【作者】 陈茂林；

【导师】 杨旭；

【作者基本信息】 华中师范大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 作为室内空气主要污染物之一，甲醛的危害是多方面的，其中之一就是气道刺激作用或称气道神经源性炎症，可以导致不良建筑物综合征（sick building syndrome,SBS）、多重化学敏感症（multiple chemical sensitivitv,MCS）等。由于室内装修的日益流行，甲醛的危害性越来越突出，已受到人们的普遍重视。甲醛刺激作用的触发性生物靶分子到底是什么?目前还不十分清楚。因此气道刺激作用的分子机理还有待阐明，不过有一些假设倒是引起了我们的注意，如Nielsen假设就是一个比较重要的、正日益受到人们重视的理论假设，它从分子机理上解释了甲醛等空气污染物是如何通过辣椒素受体的介导引起气道刺激作用和气道神经源性炎症的。但是它还只是一个假设，还需要我们做一些工作来证明它是否正确。 在Nielsen假设中有一个很重要的环节，就是甲醛作为配子体对辣椒素受体的激活，但在Dr.Nielsen提出假设时辣椒素受体还没有克隆出来，对此假设的研究自然不够方便。1997年辣椒素受体的克隆给Nielsen假设的研究带来生机，而美国加州大学旧金山分院细胞与分子药理学研究所D.J.Julius博士（克隆辣椒素受体发明人）赠送的VR1（Ⅰ型辣椒素受体）-cDNA使我们的研究工作得以开展。 这项实验中，我们首先将大白鼠VR1-cDNA质粒转入大肠杆菌中进行扩增，然后提取质粒并酶切，然后在体外转录成VR1-mRNA，接着将VR1-mRNA注射到非洲爪蟾卵母细胞中表达为蛋白质受体，建立了实验模型细胞，最后用双微电极电压钳检测此模型细胞。 当我们用辣椒素（capsaicin）（10⁵M）刺激模型细胞时，细胞出现了电流变化，而加入capsazepine（10⁵M）（Ⅰ型辣椒素受体特异性抑制剂）和capsaicin(10^?M)（辣椒素）时，模型细胞没有反应（n=3），可以认为我们的模型细胞已经成功地建立起来了。然后我们用一系列浓度的甲醛刺激模型细胞，发现甲醛确实可以激活辣椒素受体，并且在甲醛浓度与辣椒素受体的反应大小之间还可能存在量效关系。更多还原

【Abstract】 As a main indoor air pollutant, formaldehyde is very harmful. One of the harmful effects is airway irritation or airway neurogenic inflammation which results in sick building syndrome (SBS), multiple chemical sensitivity (MCS) and so on. Since indoor air formaldehyde pollution is getting more serious than before people take more attention on it. But the molecular mechanism of formaldehyde on evoked airway irritation is not clear. However, there are few of the hypotheses about it, for example, Nielsen hypothesis, which explain how formal dehyde and other air contamination beget neurogenic inflammation. Our task at present is testifying if the hypothesis is right or not .Activating of the capsaicin receptor is a important tache in Nielsen hypothesis, it might be used for testifying. But at that time capsaicin receptor was not cloned, so the condition for studying the hypothesis was immature. In 1997 cloned capsaicin receptor brought spark to studying Nielsen hypothesis, and Dr. D.J. Julius of California university, who cloned the receptor, donates of vanilloid receptor 1 (VR1) cDNA to us so as to our studying work begin.At first, the VRl-cDNA plasmids were transformed into TG1 R. coli to enlarge VRl-cDNA; then the plasmids were extracted and cut off with enzymes; subsequently, VRl-cDNAs were transcribed into VRl-inRNA by T7 or SP6 polymerase in vitro, following VRl-mRNAs were injected into Xenopus laevis oocyte to express into VR1 receptor protein; at the endthe VRl+-oocytes were tested by double electode voltage clamp.When we stimulated the oocytes with capsaicin (10-7M) , the cells were in response .to the stimuli; and with capsazepine (KT M) (specific inhibitor of VR1)+capsaicin (10-7 M), the cells had no response (n=3). So the capsaicin receptor was confirmed to express in oocytes. After we stimulated the cells with a series concentration (10-4M and 10-3M)of formaldehyde (n=3), the formaldehyde was able to activate the capsaicin receptor.更多还原