沙丁胺醇对果蝇的化蛹、羽化、寿命及抗氧化能力和DNA损伤的影响

doi:10.3864/j.issn.0578-1752.2014.19.018

摘要/Abstract

摘要： 【目的】利用重要的模式生物果蝇研究沙丁胺醇的作用机制，探索瘦肉精对生物体产生的影响及其分子机制，为进一步在生产中开发出更快速、精准且高效的瘦肉精检测方法提供重要的基础。【方法】将果蝇一龄幼虫放到不同浓度的沙丁胺醇培养基上饲喂，每天记录果蝇化蛹和羽化的情况，并统计出果蝇的化蛹率和羽化率及化蛹时间和羽化时间。收集24 h内羽化的果蝇，放入不同浓度沙丁胺醇培养基和基础培养基中，每管放入雌雄果蝇各25只，每个浓度设置4个平行实验。每天记录果蝇的存活数，每3 d更换一次培养基，直至果蝇全部死亡。分别统计雌雄果蝇半数死亡天数，平均寿命和平均最高寿命。收集不同浓度沙丁胺醇培养基上的3日龄成虫，雌雄果蝇各取15只放入离心管，研磨制成组织匀浆，离心（2 500 r/min，10 min）后取上清，然后按照试剂盒的方法测定SOD和CAT活力，同时检测MDA的含量；以果蝇3日龄成虫的中肠细胞为研究对象，采用单细胞凝胶电泳技术（Single cell gel electrophoresis, SCGE）对细胞进行处理，经EB染色后，在荧光显微镜下观察果蝇细胞DNA损伤情况并拍照，结合Cometscore^TM软件对图像进行分析。【结果】在沙丁胺醇的作用下，果蝇化蛹率和羽化率随处理浓度增加而降低，但是经统计分析发现差异不显著（P＞0.05），对果蝇化蛹时间和羽化时间也没有显著影响（P＞0.05）；用沙丁胺醇饲喂果蝇之后，对3日龄成虫体内SOD、CAT活力和MDA含量进行测定表明，随处理浓度的增加使体内MDA含量显著增加（P＜0.05），CAT的活力被抑制，呈现出显著降低的趋势（P＜0.05），促使体内抗氧化系统产生作用SOD活力显著提高（P＜0.05）；正是由于机体内抗氧化系统的保护机制产生作用，使得在一定浓度下处理的果蝇寿命呈现增加的趋势。对果蝇DNA损伤检测的试验表明，经沙丁胺醇处理之后，果蝇细胞就出现损伤，在SCGE的检测下可以观察到DNA损伤形成的拖尾，并且随着浓度的增加损伤情况也越严重，但是彗星图片中没有出现细胞核首尾分离的情况，可知沙丁胺醇并没有引起细胞凋亡。经统计分析，各处理浓度组的“彗星”尾长（Tail length, TL）、尾距（Olive tail moment, OTM）和尾部DNA百分含量（% DNA in tail），与对照组相比，均有显著差异（P＜0.05）。【结论】沙丁胺醇对果蝇化蛹和羽化没有显著影响；对果蝇抗氧化系统产生作用，抑制CAT活力，使SOD活力显著增加，使得在较高浓度处理时出现显著延长果蝇的寿命的情况；经沙丁胺醇处理之后，就会引起果蝇DNA损伤并且与对照组存在显著差异，但是并没有出现细胞凋亡的情况。

关键词: 沙丁胺醇, 果蝇, 氧化损伤, 单细胞凝胶电泳, DNA损伤

Abstract: 【Objective】The objective of this study is to explore the effect of salbutamol on the growth, development, life span, anti-oxidative capacity and DNA damage in Drosophila melanogaster, to further provide the basis for improvement of the more effective detection method.【Method】The 1st-instar larvae were fed with salbutamol and the pupate and emergence of flies were recorded, the pupation rate and the eclosion rate as well as pupation and eclosion time were counted. One day after eclosed, adult flies were fed up with normal medium and salbutamol medium, in which the concentrations of salbutamol were 0.48, 2.4,12 and 24 μg·mL^-1. Fifty flies for each medium(1﹕1 for male﹕female) and the experiment was repeated for 4 times. The number of lived flies was recorded every day. The medium was replaced every three days until all of the flies died. The 50 % lethal time, average longevity and average maximum time were recorded and calculated, respectively. The 3-day old adult flies which feed up with salbutamol or minimum medium were collected. Tissue homogenate of 30 adult flies (1﹕1 for male﹕female) were then prepared and centrifuged at 2 500 r/min and within 10 minutes. The enzyme activity kit were used to detect the enzymatic activities of SOD and CAT as well as the content of MDA. The 3-day old adult flies were used to detect the DNA damage of Drosophila mid-gut by the single cell gel electrophoresis (SCGE). The DNA damage of Drosophila cells were observed under a fluorescence microscope and photos were taken, then the images were analyzed by comet analysis software Cometscore^TM.【Result】Statistical analysis showed that the pupation rate and the eclosion rate decreased with the increasing concentration of Salbutamol, but there was no significant difference (P＞0.05). Statistical analysis also showed that the time of pupation and eclosion among the salbutamol treatments had no significant difference (P＞0.05). The enzymatic activities of SOD and CAT of the 3-day old adult flies were assayed, and the content of MDA of the 3-days old adult flies was also measured. The results showed that the content of MDA was increased significantly (P＜0.05) and the enzymatic activity of CAT was reduced significantly (P＜0.05) when the concentration of the salbutamol was increased. The antioxidant system was destroyed and the enzymatic activity of SOD increased significantly (P＜0.05). As the action of antioxidant system make the life-span of fly become longer. DNA damage detection experiments suggested that salbutamol caused the DNA damage of the fly cells. The SCGE experiment showed significant differences in the tail length (TL), olive tail moment (OTM), and tail DNA percentage (%DNA in tail) between flies grown in salbutamol treated medium and control.【Conclusion】Salbutamol has no influence on the pupation and eclosion of flies. It plays an important role in antioxidant system. Salbutamol restrains the enzymatic activity of CAT but increased enzymatic activity of SOD. High level of salbutamol can increase the life-span of flies significantly. It causes the DNA damage of the fly cells but not arouse the apoptosis.

Key words: salbutamol, Drosophila melanogaster;oxidative damage, single cell gel electrophoresis (SCGE), DNA damage

孙冰，林欣大，张春红. 沙丁胺醇对果蝇的化蛹、羽化、寿命及抗氧化能力和DNA损伤的影响[J]. 中国农业科学, 2014, 47(19): 3890-3897.

SUN Bing, LIN Xin-da, ZHANG Chun-hong. Effect of Salbutamol on the Pupation, Emergence, Life Span, Antioxidative Capacity and DNA Damage in Drosophila melanogaster[J]. Scientia Agricultura Sinica, 2014, 47(19): 3890-3897.

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