Summary

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

Published: September 16, 2020
doi:

Summary

了解环境有机氯农药(OCPs)对肝细胞线粒体功能的影响,对于探讨OCP引起代谢紊乱的机理十分重要。本文提出了检测肝线粒体功能的详细方法。

Abstract

本文提出了检测肝线粒体功能的详细方法,以便更好地了解肝细胞中环境有机氯农药(OCPs)引起的代谢紊乱的原因。HepG2细胞在同等剂量的内部接触下,在24小时内暴露在β-六氯环丙烷(+-六氯环丙烷)中。通过透射电子显微镜(TEM)对肝细胞的超结构进行了检查,以显示线粒体的损伤。线粒体功能通过线粒体荧光强度、腺苷5′-三磷酸(ATP)水平、氧消耗率(OCR)和线粒体膜电位(MMP)进一步评估,这些细胞在使用β-HCH孵育的HpG2细胞中。线粒体荧光强度后染色线粒体绿色荧光探针观察到荧光显微镜。荧光素-荧光酶反应用于确定ATP水平。在流式细胞学下检测出MMP。使用细胞外通量分析仪测量OCR。总之,这些协议用于检测肝细胞中的线粒体功能,以调查线粒体损伤。

Introduction

有机氯农药(OCPs)对健康的影响,如生殖干扰、免疫毒性、代谢变化等,此前已研究过1、2、3。,2,3检测细胞代谢和发现线粒体功能障碍的方法使科学家能够理解线粒体功能的作用(即:,线粒体STAT3水平,乳酸,丙酮酸盐,乳酸与丙酮酸盐比,共酶Q10,线粒体质子泄漏,生物能量,生物发生和动力学)在老化,肥胖,糖尿病,心血管功能,癌症和安全毒性4,54,5,6,7,6等领域7。本文介绍了评估OCPs引起的线粒体功能障碍的方法。

我们向+-六氯环氧烷(+-H环环)暴露,一种具有代表性的OCPs,剂量相当于人体内部接触24小时。首先,TEM用于观察肝细胞的超结构,如核、线粒体和内质电8。与普通显微镜相比,TEM能够探索细胞和细胞成分(细胞系或组织)的二、三D超结构、形态、化学成分以及在现代科学技术中起关键作用的自然或人工材料的功能。线粒体功能通过线粒体荧光强度、腺苷5′-三磷酸(ATP)水平、耗氧率(OCR)和线粒体膜电位(MMP)进一步评估,在使用β-HCH孵育的HpG2细胞中。Mito-跟踪器绿色是一种线粒体绿色荧光探针,可用于活细胞线粒体特异性荧光染色。肝细胞中的线粒体被线粒体染色的线托跟踪绿色溶液和线粒体荧光强度,数量和模式观察到与共理显微镜9。线粒体绿色荧光探针可用于染色活细胞。与罗丹胺123或JC-1相比,线粒体绿色荧光探针不依赖于线粒体染色的线粒体膜潜力。ATP水平由荧光素-荧光素试剂盒确定,并由蛋白质浓度进行规范化。ATP 检测试剂盒可用于检测常见溶液、细胞或组织中的 ATP 水平。此试剂盒基于萤火虫荧光素催化的荧光酶,当需要 ATP 提供能量时,可产生荧光。当萤火虫荧光酶和荧光素过量时,在一定浓度范围内,荧光的产生与ATP的浓度成正比。此外,该套件还经过专门设计,以优化 ATP 的化学发光。ATP作为最重要的能量分子,在细胞的各种生理或病理过程中起着重要的作用。ATP水平的变化可以反映细胞功能的缺陷,特别是线粒体能量的产生。通常在凋亡、坏死或处于一定毒性状态下,细胞ATP水平降低 10。JC-1 的 MSP 检测套件是使用 JC-1 作为荧光探针的试剂盒,可快速、灵敏地检测细胞、组织或纯化 MMP。它可用于早期发现凋亡。阳离子染料JC-1是一种荧光探针,用于检测MMP,可根据绿色和红色荧光比的变化在流式细胞仪下进行分析。当 MMP 高时,JC-1 在线粒体的基质中聚合,形成聚合物(J-聚合),产生红色荧光。当MMP低时,JC-1不能积累,形成单体产生绿色荧光11。因此,红与绿荧光的比例可以反映MMP的水平。细胞的OCR是正常细胞功能12的关键指标。它是研究线粒体功能的参数。细胞线粒体压力测试试剂盒为分析线粒体功能的关键参数提供了一种稳定的方法。该试剂盒提供质量控制和预测试剂,以及执行细胞线粒体应激测试的标准方法。它可用于检测所有细胞类型,包括原细胞、细胞系、悬浮细胞,也可用于小岛、线虫、酵母和孤立的线粒体。

OCR 测量可以提供对细胞的生理状态或变化的宝贵见解。它由细胞外通量分析仪确定,以检测呼吸基线、质子泄漏、最大呼吸、ATP 周转量和储备能力。简言之,在对OCR进行基线测量后,每井依次添加寡霉素(ATP,FCCP(线粒体氧化磷酸化未结合)和抗霉素甲/罗酮(氧气消耗抑制剂)后,检测到OCR。

为了促进在体外检测肝细胞中线粒体功能的更具体的方案,我们在此介绍TEM、共生显微镜、发光计、流细胞仪和细胞外通量分析仪的实验,并在未来用于线粒体损伤相关不良结果研究。

Protocol

所有实验和实验规程都按照有关准则和规定进行,并经南京医科大学地方伦理委员会批准。 1. TEM 的线粒体超结构 收集 HepG2 细胞 种子 HepG2 细胞在 100 毫米的菜肴。储存在37°C和5%CO22。 1.5 mLEP 管中具有 0.25% EDTA 的消化细胞。 在室温 (RT) 下以 1000 x g 的离心机 3 分钟。丢弃上一杯。 收集 4-6 x 105 HepG2 细胞。 …

Representative Results

HepG2细胞暴露在+-六氯上细胞中的线粒体受到明显损伤。散落的线粒体有轻微扩张,形状不规则,线粒体脊消失,线粒体结构相对异常(图1)。 平均线粒体绿色荧光强度,代表线粒体,在接触+-HCH的 HepG2 细胞(图2)和ATP水平(图3) 中下降。荧光强度和ATP水平…

Discussion

检测协议成功的关键是使用各种实验方法,这些实验方法涵盖了从表型到机制的研究。在这项研究中,HepG2细胞在DMEM中培养青霉素和链霉素和10%胎儿牛血清。当细胞达到40-50%汇合时,添加+-HCH(0,10,100纳克/mL)并孵育24小时。我们首先使用TEM,它显示了代表性的OCPs引起的肝细胞的超结构变化,+-HCH,显示了线粒体结构的损伤图1)。)此外,还进行…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家自然科学基金(第81573174号,81570574号)的支持;江苏省杰出青年基金(SBK2014010296);中国教育部研究项目(213015A);江苏高等教育机构优先学术项目发展,江苏高等教育院校旗舰专业发展;环境化学和生态毒理国家重点实验室开放项目项目(KF2015-01)。

Materials

Transmission electron microscope  FEI Tecnai G2 Spirit Bio TWIN High-contrast, high-resolution imaging, Low-dose observation and imaging, Low-temperature observation, Outstanding analytical performance, Automation for convenience and performance
Mito-Tracker Green Beyotime C1048 Mito-Tracker Green is a mitochondrial green fluorescent probe that can be used for live cell mitochondrial-specific fluorescent staining.
Laser scanning confocal microscope Zeiss 700B The design is compact, stable, light path is the shortest, high light precision, creative technology and sophisticated scanning technology together to  produce a perfect 3-dimensional specimen image.
Enhanced ATP Assay Kit Beyotime S0027 Enhanced ATP Assay Kit can be used to detect ATP (adenosine 5'-triphosphate) levels in common solutions, cells or tissues. Cells and tissue samples can be split to complete the sample preparation, detection sensitivity up to 0.1nmol / L, chemiluminescence can be sustained for 30 minutes.
Luminometer Berthold Centro LB 960 Luminometer is chemiluminescence detector, the test sample itself can be light, do not need to stimulate. Luminometer is the instrument that detects chemiluminescence.
BCA Protein Assay Kit Beyotime P0012 BCA Protein Assay Kit is one of the most commonly used methods for detecting protein concentrations.
Multimode reader TECAN InfiniteM200 Multimode reader be used to detect protein consentration.

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Cite This Article
Liu, Q., Jiang, Z., Gu, A. Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides. J. Vis. Exp. (163), e56800, doi:10.3791/56800 (2020).

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