秀丽隐杆线虫长寿的脂质补充和基因转录分析
Summary
本协议描述了 秀丽隐杆线虫液体和平板培养物中的脂质补充方法，以及来自大量或少数蠕虫和蠕虫组织的纵向研究和基因转录分析。
Abstract
衰老是一个复杂的过程，其特征是由环境和遗传贡献引起的渐进式生理变化。脂质在构成细胞膜的结构成分、储存能量和作为信号分子方面至关重要。脂质代谢和信号传导的调节对于激活不同的长寿途径至关重要。 秀丽隐杆线虫 是一种优秀而强大的生物体，可以剖析脂质代谢和信号传导对长寿调节的贡献。多项研究描述了特定脂质分子的饮食补充如何延长 秀丽隐杆线虫 的寿命;然而，补充条件的微小差异可能会导致不同实验室的科学家之间的可重复性问题。在这里，报道了秀 丽隐杆线虫 的两种详细补充方法，采用脂质补充，将细菌接种在平板上或在液体培养物中细菌悬浮液。本文还提供了使用全蠕虫裂解物或来自少数蠕虫的解剖组织进行终身脂质补充和qRT-PCR分析的寿命测定的详细信息。结合纵向研究和脂质补充转录研究，喂养测定提供了可靠的方法来剖析脂质如何影响长寿和健康老龄化。该方法还可以适用于各种营养筛查方法，以使用少量解剖组织或少数动物来评估转录本子集的变化。
Introduction
血脂
脂质是可溶于有机溶剂但不溶于水的小疏水性或两亲性分子1，2。不同的脂质分子根据其链中所含的碳数量、位置、双键数量和结合结构（包括甘油或磷酸盐）相互区分。脂质在不同细胞内和跨不同细胞发挥关键作用，以调节生物体功能，包括构成膜双层、提供能量储存和充当信号分子3，4。
首先，脂质是生物膜的结构成分，包括将内部隔室与细胞外环境分开的质膜和细胞内亚细胞膜。其次，脂质是脊椎动物和无脊椎动物能量储存的主要形式。中性脂质，包括三酰基甘油，在各种组织中长时间储存，包括脂肪组织。在线虫秀丽隐杆线虫中，肠道是主要的代谢脂肪储存器官;它的功能不仅参与营养物质的消化和吸收，还参与解毒过程，类似于哺乳动物肝细胞的活性。其他脂肪储存组织包括种系，其中脂质对卵母细胞发育至关重要，以及由皮肤样表皮细胞组成的皮下组织3，5。第三，近年来，越来越多的证据表明，脂质是参与细胞内和细胞外信号传导的强大信号分子，通过直接作用于多种受体，包括G蛋白偶联和核受体，或通过膜流动性调节或翻译后修饰间接作用6，7，8，9.进一步的研究将继续阐明脂质信号传导在促进长寿和健康方面的潜在分子机制。
模式生物对于解决在人类中研究的复杂特定生物学问题非常重要。例如，秀丽隐杆线虫是进行遗传分析以剖析与人类营养和疾病相关的生物过程的绝佳模型10。与人体生理学、复杂组织、行为模式和丰富的遗传操作工具相关的高度保守的分子途径使秀丽隐杆线虫成为非凡的模式生物11.例如，秀丽隐杆线虫在转发遗传筛选以识别表型特异性基因方面非常出色，以及通过RNA干扰12进行全基因组反向遗传筛选。
在实验室中，线虫生长在琼脂培养皿上，琼脂培养皿上接种了大肠杆菌草坪，提供蛋白质、碳水化合物、饱和和不饱和脂肪酸等常量营养素作为能量和构建块的来源，以及辅因子和维生素等微量营养素13。与哺乳动物类似，线虫从棕榈酸和硬脂酸（分别为饱和的16碳和18碳分子）合成脂肪酸分子，这些分子依次去饱和并拉长为多种单不饱和脂肪酸（MUFA）和多不饱和脂肪酸（PUFA）14，15，16，17，18。有趣的是，秀丽隐杆线虫能够从头合成所有必需的脂肪酸和参与脂肪酸生物合成、去饱和和伸长的核心酶，促进长链多不饱和脂肪酸的合成19。与其他动物物种不同，秀丽隐杆线虫可以用自己的ω-3去饱和酶将18碳和20碳ω-6脂肪酸转化为ω-3脂肪酸。此外，蠕虫具有Δ12去饱和酶，可催化油酸（OA，18：1）形成亚油酸（LA）20，21。大多数动物或植物同时缺乏Δ12和ω-3去饱和酶，因此依靠膳食摄入ω-6和ω-3来获得多不饱和脂肪酸，而秀丽隐杆线虫不需要膳食脂肪酸22。缺乏功能性去饱和酶的分离突变体已被用于研究特定脂肪酸在不同生物过程中的功能，包括繁殖、生长、寿命和神经传递。单个脂肪酸对特定生物学途径的影响可以使用遗传方法和饮食补充剂来解决16，17，23。迄今为止，脂质研究的重点是表征参与神经和发育条件下脂质合成、降解、储存和分解的基因24.然而，脂质在长寿调节中的作用才刚刚开始显现。
长寿调节中的脂质信号传导
脂质通过激活不同组织和细胞类型中的细胞信号级联，在长寿调节中起着至关重要的作用。最近的研究强调了脂质在通过脂质结合蛋白或识别膜受体 调节 转录和细胞间通讯中的积极作用25。此外，膳食脂质补充剂为剖析脂质代谢如何影响 秀丽隐杆线虫的寿命提供了极好的工具。不同的MUFAs和PUFAs已被证明通过激活转录因子来促进长寿26，27。
长寿模型，包括胰岛素/IGF-1信号传导和种系前体细胞的消融，与MUFA生物合成途径有关，MUFA补充剂，包括油酸，棕榈油酸和顺式牛痘，足以延长秀丽隐杆线虫的寿命26。尽管MUFA管理赋予的长寿效应需要进一步研究，但其潜在机制很可能由SKN-1 / Nrf2转录因子介导，SKN-1 / Nrf2转录因子是氧化应激反应和长寿调节的关键激活剂28，29。在MUFAs中，一类称为N-酰基乙醇胺（NAEs）的特定类别的脂肪酰基乙醇酰胺在不同的机制中起着至关重要的作用，包括炎症，过敏，学习，记忆和能量代谢30。特别是，称为油酰乙醇酰胺（OEA）的脂质分子已被确定为长寿的正调节因子，通过促进脂质结合蛋白8（LBP-8）易位到细胞核中以激活核激素受体NHR-49和NHR-807。补充OEA类似物KDS-5104足以延长寿命，并诱导参与氧化应激反应和线粒体β氧化的基因表达7，8。
同时，PUFA的作用也与长寿调节有关。给予PUFA ω-3脂肪酸α-亚麻酸（ALA）通过激活NHR-49 / PPARα，SKN-1 / NRF转录因子和诱导线粒体β氧化来促进长寿31。有趣的是，ALA的过氧化物产物，称为脂氧化物，激活SKN-1 / NRF，表明PUFAs及其氧化衍生物都可以带来长寿益处23。补充ω-6脂肪酸花生四烯酸（AA）和二同γ-亚麻酸（DGLA）通过自噬活化 延长 寿命，促进蛋白质质量控制，并导致浪费和有毒蛋白质聚集体的降解27，32。最近，由脂质结合蛋白3（LBP-3）和DGLA介导的细胞非自主信号传导调节已被证明通过向神经元发送外周信号来促进长寿至关重要，这表明脂质分子在系统水平的组织间通讯中具有长期作用33。本研究报告了用接种在平板上的细菌或液体培养中的细菌悬浮液进行脂质补充的每个步骤。这些方法用于评估寿命和转录分析，使用全身内容物或来自少数蠕虫的解剖组织。以下技术可以适用于各种营养研究，并提供有效的工具来剖析脂质代谢如何影响长寿和健康老龄化。
Protocol
Representative Results
Discussion
脂质补充剂已被用于衰老研究，以阐明某些脂质物种对健康衰老的直接影响6，7，23，26，27，31。然而，脂质补充过程可能具有挑战性，实验之间的任何不一致都可能导致不可重复的结果。在这里，记录了第一个详细的分步协议，以指导新科学家避?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 P. Svay 的维护支持。这项工作得到了NIH资助R01AG045183（MCW），R01AT009050（MCW），R01AG062257（MCW），DP1DK113644（MCW），March of Dimes基金会（MCW），韦尔奇基金会（MCW），HHMI研究员（MCW）和NIH T32 ES027801博士前学生研究员（MS）的支持。一些菌株由CGC提供，CGC由NIH研究基础设施计划办公室（P40 OD010440）资助。
Materials
| 1.5 mL Pestle | Genesee Scientific | 93-165P15 | For worm grinding with Trizol |
| Agarose | Sigma | A9639-500G | |
| AmfiRivert cDNA Synthesis Platinum Master Mix | GenDEPOT | R5600 | For reverse transcription from bulk worm samples |
| Applied Biosystems QuanStudio 3 Real-Time PCR | ThermoFisher | A28567 | For qRT-PCR |
| Benchmark Scientific StripSpin 12 Microcentrifuge | Benchmark Scientific | C1248 | For spin down PCR tubes |
| Branson 450 Digital Sonifier, w/ 1/8" tip | Branson Ultrasonic Corporation | 100-132-888R | |
| Chloroform | Fisher Scientific | C298-500 | |
| Cholesterol | Sigma | C8503-25G | |
| Dimethyl sulfoxide (DMSO) | Sigma | D8418-100ML | |
| Eppendorf 5424 R centrifuge | Eppendorf | 22620444R | For RNA extraction |
| Eppendorf vapo protect mastercycler pro | Eppendorf | 950030010 | For reverse transcription |
| Ethanol, Absolute (200 Proof) | Fisher Scientific | BP2818-500 | |
| Greiner Bio-One CELLSTAR, 12 W Plate | Neta Scientific | 665180 | 12-well plates for licuid feeding |
| Greiner Bio-One Petri Dish, Ps, 100 x 20 mm | Neta Scientific | 664161 | For bacterial LB plates and worm 10-cm NGM plates |
| Greiner Bio-One Petri Dish, Ps, 60 x 15 mm | Neta Scientific | 628161 | For worm6-cm NGM plates |
| Invitrogen nuclease-free water | ThermoFisher | AM9937 | |
| Isoproanol | Sigma | PX1835-2 | |
| Levamisole hydrochloride | VWR | SPCML1054 | |
| lipl-4Tg | MCW Lab | N/A | Transgenic C. elegans |
| lipl-4Tg;fat-3(wa22) | MCW Lab | N/A | Transgenic C. elegans |
| Luria Broth Base | ThermoFisher | 12795-084 | |
| Magnesium sulfate (MgSO4) | Sigma | M2643-500G | |
| MicroAmp EnduraPlate Optical 96-Well Fast Clear Reaction Plate with Barcode | ThermoFisher | 4483354 | 96-well qPCR plate |
| MicroAmp Optical Adhesive Film | Applied BioSystem | 4311971 | For sealing the 96-well qPCR plate |
| Milli-Q Advantage A10 Water Purification System | Sigma | Z00Q0V0WW | Deionized water used to make all reagents, including buffer and cultural media, unless specified as nuclease-free water in the protocol |
| N2 | Caenorhabditis Genetics Center | N/A | C. elegans wild isolate |
| NanoDrop ND-1000 Spectrophotometer | ThermoFisher | N/A | For measuring RNA concentration |
| OP50 | Caenorhabditis Genetics Center | N/A | Bacteria used as C. elegans food |
| Potasium phosphate dibasic trihydrate (K2HPO4·3H2O) | Sigma | P5504-1KG | |
| Potasium phosphate monobasic (KH2PO4) | Sigma | P0662-2.5KG | |
| Power SYBR Green cells-to-Ct kit | ThermoFisher | 4402953 | For reverse transcription and qPCR from a few worms or worm tissue |
| Power SYBR Green Master Mix | ThermoFisher | 4367659 | For qPCR from bulk worm samples |
| Pure Bright germicidal ultra bleach | KIK International LLC. | 59647210143 | 6% house bleach For worm egg preparation |
| Pyrex spot plate with nine depressions | Sigma | CLS722085-18EA | Watch glass for dissecting the worms |
| RNaseZap RNase Decontamination Solution | ThermoFisher | AM9780 | |
| Sodium cloride (NaCl) | Sigma | S7653-1KG | |
| Sodium hydroxide (NaOH) | Sigma | SX0590-3 | |
| Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) | Sigma | S9390-1KG | |
| Thermo Sorvall Legend Mach 1.6R Centrifuge | Thermo | 7500-4337 | For bacteria collection |
| Thermo Sorvall ST 8 centrifuge | Thermo | 7500-7200 | For worm egg preparation |
| TRIzol Reagent | TheroFisher | 15596018 | RNA extraction reagent |
| Turbo DNA-free kit | ThermoFisher | AM1907 | For removing DNA contamination in RNA extractions |
| Vortexer 59 | Denville Scientific INV | S7030 | |
| VWR Disposable Pellet Mixers and Cordless Motor | VWR | 47747-370 | For worm grinding with Trizol |
| VWR Kinetic Energy 26 Joules Mini Centrifuge C1413 V-115 | VWR | N/A | For worm collection. Discontinued model, a similar one available at VWR with Cat# 76269-064 |
| Worm picker | WormStuff | 59-AWP |
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