidiomycete+Schizophyllum+commune.">Antioxidative activities and chemical characterization of polysaccharides extracted from the basidiomycete Schizophyllum commune.</a> Food Science and Technology. 44 (10), 2005-2011 (2011).</li><li>Soto, M. 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=STAT3-mediated+astrocyte+reactivity+associated+with+brain+metastasis+contributes+to+neurovascular+dysfunction.">STAT3-mediated astrocyte reactivity associated with brain metastasis contributes to neurovascular dysfunction.</a> Cancer Research. 80 (24), 5642-5655 (2020).</li><li>Chrom&#253;, V., Vinkl&#225;rkov&#225;, B., &#352;prongl, L., Bittov&#225;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kjeldahl+method+as+a+primary+reference+procedure+for+total+protein+in+certified+reference+materials+used+in+clinical+chemistry.+I.+A+review+of+Kjeldahl+methods+adopted+by+laboratory+medicine.">The Kjeldahl method as a primary reference procedure for total protein in certified reference materials used in clinical chemistry. I. A review of Kjeldahl methods adopted by laboratory medicine.</a> Critical Reviews in Analytical Chemistry. 45 (2), 106-111 (2015).</li><li>Waterborg, J. H., Matthews, H. 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+Lowry+method+for+protein+quantitation.">The Lowry method for protein quantitation.</a> Methods in Molecular Biology. 32, 1-4 (1994).</li><li>Zhang, 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=Characterization+and+antioxidant+activities+of+polysaccharides+from+thirteen+boletus+mushrooms.">Characterization and antioxidant activities of polysaccharides from thirteen boletus mushrooms.</a> International Journal of Biological Macromolecules. 113, 1-7 (2018).</li><li>Barbosa, J. 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=Obtaining+extracts+rich+in+antioxidant+polysaccharides+from+the+edible+mushroom+Pleurotus+ostreatus+using+binary+system+with+hot+water+and+supercritical+CO2.">Obtaining extracts rich in antioxidant polysaccharides from the edible mushroom Pleurotus ostreatus using binary system with hot water and supercritical CO2.</a> Food Chemistry. 330, 127173 (2020).</li><li>Ma, Y. 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=Assessment+of+polysaccharides+from+mycelia+of+genus+Ganoderma+by+mid-infrared+and+near-infrared+spectroscopy.">Assessment of polysaccharides from mycelia of genus Ganoderma by mid-infrared and near-infrared spectroscopy.</a> Scientific Reports. 8 (1), 10 (2018).</li><li>Nie, 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=Immune-enhancing+effects+of+polysaccharides+MLN-1+from+by-product+of+Mai-luo-ning+in+vivo+and+in+vitro.">Immune-enhancing effects of polysaccharides MLN-1 from by-product of Mai-luo-ning in vivo and in vitro.</a> Food and Agricultural Immunology. 30 (1), 369-384 (2019).</li><li>Cerletti, C., Esposito, S., Iacoviello, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Edible+mushrooms+and+beta-glucans:+impact+on+human+health.">Edible mushrooms and beta-glucans: impact on human health.</a> Nutrients. 13 (7), 2195 (2021).</li><li>Klaus, 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=The+edible+mushroom+Laetiporus+sulphureus+as+potential+source+of+natural+antioxidants.">The edible mushroom Laetiporus sulphureus as potential source of natural antioxidants.</a> International Journal of Food Sciences and Nutrition. 64 (5), 599-610 (2013).</li><li>Kozarski, 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=Dietary+polysaccharide+extracts+of+Agaricus+brasiliensis+fruiting+bodies:+chemical+characterization+and+bioactivities+at+different+levels+of+purification.">Dietary polysaccharide extracts of Agaricus brasiliensis fruiting bodies: chemical characterization and bioactivities at different levels of purification.</a> Food Research International. 64, 53-64 (2014).</li><li>Ayimbila, F., Keawsompong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+composition+and+antioxidant+property+of+crude+polysaccharides+from+the+fruiting+bodies+of+Lentinus+squarrosulus.">Functional composition and antioxidant property of crude polysaccharides from the fruiting bodies of Lentinus squarrosulus.</a> 3 Biotech. 11 (1), 7 (2021).</li><li>de Azambuja, E., 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=Ki-67+as+prognostic+marker+in+early+breast+cancer:+a+meta-analysis+of+published+studies+involving+12,155+patients.">Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients.</a> British Journal of Cancer. 96 (10), 1504-1513 (2007).</li><li>Holubec, 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=Assessment+of+apoptosis+by+immunohistochemical+markers+compared+to+cellular+morphology+in+ex+vivo-stressed+colonic+mucosa.">Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa.</a> The Journal of Histochemistry and Cytochemistry. 53 (2), 229-235 (2005).</li><li>Borges, G. 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这项工作描述了使用成熟的技术成功地从四种不同的真菌中分离、纯化和表征 SβG 的含量。结果表明，热水提取灰 霉、黄 杨、 灵芝 和猴头孢杆菌的SMPs后，用α-淀粉酶、葡萄糖苷酶和蛋白酶水解处理后，α-葡聚糖和蛋白质的含量降低，从而显著丰富了纯SβGs的含量。
尽管如此，我们观察到大多数真菌β葡聚糖在纯化过程中不溶于水。该研究的主要?...

尽管在神经肿瘤学领域取得了新的成就，但胶质母细胞瘤患者的预期寿命仍然微不足道。针对脑肿瘤的金标准疗法基于手术、放疗和化疗的结合。然而，在过去十年中，免疫疗法已成为治疗不同类型癌症的有力策略1。因此，利用身体对肿瘤细胞的免疫反应的可能性最近已成为肿瘤学的第四大支柱。
人们早就知道，该领域最大的挑战之一是克服肿瘤微环境中发现的强免疫抑制2。特别是，在胶质母细胞瘤的情况下，这是最常见和最具侵袭性的脑癌形式之一，揭示协调这种促肿瘤场景的关键途径，并找到可以抵消免疫系统抑郁反应的新化合物，可能为未来针对这种不治之症的治疗铺平道路。
大脑拥有自己的免疫系统细胞，最相关的细胞类型是小胶质细胞。这些细胞已被证明在不同的中心疾病中具有相当复杂的行为3。在原发性脑肿瘤（例如胶质母细胞瘤）的情况下，这些细胞向支持肿瘤细胞定植脑实质的抗炎表型转移3。许多出版物增强了这些细胞在肿瘤进展过程中的主要作用。其中一个主要原因是胶质瘤相关的小胶质细胞和浸润巨噬细胞（GAMs）占肿瘤总量的三分之一，因此表明它们在脑肿瘤进展过程中的激活状态的明确影响4，5。
本着这种精神，真菌β-葡聚糖被描述为触发有效免疫反应的有效分子，包括吞噬作用和促炎因子的产生，导致有害物质的消除6，7，8，9，10。真菌β-葡聚糖通常使用来自不同蘑菇部位的提取物进行研究。然而，特定效应的归属需要其纯化以避免歧义并能够理解诸如免疫调节剂之类的分子的作用机制8。
在这项工作中，可溶性β葡聚糖从四种不同蘑菇的子实体中纯化，通常用作食用蘑菇（Pleurotus ostreatus 和 Pleurotus djamor）和药用（灵芝 和 猴头菇）蘑菇。特别是，这四种蘑菇在食品和制药工业中有很大的用途，并且是在商业企业的环境友好型循环经济中生产的（见 材料表）。
为了为真菌β-葡聚糖在脑癌治疗中的未来使用奠定基础，明确的纯化策略和临床前研究深入研究它们与免疫系统细胞的假定相互作用对于评估其作为抗肿瘤介质的潜在作用至关重要。这项工作描述了回收所选蘑菇子实体中所含的可溶性β-葡聚糖所需的许多分离和纯化步骤。一旦成功纯化，小胶质细胞就会被激活以增强其炎症表型。小鼠胶质母细胞瘤细胞（GL261）涂有不同的小胶质细胞条件培养基，先前用这些提取物处理，然后评估其对肿瘤细胞行为的影响。有趣的是，我们实验室的初步研究（数据未显示）揭示了促炎小胶质细胞如何减缓肿瘤细胞迁移和侵袭特性，不仅在胶质母细胞瘤细胞中，而且在其他癌细胞系中。这项多学科工作可能为肿瘤学研究人员提供有用的工具，以测试能够增强许多不同类型肿瘤免疫反应的有前途的化合物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well chamber slides</td>
			<td>Thermo Fisher, USA</td>
			<td>171080</td>
			<td></td>
		</tr>
		<tr>
			<td>Air-drying oven</td>
			<td>J.P. Selecta S.A., Spain</td>
			<td>2000210</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich, St. Louis</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcalase</td>
			<td>Novozymes, Denmark</td>
			<td>protease</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Thermofisher, USA</td>
			<td>A32731</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647</td>
			<td>Thermofisher, USA</td>
			<td>A32728</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade mill</td>
			<td>Retsch, Germany</td>
			<td>&#160;SM100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>MERK, Germany</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose tubing membrane</td>
			<td>Sigma-Aldrich, St. Louis</td>
			<td>D9402</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>MERK, Germany</td>
			<td>Eppendorf, 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Colocalisation pluggins</td>
			<td>ImageJ</td>
			<td></td>
			<td>(https://imagej.net/imaging/colocalization-analysis&#160;)</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MERK, Germany</td>
			<td>28718-90-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrans</td>
			<td>Pharmacosmos, Holbalk, Denmark</td>
			<td>Dextran 410, 80, 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s modified Eagle&#180;s medium, Gluta MAXTM</td>
			<td>Gibco, Life Technologies, Carlsbad, CA, USA</td>
			<td>10564011</td>
			<td></td>
		</tr>
		<tr>
			<td>Extenda (&#945;- Amylase/Glucoamylase)</td>
			<td>Novozymes, Denmark</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco, Life Technologies, Carlsbad, CA, USA</td>
			<td>A4736301</td>
			<td></td>
		</tr>
		<tr>
			<td>FT-IR spectromete</td>
			<td>Bruker-Vertex, Switzerland</td>
			<td>VERTEX 70v</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphing and analysis software</td>
			<td>GraphPad Prism (GraphPad Software, Inc.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Waters Corp, Milford, MA, USA</td>
			<td>Waters 2695 HPLC</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Eppedorf</td>
			<td>Galaxy 170S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectometer</td>
			<td>Q Exactive GC, Thermo Scientific</td>
			<td>725500</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MERK, Germany</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma-Aldrich, St. Louis</td>
			<td>P4458</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Crison, Barcelona, Spain</td>
			<td>Basic 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Gibco, Life Technologies, Carlsbad, CA, USA</td>
			<td>1010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Cleaved Caspase-3 (Asp175) Antibody</td>
			<td>Abcam, UK</td>
			<td>ab243998</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Ki-67 Monoclonal</td>
			<td>Thermofisher, USA</td>
			<td>MA5-14520</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>B&#252;chi Ib&#233;rica S.L.U., Spain</td>
			<td>El Rotavapor R-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-hydrogel linear gel-filtration column (300 mm x 7.8 mm)</td>
			<td>Waters Corp, Milford, MA, USA</td>
			<td>WAT011545</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Visible spectrophotometer</td>
			<td>Amersham Bioscience, UK</td>
			<td>Ultrospec 2100 pro</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaMount</td>
			<td>Vector Laboratories, C.A, USA</td>
			<td>H-5000-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>J.P. Selecta S.A., Spain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 7 DUO Confocal Microscope System.</td>
			<td>Zeiss, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glucan Assay Kit</td>
			<td>Megazyme, Bray, Co. Wicklow, Ireland</td>
			<td>K-BGLU</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glucans</td>
			<td>Setas y Hongos del Sur, S.L.</td>
			<td></td>
			<td>Supplied the four variants of mushrooms</td>
		</tr>
	</tbody>
</table>

isolation and purification of fungal &#946;-glucan as an immunotherapy strategy for glioblastoma

开发针对胶质母细胞瘤的有效疗法的最大挑战之一是克服肿瘤微环境中的强免疫抑制。免疫疗法已成为使免疫系统对肿瘤细胞的反应的有效策略。胶质瘤相关巨噬细胞和小胶质细胞（GAMs）是这种抗炎方案的主要驱动因素。因此，增强GAM的抗癌反应可能代表治疗胶质母细胞瘤患者的潜在联合辅助疗法。本着这种精神，真菌β-葡聚糖分子长期以来一直被称为有效的免疫调节剂。已经描述了它们刺激先天免疫活性和改善治疗反应的能力。这些调节特征部分归因于它们与模式识别受体结合的能力，有趣的是，模式识别受体在GAM中得到了极大的表达。因此，这项工作的重点是真菌β-葡聚糖的分离，纯化和随后的使用，以增强小胶质细胞对胶质母细胞瘤细胞的杀瘤反应。小鼠胶质母细胞瘤（GL261）和小胶质细胞（BV-2）细胞系用于测试从当前生物制药行业中大量使用的蘑菇中提取的四种不同真菌β-葡聚糖的免疫调节特性： Pleurotus ostreatus， Pleurotus djamor， 猴头菇和灵 芝。为了测试这些化合物，进行了共刺激测定以测量预活化的小胶质细胞条件培养基对胶质母细胞瘤细胞增殖和凋亡活化的影响。

成功纯化β-葡聚糖 表1总结了从毛竹、黄杨、灵芝和猴头孢子实体中获得的MP、SMP和SβG的质量。 从真菌中获得的MP、SMP和SβG的基本组成（总碳水化合物、β-葡聚糖和蛋白质）如表2所示。这些结果表明该方案如何允许在SMP中检索大量蛋白质含量。然而，用α-淀粉酶/葡糖淀粉酶和蛋白酶酶处理减少了蛋白质的量并增加了β-葡聚糖浓?...

Watch this Scientific Journal Video about Isolation and Purification of Fungal Î²-Glucan as an Immunotherapy Strategy for Glioblastoma at JoVE.com

Isolation and Purification of Fungal &#946;-Glucan as an Immunotherapy Strategy for Glioblastoma

本协议描述了四种不同真菌β-葡聚糖的纯化步骤和后续研究，作为增强小胶质细胞对胶质母细胞瘤细胞的抗肿瘤特性的潜在免疫调节分子。

真菌β-葡聚糖的分离纯化作为胶质母细胞瘤的免疫治疗策略

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor ...

真菌β-葡聚醣是免疫系統的重要調節劑。提高纯度和分离方案对于断言其作为癌症治疗辅助佐剂的潜在作用至关重要。使用分子生物学和蛋白质组学实验室中常见的各种设备实现了四种不同β-葡聚糖的高度。该协议的重点是测试β-葡聚糖对小胶质细胞的影响，小胶质细胞是大脑最丰富的常驻免疫细胞。调节胶质母细胞瘤中的小胶质细胞表型可以产生重要的见解。开始提取可溶性蘑菇多糖或SMP，用蒸馏水冲洗杏仁，刺头菇和灵芝的新鲜子实体五次。然后将洗涤后的子实体在传统的空气干燥炉中在50摄氏度下完全干燥约24小时，然后在刀片磨机中研磨，从每个蘑菇中获得约200克粉末。接下来，将 100 克所有四种蘑菇粉或 MP 悬浮在 1， 000 毫升蒸馏水中，并在 121 摄氏度下高压灭菌 15 分钟。在室温下孵育30分钟后，将所得悬浮液在6， 000G下在4摄氏度下离心10分钟。然后将含有不溶性蘑菇多糖或IMPs的沉淀物在50摄氏度下在空气干燥炉中干燥24小时。弃去沉淀物并将上清液在旋转蒸发器中浓缩10次。接下来，用乙醇在 4 摄氏度下沉淀含有 SMP 的浓缩物过夜。将乙醇悬浮液在6， 000G下在4摄氏度下离心15分钟后，用移液管弃去上清液，用80%乙醇洗涤沉淀三次，然后溶解并蒸馏水。将pH调节至6.5至7，温度调节至37摄氏度，并通过分别用两个单位和四个单位的α-淀粉酶和葡糖淀粉酶处理悬浮液来溶解α-葡聚糖。接下来，将pH调节至8.0，温度调节至50摄氏度，并通过用相当于每克蛋白质2.5单位的碱化酶处理悬浮液来溶解蛋白质。水解后，离心水解产物并在旋转蒸发器中清洁上清液浓缩物五次。通过加入80%乙醇再次沉淀上清液浓缩物。为了去除低分子量分子，将沉淀物溶解在蒸馏水中，并使用12， 000道尔顿截止纤维素管膜将其在蒸馏水中透析24小时。回收水溶性部分并将其冷冻干燥以产生可溶性β-葡聚糖或S β-G。分别评估总葡聚糖和α-葡聚糖。测量所得的β-葡聚糖值作为总葡聚糖和α-葡聚糖值之间的差异。要使用紫外可见分光光度计获得 S β-G 紫外光谱，请在蒸馏水中制备每毫升 1.0 毫克的每种 S β-G。将样品转移到石英比色皿中，并在室温下扫描200至400纳米区域。使用配备紫外可见检测器和Superdex TM 200 10/300 GL凝胶过滤柱的高效液相色谱或HPLC系统，通过尺寸排阻色谱或SEC估计S β-Gs的均匀性和聚合物的分子量。在傅里叶变换红外或 FTIR 光谱仪上记录每厘米 4， 000 至 500 范围内的样品红外光谱。通过用每毫升浓度为 0.2 毫克的四种不同的 β-葡聚糖涂覆 BV2 小胶质细胞 72 小时来激活小胶质细胞，这些β-葡聚糖是从 P.ostreatus、P.djamor、G.lucidum 和 H.erinaceus 中分离出来的。72小时后，用移液管收集上清液并将剩余体积通过0.20微米注射器过滤器。然后将上清液在零下80摄氏度下冷冻至少24小时。要用预活化的小胶质细胞条件培养基处理GL 261，将250微升β-葡聚糖处理的小胶质细胞培养基加入80%汇合的GL 261细胞中72小时。72小时后丢弃培养基。不同β-Gs的紫外光谱在260和280纳米处未出现紫外吸收峰，表明β-Gs缺乏核酸和蛋白质。HPLC色谱图均一性良好，主峰和单峰分别为10.9 min、灵芝10.5 min、11.3 min、8.20 min，表明馏分与均聚物一致。FTIR光谱测量了对应于共价多糖键的分子振动。它表明SS β-Gs由与最小蛋白质结合的碳水化合物组成。HPTLC和GC-MS分析了S β-Gs的单糖谱，揭示了大量D-葡萄糖，少量D-半乳糖和D-甘露糖，以及微量的D-木糖，D-鼠李糖，D-岩藻糖和L-阿拉伯糖。使用ImageJ软件中的内部脚本，量化了每个荧光通道的正像素数。该脚本使用对照样品作为每个荧光基团强度的阈值，并提供像素数。它指示了不同实验条件下每个标记物的表达。有趣的是，GL 261在暴露于不同的小胶质细胞条件培养基时在肿瘤增殖方面没有显着差异。然而，P.djamor和H.erinaceus分别显示出裂解的半胱天冬酶-3增加约6倍和9倍的强烈诱导。确保β-葡聚醣的高品位纯度对于该方案至关重要。该方案使用先进的设备，如分光光度计，HPLC。或FITR。使用这些工具增强了我们研究结果的稳健性。我们可以自信地分析β-葡聚醣对小胶质细胞的影响，并获得更可靠的结果。由于时间和世界的限制，我们将我们的方案限制在研究小胶质细胞上的β-葡聚糖。然而，来自免疫系统的其他细胞，如巨噬细胞或T细胞，可以很容易地在这个协议中。这些协议首次结合了两个不同的知识领域。首先，使用生物技术程序纯化特定产品，其次，使用细胞培养技术测试其免疫调节特性。

Research

JoVE Journal

Neuroscience

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

科研

神经科学

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Purification of Mouse Brain Vessels

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Drosophila Courtship Conditioning As a Measure of Learning and Memory

果蝇调理作为学习和记忆的措施

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Purification of the Dendritic Filopodia-rich Fraction

富德瑞迪蒂菲洛波迪亚成分的纯化

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

使用雏鸡胚胎大脑作为模型研究人类胶质母细胞瘤细胞行为

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

作者聚焦：使用雏鸡胚胎大脑作为人胶质母细胞瘤细胞行为体内和体外分析的模型  

使用小鸡胚胎大脑作为人类胶质母细胞瘤细胞行为的 体内 和 离体 分析模型

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick ...