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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Ammonium Bicarbonate (NH4HCO3) | Sigma-Aldrich | 217255 | |
| Ammonium Sulfate (NH4)2SO4 | TGI | C2388 | Toxic |
| Calcium Chloride Dihydrate (CaCl2•2H2O) | Sigma-Aldrich | C3306 | Irritating |
| Cobaltous Chloride Hexahydrate (CoCl2•6H2O) | Sigma-Aldrich | 255599 | |
| Cupric Chloride Dihydrate (CuCl2•2H2O) | Acros organics | 2063450000 | Toxic, Irritating |
| Cysteine-HCl | Sigma-Aldrich | C121800 | |
| D-biotin | Sigma-Aldrich | B4501 | |
| D-Pantothenic acid | Alfa Aesar | A16609 | |
| Disodium Ethylenediaminetetraacetate Dihydrate (Na2EDTA) | Biorad | 1610729 | |
| DL-α-methylbutyrate | Sigma-Aldrich | W271918 | |
| Ferrous Sulfate Heptahydrate (FeSO4•7H2O) | Sigma-Aldrich | F8263 | Toxic |
| Folic acid | Alfa Aesar | J62937 | |
| Glucose | Sigma-Aldrich | G8270 | |
| Hemin | Sigma-Aldrich | H9039 | |
| Hepes | Alfa Aesar | A14777 | |
| Isobutyrate | Alfa Aesar | L04038 | |
| Isovalerate | Alfa Aesar | A18642 | |
| Magnesium Chloride Hexahydrate (MgCl2•6H2O) | Sigma-Aldrich | M8266 | |
| Manganese Chloride Tetrahydrate (MnCl2•4H2O) | Sigma-Aldrich | 221279 | |
| Niacin (Nicotinic acid) | Sigma-Aldrich | N4126 | |
| Nickel(Ii) Chloride Hexahydrate (NiCl2•6H2O) | Alfa Aesar | A14366 | Toxic |
| N-valerate | Sigma-Aldrich | 240370 | |
| P-aminobenzoic acid | MP China | 102569 | Toxic, Irritating |
| Phosphoric Acid (H3PO4) | Sigma-Aldrich | P5811 | |
| Potassium Dihydrogen Phosphate (KH2PO4) | Sigma-Aldrich | P5504 | |
| Potassium Hydrogen Phosphate (K2HPO4) | Sigma-Aldrich | 1551128 | |
| Pyridoxine | Alfa Aesar | A12041 | |
| Resazurin | Sigma-Aldrich | R7017 | |
| Riboflavin | Alfa Aesar | A11764 | |
| Sodium carbonate (Na2CO3) | Sigma-Aldrich | 1613757 | |
| Sodium chloride (NaCl) | Fisher BioReagents | 7647-14-5 | |
| Sodium hydroxide (NaOH) | Fisher Chemicals | S320 | |
| Sodium Molybdate Dihydrate (Na2MoO4•2H2O) | Acros organics | 206375000 | |
| Thiamine Hydrochloride (Thiamin-HCl) | Acros organics | 148991000 | |
| Trypticase | BD Biosciences | 211921 | |
| Vitamin B12 | Sigma-Aldrich | V2876 | |
| Yeast extract | Sigma-Aldrich | 70161 | |
| Zinc Sulfate Heptahydrate (ZnSO4•7H2O) | Sigma-Aldrich | Z0251 | |
| 0.22 µm membrane filter | |||
| AMPure magnetic purification beads | Agencourt | ||
| Anaerobic chamber with incubatore | Forma anaerobic system, Thermo Scientific, USA | ||
| Bottle filter | Corning | ||
| Cheesecloth | |||
| Illumina MiSeq sequencer | Miseq reagent kit v3 | ||
| pH meter | |||
| Qiagen PowerFecal kit | Qiagen | ||
| Quantitative Insights into Microbial Ecology (QIIME) software | |||
| Qubit-3 fluorimeter | InVitrogen | ||
| Vortex | Thermoscientific | ||
| Waters-2695 Alliance HPLC system | Waters Corporation |
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