Name: an in vitro batch-culture model to estimate the effects of interventional regimens on human fecal microbiota
Uploaded: 2019-07-31T13:00:00
Description: Watch this Scientific Journal Video about An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota at JoVE.com

作者感谢糖尿病、肥胖和代谢中心以及临床和转化科学中心、威克森林医学院、国防部的资助(资助编号:W81XWH-18-0118) 提供的资金支持,克米特·格伦·菲利普斯二世心血管医学主席;国家卫生研究院资助了克劳德·佩珀老人美国中心(由P30AG12232资助);R01AG18915;R01DK114224和临床和转化科学中心(临床研究单位,由UL1TR001420资助),也得到了感谢的认可。我们还感谢志愿者提供粪便样本,以及我们的其他实验室成员在实验中的技术支持。

注意:查阅相应的材料安全数据表,并遵循相应的生物安全 2 级 (BSL-2) 培训的说明和指南。按照标准生物安全规则执行所有培养步骤,并使用无菌条件使用 BSL-2 机柜。此外,来自不同模型和人类受试者的粪便样本可能具有传播微生物传播疾病的潜在风险。发生任何伤害和感染时,立即寻求医疗援助。此外,人类和动物粪便样本的使用应经机构道德委员会批准,并必须符合使用样本和主题信息的协议。
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此处介绍的体外粪便浆发酵模型是一个简单的单组模型,用于近似不同基质和微生物菌株(如益生菌和益生菌)对人类粪便微生物群的组成及其影响代谢活动在粪便pH和SCFA水平方面。本文介绍的结果表明,与未经处理的粪便微生物群培养相比,接种硫素可降低粪便pH值,并显著提高经尿酸处理的粪便标本中的SCFA和乳酸水平(图1)。此外,肠道微生物群特征在经处理的样品和未经处理的样...

人类微生物群是一个复杂的群落,由细菌、古生物、病毒和真核微生物1组成,存在于人体内外。最近的证据已经确立了肠道微生物群和肠道微生物群(人类胃肠道中发现的微生物及其基因的全部集合)在各种人类疾病中的基本作用,包括肥胖、糖尿病、心血管疾病,和癌症1,2,3。此外,生活在我们肠道中的微生物产生广泛的代谢物,这显著影响我们的健康,也可以促进几种疾病的病理生理学以及各种代谢功能4,5.肠道微生物群的组成和功能异常变化(扰动)通常被称为"胃发育不良"。肌张力障碍通常与宿主的不健康状态相关,因此可以与与宿主的健康控制状态相关的正常(静静)微生物群落区分开来。肠道微生物群发育不良的特定模式通常存在于各种不同的疾病1,2,3,6,7。
肠道微生物群发酵未消化的食物,特别是可发酵的碳水化合物/纤维,不仅产生能量,还产生发散代谢物,包括短链脂肪酸(SCFAs)、乳酸、甲酸盐、二氧化碳、甲烷、氢气和乙醇6.此外,肠道微生物群还产生一些其他生物活性物质,如叶酸、生物素、三甲基胺-N-氧化物、血清素、色氨酸、伽马-氨基丁酸、多巴胺、去甲肾上腺素、乙酰胆碱、组胺、脱氧胆酸和4-乙基苯硫酸盐。这主要通过利用宿主-微生物利基内的内在代谢通量,这在几个身体过程,代谢功能和表观遗传变化1,8,9, 10.然而,由于缺乏简单、高效和可重复的协议,各种干预措施对此类微生物产品的影响仍然不明朗或不明确。人类肠道微生物群是一个极其复杂和多样化的生态系统,因此,关于其在人类健康和疾病病理学中的作用的许多问题仍未得到解答。许多常见的肠道微生物重组剂(如益生菌、益生菌、抗生素、粪便移植和感染)对肠道微生物群的组成和代谢功能的影响仍然在很大程度上难以实现。此外,在体内对这些影响的检查和验证是困难的,特别是因为肠道微生物群产生的大多数营养物质和代谢物在肠道中同时和迅速被吸收或处置;因此,测量体内这些代谢物(例如SCFA)的生产、量和处理仍然是一个实际挑战。事实上,动物和人类受试者等生理模型对于确定肠道微生物群系及其调节对宿主健康的作用至关重要,但由于道德、金钱或时间限制。为此,体外和/或体外模型,如在体外培育肠道微生物群,然后干预不同的微生物群调制器,可以提供节省时间和资金的机会,因此可以允许初步或大规模筛选各种成分(如益生菌、益生菌和其他介入化合物),以检查/预测其对粪便微生物群多样性、组成和代谢特征的影响。使用肠道微生物群的体外和体外系统的研究可有助于进一步理解宿主-微生物群相互作用,这些相互作用有助于宿主健康和疾病,还可能导致找到针对微生物群的新疗法。改善宿主健康,预防和治疗各种疾病1。
虽然体外肠道微生物群培养系统无法真正复制实际肠道条件,但一些实验室已努力开发这种模型,其中一些模型已在某种程度上被发现可行,并已成功用于不同的目的。最近的肠道模型之一是人类肠道微生物生态系统的模拟器,它模仿整个人类胃肠道,包括胃、小肠和结肠的不同区域。然而,这种技术上复杂的模型可能无法供全世界其他研究设施使用。因此,对于研究微生物调节剂及其对肠道微生物群和宿主健康的影响的实验室来说,仍然迫切需要开发相对简单、负担得起和实用的新替代模型。因此,使用体外(或活体)粪便微生物培养系统将有助于研究这种干预措施11、12的效果。具体来说,可以研究不同益生菌对微生物群发酵能力的影响,包括肠道微生物群多样性和成分的周期性变化、粪便pH和微生物代谢物的水平,包括SCFA和乳酸盐13.本文,以硫素(最广泛研究的益生菌成分之一)作为微生物群落调制器的范例,描述了这个简单的前体微生物群组培养系统的分步协议,以证明其用于估计使用微生物调节剂干预后,粪便微生物群和微生物代谢物的变化。

<table>
	<tbody>
		<tr>
			<td>Ammonium Bicarbonate (NH4HCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>217255</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Sulfate (NH4)2SO4 </td>
			<td>TGI</td>
			<td>C2388</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate (CaCl2&#8226;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td>Irritating</td>
		</tr>
		<tr>
			<td>Cobaltous Chloride Hexahydrate (CoCl2&#8226;6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>255599</td>
			<td></td>
		</tr>
		<tr>
			<td>Cupric Chloride Dihydrate (CuCl2&#8226;2H2O)</td>
			<td>Acros organics</td>
			<td>2063450000</td>
			<td>Toxic, Irritating</td>
		</tr>
		<tr>
			<td>Cysteine-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>C121800</td>
			<td></td>
		</tr>
		<tr>
			<td>D-biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Pantothenic acid</td>
			<td>Alfa Aesar</td>
			<td>A16609</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium Ethylenediaminetetraacetate Dihydrate (Na2EDTA)</td>
			<td>Biorad</td>
			<td>1610729</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-&#945;-methylbutyrate</td>
			<td>Sigma-Aldrich</td>
			<td>W271918</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrous Sulfate Heptahydrate (FeSO4&#8226;7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>F8263</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Alfa Aesar</td>
			<td>J62937</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma-Aldrich</td>
			<td>H9039</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Alfa Aesar</td>
			<td>A14777</td>
			<td></td>
		</tr>
		<tr>
			<td>Isobutyrate</td>
			<td>Alfa Aesar</td>
			<td>L04038</td>
			<td></td>
		</tr>
		<tr>
			<td>Isovalerate</td>
			<td>Alfa Aesar</td>
			<td>A18642</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate (MgCl2&#8226;6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese Chloride Tetrahydrate (MnCl2&#8226;4H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>221279</td>
			<td></td>
		</tr>
		<tr>
			<td>Niacin (Nicotinic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>N4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel(Ii) Chloride Hexahydrate (NiCl2&#8226;6H2O)</td>
			<td>Alfa Aesar</td>
			<td>A14366</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>N-valerate</td>
			<td>Sigma-Aldrich</td>
			<td>240370</td>
			<td></td>
		</tr>
		<tr>
			<td>P-aminobenzoic acid</td>
			<td>MP China</td>
			<td>102569</td>
			<td>Toxic, Irritating</td>
		</tr>
		<tr>
			<td>Phosphoric Acid (H3PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5811</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5504</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydrogen Phosphate (K2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>1551128</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxine</td>
			<td>Alfa Aesar</td>
			<td>A12041</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Alfa Aesar</td>
			<td>A11764</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>1613757</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher BioReagents</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher Chemicals</td>
			<td>S320</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Molybdate Dihydrate (Na2MoO4&#8226;2H2O)</td>
			<td>Acros organics</td>
			<td>206375000</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine Hydrochloride (Thiamin-HCl)</td>
			<td>Acros organics</td>
			<td>148991000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypticase</td>
			<td>BD Biosciences</td>
			<td>211921</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin B12</td>
			<td>Sigma-Aldrich</td>
			<td>V2876</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>70161</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Sulfate Heptahydrate (ZnSO4&#8226;7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>Z0251</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m membrane filter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure magnetic purification beads</td>
			<td>Agencourt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerobic chamber with incubatore</td>
			<td>Forma anaerobic system, Thermo Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle filter</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cheesecloth</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina MiSeq sequencer</td>
			<td>Miseq reagent kit v3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen PowerFecal kit</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quantitative Insights into Microbial Ecology (QIIME) software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit-3 fluorimeter</td>
			<td>InVitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waters-2695 Alliance HPLC system</td>
			<td>Waters Corporation</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro batch-culture model to estimate the effects of interventional regimens on human fecal microbiota

肠道微生物群在几种人类疾病中正在显现的作用要求新的工具、技术和技术取得突破。需要进行这种改进,以破译微生物调节剂的利用,为人类健康带来益处。然而,大规模筛选和优化调制器以验证微生物群落调制和预测相关健康益处可能实际上很困难,因为需要大量的动物和/或人类受试者。为此,体外或体外模型可促进微生物调节剂的初步筛选。在此,它被优化和演示了一个前体粪便微生物群培养系统,可用于检查肠道微生物调节剂的各种干预措施的影响,包括益生菌,益生菌和其他食品成分,除了营养药物和药物,关于人类肠道微生物群的多样性和组成。Inulin 是研究最广泛的益生菌和微生物重组剂之一,这里以它为例,研究其对健康粪便微生物群成分及其代谢活动(如粪便 pH 和有机酸的粪便水平)的影响包括乳酸和短链脂肪酸(SCFA)。该议定书可能有助于旨在估计调制器不同干预措施对粪便微生物群谱的影响和预测其健康影响的研究。

该规程用于演示特定益生菌(即,在粪便pH值的变化以及乳酸和SCFA在健康人类受试者粪便中的浓度变化方面,硫素对微生物群组合物和代谢活动的影响)。不同的时间点后处理与硫宁)。粪便pH、乳酸和SCFA的粪便水平(图1)和微生物群组合物(图2和图3)在0(基线)、9和24小时孵育时测量,无论是否使用硫素。研究结果表...

Watch this Scientific Journal Video about An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota at JoVE.com

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

该协议描述了人类粪便微生物群的体外批量培养发酵系统,使用硫素(一种众所周知的益生菌和研究最广泛的微生物群调制器之一)来证明该系统在估计特定效果时的使用粪便微生物群成分和代谢活动的干预措施。

评估介入疗法对人体粪便微生物群影响的体内批处理培养模型

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

考虑到肠道微生物群在各种人类疾病中的新作用，一些肠道微生物组调节剂，如益生菌、益生菌、饮食和药物，可以帮助改善肠道微生物群相关的健康问题。然而，没有快速和负担得起的筛查系统可用于预测这种肠道微生物组调节剂的影响，但该系统非常简单，足以预测特定干预措施如何影响肠道微生物群，最终影响宿主健康。该协议简单、经济、实用，适合研究肠道微生物组调制器及其对微生物群和宿主健康的影响的实验室。维持无菌和厌氧条件对于这个实验至关重要。此外，粪便标本应使用重新收集后立即。否则，如果稍后计划进行实验，样品在收集后立即储存在零下 80，而不会暴露在环境温度和空气中。在等分准备和移液过程中处理精度对于保持精度和可重复性也很重要。白天需要不到12个小时。如果他们在24小时内需要样品，我们可以在一夜之间建立一种文化。然后，对短链脂肪酸和微生物群进行测定。这个过程将由两个伟大的大学学生，肖库·艾哈迈迪和拉比纳·马亚利来自我的实验室进行演示。在烟气罩下准备发酵介质，在磁铁混合物的试剂瓶中，混合溶液A、溶液B、微量矿物质溶液、水溶性维生素溶液、叶酸生物素溶液、核黄素溶液、血脂溶液、短链脂肪酸混合物和雷沙祖林。然后加入瓶酵母提取物，碳酸钠，半胱氨酸盐酸单水合物，并尝试。加入296.1毫升蒸馏水，用一个正常的盐酸盐或氢氧化钠调整pH值，以确保其在7.0左右。将烧杯转移到无菌工作站，并使用孔径为 0.22 微米的真空过滤器过滤介质进行消毒。准备一升厌氧稀释液，在电离水中溶解五克氯化钠、2克葡萄糖和0.3克半胱氨酸盐酸单水合物。将带瓶盖的试剂瓶在121.1摄氏度下下15分钟，并存放在厌氧室内。在发酵实验开始前，将发酵实验所需的所有材料、溶液和工具留在厌氧室中至少48小时，以确保去除任何残留的氧气。在发酵实验前至少24小时，重达300毫克的伊努林，然后转移到50毫升的管中，然后放在厌氧室内。将26毫升灭菌发酵介质加入管中，并加入漩涡混合。允许纤维水化约24小时。发酵实验当天，准备粪便。首先，在50毫升锥形管中称重5克新鲜粪便样品。向管中加入厌氧稀释溶液，达到 50 毫升的最终体积。漩涡15分钟，或直到完全均匀。将接种的管子保持在37摄氏度的厌氧室内，并每小时轻轻反转一次，以重新填充纤维和接种。三、六、九或二十四小时后，拿出适当数量的发酵介质，在摄氏四度下以14，000次g离心，10分钟。立即将上清液和颗粒冷冻在液氮中，然后捕捉冷冻后，将上清液储存在零下80摄氏度，用于短链脂肪酸分析，以及用于微生物群分析的颗粒。该协议用于演示特定益生菌的效果。健康人类受试者的粪便，在治疗伊努林后，在9小时内表现出相对稳定的pHH，而24小时后则急剧下降。与未处理的粪便微生物群相比，接种 inulin 显著增加了未处理的粪便标本中短链脂肪酸和乳酸盐的含量。主要坐标分析显示，在未经处理的胰蛋白样品和未经处理的样品之间，对加权和未加权的UniFrac的β多样性测量有不同的微生物群组成。通过PD全树、物种丰富度、可操作分类单位、物种均匀度以及主要植物和基因的相对丰度，阿尔法多样性与植物遗传多样性的指数，都呈现了经治疗的未处理样本和未经治疗的样本之间的不同肠道微生物特征。分类包层图，源自16S序列的线性鉴别效应大小分析，代表了不同组样本之间的差异性丰富分类。请仔细执行所有步骤。最重要的是保持严格的无菌和厌氧条件。上那和从实验可用于处理短链脂肪酸水平的变化，由于纤维的发酵为基础的。此外，颗粒中可用于分析肠道微生物群组成的变化。用于制作培养介质的一些化学品具有潜在危险性。请使用 MSDS 工作表并采取相应的预防措施。

Research

JoVE Journal

Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

科研

免疫与感染

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号：对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

味觉信号蛋白废除对炎症性肠病小鼠肠道炎症的影响

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

内生菌与人类肠道微生物群使用体外浴发酵系统的相互作用分析

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...