Name: system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis
Uploaded: 2017-04-05T14:30:00
Description: Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

This work was supported by BC Lung Association and Mitacs.

1.细菌菌株和生长培养基<ol><li>通过溶解25克牛血清白蛋白的10.0克右旋糖，并在460毫升去离子水4.05克的氯化钠使白蛋白右旋糖和盐储备溶液（ADS）。在4℃下过滤灭菌的ADS和存储。 </li><li>通过添加4.7克7H9粉末和2mL甘油至900毫升的精制水使7H9肉汤。高压釜中在121℃的7H9肉汤10分钟，并允许其继续之前冷却到室温。通过加入100mL的ADS和0.5mL吐温80至900毫升7H9肉汤使7H9ADST。储存于4℃。 </li><li>称取50毫克?...

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这项研究的目标是创建使用结核分枝杆菌的人的细胞内感染模型的简单和具有成本效益的方法HTS。结核病是人类疾病，其特征感染肺泡巨噬细胞的由结核分枝杆菌 。由于生物安全问题，包括细菌和宿主细胞两者的生物模型研究已经在过去使用。然而，已经表明，替代细菌和非人体模型的使用是在药物开发的命中对铅的成功不能预测，表明药物筛选最好用结核分枝杆菌感染<sup c...

结核分枝杆菌 ，结核病（TB）的病原体，是全世界发病率和死亡率的主要原因。药物敏感的TB是可治疗的疾病，需要一段6个月多种抗生素。尽管是一种可治疗的疾病，结核病的死亡率，估计是在2015年1 150万。在过去的10年来，一直在增加了耐药结核分枝杆菌的发病率的担忧。多药耐药TB（MDR-TB）被定义为TB即至少对异烟肼（INH）和利福平（RMP），最MDR-TB菌株也耐选择二线结核病药物，从而限制了治疗选择耐。耐药性的作用创造了治疗患者共感染人类免疫缺陷病毒（HIV），一个更大的挑战;全球40万例，到2015年，死亡1例HIV相关结核。令人失望的是，美国食品和药物ADMINIST比已批准对MDR-TB，bedaquiline只有一个新的抗结核药物，在过去40年中2。在找到更好的和更短的结核病治疗方法的进步，迫切需要以对抗结核病和耐多药结核病的斗争中保持领先地位。 传统上，TB药物屏幕在生长培养基中体外生长条件，从而被添加到生长的培养物的化合物和它们在消灭微生物效力是由固体培养基上计数菌落形成单位（CFU）测定条件下进行。作为CFU计数是劳动密集，耗时且昂贵，各种间接的方法已经被开发来缓解这个问题。这样的方法包括阿尔玛蓝活力试验3，荧光4的从绿色荧光蛋白（GFP），或从荧光素酶表达菌发光5的测定，总三磷酸腺苷的估计（ATP）6,7。 典型TB的特征是肺，其中所述细菌驻留和复制肺泡巨噬细胞8的吞噬体内部的结核分枝杆菌感染。简单的在肉汤表型屏幕可能适合胞外病原体;然而，在历史的角度看，打使用这种方法鉴定的针对结核分枝杆菌的化合物往往不能期间感染模型下游验证步骤，以达到预期。我们建议，抗结核药物在细胞内宿主细胞感染模型表现最好。然而，细胞内的机型具备高通量筛选（HTS）开发的许多技术和生物屏障。一大障碍是感染过程的复杂性，由许多步骤和由精心制作的洗涤除去在中间外细菌的举例说明。第二个主要障碍是漫长的时间再quirements，生长检测，通常由CFU上培养板计数完成，是一个过程，需要超过3周才能完成。一种解决方案，以取代CFU计数已经提供通过自动化荧光显微镜结合荧光的细菌。然而，这种解决方案需要一个初始设备投资是遥不可及的许多研究实验室。一个简单的，低成本的，与疾病相关的HTS方法将大大提高药物发现过程。 在这项研究中，我们报告一个新的，模块化的HTS系统，其目的在于提供适合于确定针对细胞内结核分枝杆菌的化合物的酶活性的快速，高度可扩展的，而经济，测定。该系统由三个模块组成：（ⅰ）细胞内，（ⅱ）细胞毒性，和（iii）中的发酵液测定法。将合并的最终结果提供化合物性质的全面描述，以作为动作的电位模式的附加信息。这SCreening系统已经结合靶向的作用模式不同的化合物库，包括药物的协同作用9的分析多个项目被使用，自噬10的刺激，和结核分枝杆菌的抑制-secreted毒力因子（未发表）。的未知作用模式化合物也已研究了11。该方法的修改版本也通过了我们的工业伙伴作为初筛方法来标识对细胞内结核分枝杆菌 11的新化合物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma-Aldrich</td>
			<td>R5886</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9</td>
			<td>Becton, Dickinson and Company</td>
			<td>271210</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween80</td>
			<td>Fisher Scientific</td>
			<td>T164</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine pH7</td>
			<td>Affymetrix</td>
			<td>10857</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>BP350</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Sigma-Aldrich</td>
			<td>M2128</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide (DMF)</td>
			<td>Fisher Scientific</td>
			<td>D131</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Hydrocholoric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>351279212</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>351269</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin</td>
			<td>Alfa Aesar</td>
			<td>B21187</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1</td>
			<td>American Type Culture Collection</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>M. tuberculosis H37Rv</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well flat bottom white plate</td>
			<td>Corning</td>
			<td>3917</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well flat bottom clear plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent plate sealer</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-0580</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Biomate 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate spectrophotometer</td>
			<td>Biotek</td>
			<td>Epoch</td>
			<td></td>
		</tr>
		<tr>
			<td>luminometer</td>
			<td>Applied Biosystems</td>
			<td>Tropix TR717</td>
			<td></td>
		</tr>
	</tbody>
</table>

system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

使用结核分枝杆菌表达荧光素酶基因的高通量筛选的细胞内 图2A和表1包含由光度计收集的原始数据，以相对发光单位（RLU）来表示，表现出对结核分枝杆菌的TB药利福平THP-1细胞内的浓度的增加的效果。 图2A是在RLU测定各种浓度的利福平的原始发光的散点图。误差棒表示平均值?...

Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

我们已经开发了模块化高通量筛选系统，用于发现新化合物对结核分枝杆菌 ，靶向细胞内和细胞在肉汤中生长的细菌以及细胞毒性对哺乳动物宿主细胞。

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Research

JoVE Journal

Immunology and Infection

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

科研

免疫与感染

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...