【作者】 狄慧；

【导师】 胡满；

【作者基本信息】 河北农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 目的:通过对FOS蛋白的检测,来确定由光照刺激引起的c-fos基因在母鸡间脑内的表达,由此来探究光照对鸡生殖性能——即母鸡产蛋量的影响,为进一步了解光照对鸡产蛋的影响机理打下基础。方法:首先,对母鸡进行光照刺激,将母鸡右眼遮光7天后接受201x的光照刺激1.5h,暗适应1.5h。经遮光和光照刺激后,用两种方法检测FOS蛋白:1)免疫组化方法,灌流固定,取脑制作石蜡切片。通过免疫组化方法检测c-fos基因在间脑的表达,确定FOS蛋白出现最多的神经核团:2)蛋白印迹法,对母鸡进行光照刺激后,直接断头,将鸡头在冰上迅速冷却,然后在低温环境下剥离出鸡脑。将剥离出来的鸡大脑用锡纸包好,迅速放于-80℃冰箱中冷冻。将冷冻后的鸡脑捣碎,10000转/秒离心,提取上清液,测出提取液中的蛋白含量,然后用蛋白印迹法来验证提取液中是否含有FOS蛋白,以此来再次验证FOS蛋白在鸡脑中的存在。结果:1)免疫组化结果显示,经DAB显色后Fos样免疫反应阳性物质呈棕黄色颗粒,细胞核与胞浆都有Fos蛋白的表达,但主要位于细胞核内,间脑内有大量Fos样免疫反应阳性神经元,主要见于左侧,分布于圆核(ROT)、室旁核(PVN)、前背外侧核(DLA)、外侧膝状核腹侧部(GLv)和缰核(HB)等核团内,其中ROT、和PVN内免疫反应阳性神经元最多。2)蛋白印迹法(western blot)检测的结果显示,49kD到90kD间有明显特异条带,即FOS蛋白条带,证明在母鸡脑蛋白提取液中有FOS蛋白存在。结论:在这些核团中,Fos样免疫反应阳性神经元多,而且多呈强阳性或阳性,这表明这些核团的神经元在光信息光信息传递中起重要作用。而这些神经核团与鸡生理和生产机能调节又密切相关,以此来推论出,光照通过这些核团接受光刺激从而影响产蛋量的。更多还原

【Abstract】 Objective: To investigate the pathway of optical information in diencephalon so as to do some basic work for illuminating the mechanism on the effect of light on egg-laying.Methords: After wearing a blinder to keep dark for 7 days, the right eyes received a light stimulation of 20Lux for 1.5h and kept in dark again for 1.5h. Following perfusing with fixative, their brains were removed and were made into continuous paraffin sections. Using immunohistochemical method, expression of c-fos gene in diencephalon was examined. The second method is western blotting. After the stimulation of light, the heads of hens were cut down and the brains were removed and rapidly chilled in the -80℃fridge. After breaking up the brains, their protein were extracted. Whether the FOS protein was present in the extract was detected by western blotting method.Results: 1) The result of immunohistochemistry: The result showed that a number of Fos-like immunoreactive positive neurons were seen in diencephalons. They were mainly present in left side, distributing in nucleus rotundus (ROT), paraventricular nucleus (PVN), Nucleus dorsolateralis anterior (DLA), Nucleus geniculatus lateralis, pars ventralis (GLv)and habenular nucleus (HB) etc. especially being numerous in ROT and PVN. 2) The result of western blotting showed that obviously there is specific a band between the bands of 49kD to 90 kD. The band is correspond to the FOS protein band. It proves the FOS protein being in the extract of brains.Conclusion: It indicates that the neurons in these nuclei play important roles in the conveying course of optical information.更多还原