秀丽隐杆线虫体内脂肪染色方法的探究

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神奇的模式生物—秀丽隐杆线虫

神奇的模式生物—秀丽隐杆线虫摘要：本文对秀丽隐杆线虫的模式生物一般特征入手，介绍了线虫形态学、生物学特征和繁殖、基因组和遗传学等方面的内容。

关键词：秀丽隐杆线虫模式生物基因组最近，秀丽隐杆线虫用于生物实验材料倍受科学家们的关注。

进入21世纪以来，已经有六位科学家利用秀丽隐杆线虫为实验材料揭开了生命科学领域的重大秘密而获得了诺贝尔奖。

1974年英国科学家悉尼·布雷内（Sydney Brenner）第一次把秀丽隐杆线虫作为模式生物，成功地分离出线虫的各种突变体，发现了在器官发育过程中的基因规则而获得了2002年诺贝尔生理学或医学奖。

与悉尼·布雷内共同分享诺贝尔奖的有两名科学家，其中一位科学家是英国约翰·苏尔斯顿（John E. Sulston），通过显微镜活体观察线虫的胚胎发育和细胞迁移途径，于1983年完成线虫从受精卵到成体的细胞谱系。

另一位科学家是美国的罗伯特·霍维茨（H. Robert Horvitz），是利用秀丽隐杆线虫作为研究对象进行了“细胞程序性死亡”研究。

克雷格·梅洛（Craig C. Mello）和安德鲁·菲尔和（Andrew Z. Fire）利用秀丽隐杆线虫实验发现一种全新的基因调控方式—RNA干扰（RNAi）而获得2006年诺贝尔生理学或医学奖。

此外，Martin Chalfie证明了GFP(绿色荧光蛋白)作为多种生物学现象的发光遗传标记的价值。

在最初的一项实验中，他用GFP使秀丽隐杆线虫的6个单独细胞有了颜色，由此获得了2008年化学奖。

究竟什么原因使秀丽隐杆线虫成为如此富有盛名的实验材料？1.秀丽隐杆线虫一般特征秀丽隐杆线虫是一种食细菌的线形动物，学名是Caenorhabditis elegans，通常缩写成C.elegans其成体长仅1mm，全身透明，以细菌为食，居住在土壤中，被称为“自由生活线虫”。

1.1分类地位秀丽隐杆线虫属于线虫门（Phylum nematoda）、侧尾腺纲（Secernentea）、小杆线虫目（Rhabditida）小杆线虫科（Rhabditidae）小杆线虫属（Caenorhabditis）。

茯砖茶上调秀丽隐杆线虫NHR-49和ACO基因促进脂质分解代谢

茯砖茶上调秀丽隐杆线虫NHR-49和ACO基因促进脂质分
解代谢
潘联云;刘本英
【期刊名称】《茶叶通讯》
【年(卷),期】2024(51)1
【摘要】茯砖茶通过抑制脂质合成的降脂作用已在多项研究中报道,而关于其调控脂质分解代谢的研究还较少。

以秀丽隐杆线虫为模式生物,通过脂质染色、突变体、RNA干扰和qPCR检测分析茯砖茶水提物对秀丽隐杆线虫脂质分解代谢的调控作
用及其机制。

研究发现,茯砖茶水提物能够通过上调核激素受体nhr-49和顺乌头酸酶aco-1的基因表达提高脂肪酸β氧化和三羧酸循环,促进脂质氧化分解,从而减少秀丽隐杆线虫脂肪含量。

【总页数】7页(P78-84)
【作者】潘联云;刘本英
【作者单位】云南省农业科学院茶叶研究所/云南省茶学重点实验室
【正文语种】中文
【中图分类】S571.1;TS272
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秀丽隐杆线虫研究综述

秀丽隐杆线虫研究综述一、本文概述秀丽隐杆线虫（Caenorhabditis elegans，简称C. elegans）是一种微小的、透明的、生活在土壤中的线虫，自20世纪60年代以来，它已成为生物学研究的重要模型生物之一。

由于其生命周期短、繁殖迅速、基因组小且相对简单等特点，秀丽隐杆线虫被广泛用于研究细胞生物学、发育生物学、神经生物学、遗传学、基因组学等多个领域。

本文旨在对秀丽隐杆线虫的研究进行全面的综述，从基础生物学特性、基因组学进展、到其在各个领域的应用研究，以期为读者提供一个清晰、全面的秀丽隐杆线虫研究图景。

二、秀丽隐杆线虫的基本生物学特性秀丽隐杆线虫（Caenorhabditis elegans，简称C. elegans）是一种具有独特生物学特性的小型线虫，其身体长度仅约1毫米，属于线虫动物门、无尾感器纲、小杆目、小杆科。

自1974年被悉尼·布伦纳（Sydney Brenner）选为遗传学研究的模式生物以来，秀丽隐杆线虫已成为生物学和医学领域广泛研究的对象。

生命周期与繁殖：秀丽隐杆线虫的生命周期大约为3天，在适宜的环境下，它们能以极快的速度繁殖。

它们通常以细菌为食，尤其是大肠杆菌（Escherichia coli），并通过摄取这些细菌来获取所需的营养。

成年线虫通过自交或雌雄同体交配繁殖，产生的后代数量巨大，每个成虫一生可以产生多达300个子代。

基因组与遗传学：秀丽隐杆线虫的基因组相对较小，约含有1亿个碱基对，使其成为研究基因功能和基因相互作用的理想模型。

由于其生命周期短、繁殖迅速，科学家能够迅速地进行遗传筛选和基因编辑，以研究特定基因的功能。

神经系统与行为：秀丽隐杆线虫拥有相对简单的神经系统，仅由302个神经元组成。

尽管如此，这些神经元足以控制线虫的各种复杂行为，如觅食、逃避、交配等。

这使得秀丽隐杆线虫成为研究神经生物学和行为学机制的重要工具。

衰老与疾病模型：秀丽隐杆线虫因其短寿命和快速的生理变化而成为研究衰老机制的理想模型。

秀丽隐杆线虫作为病原菌宿主模型的研究概述

制。目前，以犆．犲犾犲犵犪狀狊为模式宿主进行研究的病原菌有真菌、细菌、病毒等，有５０多种（表１）。其中
模式生物用于研究动物发育和行为的模式动物，现研究较深入的主要是一些人类病原菌，如铜绿假单
已经发展成为研究动物发育、神经、衰老、毒理学、脂肪沉积和天然免疫等方面重要的模式生物。［１］近些年来，以犆．犲犾犲犵犪狀狊作为病原菌宿主模型来研究病原菌与宿主的相互作用逐渐成为了一个新的热点，犆．犲犾犲犵犪狀狊作为病原菌宿主模型有很多优势，个体小，成虫的长度大约１．５ｍｍ；生长快，３ｄ～３．５ｄ就
可以长成成虫；繁殖快，成熟的线虫每次可以获得３００个～３５０个子代个体；培养简单，可以直接以要研究的细菌为食物［１］；基因组测序已经完成；基因操作系统完善，突变体数量齐备，转基因线虫和ＲＮＡｉ技术成熟；表型易观察等特征。［２］目前，犆．犲犾犲犵犪狀狊作为病原菌宿主模型，在病原菌的致病机制和宿主防御病原菌天然免疫等方面取得了一系列的进展。本文就近些年来利用线虫作为宿主模型，在重要病原菌的致病机制和线虫天然免疫信号通路取得的进展进行综述。
鞠守勇等：秀丽隐杆线虫作为病原菌宿主模型的研究概述
铜绿假单胞菌（犘狊犲狌犱狅犿狅狀犪狊犪犲狉狌犵犻狀狅狊犪，ＰＡ）在自然界分布广泛，是医院内感染的主要病原菌之

小虫子带来的长寿启示

小虫子带来的长寿启示笔者主要的研究方向是衰老和代谢。

我们的实验室是从一只只小虫子入手，研究人类关心的大问题。

小蟲子担大任线虫的学名叫做秀丽隐杆线虫，广泛分布在自然界，即使在室外的土壤里，也能够分离到线虫。

线虫的成虫约1毫米长，一般需要借助显微镜来进行研究观测。

线虫从卵里孵化出来后，会经过四个幼虫期，然后变成有生殖能力的成虫。

在产卵期结束之后，成年线虫慢慢进入衰老期直到死亡。

线虫的每一个阶段都有明显的状态表现。

比如结束产卵期之后，成年虫子就开始衰老。

当它衰老的时候，运动会明显减慢，进食也明显减少。

线虫的衰老，在很多形式上和人有相似之处。

所以我们觉得借助线虫进行衰老研究，获得的研究成果，或许能够应用到高等生物上。

节食或能延寿我们怎样利用线虫来研究衰老呢？答案是借助基因突变的形式。

在自然界，基因突变在进化过程中经常发生。

通过化学诱变增加基因突变的频率，科学家确实发现了有基因突变的线虫能够显著延长寿命。

这个基因叫做daf-2。

有这种突变基因的线虫，活到80天左右才全部死掉。

它们的寿命是一般线虫寿命的2.5倍。

在这些长寿的线虫中，除了daf-2基因，科学家还筛选到其他基因，例如，其中一个基因叫做eat-2。

Eat中文意思是“吃”。

这个基因突变导致的结果是什么呢？就是线虫吃得少了，间接地模拟了一种热量限制性进食。

通俗地讲，就是适当地节食。

关于线虫的研究发现告诉我们，适当节食，或者是热量限制性进食，可以延长一个物种的寿命。

线虫上的这个发现，科学家们通过研究证实，对于高等生物（如猴子）同样适用。

未来减肥不再难我们用一种叫做油红O的染料，对线虫的脂肪进行染色。

因为线虫通体透明，所以可以直接在显微镜下观察。

研究发现，正常喂食的线虫，它的脂肪染色是比较深的红色，说明体内此时储存着大量的脂肪。

被饿了12个小时的线虫，知道自己饿了，便开始启动自身脂肪的分解，染色的红色部分就变浅了。

我们还发现，此时与脂肪分解相关的基因表达量也升高了，说明这些基因开始行使功能，促进脂肪分解。

秀丽隐杆线虫脂肪染色的方法比较

秀丽隐杆线虫脂肪染色的方法比较冯婉娟;黄文明;许想平;吴政星【摘要】Obesity and its related diseases are affecting an increasing number of people.Many key fat regulatory genes and fat metabolism pathways found in mammals are conserved inC.elegans.Therefore,C.elegans is a powerful genetic model for fat biology research.Different methods were used to quantitate the fat stores,and the results turn out different.According to the test in fixed worms during fasting or gene mutation,the dye such as Oil Red O,Sudan Black B,Nile Red and C1-BODIPY-C12 could quantitate fat stores correctly.%肥胖及其相关的疾病影响了越来越多的人.在哺乳动物中调节脂肪代谢的因子和脂肪代谢途径在线虫中也是保守存在的,因此线虫是脂肪研究的良好动物模型.在研究线虫脂肪代谢中,存在多种脂肪染色方法,而不同方法所表征的脂肪含量存在一定的差异,通过各种染色方法检测基因突变或者饥饿引起的脂肪含量变化.实验结果表明,采用油红、苏丹黑、尼罗红染料的固定染色法均能够正确地表征线虫的贮存脂肪.【期刊名称】《生命科学研究》【年(卷),期】2012(016)001【总页数】6页(P9-14)【关键词】线虫;脂肪染色;荧光染料;油红;苏丹黑【作者】冯婉娟;黄文明;许想平;吴政星【作者单位】华中科技大学生命科学与技术学院中国湖北武汉430074;华中科技大学生命科学与技术学院中国湖北武汉430074;华中科技大学生命科学与技术学院中国湖北武汉430074;华中科技大学生命科学与技术学院中国湖北武汉430074【正文语种】中文【中图分类】Q504Fat metabolic pathways are conserved between C.elegans and mammals.These pathways include fatty acid synthesis,fatty acid elongation and desaturation,mitochondrial and peroxisomal β-oxidation of fatty acids,and amino acid metabolism[1～5].In addition,C.elegans store fat in droplets in their intestinal cells and hypodermal cells[4],and these fat stores can be directly visualised by microscopy in intact animals due to the transparent bodies of C.elegans.Thus,C.elegans is a powerful system for analyzing the mechanisms of fat storage.There are several methods for examining fat storage and metabolism in C.elegans.An early method for visualising fat storage is using a classic lipophilic dye,Sudan Black B,to stain fixed animals[6,7].In using this stain,lipid droplets become visiible in intestinal and hypodermal tissues.Oil Red O is also a classic lipophilic dye that stains lipid droplets red[8].Aside from colourimetric dyes,another way to stain fat is through the use of lipophilic fluorescent dyes.For example,Nile Red and C1-BODIPY-C12 both fluoresce when in hydrophobicenvironments and thus are used to stain intracellular lipid droplets and can be used to examine fat content in intactliving animals[9,10].When fed C1BODIPY-C12,worms show fluorescence not only fat stores but also other organelles containing lipids.Though these mentioned dyes label fat storage compartments,there is controversy surrounding them,mainly that their results are inconsistent[9,11].An alternative dye-labelling assay for quantifying fat stores is coherent anti-Stokes Raman scattering(CARS)microscopy[12,13],which is a label-free chemicalimagingtechniquethatrelieson intrinsic molecular vibration as a contrast mechanism.Both hypodermal and intestinal fat stores are visualized using this technique.However,the disadvantage of CARS microscopy is that specialised equipment is required and that the assay is very expensive.In this paper,we confirmed that Nile Red and C1-BODIPY-C12 could stain fat storage as efficiently as Oil Red O in fixed worms.Then,we used fixed staining with different dyes to label fat stores in well-fed and fastingC.elegans,where fat stores are present orconsumed,respectively.Finally,using different dyes we tested several mutants which are insulin pathway related or a nuclear hormone receptor of stly,we compared the use of different dyes in fat storage quantification to facilitate researchers who could benefit from such analysis.Nematode strains were obtained from the C.elegans geneticcentre(CGC)unless otherwise stated.All strains were maintained at20℃us ing standard methods[14].The strains used in this study were as follows:wild-type(N2),daf-2(e1370),nhr-49(gk405)and ZXW3 hkdEx3[sur-5::atgl-1::gfp；Rol-6].Oil Red O staining was performed as previously described[9].To permeabilize the cuticle,worms were suspended and washed twice with PBS(phosphate buffered saline)and then suspended in 120 μL ofPBS.Next,an equal volume of 2× MRWB(Modified Ruvkun’s witches brew)buffer containing 2%paraformaldehyde was added,and animals in suspension were rocked for an hour(composition:160 mmol/L KCl,40 mmol/L NaCl,14 mmol/L Na2EGTA,1 mmol/L spermidine HCl,0.4 mmol/L spermine,30 mmol/L Na PIPES at pH 7.4,0.2%βmercaptoethanol).After permeabilizing,worms were re-suspended and dehydrated in60%isopropanol for 15 min at room temperature.After allowing worms to settle,isopropanol was removed,and approximately 1 mL of 60%Oil Red O solution(Cat.No.09755,Sigma-Aldrich,St.Louis,MO,USA)was added to each sample.Samples were incubated overnight while rocking.Oil Red O was prepared as follows:0.5 g of Oil Red O powder was dissolved in 100 mL of isopropanol and equilibrated for several days.Animals were mounted and imaged using an Olympus microscope outfitted with DIC optics.For Sudan Black staining,young adult animals were fixed in2%paraformaldehyde in M9 buffer(3 g KH2PO4,6 g Na2HPO4,5 g NaCl,1 mL 1 mol/L MgSO4,adding H2O to 1 L)with rocking for an hour.Fixed worms were then washed with M9 and dehydrated through an ethanol series (25%,50%,and 70%ethanol).Staining was performed overnight in a 50%saturated solution of Sudan black B in 70%ethanol[15].Stained animals were visualised with an Olympus microscope outfitted with DIC optics.Approximately 500～1 000 nematodes were suspended in 1 mL of water,and 50 μL of freshlyprepared 10% paraformal dehyde solution was then added.Animals and paraformaldehyde solutions were mixed and rocked for an hour.Afterward,rocking was stopped,and worm solutions were allowed to settle.Then,1 mL of 100 μg/L Nile Red in M9 was added to the worm pellet,and the entire solution was incubated for 6 h at room temperature with occasional gentle agitation.Worms were allowed to settle again and then were washed once with M9 buffer.After most of the staining solution had been removed,the fixed worms were mounted onto 2%agarose pads for microscopic observation and photography[15].A stock solution (2 g/L)was made by dissolving C1-BODIPY-C12 in DMSO (Dimethyl sulfoxide).The stock solution was then diluted in 1X PBS to a final concentration of 100 μg/L C1-BODIPYC12,and fixed worms were incubated 6 h in the working solution.After most of the staining solution had been removed,the fixed worms were mounted onto 2%agarose pads for microscopic observation and photography[16].Fixe d Nile Red and C1-BODIPY-C12 stained worms were visualised using an Olympus IX 71 inverted microscope (Olympus,Japan)using a 40x objective(Olympus UPlanSApo series).Fourteen bit images were taken with a CCD camera(Andor DV885).Nile Red yellow (referred to as Nile Red 580)was visualised with 586/20 nm emission filters with excitation light of 491 nm.Image analysis was performed using Image J software(Wayne Rasband,USA).A recent study claimed that Nile Red and C1-BODIPY-C12 did not show fatstorage staining[12].When C.elegans are fed Nile Red,the dye accumulates in lysosome-related organelles[17].Therefore,it was concluded that fixed Nile Red is a better proxy for fat storage visualisation than fed Nile Red.To confirm that fixed C1-BODIPY-C12 and fixed Nile Red are both good indicators of fat stores,we co-localised fixed Nile Red and fixed C1-BODIPY-C12 images.As shown in Fig.1a,we found that fixed Nile Red and fixed C1-BODIPY-C12 co-labelled a population of structures in gut epithelial organelles.Additionally,fixed Nile Red co-localised with Oil Red O and ATGL-1::GFP (Fig.1b and 1c).ATGL-1 encodes a homology of mammalian adipose triglyceride lipase,which is a lipid dropletmarker[18].So fixed Oil Red O staining is a reliable method to measure fat stores.Thus fixed Nile Red and fixed C1-BODIPY-C12 are sufficient dyes to visualise fat stores properly in C.elegans models.We also conducted similar assays which fed Nile Red and ATGL-1::GFP were assessed for co-localisation.According to Fig.1d,fed Nile Red was not co-localised with ATGL-1::GFP,it suggested a poor indicator of fat stores did fed Nile Red act. Mammals consume fat stores to fulfil their energy requirements upon starvation.We used different dyes to examine fat storage changes between well-fed and starved worms.After 12 h of starvation,worms were fixed and stained using Sudan Black B,Oil Red O,Nile Red and C1-BODIPYC12,respectively.In Oil Red O stained worms,well-fed animals had notable staining,while starved animals exhibited reduced staining(Fig.2).Sudan Black-stained worms show similar results(Fig.2).As for fixed Nile Red worms,the fluorescence intensity was brighter in well-fed wormsthan in 12 h starved worms (Fig.2).The situation was the same in fixed C1-BODIPY-C12-stained worms (Fig.2).Taken together,fasting caused fat storage consumption in C.elegans just as in mammals,and different dye-labelled assays showed the reflected fat store consumption in starved worms compared to well-fed worms.The insulin pathway is an important signal pathway to control lipid metabolism.The gene daf-2 encodes an insulin-receptor.Mutation of this gene results in a temperature-sensitive and constitutive dauer(a diapause stage of nematode worms whereby the larva can survive harsh conditions)formation.When daf-2 mutants are grown at permissive temperatures,the adults exhibit increased lifespan and enhanced fat storage[15].To test if the different fat staining assays can reflect the role of DAF-2 in neutral fat mass regulation,we examined daf-2(e1370)mutants using different dye-labelled assays.According to Fig.3,fat content stained by Oil Red O and Sudan Black B in daf-2 loss-of-function mutants were much higher than that of the wild type.Fluorescence dye assays with Nile Red and C1-BODIPY-C12 were performed and fluorescence intensities of fat stores were then quantitated.Results in Fig.4 showed that fluorescence intensity increased in daf-2 mutants compared to that of wild-type worms in both fluorescence dye assays.This increase suggested that the lack of functional daf-2 caused accumulation of fat stores as previously described[15].Taken together,these results suggest that different assays can represent fat masses in C.elegans.The gene nhr-49 encodes a nuclear hormone receptor (NHR),and a recent study indicated that nhr-49 acts as akey regulator of fat usage by modulating fat consumption and fatty acid composition in C.elegans[3,19,20].However,this conclusion rested on the Nile Red live fed staining and was later proven to be a poor indicator of fat content.Therefore,we tested the fat stores of nhr-49 mutants in several assays.Our data showed that Oil Red O and Sudan Black-labelled nhr-49 mutants exhibited normal fat stores as in wild-type animals.We also labelled wild-type and nhr-49 mutants with Nile Red and C1-BODIPY-C12.Results were similar to Oil Red O and Sudan Black staining.This similarity might be due to the fact that NHR-49 protein regulates fatty acid metabolism and only negligibly affects fat stores.Different assays using dye labels could be used to monitor fat storage changes caused by fasting or gene mutation[17].In this paper,we compared fat stores in well-fed and starved worms and specific genetic mutants.As anticipated,fat stores were consumed when wormsfasted.Additionally,all dye-labelled assays used in this study showed increases in fat stores in daf-2 mutants.It is universally acknowledged that daf-2 mutants show this phenotype.For nhr-49 mutants,some research has suggested that nhr-49 animals display abnormally high fatcontent[3,19].However,this conclusion depends on the Nile Red staining in which the dye was fed to animals,which has been proven to be a poor indicator of fat content[17].We tested the fat stores of nhr-49 mutants in several assays and concluded that nhr-49(gk405)mutants exhibit normal abilities to store fat.We summarised different assays of fat labelling with dyes in Table 1.Generally,Oil Red O,Sudan Black B,fixed Nile Red,and fixed C1-BODIPY-C12 are successful in accurately representing fat stores.However,classical fixed Sudan black dye has been shown easily to unsuccessfully label fat stores in C.elegans due to the ethanol-based wash-ing steps during the fixing procedure[15].Oil Red O was supported as an appropriate method to stain and quantify the main fat stores in C.elegans[9].The limitation of this method is that fixation can be variable and difficult to quantify.Certain studies in-dicated that fluorescent dyes,such as Nile Red and C1-BODIPY-C12,could be used to examine fat content in intact living animals.The advantage of Nile Red is the ability to use it in high-throughput screens designed to identify gene inactivations as-sociated with fat reduction or accumulation.Howev-er,more recent research has suggested that fluores-cent dyes fed to worms could not represent neutral lipid content in live worms[12].Therefore,we chose C1-BODIPY-C12 and Nile Red to stain fat stores when used in fixative assays.Both fixed Nile Red and fixed C1-BODIPY-C12 staining performed with short incubation times led to very efficient staining of lipid droplets.Additionally,C1-BODIPY-C12 is more specific than Nile Red staining of lipid droplets,and this method may be suitable for screening assays.Acknowledgement:We thank Caenorhabditis Genetic Centre for wild-type,nhr-49(gk405),and daf-2(e1370)stains and Roy R for sur-5::atgl-1::gfp construct.[1]MCKAY R M,MCKAY J P,AVERY L,et al.C elegans:a model for exploring the genetics of fat storage[J].Developmental Cell,2003,4(1):131-142.[2]WANG J,KIM S K.Global analysis of dauer gene expression in Caenorhabditis elegans[J].Development,2003,130(8):1621-1634.[3]Van GILST M R,HADJIVASSILIOU H,JOLLY A,et al.Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition inC.elegans[J].PLoS Biology,2005,3(2):e53.[4]ASHRAFI K.Obesity and the regulation of fatmetabolism[J].WormBook,2007,1-20.[5]HOLT S J,RIDDLE D L.SAGE surveys C.elegans carbohydrate metabolism:evidence for an anaerobic shift in the long-lived dauerlarva[J].Mechanisms of Ageing and Development,2003,124(7):779-800.[6]RALSER M,BENJAMIN I J.Reductive stress on life span extension inC.elegans[J].BMC Research Notes,2008,(1):19.[7]OGG S,RUVKUN G.The C.elegans PTEN homolog,DAF-18,acts in the insulin receptor-like metabolic signaling pathway[J].MolecularCell,1998,2(6):887-893.[8]SOUKAS A A,KANE E A,CARR C E,et al.Rictor/TORC2 regulates fat metabolism,feeding,growth,and life span in Caenorhabditiselegans[J].Genes&Development,2009,23(4):496-511.[9]O'ROURKE E J,SOUKAS A A,CARR C E,et al.C.elegans major fats are stored in vesicles distinct from lysosome-related organelles[J].Cell Metabolism,2009,10(5):430-435.[10]ASHRAFI K,CHANG F Y,WATTS J L,et al.Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes[J].Nature,2003,421(6920):268-272.[11]WANG M C,O'ROURKE E J,RUVKUN G.Fat metabolism links germline stem cells and longevity in C.elegans[J].Science,2008,322(5903):957-960.[12]YEN K,Le TT,BANSAL A,et al.A comparative study of fat storage quantitation in nematode Caenorhabditis elegans using label and labelfree methods[J].PLoS One,2010,5(9):e12810.[13]KLAPPER M,EHMKE M,PALGUNOW D,et al.Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches[J].Journal of Lipid Research,2011,52(6):1281-1293.[14]BRENNER S.The genetics of Caenorhabditiselegans[J].Genetics,1974,77(1):71-94.[15]KIMURA K D,TISSENBAUM H A,LIU Y,et al.daf-2,an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditiselegans[J].Science,1997,277(5328):942-946.[16]ZHANG S O,BOX A C,XU N,et al.Genetic and dietary regulation of lipid droplet expansion in Caenorhabditis elegans[J].Proceeding of the National Academy of Sciences of the United States of America,2010,107(10):4640-4645.[17]BROOKS K K,LIANG B,WATTS J L.The influence of bacterial diet on fat storage in C.elegans[J].PLoS One,2009,4(10):e7545.[18]NARBONNE P,ROY R.Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-termsurvival[J].Nature,2009,457(7226):210-214.[19]Van GILST M R,HADJIVASSILIOU H,YAMAMOTO K R.A Caenorhabditiselegans nutrient response system partially dependent on nuclear receptor NHR-49[J].Proceeding of the National Academy of Sciences of the United States of America,2005,102(38):13496-13501.[20]HORIKAWA M,SAKAMOTO K.Fatty-acid metabolism is involved in stress-resistance mechanisms of Caenorhabditis elegans[J].Biochemical and Biophysical Research Communications,2009,390(4):1402-1407.【相关文献】[1]MCKAY R M,MCKAY J P,AVERY L,et al.C elegans:a model for exploring the genetics of fat storage[J].Developmental Cell,2003,4(1):131-142.[2]WANG J,KIM S K.Global analysis of dauer gene expression in Caenorhabditiselegans[J].Development,2003,130(8):1621-1634.[3]Van GILST M R,HADJIVASSILIOU H,JOLLY A,et al.Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C.elegans[J].PLoSBiology,2005,3(2):e53.[4]ASHRAFI K.Obesity and the regulation of fat metabolism[J].WormBook,2007,1-20.[5]HOLT S J,RIDDLE D L.SAGE surveys C.elegans carbohydrate metabolism:evidence for an anaerobic shift in the long-lived dauer larva[J].Mechanisms of Ageing and Development,2003,124(7):779-800.[6]RALSER M,BENJAMIN I J.Reductive stress on life span extension in C.elegans[J].BMC Research Notes,2008,(1):19.[7]OGG S,RUVKUN G.The C.elegans PTEN homolog,DAF-18,acts in the insulin receptor-like metabolic signaling pathway[J].Molecular Cell,1998,2(6):887-893.[8]SOUKAS A A,KANE E A,CARR C E,et al.Rictor/TORC2 regulates fatmetabolism,feeding,growth,and life span in Caenorhabditiselegans[J].Genes&Development,2009,23(4):496-511.[9]O'ROURKE E J,SOUKAS A A,CARR C E,et al.C.elegans major fats are stored in vesicles distinct from lysosome-related organelles[J].Cell Metabolism,2009,10(5):430-435.[10]ASHRAFI K,CHANG F Y,WATTS J L,et al.Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes[J].Nature,2003,421(6920):268-272.[11]WANG M C,O'ROURKE E J,RUVKUN G.Fat metabolism links germline stem cells and longevity in C.elegans[J].Science,2008,322(5903):957-960.[12]YEN K,Le TT,BANSAL A,et al.A comparative study of fat storage quantitation innematode Caenorhabditis elegans using label and labelfree methods[J].PLoSOne,2010,5(9):e12810.[13]KLAPPER M,EHMKE M,PALGUNOW D,et al.Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches[J].Journal of Lipid Research,2011,52(6):1281-1293.[14]BRENNER S.The genetics of Caenorhabditis elegans[J].Genetics,1974,77(1):71-94.[15]KIMURA K D,TISSENBAUM H A,LIU Y,et al.daf-2,an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditiselegans[J].Science,1997,277(5328):942-946.[16]ZHANG S O,BOX A C,XU N,et al.Genetic and dietary regulation of lipid droplet expansion in Caenorhabditis elegans[J].Proceeding of the National Academy of Sciences of the United States of America,2010,107(10):4640-4645.[17]BROOKS K K,LIANG B,WATTS J L.The influence of bacterial diet on fat storage inC.elegans[J].PLoS One,2009,4(10):e7545.[18]NARBONNE P,ROY R.Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-term survival[J].Nature,2009,457(7226):210-214.[19]Van GILST M R,HADJIVASSILIOU H,YAMAMOTO K R.A Caenorhabditis elegans nutrient response system partially dependent on nuclear receptor NHR-49[J].Proceeding of the National Academy of Sciences of the United States of America,2005,102(38):13496-13501.[20]HORIKAWA M,SAKAMOTO K.Fatty-acid metabolism is involved in stress-resistance mechanisms of Caenorhabditis elegans[J].Biochemical and Biophysical Research Communications,2009,390(4):1402-1407.Using Different Dyes to Label Storage Fat in C.elegans。

四川省绵阳市绵阳中学2024-2025学年高三上学期高考适应性月考卷 (一) 生物学试题(含答案)

四川省绵阳中学2025届高考适应性月考卷（一）生物学注意事项：1．答题前，考生务必用黑色碳素笔将自己的姓名、准考证号、考场号、座位号在答题卡上填写清楚。

2．每小题选出答案后，用2B 铅笔把答题卡上对应题目的答案标号涂黑。

如需改动，用橡皮擦干净后，再选涂其他答案标号。

在试题卷上作答无效。

3．考试结束后，请将本试卷和答题卡一并交回。

满分100分，考试用时75分钟。

一、选择题：本题共15小题，每题3分，共45分。

在每小题给出的四个选项中，只有一项是符合题目要求的。

1. 淀粉肠是一种常见的夜市小吃。

依据国家标准，和普通火腿肠对比，淀粉肠中淀粉和脂肪含量更高，蛋白质含量更低。

下列相关叙述正确的是（）A. 淀粉肠中有机化合物的共有元素为C 、H 、O 、NB. 淀粉肠的制作原料多为鸡肉，富含不饱和脂肪酸C. 油炸淀粉肠时，会破坏蛋白质中的肽键D. 与普通火腿肠相比，淀粉肠食用后更容易引起血糖上升2. 下列有关同位素标记技术的实验描述，正确的是（）A. 人和小鼠细胞膜蛋白融合实验使用同位素标记技术证明细胞膜具有流动性B. 赫尔希和蔡斯使用32P 、35S 同时标记T2噬菌体，证明DNA 是噬菌体的遗传物质C. 用3H 标记亮氨酸的R 基，可以追踪分泌蛋白的合成、加工、运输路径D. 鲁宾和卡门使用18O 分别标记H 2O 和CO 2，通过检测放射性研究光合作用中氧气的来源3. 黑藻是沉水草本植物，其叶肉细胞中含有丰富的叶绿体和中央大液泡，是一种易获得的理想实验材料。

下列相关叙述错误的是（）A. 用显微镜观察黑藻细胞质的流动时，视野内细胞中叶绿体的流动方向不一定相同B. 用黑藻做质壁分离和复原实验时，可以同时观察到液泡体积和颜色的变化C. 用黑藻提取和分离光合色素时，加入碳酸钙有利于滤纸条上出现4条色素带D. 用黑藻测定叶绿体产生O 2的最大速率时，需要先测出黑暗条件下的呼吸速率4. 古核生物是一类特殊的生物，在细胞结构和新陈代谢方面与细菌相似，而转录的过程则更接近真核生物。

秀丽线虫的研究和饲养

注射时将将琼脂糖固定垫放
线虫挑至琼脂糖固定垫上，调整线虫使性腺暴露，滴加注射油覆
在载物台上，40x物镜下找到线虫，使性腺聚焦在正确的平面．操作微操或轻移滑动载物台，将注射针尖刺入性
盖整个虫体．固定好后的操作要迅速，否则线虫容易脱水而死
腺．启动微量加压器进行注射，能观察到注射液在性腺中快速流动，注射后的性腺被液体充满．
显微注射后的整合
•目前常用的整合方法有：用 y射线和 X射线照射，或用光敏剂补骨脂素加长波紫外线照射整合(TMP/UV integration)．基本策略是大量筛选经 •射线照射过的转基因线虫，一般挑取数百只 F1 代繁殖，筛选 F4 代，检测是否有 100%的转基因表达，若是则说明整合成功．一般一次整合能得到若干个独立种系，可选择最好的一个进行实验．
秀丽隐杆线虫的饲养及研究用途
精品课件
Hale Waihona Puke By 王传杰介绍
• 秀丽隐杆线虫（Caenorhabditis elegans）,属于线形动物门、线虫纲。体形非常小,成虫只有1mm左右。线虫是细胞定数动物，两性成虫只有 9 5 9 个体细胞，雄性成虫只有 1 0 3 1 个体细胞，其中 1 3 1个细胞注定要接一定的发育程序陆续死亡。神经系统解剖结构十分简单，仅有 3 0 2个细胞，约占整个动物体细胞总数的三分之一。它身体透明,能感知气味和味道 ,对光线、温度有反应。研究者很容易在显微镜下对其细胞和组织进行跟踪观察
精品课件
在体视显微镜下，滴加恢复缓冲液至注射后的线虫正上方，由于与油互不相溶，缓冲液会渗入油下使线虫浮起．一般等待 2～ 5 min，线虫活力恢复，身体开始游动，即可挑至培养板上，20℃ 常规培养．

三七醇提物对秀丽隐杆线虫的降脂作用

2020第二十二卷第五期★Vol.22No.5三七醇提物对秀丽隐杆线虫的降脂作用＊黄壮，李静，杜鸿志，韩林涛，洪怡，黄必胜，曹艳＊＊（湖北中医药大学药学院省部共建中药资源和中药复方教育部重点实验室武汉430065）摘要：目的研究三七醇提物降低秀丽隐杆线虫脂肪沉积的作用以及可能的分子机制。

方法本研究采用秀丽隐杆线虫为模式生物，三七醇提物以不同剂量给药后，观察对线虫的寿命、生殖和抗逆性的影响；采用油红O染色法，观察了线虫体内脂肪沉积的变化；同时，检测了线虫体内的甘油三酯含量和脂肪代谢相关的基因acs-2、pod-2、fat-5的表达水平。

结果与对照组比较：三七醇提物不同给药剂量组均能延长线虫的寿命，增强抗逆性，且高剂量组更为显著（P<0.01）；对线虫生殖能力没有影响；油红O染色结果显示，三七醇提物可显著降低线虫肠道脂肪沉积（P<0.01）；三七醇提物高剂量组还能显著降低甘油三酯的含量（P<0.05），并影响与脂肪代谢相关基因表达水平，其中acs-2mRNA被上调，pod-2、fat-5mRNA被下调（P<0.05）。

结论三七醇提物在不影响线虫生命特征的前提下，可减少线虫体内脂肪沉积，对甘油三酯水平及脂肪代谢相关的基因表达水平有调控作用。

关键词：三七秀丽隐杆线虫降脂doi:10.11842/wst.20190710010中图分类号:R284文献标识码:A三七[Panax notoginseng(Burk.)F.H.Chen]为五加科多年生草本植物，是我国传统名贵的中药材，药用部分主要为根茎，至今已有六百多年的用药历史。

三七，味甘、微苦、性温，具有化瘀止血，活血定痛的功效。

主治出血证，跌打损伤，瘀血肿痛。

归肝经、胃经、心经、肺经、大肠经[1]。

现代药理研究表明，三七具有活血止血、免疫调节、延缓衰老、改善记忆、促进生长发育、抗疲劳、降血脂等多种药理活性[2-4]。

据报道临床使用中，三七单味药使用或与其他中药配伍使用，均表现出一定的降脂作用[5-6]。

线虫脂肪模型研究

线虫脂肪模型研究徐蔓玲;赵阳;贾熙华;季宇彬【摘要】Obesity is a kind of chronic disease with the fatty acid accumulation increased . Studying on the model of organism can discover the obesity disease -causing genes or related pathogenic factors , and find potential drug targets .The progress of research on Caenorhabdi-tis elegans as a model for fatty was summarized in this paper .The key genes and core path-ways in fatty acid metabolism was involved .The data obtained in Caenorhabditis elegans on fatty storage control will contribute largely to the study on metabolism related diseases , such as obesity in human beings .C.elegans may be invaluable in the development of reducing fat and body weightdrugs in the future .%肥胖是一种脂肪酸积累增加的慢性疾病，采用研究模式生物的方法可以有助于探明肥胖产生的致病基因或致病相关因子，进而寻找可能的药物靶点。

综述了国内外对线虫脂肪模型的研究进展，分析了线虫体内脂肪酸代谢途径的关键基因和核心通路。

丙醇二酸降低秀丽隐杆线虫体内脂肪含量的研究

ＡＴＧＧＴＣＧＣＴＣＡＴＴＣＣＴＣＡ
ＡＴＴＧＧＡＴＧＣＧＴＧＧＣＴＣＴＴ
ＧＴＣＡＡＡＧＣＡＧＣＡＡＴＣＣＣＴ
ＡＡＴＡＣＣＡＴＣＣＡＡＧＡＡＧＣＣＣ
ＡＴＧＧＣＡＡＡＴＧＧＣＴＣＴＡＣＣ
ＡＴＴＣＡＴＣＧＴＧＣＴＣＴＣＣＧＴ
ＧＧＴＡＴＴＡＣＧＡＧＡＧＣＡＡＧＡＧＣＡＴ
ＧＧＴＡＴＴＡＣＧＡＧＡＧＣＡＡＧＡＧＣＡＴ
摘要: 目的: 探索丙醇二酸对秀丽隐杆线虫体内脂肪的影响及调节机制ꎮ 方法: 在ＮＧＭ培养基中加入不同浓
度丙醇二酸饲养线虫ꎬ 油红Ｏ和尼罗红染色法展示线虫体内脂肪颗粒大小、数量和相对脂肪含量ꎬ 测定线虫身体摆
动频率、吞咽频率、４８ｈ存活率、身宽、体长等指标ꎮ ＱＰＣＲ检测ｆａｔ￣１至ｆａｔ￣７、ｓｂｐ￣１和ｎｈｒ￣４９基因相对表达量ꎬ 荧
酸处理ꎬ 线虫体长无明显差异ꎬ 身宽分别降至５３１、
５７０１、５６０３ μｍ (Ｐ<００５ꎬ 表２)ꎮ 综上表明ꎬ 丙醇二酸
对线虫无明显毒性ꎬ 不影响线虫的运动和吞咽ꎮ 不同浓度
丙醇二酸不影响线虫的身长ꎬ 但均显著降低体宽ꎬ 这可能
与其降低线虫体内脂肪含量有关ꎮ
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实验重复３次ꎮ
２２丙醇二酸处理线虫部分指标变化
ＲＮＡꎬ 并测定其含量ꎮ 根据ＲＮＡ含量ꎬ 按逆转录试剂
率分别为６０４２、１５０２５次/ ｍｉｎꎮ 各剂量组与对照组相比ꎬ
转录水平测定ꎮ 以ａｃｔ￣１为内参基因ꎬ 用２
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并列第一作者: 刘晓颖 (２０００— )ꎬ 女ꎬ 在读硕士研究生ꎬ 研究方向: 营养及食品安全检测ꎻ 王润圆 (１９９５— )ꎬ 女ꎬ 在读硕士研究生ꎬ 研究方向: 营养及

秀丽隐杆线虫研究情况

秀丽隐杆线虫研究情况
秀丽隐杆线虫被应用于实验研究至今已逾30年，因为易于实验室培养、基因易处理、解剖学结构简单以及可以提供广泛的遗传学和基因组信息，已成为一种重要的研究细菌和真菌的哺乳动物替代模型。

与黑腹果蝇一样，秀丽隐杆线虫将天然免疫作为防御微生物感染的唯一防线。

Mylonakis等研究发现，一些对哺乳动物起作用的新生隐球菌毒力因子在杀死秀丽隐杆线虫的过程中同样有效，这些基因包括信号转导途径GPA1、PKA1、PKR1、 RAS1和漆酶等；而那些对哺乳动物毒力较低的因子在秀丽隐杆线虫模型中致病性亦较弱。

还有作者通过秀丽隐杆线虫模型研究荚膜、黑色素、调节通路等毒力因子来鉴定毒力减低的新生隐球菌，结果发现rom2基因突变的隐球菌在37℃时失去繁殖及生长的能力，并无法生成细胞壁和难耐高渗。

多数秀丽隐杆线虫是可以自身繁殖的雌雄同体动物，偶尔也可见到雄性单体。

实验结果证实野生雄性线虫较雌雄同体线虫对真菌的抵抗力增强，而且这种抵抗力的增强归因于应激反应激活因子DAF-16的参与，而不是由于行为或生殖方式的不同。

重金属暴露可以改变秀丽线虫的脂肪积累

重金属暴露可以改变秀丽线虫的脂肪积累【摘要】目的:研究重金属暴露对秀丽线虫脂肪积累的急性中毒效应,鉴定重金属暴露秀丽线虫中发生表达模式改变的与脂肪酸代谢相关的基因。

方法:银、镉、铬、铜、汞、锰和锌作为测试重金属,Nile Red荧光探针染色分析重金属暴露线虫肠道的脂肪酸含量变化,并测定重金属暴露线虫中脂肪酸代谢相关基因的转录水平变化。

结果:在所分析重金属中,高浓度银、铬和铜暴露可以显著诱导线虫肠道脂肪积累的增加,而高浓度镉暴露可以显著降低线虫肠道脂肪的积累。

而且,与对照组相比,高浓度的银和铬暴露不明显影响基因pod2、gei7、lbp8、gpd3、W05G11.6、C03H5.4和C07E3.9的表达水平,而在暴露的线虫动物中,基因fat5、fat6和fat7的表达水平显著升高,lbp1、acs2和ech1的表达水平显著降低。

结论:高浓度银、铬、铜和镉暴露可以导致异常的秀丽线虫肠道的脂肪积累变化,其中高浓度银和铬暴露导致线虫脂肪积累增加的主要原因可能归因于暴露动物体内脂肪酸去饱和与线粒体β氧化代谢途径以及脂肪酸结合能力的异常。

【关键词】脂肪积累; 金属暴露; 代谢途径; Nile Red染色; 秀丽线虫Bioassays, usually used in monitoring scheme in toxicological studies, can be explored to monitor the acute toxicity of aquatic effluents according to the guideline set by regulatory authority. Physical and chemical methods can also beused for such monitoring by employing sophisticated equipment. However, considering the facts that field samples often contain unidentified components, and referencing of all toxicants may not be available, whole sample analysis with bioindicator animals was proposed by the U.S. Environment Protection Agency to circumvent such a limitation[1]. A free living nematode, Caenorhabditis elegans, a nonparasitic bacterial feeder that lives in soil interstitial, satisfies all the criteria for bioindicator[2]. Because of its convenient handling in the laboratory, its sensitivity to different kinds of stresses, and its abundance in soil ecosystems, C. elegans is widely used in ecotoxicological studies using different exposure media, including soil and water[3-4].The ability of an organism to regulate the production, storage, and release of energy is crucial for health and survival, and a major source of energy is stored as fat, which is required for the life cycle of organisms[5]. Fat storage regulation is a fundamental process, and abnormalities in fat storage will underline important human diseases[6]. The alarming worldwide increase in obesity has intensified the research to identify genes that control the differentiation and function of fat storing tissues, and to examine environmental clues thatinduce the severe abnormalities in fat accumulation[5]. Previous studies have suggested that the lipid metabolism can be altered in acute and chronic lead(Pb) exposed humans and animals[7], and cadmium(Cd) exposure can result in the impaired lipid storage in European eel[8]. Model organisms are a powerful resource for the discovery of genes and environmental clues critical to human health and disease. So far, it has been proven that the C. elegans is an excellent model to analyze the mechanisms of fat storage[5]. Worms have homologs of many mammalian lipogenic and lipolytic enzymes, and genes of encompassing a wide range of components of the mammalian fat regulation cascade[5,9].Thus, the current literature regarding C. elegans provides insight concerning the relative sensitivity of the fat storage endpoint. In the present study, the toxicological effects of metal exposure on fat storage were examined in nematodes. Silver(Ag), Cd, chromium(Cr), copper(Cu), mercury(Hg), manganese(Mn), and zinc(Zn) were chosen as test metals both because of the availability of toxicological data in the literature regarding nematodes and other organisms and because of their abundance in the environment. The aims of the present study were to evaluate the acute toxicity of metalexposure on fat storage of nematodes, and to identify the sensitive genes required for the metabolism of fatty acid in metal exposed nematodes.1 Materials and methods1.1 ChemicalsThe metal concentrations used in this study were selected as previously described[10-13]. Three concentration levels of AgNO3, CdCl2, CrCl2, CuSO4, HgCl2, MnCl2, and ZnCl2 solutions were used in the current study, and they were 2.5, 75, and 200 μmol·L-1, respectively. All the chemicals were obtained from Sigma Aldrich(St. Louis, MO, USA).1.2 StrainsAll nematodes used were wild type Bristol strain N2, originally obtained from the Caenorhabditis Genetics Center(CGC). They were maintained on nematode growth medium(NGM) plates seeded with Escherichia coli OP50 at 20 ℃as described[14]. Gravid nematodes were washed off the plates into centrifuge tubes and were lysed with a bleaching mixture(0.45 mol·L-1 NaOH, 2% HOCl). Age synchronous populations of N2(L4larvae stage) were obtained by the collection as described[15]. The L4larvae stage nematodes were washed with double distilled water twice, followed by washing withmodified K medium once(50 mmol·L-1 NaCl, 30 mmol·L-1 KCl, 10 mmol·L-1 NaOAc, pH 5.5)[16]. Exposures were performed in 12well sterile tissue culture plates. All exposures were 24 h, and were carried out in 20 ℃ incubator in the presence of food.1.3 Nile Red stainingThe method was performed as described[17]. Nile Red(5H benzo[α]phenoxazine5one, 9diethylamino) powder(N1142 Molecular Probes) was dissolved in acetone at 500 μg·ml-1, diluted in phosphate buffered saline(PBS) and added on top of NGM plates already seeded with OP50 bacteria, to a final concentration of 0.05 μg·ml-1. Nematodes were then grown on plates with Nile Red as eggs, and photographed with epifluorescence(rhodamine channel). Their staining phenotypes were assessed prior to starvation at the young adult stages. Fat content was monitored and evaluated using ImageJ Software(NIH Image) by determining average intensity in each animal s intestine. More than 30 nematodes were counted for the statistical analysis.1.4 Analysis of gene expression in metal exposed nematodesPoly(A)+RNA of nematodes was prepared and analyzed asdescribed[18-19]. The frozen nematodes pellets were harvested from 1 liter of mixed stage liquid cultures. A total of 1 μg of poly(A)+RNA per lane was separated by electrophoresis on a 1.2% agarose gel containing 2 mol·L-1 formaldehyde and 0.02 mol·L-1 MOPS, and transferred onto a nylon membrane by a mild alkaline transfer method. Northern hybridizations of transferred poly(A)+RNA were performed using PCR product probes labeled with32P dCTP at 65 ℃overnight. Gene specific primers were designed for gpd3(gpd3f, 5′ATGACCA AGCCAAGTGTC3′; gpd3r, 5′TAGGCCTTGGTAGCAATG3′), for W05G11.6(W05G11.6f, 5′GAGTGTCGTTCTCTGCG3′; W05G11.6r, 5′CCTGGCCACAAGAACTTG3′), for C03H5.4(C03H5.4f, 5′GCCAGAAACTCTGAATAC3′; C03H5.4r, 5′CTCAACATCTTCTTCTGC3′), for C07E3.9(C07E3.9f, 5′TTCTAGTGTTTCTAGCCG3′; C07E3.9r, 5′CAATGTTGGAAATGTTTC3′), for acs 2 (acs2f, 5′CCTCGCTCTACACTCTCG3′; acs2r, 5′GACGACATGAGCATCAGC3′), for ech1(ech1f, 5′GATTCTTGACGTTGTCCG3′; ech1r, 5′ACAGATTCGTACACTCCG3′), for gei7(gei7f, 5′CTATTGCACACTGAGCCA3′; gei7r, 5′GGCTCCAGAGTTCATAGC3′), for fat5(fat5f, 5′GAGATCCGACAAATGCAG3′; fat5r, 5′CGAACTTCTTGCACTGTC3′), for fat6(fat6f, 5′CTCTTCACTTCGCTGCAG3′; fat6r, 5′GGCCGATAATCTCATCAC3′), for fat7(fat7f, 5′GCATTGCCAAGAAGATTG3′; fat7r, 5′CAAGACCAAGAACAGCTG3′), for pod 2 (pod2f, 5′GGAGCAGTTCATTCACTC3′; pod2r, 5′GTCGAGCACATCGTATTC3′), for lbp 1 (lbp1f, 5′TCGCTCTTCTCCTGGTTC3′; lbp1r, 5′GTCGTTTGTAGAATCGGC3′), and for lbp8 (lbp8f, 5′ATGGTTTCCATGAAAGAG3′; lbp8r, 5′TCTAATTTTGAAAGCGAG3′). act 1 was used to determine the equal loading for each sample, and primers specific for act 1 were used in control reactions(act1f, 5′CGAAGCTTACCGTCCCAATCTACGAAG3′; act1r, 5′TGAGAATTCGAAGCACTTGCGGTGAAC3′). Amplification of all DNA fragments for RT PCR was performed for 30 cycles in a Perkin Elmer 480 thermal cycler(Perkin Elmer, Waltham, USA) using a 55 ℃ annealing temperature and a 1min extension.1.5 Statistical analysisAll data in this article were expressed as means ± S.D. Graphs were generated using Microsoft Excel(Microsoft Corp., Redmond, WA). One way analysis of variance(ANOVA) followed bya Dunnett s t test was used to determine the significance of the differences between the groups. The probability levels of 0.05 and 0.01 were considered statistically significant.2 Results2.1 Alterations of fatty acid in intestine of metal exposed nematodes In nematodes, the intestine is the major site of energy stores, and the principal site of fat storage. Addition of Nile Red to E. coli OP50, the laboratory diet of C. elegans, can cause uptake and incorporation of the dye Nile Red into lipid droplets in intestinal cells[17,20]. We explored the vital dye Nile Red to visualize fat storage droplets in intestinal cells in control and metal exposed living nematodes. As shown in Fig 1, exposure to all examined seven metals at the concentration of 2.5 μmol·L-1 for 24 h in the presence of food did not obviously influence the fat storage in intestine of nematodes. Exposure to metals of Hg, Mn, and Zn at higher concentrations(75 μmol·L-1 and 200 μmol·L-1) also did not noticeably affect the fat storage in intestine of nematodes. Similarly, exposure to metals of Cd and Cu at the concentration of 75 μmol·L-1 could not alter fat storage in nematodes. In contrast to these, exposure to Ag and Cr at the concentrations of 75 μmol·L-1 and 200 μmol·L-1 and exposure to Cu at the concentration of 75 μmol·L-1 significantly(P<0.01) increased the fat storage in intestine of nematodes. Different from the increased fat storage phenotype observed in Ag, Cr, and Cu exposed nematodes, exposure to Cd at the concentration of 200 μmol·L-1 moderately but significantly(P<0.01) decreased the fat storage in intestine of nematodes. Therefore, exposure to Ag, Cr, Cu, and Cd at high concentrations will influence the fat storage to different degrees in exposed nematodes.A. Pictures of Nile Red staining.B. Quantification of Nile Red fluorescence in control and metal exposed nematodes. Control, without metal exposure. Bars represent mean ±S.D. a. P<0.01 vs 0 μmol·L-1(control)Fig 1 Fat storage in control and metal exposed nematodes as monitored by Nile Red staining 2.2 Expression patterns of gene required for the fatty acid metabolism in Ag and Cr exposed nematodes Next, we examined the expression patterns of genes required for the fatty acid metabolism in control and metal(Ag and Cr) exposed nematodes by analyzing the transcription levels of genes. pod 2 encodes an acetyl CoA carboxylase, and by sequence similarity, POD 2 is predicted to catalyze the first step in fatty acid biosynthesis. gei7 encodes a predicted isocitrate lyase/malate synthase, an enzyme known to functionin the glyoxylate cycle. As shown in Fig 2A, exposure to Ag and Cr at the concentration of 200 μmol·L-1 did not affect the expression levels of genes of pod 2 and gei7 compared with control. Inhibition of fat5, fat6, and fat7 genes encoding delta9 fatty acid desaturation enzymes is associated with reduced fat levels in nematodes[21]. In contrast, as shown in Fig 2B, the fat5, fat6, and fat7 expression was dramatically(P<0.01) increased in 200 μmol·L-1 Ag and Cr exposed nematodes compared with control. The genes of lbp 1 and lbp8 regulate the fatty acid binding and transportation in nematodes[22]. As shown in Fig 2C, exposure to Ag and Cr at the concentration of 200 μmol·L-1 did not influence the expression of lbp8, whereas the expression of lbp 1 was significantly(P<0.01) decreased by the Ag and Cr exposure at the concentration of 200 μmol·L-1 compared with control. gpd3 encodes a predicted glyceraldehyde 3phosphate dehydrogenase, and its function is involved in the glycolysis regulation[22]. W05G11.6 encodes a phosphoenolpyruvate carboxykinase, and its function is involved in the gluconeogenesis regulation in nematodes[22]. As shown in Fig 2D, no obvious alteration of gpd 3 and W05G11.6 expression was observed in 200 μmol·L-1 Ag and Cr exposed nematodes comparedwith control. Genes of C03H5.4 and C07E3.9 encode secreted phospholipase A2, and they regulate the phospholipids biosynthesis in nematodes[22]. As shown in Fig 2E, exposure to Ag and Cr at the concentration of 200 μmol·L-1 did not noticeably alter the C03H5.4 and C07E3.9 expression compared with control. Down regulation of acs 2 and ech1, mitochondrial βoxidation genes, causes fat accumulation[22]. As shown in Fig 2F, the expression of acs2 and ech 1 were all significantly(P<0.01) suppressed by Ag and Cr exposure at the concentration of 200 μmol·L-1 compared with control. Therefore, our results suggest that the observed formation of increased fat storage in Ag and Cr exposed nematodes was largely due to the abnormalities of fatty acid desaturation, mitochondrial βoxidation, and fatty acid binding.A. Expression patterns of pod 2 and gei7 in control and metal exposed nematodes.B. Expression patterns of fat5, fat 6 and fat7 in control and metal exposed nematodes.C. Expression patterns of lbp 1 and lbp8 in control and metal exposed nematodes.D. Expression patterns of gpd 3 and W05G11.6 in control and metal exposed nematodes.E. Expression patterns of C03H5.4 and C07E3.9 in control and metal exposed nematodes.F.Expression patterns of acs 2 and ech 1 in control and metal exposed nematodes. Control, without metal exposure. Bars represent mean ±S.D. a. P<0.01 vs 0 μmol·L-1(control) Fig 2 Expression patterns of genes required for the fat storage in 200 μmol·L-1 metal(Ag and Cr) exposed nematodes 3 DiscussionIn the present study, we investigated the acute toxicity on fat storage from metal exposure. Our data demonstrated that exposure to low concentration, such as 2.5 μmol·L-1, of examined metals(Ag, Cd, Cr, Cu, Hg, Mn, and Zn) can not induce the severe abnormality of fat storage in nematodes. Considering the fact that the concentrations of examined metals in our surviving environment are usually not more than the value of 2.5 μmol·L-1, the metals in the normal environment can not cause the severe impairment on the fat storage in human beings. However, our data further suggest that exposure to 200 μmol·L-1 of Ag, Cr, and Cu can result in the severe increase of fat storage, and exposure to 200 μmol·L-1 of Cd can cause the severe decrease of fat storage in exposed nematodes. Especially, the noticeable increase of fat storage can also be observed in nematodes exposed to 75 μmol·L-1 of Ag and Cr. These observations using the Nile Red staining method were confirmed by the Sudan Black stainingmethod(data not shown). Therefore, these data suggest that exposure to high concentrations of metals, including Ag, Cr, Cu, and Cd, will result in the abnormality of fat storage in intestine of nematodes. Previous study has suggested that eels contaminated by Cd showed a lower body weight growth with a lower efficiency of lipid storage compared to control, which is mainly explained by an increased utilization of triglycerides since Cd exposure did not trigger a reduced fatty acid synthesis[8].More interestingly, different metals will have different adverse effects on the fat storage in exposed organisms. For example, exposure to Ag, Cr, and Cu will induce the increased fat storage, whereas exposure to Cd will induce the reduced fat storage. In C. elegans, the synergistic, additive, or neutralizing toxicity of multiple heavy metals has been revealed by a biological assay using a transgenic derivative nematode[2]. In addition, the combinational toxicity from multiple metals can also be detected in a paper recycling industrial waste water[23]. Our data strongly imply that the similar synergistic, additive, or neutralizing toxicity on fat storage from metal exposure may be also formed in nematodes.C. elegans is an attractive animal model for the study of the ecotoxicological relevance of chemical induced, genelevel responses[3]. In the current work, we further examined the expression patterns of genes required for fatty acid metabolism in Ag and Cr exposed nematodes. The reason to select the meals of Ag and Cr was that the sharp increase of fat storage can be observed in nematodes exposed to high concentrations of Ag and Cr; however, only moderate defects of fat storage can be formed in Cu and Cd exposed nematodes. The expression patterns of genes required for the fatty acid metabolism suggest that exposure to Ag and Cr at the concentration of 200 μmol·L-1 will not obviously influence the expression of pod2, gei7, lbp 8, gpd3, W05G11.6, C03H5.4 and C07E3.9 compared with control, which suggests that at least the metabolic pathways of fatty acid synthesis, phospholipids synthesis, glycolysis, and gluconeogenesis can not be severely altered by exposure to Ag and Cr. In contrast, the increased expression of fat5, fat 6, and fat7 can be observed in Ag and Cr exposed nematodes at the concentration of 200 μmol·L-1 compared with control, indicating the alteration of delta9 fatty acid desaturation by exposure to Ag and Cr. In addition, the decreased expression of acs 2 and ech 1 can be observed in Ag and Cr exposed nematodes at the concentration of 200 μmol·L-1 compared with control, indicating the disruption of mitochondrial βoxidation from Ag and Cr exposure. These data suggest that the observed increased fat storage in Ag and Cr exposed nematodes is the combinational effect of delta9 fatty acid desaturation with mitochondrial βoxidation. That is, exposure to Ag and Cr will influence both the desaturation and the breakdown processes of fatty acid in nematodes.Acknowledgements Strain used in this work was provided by the Caenorhabdits Genetics Center(funded by the NIH, National Center for Research Resource). This work was supported by the grants from the National Natural Science Foundation of China(No. ) and the Program for New Century Excellent Talents in University.【参考文献】[1] WEBER C I. Methods for measuring the acute toxicity of effluents to freshwater and marine organisms [M]. 4th ed. Cincinnati: Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, OH. EPA/600/490/027F, 1993.[2] CHU K W, CHOW K L. Synergistic toxicity of multiple heavy metals is revealed by a biological assay using a nematode and its transgenic derivative [J]. Aquat Toxicol, 2002,61:5364.[3] ROH J, LEE J, CHOI J. Assessment of stress related gene expression in the heavy metal exposed nematode Caenorhabditis elegans: a potential biomarker for metal induced toxicity monitoring and environmental risk assessment [J]. Environ Toxicol Chem, 2006,25:29462956.[4] LEUNG M C K, WILLIAMS P L, BENEDETTO A, et al. Caenorhabditis elegans: an emerging model in biomedical and environmental toxicology [J]. Toxicol Sci, 2008,106:528.[5] MCKAY R M, MCKAY J P, AVERY L, et al. C. elegans:a model for exploring the genetics of fat storage [J]. Dev Cell, 2003,4:131142.[6] CAMPBELL P, DHAND R. Obesity [J]. Nature, 2000,404:631671.[7] ADEMUYIWA O, AGARWAL R, CHANDRA R, et al. Lead induced phospholipidosis and cholesterogenesis in rat tissues [J]. Chem Biol Interact, 2009,179(23):314320.[8] PIERRON F, BAUDRIMONT M, BOSSY A, et al. Impairment of lipid storage by cadmium in the European eel(Anguilla anguilla). Aquat Toxicol, 2007,81:304311.[9] C. Elegans Sequencing Consortium. Genome sequence of the nematode C. elegans: a platform for investigating biology [J]. Science, 1998,282(5396):20122018.[10] WANG Y, XIE W, WANG D Y. Transferable properties of multi biological toxicity caused by cobalt exposure in Caenorhabditis elegans[J]. Environ Toxicol Chem, 2007,26:2405 2412.[11] WANG D Y, XING X J. Assessment of locomotion behavioral defects induced by acute toxicity from heavy metal exposure in nematode Caenorhabditis elegans[J]. J Environ Sci, 2008,20:11321137.[12] WANG D Y, WANG Y. Phenotypic and behavioral defects caused by barium exposure in nematode Caenorhabditis elegans[J]. Arch Environ Contam Toxicol, 2008,54:447453.[13] DU M, WANG D Y. The neurotoxic effects of heavy metal exposure on GABAergic system in nematode Caenorhabditis elegans[J]. Environ Toxicol Pharmacol, 2009,27:314320.[14] BRENNER S. The genetics of Caenorhabditis elegans[J]. Genetics, 1974,77:7194.[15] DONKIN S, WILLIAMS P L. Influence of developmental stage, salts and food presence on various end points using Caenorhabditis elegans for aquatic toxicity testing [J]. Environ Toxicol Chem, 1995,14:21392147.[16] WILLIAMS P L, DUSENBERY D B. Aquatic toxicity testing using the nematode Caenorhabditis elegans[J]. EnvironToxicol Chem, 1990,9:12851290.[17] ASHRAFI K, CHANG F Y, WATTS J L, et al. Genomewide RNAi analysis of Caenorhabditis elegans fat regulatory genes [J]. Nature, 2003,421:268272.[18] YANASE S, ISHII N. Cloning of the oxidative stressresponsive genes in Caenorhabditis elegans [J]. J Rad Res, 1999,40:3947.[19] YANASE S, YASUDA K, ISHII N. Adaptive resistance to oxidative damage in three mutants of Caenorhabditis elegans(age1, mev 1 and daf16) that affect life span [J]. Mech Ageing Dev, 2002, 123:15791587.[20] GREENSPAN P, FOWLER S D. Spectrofluorometric studies of the lipid probe, Nile Red [J]. J Lipid Res, 1985,26:781789.[21] BROCK T J, BROWSE J, WATTS J L. Genetic regulation of unsaturated fatty acid composition in C. elegans[J]. PLos Genet, 2:e108[22] van GILST M R, HADJIVASSILIOU H, JOLLY A, et al. Nuclear hormone receptor NHR49 controls fat consumption and fatty acid composition in C. elegans[J]. PLos Biol, 2005,3:e53.[23] WANG X Y, SHEN L L, YU H X, et al. Toxicity evaluation in a paper recycling mill effluent by couplingbioindicator of aging with the toxicity identification evaluation method in nematode Caenorhabditis elegans[J]. J Environ Sci, 2008,20:13731380.。

染色法鉴别秀丽隐杆线虫生命状态探究

染色法鉴别秀丽隐杆线虫生命状态探究高学娟;尹文彦;陆娟;屈长青【摘要】利用0.5%的曙红、10%的番红花、0.5%的亚甲基蓝和红墨水,分别对死伤秀丽隐杆线虫进行染色观察,进而找出一种清晰可见、简单易行的鉴别死伤线虫的方法.【期刊名称】《广东农业科学》【年(卷),期】2010(037)009【总页数】2页(P189,195)【关键词】秀丽隐杆线虫;解冻复苏;死活鉴定【作者】高学娟;尹文彦;陆娟;屈长青【作者单位】阜阳师范学院生命科学学院,安徽,阜阳,236041;阜阳师范学院生命科学学院,安徽,阜阳,236041;阜阳师范学院生命科学学院,安徽,阜阳,236041;阜阳师范学院生命科学学院,安徽,阜阳,236041;抗衰老中草药安徽省工程技术研究中心,安徽,阜阳,236041【正文语种】中文【中图分类】Q959.17秀丽隐杆线虫(Caenorhabditis elegans)属于线形动物门、线虫纲动物。

个体非常小，成虫只有1 mm 左右[1]。

英国Brenner于1963年发现并对其生长发育状况进行了研究[2-3]，庞林海等人发现秀丽隐杆线虫在16℃的NGM培养基生长良好[4]。

作为一种模式生物，秀丽隐杆线虫现已被广泛应用于生物学和医学领域[5]。

秀丽隐杆线虫个体小，而且具有假死习性，要确定它的死活十分困难[6]。

传统的做法是在解剖镜下用光、热刺激或用细针触动看其是否活动来确定，但这种方法存在着很大的主观性和随机性。

因此，在研究线虫的过程中面临的一大难题就是如何快速、准确地判断被试线虫的死活，这同时也是植物性杀线虫剂活性筛选中的一个难题[7]。

本试验选用0.5%曙红，10%番红花，0.5%亚甲基蓝和红墨水等各种试剂通过着色情况鉴别线虫死活，进而找出一种清晰可见、简单易行的鉴别死伤线虫的方法。

1 材料与方法1.1 试验材料供试秀丽隐杆线虫由中科院等离子体物理研究所吴李君研究员惠赠；大肠杆菌作为秀丽隐杆线虫的食饵。

秀丽隐杆线虫体内脂滴的油红O染色观察

秀丽隐杆线虫体内脂滴的油红O染色观察姬云涛;王亦舒;李星辰;屈长青【摘要】目的通过油红O染色对秀丽隐线虫(Caenorhabditis elegans)肠道脂类物质沉积的形态学变化进行观察.方法在NGM培养基中加入油红O染液,对正常生理状态中的秀丽隐杆线虫体内的脂滴进行染色.结果 72 h后发现线虫的肠道普遍被染成红色.结论油红O染液可以对活体线虫的脂滴进行染色,为后续研究线虫脂肪沉积的调控机制奠定基础.【期刊名称】《中国生化药物杂志》【年(卷),期】2011(032)002【总页数】2页(P141-142)【关键词】秀丽隐杆线虫;油红O;脂滴;染色【作者】姬云涛;王亦舒;李星辰;屈长青【作者单位】抗衰老中草药安徽省工程技术研究中心,安徽,阜阳,236041;安徽师范大学生命科学学院,安徽,芜湖,241000;抗衰老中草药安徽省工程技术研究中心,安徽,阜阳,236041;抗衰老中草药安徽省工程技术研究中心,安徽,阜阳,236041【正文语种】中文【中图分类】R965.1秀丽隐杆线虫(Caenorhabditis elegans)属于线虫动物门、线虫纲动物，是研究发育生物学、神经生物学和老年衰老学的理想模型生物［1］。

两性成虫只有959个体细胞，雄性成虫只有1 031个体细胞。

其肠道是由咽和肠两部分组成，是脂类物质沉积的主要场所，由于线虫虫体透明，所以在显微镜下可以很清楚地看到从头部到尾部的整个肠道。

脂肪是重要的生命物质，但脂肪过多对动物也会带来很大的危害。

所以对动物体内的脂肪组织进行分析研究具有极为重要的意义。

油红O为脂溶性染料，在脂肪内能高度溶解，进而着色，能较好的显示组织中的脂肪滴［2］。

本文以油红O为染色试剂，对秀丽隐杆线虫体内的脂肪组织进行染色，为后续研究线虫脂肪沉积的调控机制奠定基础。

N2野生型秀丽隐杆线虫，由中科院等离子体物理研究所吴李君研究员惠赠;大肠杆菌OP50作为秀丽隐杆线虫的食物。

外源添加L-苹果酸降低秀丽隐杆线虫体内脂肪含量

外源添加L-苹果酸降低秀丽隐杆线虫体内脂肪含量王润圆;赵歆;张梦媛;曾琳;邹伟;王琦【期刊名称】《中国食物与营养》【年(卷),期】2022(28)8【摘要】目的:外源添加L-苹果酸研究其在秀丽隐杆线虫体内对脂肪代谢的影响及调节机制。

方法:含L-苹果酸的NGM培养基饲养秀丽隐杆线虫,油红O(ORO)和尼罗红(NR)染色法展示秀丽隐杆线虫体内脂肪,测定线虫体内甘油三酯和脂肪酸含量,qPCR检测fat-5、fat-6、fat-7和sbp-1基因mRNA,同时荧光显微镜观察fat-6:GFP、fat-7:GFP荧光强度的变化。

结果:外源添加200、500、1000μg/mL L-苹果酸,秀丽隐杆线虫体内脂肪降至83.33%、87.67%、76.67%,甘油三酯(TG)降至0.49、0.61、0.82倍。

脂肪酸含量发生变化,C16:1n-7/C16:0和C18:1n-9/C18:0比例降低,抑制硬脂酸C18:0向油酸C18:1n-9的转化,抑制棕榈酸C16:0向棕榈油酸C16:1n-7的转化。

1000μg/mL L-苹果酸饲养线虫,fat-5、fat-6、fat-7、sbp-1 mRNAs下调为0.46、0.57、0.63、0.50倍;fat-6:GFP和fat-7:GFP荧光强度减弱至0.36和0.28倍。

结论:L-苹果酸抑制转录因子sbp-1表达,进而降低fat-5、fat-6、fat-7基因转录,抑制硬脂酸C18:0向油酸C18:1n-9的转化,抑制棕榈酸C16:0向棕榈油酸C16:1n-7的转化,导致线虫脂肪储存的减少。

【总页数】6页(P43-48)【作者】王润圆;赵歆;张梦媛;曾琳;邹伟;王琦【作者单位】昆明医科大学公共卫生学院【正文语种】中文【中图分类】TS2【相关文献】1.应用活性碳吸附法降低L-苹果酸中富马酸含量研究2.添加外源碳调节植烟土壤氮素供应降低上部烟叶烟碱含量的研究3.L-阿拉伯糖促进秀丽隐杆线虫的生长发育及降低脂肪合成4.HPLC测定混合型饲料添加剂中L-苹果酸含量5.外源添加物降低镉胁迫下香菇体内镉含量及对酶活性的影响因版权原因，仅展示原文概要，查看原文内容请购买。

不同浓度亳菊水提物对秀丽隐杆线虫脂滴凝聚影响观察

不同浓度亳菊水提物对秀丽隐杆线虫脂滴凝聚影响观察屈长青;宫雯珺;常好才;姬云涛【摘要】Objective To explore the influence of the aqueous extract from Flos Chrysanthemi (the flower of Chrysanthemum morifolium Ramat. ) Boju on lipidosis of Caenorhabditis elegans. Methods Different concentrations of the aqueous extract from Boju were used to prepared NGM media for nematodes. After oil red O staining and isopropyl alcohol extraction, OD values were detected by spectrophotometer. Results The absorbency value showed a certain curve which changed with the increase in the concentration, and its mechanism needs to be studied further.%目的探讨亳菊对秀丽隐杆线虫脂肪沉积的影响.方法用不同浓度亳菊水提取液配制NGM培养基,培养同步化秀丽隐杆线虫,成虫经油红O染色,异丙醇萃取,测定OD值,并与普通NGM培养基培养的同步化线虫相比较.结果随着浓度的提高,线虫体内的脂肪沉积量呈现出一定的曲线变化,其具体机制有待于进一步研究.【期刊名称】《中国组织化学与细胞化学杂志》【年(卷),期】2012(021)006【总页数】3页(P528-530)【关键词】亳菊;水提物;秀丽隐杆线虫;同步化;油红O【作者】屈长青;宫雯珺;常好才;姬云涛【作者单位】抗衰老中草药安徽省工程技术研究中心;阜阳师范学院生命科学学院,安徽阜阳236041;阜阳师范学院生命科学学院,安徽阜阳236041;阜阳师范学院生命科学学院,安徽阜阳236041;阜阳师范学院生命科学学院,安徽阜阳236041【正文语种】中文【中图分类】R917肥胖已经成为危害人类健康的一个重要的危险因素，给人类社会造成日益沉重的经济负担［1］。

罗勒水提物对秀丽隐杆线虫脂肪沉积的影响

罗勒水提物对秀丽隐杆线虫脂肪沉积的影响
屈长青;徐林丽;陆娟;姬云涛
【期刊名称】《中国生化药物杂志》
【年(卷),期】2012(033)002
【摘要】目的研究罗勒水提物对秀丽隐杆线虫脂肪沉积的影响.方法用罗勒水提物配制NGM培养基培养同步化秀丽隐杆线虫,经油红0染色后,裂解虫体,在490 nm波长处测定吸光度值.结果在罗勒水提物配制的培养基生长的线虫吸光度值较小,与对照组相比差异显著.结论罗勒水提物可减少线虫体内脂质含量,但其机制有待于进一步研究.
【总页数】2页(P165-166)
【作者】屈长青;徐林丽;陆娟;姬云涛
【作者单位】阜阳师范学院生命科学学院,安徽阜阳236041;抗衰老中草药安徽省工程技术研究中心,安徽阜阳236041;阜阳师范学院生命科学学院;阜阳师范学院生命科学学院;阜阳师范学院生命科学学院
【正文语种】中文
【中图分类】R285
【相关文献】
1.罗勒水提取物对ADP、凝血酶诱导的大鼠血小板聚集的影响 [J], 依巴代提·托合提;玛依努尔·吐尔逊;毛新民;苏巴提·吐尔地
2.不同浓度亳菊水提物对秀丽隐杆线虫脂滴凝聚影响观察 [J], 屈长青;宫雯珺;常好
才;姬云涛
3.罗勒水提取物涂膜对珍珠番石榴耐藏性的影响 [J], 许银冰; 肖智丽; 马晓敏; 曾健浩; 陈晓琪; 李运生; 张福平
4.罗勒水提取物涂膜对珍珠番石榴耐藏性的影响 [J], 许银冰; 肖智丽; 马晓敏; 曾健浩; 陈晓琪; 李运生; 张福平
5.罗勒水提物对阿霉素引起的大鼠心肌毒性的影响 [J], 依巴代提.托乎提;玛依努尔.吐尔逊;苏巴提.吐尔地;艾尼瓦尔.吾买尔
因版权原因，仅展示原文概要，查看原文内容请购买。

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秀丽隐杆线虫，由抗衰老中草药安
徽省工程技术研究中心提供；大肠杆菌ＯＰ０Ｓ，作为秀丽隐杆线虫的食饵。
拍照（图１图２和图３，相比之下，图３可显如、）示出明显的红色脂滴，表明１ＴｉｎＸ１０和油ｒｏ一０ｔ红。浸泡３ｍｉ处理染色效果较好，便于观察。０ｎ的
讨论
１．材料
结果
虫成体的染色便于对其镜下观察，利用特异性染色试剂对各种组织成分着色，可以对组织成分准确定位，为许多实验研究提供了便利。油红０（ｌＯｉＲｄＯ）是一种油溶性偶氮染料，易溶于苯，溶于ｅ
抗衰老和基因组学研究的重要模式生物ｌ。对线３］
用ＰＳ冲洗３次。弃去ＰＳＢＢ，用１Ｔｒｏ一∞ ｉｎＸ１ｔ
、
异丙醇油红Ｏ溶液浸泡３ｍｉ。用ＰＳ冲洗３０ｎＢ
次。用异丙醇镜下分化至清晰，观察拍照。
［图分类号］中Ｒ３９２［献标识码）文ＡＤＯＩ１．８０ｚｚｈ．０００．１：０３７／ｇｚｘ２１．６０７
秀丽隐杆线虫（．ｌｇｎ）属于线形动物门、Ｃｅａｓｅ线虫纲动物，其成虫体长１ｍｍ，身体透明，生活
探索性的使用了１Ｔｒｏ一０，利用其透过作ｉｎＸ１０ｔ用使油红０更好的对虫体着色，得到了较好的效
果，为以后研究线虫脂肪调控奠定了基础。
材料和方法
Ｘ１０和油红Ｏ共同染色３ｍｉ，ＰＳ冲洗后观察００ｎＢ
乙醇和丙酮，目前被认为是最优良的脂肪染色染
经过多次试验探索发现，在油红Ｏ染色之前，
需要将ｌ多聚甲醛固定液和ＰＳ冲洗液离心弃Ｂ去，否则会产生沉淀杂质而影响镜下观察。通过分别设计对照组：① 不用１ＴｉｎＸ１０ｒｏ一０，仅用油ｔ
红Ｏ处理，染色３ｍｉ０ｎ、② 用１ＴｒｔｎＸ０ｉ１０处ｏ
料］，在脂肪内能高度溶解，从而染色，能保存
组织中的脂肪滴，染色效果好，颜色鲜红。本实验
理１ｍｉ，再用油红。染色３ｍｉ５ｎ０ｎ和 ③ １Ｔｉｎｒｏｔ
秀丽隐杆线虫体内脂肪染色方法的探究
徐林丽高学娟屈长青
（阜阳师范学院生命科学学院，安徽；抗衰老中草药安徽省工程技术研究中心，安徽２６４）３０１
［要］近几十年来，秀丽隐杆线虫（ａｎｒａｄｔｌａｓ因其结构简单、通体透明、生命周期短和易于培养，摘Ｃｅｏｈｂｉｓｅｅｎ）ｉｇ常作为一种模式生物被广泛用于现代发育生物学、遗传学、抗衰老和及脂肪调控等方面的研究。本文探索了一种对秀丽隐杆线虫体内脂肪的油红Ｏ染色方法，利用１Ｔｒｏ一０ｉｎＸ１０的透过作用，线虫体内脂滴可被油红０更好的着色，镜下观察ｔ颜色鲜红，染色效果较好，为以后研究线虫脂肪调控奠定了基础。［键词］秀丽隐杆线虫；关成体；染色
第１９卷第６期２ｌＯ０年１２月
中国组织化学与细胞化学杂志
ＣＨＩＮＥＳＪＥＯＵＲＮＡＬＯＦＳＨＩＴＯＣＨＥＭＩＳＴＲＹＡＮＤＣＹＴＯＣＨＥＭＩＴＲＹＳ
Ｖ０１９．Ｏ．１Ｎ．６Ｄｅｅｂｒ２０ｃｍｅ．０１
油红ｏ、异丙醇、１ＴｒｔｎＸ一。。％ｉ１Ｏｏ
３．方法用Ｍ９缓冲液将秀丽隐杆线虫洗至
１５ｌ的பைடு நூலகம் 心管中，在１多聚甲醛室温或４固定．肚 ℃
在世界各地的泥土中，以大肠杆菌为食饵，容易人工养殖，生命周期（ｉｙｌ）短ｌ，对人、动物１ｅｃｃｅｆ】］
和植物没有危害ｌ。Ｃｅｅａｓ成体内每一个细胞２］．ｌｇｎ
１－０ｎ５３ｍｉ，然后置于８ ℃ 冰箱中冷冻１ｒｉ，于０５ｎａ４ ℃水浴中迅速解冻。低速离心１ｎ３ｍｉ，弃上清。
的形成都可以被追踪，而且其胚胎发育过程中细胞分裂、分化以及细胞的死亡具有高度的程序性，因此Ｃｅｅａｓ已经成为现代发育生物学、遗传学、．ｌｎｇ