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经研究发现首个与失眠有关的生化标记。该标记是唾液中的一种酶，学名淀粉酶。当延长睡眠剥夺时间时，这种淀粉酶的活性就会随之增高。

研究人员希望淀粉酶，这一首个生物标记的发现能有助于睡眠障碍的诊断和治疗，并在将来有助于评估驾车嗜睡发作或在其他危险情况下嗜睡发作的风险。

“在我们为假期打点行囊和为走访远亲做好长时间开车准备的当口，我希望这个发现会让大家停下来好好思考不睡觉所带来的危险和代价。”神经生物学助理教授，该篇研究报告的第一作者普尔•J•肖博士说道，“如果在回奶奶家的路上，当你横跨河流、穿越树林时感到昏昏欲睡，那么最好把车靠一下边，找个地方小睡一会，而不要一直开车，以免发生严重车祸。”

该研究报告于本周发表于网络版的国家科学院学报。肖博士的实验室首次成功将果蝇置于一种与睡眠状态无异的静息状态。研究人员发现果蝇有几个静息时期，在这些时期要唤醒这些小东西需要较为强烈的刺激。象人类一样，一整天都不能睡觉的果蝇会试图在第二天睡更长的时间以弥补损失掉的睡眠时间。这一现象称为睡眠冲动增多现象或睡眠代偿现象。

为了寻找睡眠代偿生物标记，肖博士决定从唾液入手。唾液唾手可得，而且含有多种在血液和尿液中发现的物质，这使得临床医生愈来愈青睐于用唾液作为诊断疾病的检测物。肖博士的研究小组同样钟意于以唾液作为研究对象，因为调节唾液分泌的大脑区域所接收的信号由调节睡眠冲动的大脑区域所发送。

研究初始阶段，肖博士让果蝇经受不同程度的睡眠剥夺并运用微排列法测定多种不同基因中出现的活性改变。淀粉酶水平在失眠后的改变始终一致。存在于唾液中的淀粉酶属于酶的一个系谱，可分解淀粉。

为了证实淀粉酶与失眠之间的联系，肖博士的实验室对不同种类的果蝇在睡眠被剥夺后施予监控。这些果蝇的基因事先都被进行改造，为的是调节睡眠冲动。

在一项重要测试中，有一只果蝇的淀粉酶水平并未升高。这是一只基因被改造过的果蝇，为的是让它能经受比正常果蝇为时更长的睡眠剥夺，却不引起睡眠代偿现象。当研究人员将相同的突变果蝇保持清醒状态长达9或12小时时，这些果蝇的淀粉酶水平就会增高。通常情况下，这么长的时间足以使正常果蝇出现睡眠代偿现象。

肖博士说：“这一发现证明我们过去所发现的淀粉酶活性水平增高不仅仅是由不寐所导致。”

相对于控制正常睡眠的淀粉酶水平来讲，人类28小时不眠不休也会引起淀粉酶水平的增高。

肖博士之前的研究表明咖啡因和脱氧麻黄碱可用于维持果蝇的清醒状态。咖啡因能引起睡眠代偿现象，导致精疲力竭的果蝇补睡时间延长，而脱氧麻黄碱则不会出现这样的问题。研究人员监控到果蝇的淀粉酶水平随药物的不同而发生改变，他们发现咖啡因能够增强淀粉酶活性，而脱氧麻黄碱则不会。

给果蝇服用立效除草剂未见有淀粉酶水平的升高，这表明淀粉酶活性改变与压力无关。缺少淀粉酶基因的果蝇其睡眠苏醒的周期正常，这表明虽然淀粉酶与睡眠冲动关系密切，但却与睡眠冲动调节无太大关联。

“我们欣喜地看到淀粉酶水平与睡眠代偿现象关系密切，但为使诊断测试更准确，我们将可能会着手寻找更多的生物标记”肖博士说道，“如此一来，我们将沿用我们所开发出来的程序步骤。这些程序步骤原先被用于寻找随睡眠代偿程度而改变的其他物质。”

史蒂芬•L•敦特礼是个经常与肖博士合作研究的医学博士，他是华盛顿大学睡眠医学中心的主任及神经学副教授。

“尽管睡眠代偿会诱发严重疾病以及产生严重的公共健康后果，诊断睡眠代偿依靠地是不可靠的主观等级评定方法和花费不菲的，而且通常是不太切合实际的实验室睡眠测试法。”敦特礼说，“简单、易得的人类睡眠代偿生物标记将会刮起一场革命旋风，不论是在研究失眠的原因和后果方面，还是为临床医生提供实用的新器械以帮助诊断睡眠障碍病人和评估疗效方面。”

在肖博士看来，嗜睡生物标记同样对于动物睡眠研究有所帮助。

“象虎鲸这样的鲸鱼，可以很长时间都不睡觉。我们很想知道为什么以及虎鲸是否有睡眠代偿现象”肖博士说，“目前为止，研究失眠对大脑影响的主要方法是通过电极的联导，而对虎鲸也用同样方法的话，未免稍显笨拙。希望我们的生物标记研究成果将使得对于这些现象的研究变得更简单易行。”

Public release date: 11-Dec-2006
Contact: Michael C. Purdy
purdym@wustl.edu
314-286-0122
Washington University School of Medicine

First biomarker for human sleepiness identified in fruit flies

Scientists have identified the first biochemical marker linked to sleep loss, an enzyme in saliva known as amylase, which increases in activity when sleep deprivation is prolonged.

Researchers hope to make amylase the first of a panel of biomarkers that will aid diagnosis and treatment of sleep disorders and may one day help assess the risk of falling asleep at the wheel of a car or in other dangerous contexts.

"As we prepare for the holiday season and long drives to distant relative's houses, I hope this finding will get people thinking about the dangers and costs of sleep deprivation," says lead author Paul J. Shaw, Ph.D., assistant professor of neurobiology. "If you're feeling sleepy on your way over the river and through the woods to grandmother's house, it's much better to pull over and find a place where you can sleep for a while than to continue on and risk a serious accident."

The study appears this week in the online edition of Proceedings of the National Academy of Sciences. Shaw's lab was the first to show that fruit flies enter a state of inactivity comparable to sleep. They demonstrated that the flies have periods of inactivity where greater stimulation is required to rouse them. Like humans, flies deprived of sleep one day will try to make up for the lost time by sleeping more the next day, a phenomenon referred to as increased sleep drive or sleep debt.

To identify a marker for sleep debt, Shaw decided to look in saliva. Easily accessible, saliva contains many of the substances found in blood and urine, making it an increasingly popular target for diagnostics. Saliva was also an attractive target for Shaw's lab because the brain areas that regulate sleep drive are known to send signals to the brain areas that regulate salivation.

To start his search, Shaw subjected the flies to different kinds of sleep deprivation and used microarrays to look for changes in activity in many different genes. Amylase levels consistently changed after sleep loss. Amylases are a family of enzymes found in the saliva that break down starch.

To verify amylase's connection to sleep loss, Shaw's lab monitored its activity level after sleep deprivation in different fruit fly lines genetically altered to modify their sleep drive.

In one key test, amylase did not increase in a fly modified to endure sleep deprivation longer than normal flies without incurring sleep debt. When scientists kept the same mutant flies awake for extended nine or 12 hour stretches that normally cause them to incur sleep debt, their amylase levels increased.

"This helped prove that the increases in amylase activity level we were seeing weren't just triggered by wakefulness," Shaw says.

Humans kept awake for 28 hours also had increased amylase levels versus controls allowed to sleep normally.

Shaw's lab previously showed that they can use caffeine and methamphetamine to keep flies awake. Caffeine inflicts sleep debt, causing flies to sleep for extended periods when it wears off, while methamphetamine does not. When they monitored fly amylase levels in response to these drugs, they found caffeine drove amylase activity up while methamphetamine did not.

Flies dosed with the herbicide paraquat did not have increased amylase levels, suggesting changes in amylase activity were not related to stress. Flies lacking the gene for amylase had normal sleep and waking cycles, showing that while amylase is tightly linked to sleep drive, it is not actively involved in its regulation.

"We're very pleased with how tightly amylase levels correlate with sleep debt, but for a good diagnostic test we're likely going to need more than one biomarker," Shaw says. "So we're going to continue to use the processes that we've developed to look for other substances that change in connection with the level of sleep debt."

Stephen L. Duntley, M.D., associate professor of neurology and director of the Washington University Sleep Medicine Center, is a frequent research collaborator with Shaw.

"Despite the tremendous medical and public health consequences of sleep debt, its measurement in humans relies upon unreliable subjective rating scales and expensive, often impractical sleep laboratory testing," Duntley says. "Simple, easily accessible biomarkers for sleep debt in humans would revolutionize our ability to conduct research on the causes and consequences of sleep deprivation and provide clinicians with valuable new tools for diagnosing and assessing treatment efficacy in patients with sleep disorders."

According to Shaw, sleepiness biomarkers will also prove useful to studies of sleep in animals.

"Cetaceans like killer whales, for example, are known to go for extended periods of time without sleep, and we'd like to know more about how that works and whether they incur sleep debt," Shaw says. "Until now, the main way to study sleep deprivation's effects on the brain has been to attach electrodes, which can be a bit awkward when your target is a killer whale. Hopefully the markers we develop will make these kinds of phenomena much easier to study."

http://www.eurekalert.org/pub_releases/2006-12/wuso-fbf121106.php

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