抗K物质单克隆抗体的制备

速激肽族肽,包括K物质、P物质、神经素B等都具有神经递质的功能,它们越来越引起人们的关注。免疫学方法诸如:放射免疫测定法、免疫组织化学法等对神经肽的分布、定位和生物学功能等方面的研究提供了极大的方便。我们以K物质-甲状腺球蛋白偶联物免疫动物——Wistar大鼠和Balb/c小鼠。Wistar大鼠对K物质-甲状腺球蛋白的免疫应答能力显著高于Balb/c小鼠。通过Wistar大鼠脾细胞与小鼠骨髓瘤细胞进行异种细胞融合,获得了一株稳定地分泌抗K物质的单克隆抗体的细胞系(SE-1)。用RIA检测该单克隆抗体的特异性,除与P物质有一些交叉外,与其余我们所用的脑肽均不反应。     

甲硫氨酸脑啡肽类似物对大鼠增重作用的观察

根据鲍威斯(Bowers)等报道,在大鼠垂体离体培养液中,如加入甲硫氨酸脑啡肽类似物(酪·D-色·甘·苯丙·甲硫酰胺Tyr·D-Trp·Gly·Phe·MetNH2)能促进垂体细胞分泌生长激素。此外,还证实它对促黄体生长激素(LH)、促甲状腺激素(TSH)、催乳激素(PRL)和促肾上腺皮质激素(AC/H)无交叉反应。     

克伦特罗单克隆抗体的纯化及其生物学特性的研究

目的：将自制克伦特罗（CL）单克隆抗体纯化并研究其生物学特性,进行性质鉴定并建立检测标准曲线。方法：用ELISA法测定克伦特罗单克隆抗体的亲和常数和抗体活性,ELJSA测定单克隆抗体与BSA的交叉反应及与几种结构和功能类似物的交叉反应,然后采用间接竞争ELISA方法建立检测标准曲线。将制备的含克伦特罗单克隆抗体的小鼠腹水用盐析法和免疫亲和柱层析法进行抗体纯化。结果：经ELISA法测定,单克隆抗体亲和常数为2．90×10mmol／L,抗体效价最高达10^6。单克隆抗体对BSA无反应,对几种结构和功能类似物的交叉反应率均小于0．005％。建立的标准曲线R2=0．9812,最低检测限为1．0ng／ml。结论：建立了间接竞争ELISA检测cL的标准曲线。自制的克伦特罗单克隆抗体亲和力好,特异性高。为以后实际样品的检测及制备CL免疫检测试纸条和试剂盒奠定了基础。     

脑啡肽的放射免疫测定

1975年发现亮氨酸脑啡肽(LEK)和甲硫氨酸脑啡肽(MEK)。其测定以放射免疫(RIA)最佳。作者提议并合成了多聚赖氨酸琥珀酰脑啡肽免疫原,EK结合率81～90%,每2.3或3.2个赖氨酸残基连上一个EK。免疫3或6个月时获得抗EK血清(ALS或AMS),工作稀度可达1:5000,K_(eff)10~9升/克分子。试用改进的氯胺T标记法及DEAE-Sephadex A-25柱层析制备高比放射性~(125)I-EK免疫活性良好。双抗体法分离抗体结合的和游离的~(125)I-EK。竞争抑制曲线:灵敏度LEK～11微微克,MEK～130微微克;精密度1.8%及1.4%。β-内啡肽,八种肽类激素、吗啡、纳洛酮无明显干扰;交叉反应MEK对ALS为3.3%、LEK对AMS为<1%。脑组织用0.0N盐酸抽提,中和后,直接作RIA。本方法已通过多种方法学考验。正常大鼠脑中EK浓度以下丘脑为最高,尾核次之。本法是灵敏、可靠和特异的。     

P2X7受体短肽单克隆抗体的制备及鉴定

旨在制备与鉴定鼠抗P2X7受体的蛋白单克隆抗体.以人P2X7受体的胞外段制备短肽作为抗原,皮下注射免疫Balb/c小鼠.分离小鼠脾脏B淋巴细胞与骨髓瘤细胞融合并培养,挑选阳性杂交瘤细胞,扩大培养,制备和鉴定P2X7其生物学效应.结果显示,获得1株稳定分泌抗人P2X7受体的单克隆抗体的杂交瘤细胞株,所分泌的单抗类型重链为IgG1,轻链为κ;该株杂交瘤细胞腹水效价为1∶6.4×104;传30代及液氮中保存6个月,抗体效价稳定;Western blotting检测证明该单抗与人细胞表面的P2X7受体蛋白特异地结合.所制备的抗人P2X7受体的单克隆抗体具有高度的特异性及稳定性,为针对P2X7受体为靶点的抗体药物的开发应用、疾病的辅助诊断奠定了基础.     

I型马立克氏病毒38kD磷蛋白与Ⅱ和Ⅲ型病毒间的交叉免疫反应

由致病性Ⅰ型马立克氏病病毒(MDV)特异性的单克隆抗体H_(19)制备亲和层析柱,从感染重组杆状病毒BP38Ⅱ的昆虫细胞系Sf9细胞中提纯MDV的pp38基因表达产物,以此免疫小鼠制备抗血清,测定其与不同型MDV毒株感染的鸡胚成纤维细胞免疫反应性,同时也用不同型MDV毒株(HVT,Z4)制备的抗血清和自然感染发病鸡血清分别与感染重组病毒的Sf9细胞进行免疫交叉反应.试验结果表明,完整的Ⅰ型MDV来源的pp38基因产物与所试Ⅱ型Z4株和Ⅲ型HVT Fc126毒株在抗原性上有显著交叉反应.这一结果表明,在Ⅱ型和Ⅲ型MDV毒株中可能存在着pp38的类似物.     

白xuan胃肠道内分泌细胞的免疫组织化学定位

为了解白xuan的消化生理过程,利用ABC免疫组织化学方法,对白xuan胃肠道5种内分泌细胞：5-羟色胺(5-HT)、脑啡肽、P物质、胃动素和抑胃素的分布进行了研究。结果表明：5-HT细胞主要分布于十二指肠及其以下部位,尤其是盲肠和直肠;而腺胃、肌胃和幽门中均未发现该类细胞。脑啡肽和P物质细胞数量明显较5-HT少,它们稀疏分布于十二指肠及其以下部位,无显著的分布积聚区。抑胃素和胃动素在整个胃肠道中均无分布。分析表明白xuan胃肠道5种内分泌细胞的分布与其他鸟类相似,但与其他脊椎动物明显不同。     

白yuan胃肠道内分泌细胞的免疫组织化学定位

为了解白yuan的消化生理过程,利用ABC免疫组织化学方法,对白yuan胃肠道5种内分泌细胞：5-羟色胺(5-HT)、脑啡肽、P物质、胃动素和抑胃素的分布进行了研究。结果表明：5-HT细胞主要分布于十二指肠及其以下部位,尤其是盲肠和直肠;而腺胃、肌胃和幽门中均未发现该类细胞。脑啡肽和P物质细胞数量明显较5-HT少,它们稀疏分布于十二指肠及其以下部位,无显著的分布积聚区。抑胃素和胃动素在整个胃肠道中均无分布。分析表明白yuan胃肠道5种内分泌细胞的分布与其他鸟类相似,但与其他脊椎动物明显不同。     

单克隆抗体亲和层析技术

亲和层析技术中用常规抗血清作配体时,由于抗血清成份复杂、亲和力多样化,特异性强的制剂又不易得,因而此层析技术的应用受到限制.单克隆抗体(McAb)技术的建立,为亲和层析技术的应用带来了突破.因为McAb作为亲和层析技术中的配体有以下几个优点:(1)可以利用粗制抗原制备分泌单一抗体的杂交瘤细胞株,在体外或体内大量生产McAb;(2)由于McAb是针对单一抗原决定簇的,该决定簇是某一抗原物质所特有或是多种抗原物质所共有时,可没有任何交叉反应或有完全的交叉反应.     

白鹮胃肠道内分泌细胞的免疫组织化学定位

为了解白的消化生理过程 ,利用ABC免疫组织化学方法 ,对白胃肠道 5种内分泌细胞 :5 -羟色胺 (5 HT)、脑啡肽、P物质、胃动素和抑胃素的分布进行了研究。结果表明 :5 HT细胞主要分布于十二指肠及其以下部位 ,尤其是盲肠和直肠 ;而腺胃、肌胃和幽门中均未发现该类细胞。脑啡肽和P物质细胞数量明显较 5 HT少 ,它们稀疏分布于十二指肠及其以下部位 ,无显著的分布积聚区。抑胃素和胃动素在整个胃肠道中均无分布。分析表明白胃肠道 5种内分泌细胞的分布与其他鸟类相似 ,但与其他脊椎动物明显不同。     

尾核P物质对胃运动的抑制效应系通过黑质,迷走背核经迷走神经所介导

本工作观察损毁下丘脑外侧区,黑质,迷走背核及其传出神经对尾核微量注射Ｐ物质引起的胃肌电快波和胃运动抑制效应的影响。实验结果：该抑制效应不依赖于下丘脑外侧区的完整但可被损毁黑质,迷走背核或迷走上所消除。用利血平耗竭交感神经递质则不影响该效应。这些结果表明：尾核ＳＰ的抑胃效应系通过黑质、迷走背核经迷走神经所传出。     

黄雀发声核团与部分听觉中枢内P物质的分布和性双态比较

用免疫组织化学方法研究P物质在雌雄黄雀发声控制核团和听觉中枢内的分布,结合计算机图像分析仪检测SP免疫阳性细胞和末梢的灰度值,并作雌雄比较。结果如下:1.在发声学习中枢嗅叶X区有大量的SP阳性神经末梢和一些神经细胞。2.在发声控制核团前脑高级发声中枢(HVc)、古纹状体栎核、发声学习中枢新纹状体巨细胞核和丘脑背内侧核外侧部内有许多的SP免疫阳性细胞。3.在发声控制中枢中脑背内侧核和延髓舌下神经核气管呜管部、听觉中枢丘脑卵圆核的壳区、中脑背外侧核壳区及中脑丘间核等有密集的SP免疫阳性神经末梢和纤维分布;雄性发声中枢内SP的分布比雌性丰富,两者有显著的差异。结果表明:SP的分布在雌雄发声中枢之间存在显著的性双态;SP广泛分布于黄雀发声控制核团和部分听觉中枢内,提示SP可能在发声控制及听觉中枢内具有重要的生理功能。     

Neuronal location of the bombesin-like immunoreactivity in the central nervous system of the rat

The immunohistochemical distribution of bombesin-like immunoreactivity in the central nervous system of the rat was revealed using a rabbit antibody against [Glu⁷]bombesin(6–14). In radioimmunoassay, the antibody had minimal cross reactivity with substance P thereby enhancing the significance of histochemical controls proving that the immunoreactivity detected was related to bombesin but not to substance P. Bombesin-immunoreactive neurons were detected in several brain structures including the hypothalamus, interpeduncular nucleus, central grey, dorsolateral tegmental nucleus, dorsal parabrachial nucleus, nucleus of the solitary tract and trigeminal complex. In the spinal cord, intense immunoreactivity was found in the superficial layers of the posterior horn. Since in this area the reaction diminished after rhizotomy the location of the peptide in afferent neurons was considered. In the anterior horn the bombesin-like immunoreactivity located in nerve terminal-like structures was unchanged after rhizotomy suggesting that the cell bodies were located in CNS.     

Monoclonal Antibodies to Substance P: Production, Characterization of Their Fine Specificities, and Use in Immunocytochemistry

Five hybrid clones secreting antibodies to the neuropeptide substance P have been obtained by somatic cell fusion of mouse myeloma cells with splenocytes from immunized mice of the Biozzi strain. To perform rapid and sensitive screening tests as well as to study the fine specificities of each monoclonal antibody, we developed a new enzyme immunoassay of substance P using acetylcholinesterase as label. All five monoclonal antibodies were directed to the C-terminal pentapeptide of substance P, especially to the Phe7 residue. They cross-reacted with neurokinin A and to some extent with neurokinin B but not with other nontachykinin mammalian peptides. One monoclonal antibody (SP 14) was used for immunocytochemical experiments in the rat spinal cord and spinal ganglion, both at the light and electron microscopic levels. A strong specific neurokinin-like immunoreactivity was observed in cell bodies, nerve fibers, and terminals, with a very low background staining. Finally, the affinities of several analogues of substance P for SP 14 monoclonal antibody were shown to be correlated with their biological activities, as measured by their hypotensive effects in vivo. These findings suggested a strong structural resemblance between the combining site of the antibody and that of the physiological substance P receptor.     

Effect of substance P on neonatally axotomized noradrenaline neurons in rat brain

Transection (axotomy) of the dorsal tegmental noradrenaline bundle in the neonatal stage leads to a permanent degeneration of noradrenaline nerve terminal projections distal to the lesion (e.g. in the neocortex), while projections proximal to the lesion increase their nerve terminal density (e.g. in the cerebellum). These structural changes are reflected by marked reductions and elevations respectively of the endogenous noradrenaline levels, [3H]-noradrenaline uptake in vitro and nerve density as demonstrated by fluorescence histochemistry. Intracisternal administration of substance P after the transection did not alter these noradrenaline parameters in the neocortex, whereas dose-dependent and significant increases were found in the cerebellum and pons-medulla. The results indicate that substance P may have a growth-stimulatory effect on damaged locus coeruleus noradrenaline neurons in the CNS during ontogeny.     

黄雀发声核团与部分听觉中枢内P物质的分布和性双态比较

用免疫组织化学方法研究P物质在雌雄黄雀发声控制核团和听觉中枢内的分布,结合计算机图像分析仪检测SP免疫阳性细胞和末梢的灰度值,并作雌雄比较。结果如下：1．在发声学习中枢嗅叶X区有大量的SP阳性神经末梢和一些神经细胞。2．在发声控制核团前脑高级发声中枢(HVc)、古纹状体栎核、发声学习中枢新纹状体巨细胞核和丘脑背内侧核外侧部内有许多的SP免疫阳性细胞。3．在发声控制中枢中脑背内侧核和延髓舌下神经核气管鸣管部、听觉中枢丘脑卵圆核的壳区、中脑背外侧核壳区及中脑丘间核等有密集的SP免疫阳性神经末梢和纤维分布;雄性发声中枢内SP的分布比雌性丰富,两者有显著的差异。结果表明：SP的分布在雌雄发声中枢之间存在显著的性双态;SP广泛分布于黄雀发声控制核团和部分听觉中枢内,提示SP可能在发声控制及听觉中枢内具有重要的生理功能。     

Effects of illumination and enucleation on substance-P-immunoreactive structures in subcortical visual centers of golden hamster and Wistar rat

The undecapeptide substance P is found in different entities of the visual system that control eye movement and synchronize endogenous rhythms with the light cycle (i.e., superior colliculus, suprachiasmatic nucleus, intergeniculate leaflet). Immunocytochemical methods were used to compare the reactivity to substance P in the brain of five groups of golden hamsters and two groups of Wistar rats: (1) untreated hamsters kept under 14L:10D and sacrificed at noon; (2) identically maintained animals sacrificed at midnight; (3) enucleated animals kept under control conditions; (4) hamsters kept under constant darkness; (5) hamsters kept under the same conditions as the controls, but intraventricularly injected with colchicine. The results obtained in golden hamsters of groups (1) and (3) were compared with findings in Wistar rats treated accordingly [groups (6) and (7)]. Substance P-immunoreactive perikarya were found in the suprachiasmatic nucleus and superior colliculus of hamsters and Wistar rats. Substance P-immunoreactive nerve fibers were abundant in the hypothalamic area ventral to the paraventricular nucleus, in the intergeniculate leaflet, in some thalamic nuclei, and in the superior colliculus. Immunoreactivity to substance P in the suprachiasmatic nucleus and intergeniculate leaflet did not vary among the experimental groups. However, a conspicuous decrease in reactivity to substance P was observed in the superficial layers of the superior colliculus of enucleated hamsters and rats, compared with all other groups. These results indicate that substance P immunoreactivity in the superior colliculus, but not that in the suprachiasmatic nucleus or intergeniculate leaflet, depends on the integrity of the retinal projection.     

Peripheral axotomy of the rat mandibular trigeminal nerve leads to an increase in VIP and decrease of other primary afferent neuropeptides in the spinal trigeminal nucleus

In the vasoactive intestinal polypeptide (VIP)-rich lumbosacral spinal cord, VIP increases at the expense of other neuropeptides after primary sensory nerve axotomy. This study was undertaken to ascertain whether similar changes occur in peripherally axotomised cranial sensory nerves. VIP immunoreactivity increased in the terminal region of the mandibular nerve in the trigeminal nucleus caudalis following unilateral section of the sensory root of the mandibular trigeminal nerve at the foramen orale. Other primary afferent neuropeptides (substance P, cholecystokinin and somatostatin) were depleted and fluoride-resistant acid phosphatase activity was abolished in the same circumscribed areas of the nucleus caudalis. The rise in VIP and depletion of other markers began 4 days postoperatively and was maximal by 10 days, these levels remaining unchanged up to 1 year postoperatively. VIP-immunoreactive cell bodies were absent from trigeminal ganglia from the unoperated side but small and medium cells stained intensely in the ganglia of the operated side after axotomy. These observations indicate that increase of VIP in sensory nerve terminals is a general phenomenon occurring in both cranial and spinal sensory terminal areas. The intense VIP immunoreactivity in axotomised trigeminal ganglia suggests that the increased levels of VIP in the nucleus caudalis are of peripheral origin, indicating a change in expression of neuropeptides within primary afferent neurons following peripheral axotomy.     

Immunohistochemical localization of substance P in the pineal gland of the domestic pig

An immunohistochemical study of the pig pineal gland was carried out using polyclonal rabbit antiserum raised against substance P (SP). The pineal glands were taken from the newborn, 21-day- and 7-month-old female pigs. Immunoreactive nerve fibers were observed in the pineal gland as well as in the posterior commissure and habenular areas. The bundles of SP-immunoreactive fibers were also seen in the subependymal layer of the pineal tissue. The single SP-immunoreactive nerve fibers and few small bundles of nerve fibers were located with equal density throughout the pineal gland, in the connective tissue septa and in the parenchyma. SP-immunoreactive cell bodies were observed in the medial habenular nucleus. The obtained results point to this nucleus as one of the central sources of SP innervation in the pig pineal gland. The study did not show any differences in the distribution and the density of SP-immunoreactive nerve fibers between newborn, 21-day- and 7 month-old pigs.     

Lack of functional and morphological susceptibility of the greater superficial petrosal nerve to developmental dietary sodium restriction

Restriction of dietary sodium during gestation has major effects on taste function and anatomy in the offspring. The chorda tympani nerve of offspring that are maintained on sodium-reduced chow throughout life (NaDep) has reduced neurophysiological responses to sodium and altered morphology of its terminal field in the nucleus of the solitary tract. There are many anatomical and physiological similarities between the chorda tympani nerve that innervates taste buds on the anterior tongue and the greater superficial petrosal nerve (GSP) that innervates taste buds on the palate. To determine if the GSP is similarly susceptible to the effects of dietary sodium restriction, the present study examined neurophysiological responses and the terminal field of the GSP in NaDep and control rats. Neurophysiological responses of the GSP to a variety of sodium and non-sodium stimuli did not differ between NaDep and control rats. Furthermore, the volume and shape of the GSP terminal field in the nucleus of the solitary tract did not differ between the groups. Therefore, despite the high degree of functional and anatomical correspondence between the chorda tympani nerve and the GSP, the GSP does not appear to be susceptible to the effects of lifelong dietary sodium restriction.