【作者】 季华；

【导师】 凌树才；

【作者基本信息】 浙江大学 ， 神经生物学， 2006， 硕士

【摘要】 【背景和目的】 P物质（SP）广泛分布于中枢神经系统和外周神经系统中,是由中枢和外周初级传入神经末梢释放的一种神经递质。哺乳动物的SP由前速激肽原（Preprotachykinin,PPT）A基因编码。PPT—A基因同样编码其他速激肽如neurokinin A,neuropeptide B等。速激肽受体也被分为速激肽NK₁受体,速激肽NK₂受体,速激肽NK₃受体。速激肽NK₁受体与SP有着最强亲和力,即P物质受体（SPR）。SP与SPR特异结合后使受体内化,通过激活磷脂酶C（PLC）并水解磷酸肌醇（PIP₂） 生成三磷酸肌醇（IP₃）和二酯酰甘油（DAG）两种第二信使发挥生物学效应。 已有的实验发现在小鼠胚胎时期脑内已有SP表达,但其与中枢神经系统发育的关系仍不清楚,而且关于SPR在小鼠胚胎时期脑发育过程中的表达也未见报道,因此调查SP与SPR在小鼠胚胎时期脑的分布情况及其相互间关系,可以为进一步研究SP在中枢神经系统发育阶段所起的作用提供形态学依据。本研究采用免疫组织化学染色方法结合western blotting半定量方法,使用特异性SP和SPR抗体,观察SP和SPR在小鼠胚胎发育时期脑内表达的变化,推测胚胎发育阶段SP在中枢神经系统中所起的作用。 【材料与方法】 1.实验动物及组织切片制备 分别取E11（胚胎11天）,E13,E15,E17,E19和P0（新生）ICR品系小鼠脑经冠状切面进行连续冰冻切片。 2.酶标免疫组织化学 将冰冻切片分别滴加兔抗SP多克隆抗体和抗SPR多克隆抗体,生物素标记的羊抗兔IgG,加入抗生物素蛋白—辣根过氧化物酶复合物,二甲基联苯胺呈色观察。更多还原

【Abstract】 Substance P expression is confined to central nervous system （CNS） and the peripheral nervous system （PNS）. Today it is fully recognized that substance P is released from both the central and peripheral endings of primary afferent neurons and functions as a neurotransmitter.Mammalian substance P derives from the preprotachykinin- A （PPT-A） gene, The PPT-A gene also encodes for other tachykinins, including neurokinin A, neurokinin B. The three tachykinins have their receptors such as NK₁ receptor, NK₂ receptor and NK₃ receptor. NK₁ receptor has the highest affinity with substance P, also be called as substance P receptor. When substance P binding NK₁ receptor, NK₁ receptor internalization, activate phosphatidolipaseC and hydrolyze phosphinositides, generate two second message IP3 and DAG to induce biological effect.Finished experimentation show substance P is already expression in embryo brain prenatal, but also have no idea with its role in the development of central nerves system. There also have no report about the expression of substance P receptor in the development of central nerves system. So it is very necessary to investigate the expression and localization of substance P and substance P receptor, can provide the morphology proof in research about substance P’function in the development of central nerves system.Materials and Methods1. Animals and histological section preparationAnimals: El l（Embryonic mice at 1 Id）, E13, E15, E17, E19 and PO （new born） Sections: All of mice brians were cut in cryostat coronal sections.2. ImmunohistochemistryFor immunocytochemistry, sections were incubated with polyclonal rabbit-anti-SP or SPR antibody, biotinylated goat-anti-rabbit secondary antibody and ABC complex, respectively.3. Western blottingEll, E13, E15, E17, E19 and PO mice brians were removed, respectively. After homogenation, they were taken to PAGE, and transferred to nitrocellulose membrane. Membranes were incubated with polyclonal anti-SP or SPR antibody, biotinylated goat-anti-rabbit secondary antibody and ABC complex, respectively.Resultsl.SP and SPR expression in the development of embryo mice brainOn Ell, SP-immunoreactive neurons were detected in the marginal zone of caudal epithalamus neuronepithelium, while positive-fibers were not seen. Some SPR-immunoreactive cells were seen in the ventral hypothalamus, pons and cingulate cortex.On El 3, SP-immunoreactive neurons were found in the striatum, globus pallidus and septal area. SPR-immunoreactive neurons were seen in the medullary raphe, olfactory brain, hypothalamus. Positive neural fibers were seen in the ventral tegmental nucleus （VTG）.On El5, the dense stained SP-immunoreactive neurons existed at olfactory brain, septal area, piriform cortex and diagonal band, and also appears at striatum, globus pallidus and nucleus accumbens. The distribution of SPR-immunoreactive neurons was found to be similar to the distribution of SP. SPR-immunoreactive neurons were found in the olfactory brain, septal area, piriform cortex, hippocampus, amygdala and striatum,especially at medulla oblongata. The neurons were faintly stained.On El7, SP-immunoreactive cells beam was found from entopeduncular nucleus and bed nucleus of the stria terminalisto the striatum through the internal capsule and globus pallidus. Dense stained largeround or fusiform cells with distinct fibre projections were observed. SP-immunoreactive neurons also can be found in the hippocampus and gyrus dentatus, and SP-immunoreactive neurons terminus were found in the medial amygdaloid necleus. SPR-immunoreactive cells were also found in the same region such as striatum and globus pallidus. SPR-immunoreactive fibers were found in the amygdale, ventral thalamus, lateral hypothalamus and the raphe region of medulla oblongata such as nucleus raphes dorsalis, locus caeruleus, central gray and superior raphe nucleus.On El9, lots of SP-immunoreactive termini were observed in the entopeduncular nucleus, Forel region, thalamus, anterior colliculi, periaqueductal gray, reticular formation, inferior olivary nucleus, ventral tegmental nucleus and locus caeruleus. SP-immunoreactive cells were found in the epithalamus, ganglion habenulae, entopeduncular nucleus, central gray, dorsal tegmental nucleus and interpeduncular nucleus.On PO, SP-immunoreactive cells were found in the hypothalamus, entopeduncular nucleus, striatum, amygdale, reticular nucleus of raphe in medulla oblongata, nucleus raphes dorsalis and zona incerta, cells were densely stained and accompanied with lots of terminus. SPR-immunoreactive fibres appeared in the amygdala, striatum, nucleus accumbens, central gray and ventral tegmental nucleus. 2. Western blottingWe found SP began expressed at Ell and gradually increasing. Then in El5 and El7 increased significantly until birth, SPR began expressed at Ell and have stable expression until birth.Conclusions1. Substance p began expressed at neostriatum in the early embryo development, while substance P receptor expressed at raphe of medulla oblongata;2. In the midanaphase of embryo development SP and SPR were found in the same place such as striatum and raphe of medulla oblongata, but the shape of positive-cells was different.3. On early stage of developmental 1) SP expression can be found and gradually increased, at El5 and El7 the expression increased significantly until birth. The expression of SPR also can be found on Ell and maintained stable expression, before birth the expression increased.4. It suggested that SP may play a role in central nervous system genesis and mature mechanism.更多还原