麝香、麝香酮对大鼠视网膜神经细胞毒性的对比考察

为考察麝香、麝香酮对体外培养大鼠视网膜神经细胞的最大无毒浓度(TC0),比较两者的细胞毒性,分别将倍比稀释的麝香、麝香酮试液加入大鼠视网膜神经细胞中培养观察,用MTT法测得存活细胞OD值,经公式计算麝香与麝香酮对细胞的最大无毒浓度(TC0)及对50%细胞产生毒性的浓度(TC50)。结果表明,麝香、麝香酮对大鼠视网膜神经细胞的TC0分别为1.29、7 mg/mL;麝香对其的TC50为8.7 mg/mL。说明麝香对体外培养大鼠视网膜神经细胞的毒性高于麝香酮。     

利用气相色谱/质谱联用分析林麝麝香中麝香酮和甾类成分

采用气相色谱/质谱(GC/MS)联用的方法,对林麝麝香中的甾体成分进行分析,确定了林麝麝香样品含有多种甾体成分的结构,并分析了不同来源的林麝麝香的麝香酮及甾体成分.通过检索NIST05质谱库,进一步确定了麝香中含有16种甾体成分.利用外标法、标准曲线法同时测定了麝香样品中3种甾类成分(胆固醇、苯胆烷醇酮及麝香酮)的含量,麝香酮的定量分析显示所有样品麝香中麝香酮含量均较高(30.1～45.2 mg/g),但甾类成分含量波动较大.聚类分析显示,9个麝香样品聚为两支.利用GC/MS技术检测麝香成分的方法,可以提供麝香较为全面的甾类信息,可高效准确地对麝香进行质量分析.     

麝香中脂溶性成分的提取与麝香质量鉴别

采用薄层层析和气相色谱技术,比较了超声、冷浸和热回流3种提取方法及乙醇、乙醚、正己烷3种溶剂对麝香提取液中脂溶性成分及麝香酮含量的影响。结果显示用不同提取方法和溶剂提取的麝香脂溶性成分经薄层层析后均呈现6或7个斑点。而经气相色谱分析,其结果却有较大的差别,麝香乙醚提取液的色谱峰较乙醇和正己烷更丰富,超声提取的效果较冷缦和热回流好,色谱峰达到20个。麝香酮的定量分析显示冷浸法提取的麝香酮含量高于超声和热回流提取。通过薄层层析和气相色谱分析,对10个麝香样品的质量进行了鉴别。     

饲料营养对林麝麝香产量和品质的影响

上海地区引种林麝取得成功后,首次进行了饲料营养对麝香产量和品质影响的研究,试验麝饲养在上海市郊崇明岛东平林场,进行分期对比试验,分别用青绿饲料,青绿饲料加代蛋白饲料,青绿饲料加高蛋白饲料,配合饲料饲喂受试麝,考察饲料营养对麝香产量和质量的影响。结果表明,饲料营养显著影响麝香产量和品质,单独饲青绿饲料不能满足泌香的营养需要,平均产香量３．２１ｇ,麝香色深而湿,品质差,提高营养水平后,麝香产量显著提高     

离体培养嗅鞘细胞促进新生大鼠螺旋神经节细胞的存活

本文旨在探索离体实验中的嗅鞘细胞(olfactory ensheathing cells,OECs)有无促进耳蜗听觉传入神经元——螺旋神经节细胞(spiral ganglion cells,SGCs)存活作用及其可能机制。取成年大鼠嗅球和新生大鼠蜗轴组织块进行OECs与SGCs的培养,采用差速贴壁法纯化培养OECs。实验分OECs与SGCs共培养组和SGCs单独培养组。倒置相差显微镜下观察OECs和SGCs生长状态,神经营养因子受体p75免疫组织化学法鉴定OECs,神经元特异性标志物βIII-tubulin标记SGCs。为了研究OECs与SGCs共培养体系中,前者促进后者存活的可能机制,共培养组中分别加入脑源性神经生长因子(BDNF,500pg/mL)和BDNF抗体(IgY型,50μg/mL),对照组为未加任何处理的共培养组,然后检查各培养组中SGCs存活数量和存活时间。结果显示,OECs贴壁培养7d后形成一细胞单层,在OECs与SGCs共培养体系中,SGCs在OECs形成的细胞单层的表面生长,并伸出长突起,呈现典型的双极神经元形态;在培养的前6天内,随着培养时间的增加,两组中的SGCs都较接种前减少,但共培养组中SGCs存活数量明显高于SGCs单独培养组(P0.01);单独培养组的SGCs数量在培养的第6天出现大幅度减少,在培养的第9天几乎没有生长;共培养组的SGCs数量未见明显变化(P0.05);共培养中加入BDNF对OECs促进SGCs存活无明显影响,而加入BDNF抗体(IgY)后存活的SGCs减少(P0.01)。本研究结果提示,OECs与SGCs共培养能够促进新生大鼠SGCs存活和突起生长,延长存活时间,OECs分泌BDNF可能是促进SGCs存活的机制之一。     

中药麝香胶囊抑瘤作用的实验研究

目的探讨中药麝香胶囊对小鼠实验性肿瘤的疗效。方法昆明小鼠分别接种艾氏腹水癌、S-180、肝癌细胞株（hepatocellular carcinoma,HCC）三种癌细胞株,接种24h后开始给予中药麝香胶囊,每日1次,共10d。分高、中、低三个剂量给药（4、2、1g／kg以主药麝香药量计）。阳性对照以天仙丸胶囊（1g／kg）一次性灌服。阴性对照组以同体积生理盐水灌服。接种实体瘤动物于第10天处死,称瘤重,计算抑瘤率;接种腹水瘤动物,观察存活时间,计算生命延长率。结果中药麝香胶囊对实体癌有一定的抑瘤作用,但未达到药典规定的抑瘤率30％的要求;对腹水癌也有一定的抑瘤作用,但也未达到药典规定生命延长率50％的要求;统计学分析（P〉0．05）差异没有显著性。结论中药麝香胶囊抑瘤作用不明显,其配方及剂型有待进一步研究与改进。     

气相色谱法测定寨香祛痛凝胶的4 种有效成分的含量

目的:采用气相色谱法,程序升温方式同时测定麝香祛痛凝胶中主要成分麝香酮、樟脑、薄荷脑、冰片的含量。方法:采用玻璃柱3 m×3．2 mm,担体Chro2mosorb W 60-80目,涂布6％聚乙二醇(PEG)-20M,2％苯基(50％)甲基硅酮(OV-17)。从80℃到180℃程序升温;载气:高纯氮,流量:50ml·min-1;FID检测器;进样体积6μL。结果:试验表明,樟脑、薄荷脑、冰片、麝香酮分别在1-8μg,0．6-4．8μg,1-8μg,0．15-1．2μg范围内呈良好的线性关系,回归方程分别为Y=18 062 X-483(r=0．999 9),Y= 9 829 X 61(r=0．999 6),Y=21 006X 562(r=0．999 1),Y=286 986X 406(r=0．999 4)。平均回收率分别为99．78％,100．9％,98．81％,98．68(n=5)。结论:该法可靠简便,结果准确,可作为控制麝香祛痛凝胶质量的方法。     

麝香水溶物对大鼠神经干细胞的生长、分化和电转染率的影响

目的：研究麝香水溶物对培养的大鼠神经干细胞的生长、分化和电转染率的影响。方法：在培养基中加入不同浓度的麝香水溶性提取物后,观察大鼠神经干细胞的生长分化情况;利用表达增强型绿色荧光蛋白的质粒pEGFP-C1,对麝香水溶物处理的神经干细胞进行电穿孔转染,调查电转染率。结果：麝香水溶物处理后的大鼠神经干细胞的细胞团分散,神经突起增多、变长,贴壁细胞增加,细胞形态呈多样性。在0.3‰浓度下,神经干细胞有向神经胶质样细胞分化的趋势。对于麝香处理后变化的细胞,再转到正常培养基中后,细胞基本都能恢复到正常的神经干细胞形态,浓度较高（3‰）时,细胞的恢复能力下降,部分细胞因细胞膜受损严重而死亡。电转染结果表明,麝香处理后发生变化的细胞对pEGFP-C1的电转染率明显提高。结论：麝香水溶物能促进大鼠神经干细胞团的分散和细胞贴壁、变形,并有向神经胶质样细胞分化的趋势。同时可以提高神经干细胞对pEGFP-C1的电转染率。     

麝香多肽分离纯化及其抗炎作用机制研究CSCD

从天然麝香中提取、纯化麝香多肽,利用体外细胞模型筛选有抗炎活性的麝香多肽流分,并研究麝香多肽对小鼠急性肺损伤的保护作用及作用机制。采用冰浴超声、离子交换色谱法从天然麝香中提取、纯化获得5个流分,即为SXP1、SXP2、SXP3、SXP4、SXP5。在LPS诱导的THP-1巨噬细胞炎症模型中,SXP4(100μg/mL)可以显著抑制TNF-α和IL-1β产生,表现出明显的抗炎活性。SDS-PAGE初步检测SXP4主要分布在10~26 kDa范围内。在LPS诱导的急性肺损伤小鼠模型中,SXP4(5、15、50 mg/kg)可减少小鼠血清TNF-α和IL-6的含量(P<0.05或P<0.01);改善小鼠肺组织中炎症细胞浸润和改善肺泡壁增厚情况,减轻小鼠肺组织病理损伤;减少小鼠肺组织中IL-1β、IL-18产生,抑制NLRP3、ASC、Caspase-1、Gasdermin D蛋白表达(P<0.01)。综上,麝香多肽SXP4具有较强的抗炎活性,可减轻急性肺损伤小鼠的肺组织病理损伤,其机制可能与SXP4抑制了小鼠体内NLRP3/Caspase-1介导的细胞焦亡有关。     

NGF 对大鼠胚胎中脑神经细胞体外增殖和分化的影响

目的:探讨神经生长因子(nerve growth factor,NGF)对大鼠胚胎中脑神经细胞体外增殖和分化的影响。方法:在体外分离培养大鼠胚胎中脑神经细胞的培养液中加入不同浓度(10、50、100、200ng/ml)的NGF,培养不同时间,以不加神经营养因子的细胞为对照组,通过MTT法检测细胞活性,神经元特异性烯醇化酶免疫细胞荧光技术鉴定神经细胞,光镜下形态学观察各组大鼠中脑神经细胞体外增殖和分化情况。结果:胚胎中脑神经细胞胞体增大、突起延长且有丰富的神经纤维连结成网络状,细胞集落数增加,显示出剂量-效应关系。结论:一定剂量的NGF能促进大鼠中脑神经细胞分化和增殖,增强其活性。     

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.     

Effect of Purified Murine NGF on Isolated Photoreceptors of a Rodent Developing Retinitis Pigmentosa

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.     

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.     

Nerve growth factor collaborates with myocyte‐derived factors to promote development of presynaptic sites in cultured sympathetic neurons

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle‐containing growth cones. In conjunction with previously published results showing that NGF has long‐term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle–containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte‐derived cell surface or matrix‐associated factors. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 460–476, 2000     

Leukemia inhibitory factor receptor signaling negatively modulates nerve growth factor-induced neurite outgrowth in PC12 cells and sympathetic neurons

Nerve growth factor (NGF) is required for the development of sympathetic neurons and subsets of sensory neurons. Our current knowledge on the molecular mechanisms underlying the biological functions of NGF is in part based on the studies with PC12 rat pheochromocytoma cells, which differentiate into sympathetic neuron-like cells upon NGF treatment. Here we report that the expression of leukemia inhibitory factor receptor (LIFR), one of the signaling molecules shared by several neuropoietic cytokines of the interleukin-6 family, is specifically up-regulated in PC12 cells following treatment with NGF. Attenuation of LIFR signaling through stable transfection of antisense- or dominant negative-LIFR constructs enhances NGF-induced neurite extension in PC12 cells. On the contrary, overexpression of LIFR retards the growth of neurites. More importantly, whereas NGF-induced Rac1 activity is enhanced in antisense-LIFR and dominant negative-LIFR expressing PC12 cells, it is reduced in LIFR expressing PC12 cells. Following combined treatment with NGF and ciliary neurotrophic factor, sympathetic neurons exhibit attenuated neurite growth and branching. On the other hand, in sympathetic neurons lacking LIFR, neurite growth and branching is enhanced when compared with wild type controls. Taken together, our findings demonstrate that LIFR expression can be specifically induced by NGF and, besides its known function in cell survival and phenotype development, activated LIFR signaling can exert negative regulatory effects on neurite extension and branching of sympathetic neurons.     

TrkA cross-linking mimics neuronal responses to nerve growth factor.

TrkA, a tyrosine kinase receptor, is an essential component of the nerve growth factor (NGF) response pathway. The binding of NGF to the receptor induces receptor autophosphorylation and activation of intracellular signaling pathways, resulting in diverse biological effects. We prepared polyclonal antibodies against the entire extracellular domain of rat trkA produced using a baculovirus expression system. These antibodies specifically recognize rat trkA on antigen blots and in immunoprecipitations. Both IgG and Fab fragments block binding of NGF to trkA expressed by the PC12 cell line. In NGF binding studies using anti-trkA and anti-low-affinity NGF receptor (LNGFR) immunoglobulin (Ig) G, essentially all binding of NGF can be inhibited. The results imply that > or = 97% of the NGF binding sites on PC12 cells are accounted for by trkA and the LNGFR. The binding data also argue that all low-affinity NGF binding sites on PC12 cells reflect interactions with the LNGFR, while all high-affinity sites are trkA dependent. A fraction of the high-affinity (or slow) binding sites seem to require both trkA and the LNGFR. Although the monovalent anti-trkA Fab fragments inhibited the biological effects of NGF, such as induction of tyrosine phosphorylation, and survival and neurite outgrowth of sympathetic neurons, the IgG preparation was not effective as an inhibitor. Instead, the IgG fraction by itself was almost as effective as NGF at stimulating receptor activation, cell survival, and neurite outgrowth. Thus, it appears oligomerization of trkA by antibody-induced cross-linking is sufficient to produce the known cellular effects of NGF.     

Binding constants of soluble NGF-receptors in rat oligodendrocytes and astrocytes in culture

The neuronotrophic factor NGF binds to peripheral neurons of the dorsal root ganglion and the sympathetic nervous system. NGF binds to a cell surface receptor, NGFR, on these cells and displays Kd's of 10(-9) and 10(-11)M. NGF receptors have also been reported for basal forebrain magnocellular neurons. In addition, NGF specifically binds to NGFR on Schwann cells although the biological significance of this binding is not known. Here we report that NGF binds in a saturable and specific fashion to receptors on cultured isolated populations of rat astrocytes but not to oligodendrocytes. The binding to astrocytes in culture displayed a Kd of 2.7 +/- 1.0 nM with 36,000 receptors per cell.