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几种小分子对NGF诱导的PC12细胞轴突生长作用的研究
Studies on the Effects on Ngf-Induced PC12Cells Neurite Outgrowth of Some Small Molecular Compounds
【作者】 刘莉;
【导师】 高锦明;
【作者基本信息】 西北农林科技大学 , 化学生物学, 2012, 博士
【摘要】 神经生长因子(NGF)是一种对神经细胞起营养作用的多肽分子,在神经元的生长发育、轴突的生长、递质的合成及细胞的凋亡等阶段起着重要作用,是人和动物体内必不可少的多功能因子。它与多种疾病的发生、发展有关,且无论是它本身水平的改变或是它的受体水平或功能的改变都对某些疾病的病理生理学具有重要影响。多项研究证实,NGF对神经退行性疾病,糖尿病性神经病,神经损伤,炎症和某些癌症具有潜在治疗作用。但作为制剂,NGF的药代动力学差,分子质量较大,半寿期短;不稳定,不易跨越血脑屏障;另外,NGF有两个能执行相反作用的受体Trk A和p75NTR,能激活多个信号通路,产生不同的反应;再加上其较高的价格,使NGF很难大规模应用于神经退行性疾病、糖尿病性神经病及神经损伤等的临床治疗。因此,人们以寻找具有类似NGF活性的,或是能促进NGF在细胞内合成分泌的,或是具有NGF激动剂或拮抗剂活性的药代动力学高,副作用少的小分子化合物作为解决这一难题的有效途径。为了寻找具有促神经分化活性的小分子,本文以大鼠嗜铬细胞瘤细胞系PC12(ratpheochromocytoma cells)作为研究模型,在细胞形态观察、细胞分化率分析的基础上,对鳞盖肉齿菌Sarcodon scabrosus (Fr.) karst子实体中分离得到的一系列化合物进行了活性筛选,选择结构相似的鸟巢烷型二萜类化合物和两种原小檗碱类化合物及其氢化产物作为研究对象,采用形态观察、细胞计数、ELISA,Western blotting等方法对其构效关系及分子机理进行了系统研究。获得了以下主要创新性结果:1.本文首次对20个小分子化合物(包括6个新化合物和一个人工合成的化合物)对NGF诱导的PC12细胞轴突生长的作用进行了研究,首次发现sarcodonin G和sarcodonin A具有NGF依赖的促PC12细胞轴突生长活性。通过对PC12细胞p-TrkA和p-ERK的检测、形态学统计分析以及化合物的结构分析,发现多个官能团对鸟巢烷型二萜类化合物的NGF依赖的促PC12轴突生长活性有重要影响,从分子水平阐明化合物构效关系。同时本文还证明sarcodonin G和sarcodonin A都是通过NGF/Trk A体系触发反应,并经由ERK1/2信号转导通路促进NGF依赖的PC12细胞轴突生长。本文以活性先导化合物的发现为基础,为鳞盖肉齿菌的开发利用提供了实验依据;为设计具有临床应用价值的神经营养因子激动剂提供了理论依据。2.本文首次发现一个新化合物scabronine M具有抑制NGF诱导的PC12细胞轴突生长的活性,且该化合物是通过NGF/Trk A体系触发反应,部分影响ERK1/2途径,实现对NGF依赖的PC12细胞轴突生长的剂量依赖性抑制作用。本文以活性先导化合物的发现为基础,为鳞盖肉齿菌的开发利用提供了实验依据;为设计具有临床应用价值的神经营养因子抑制剂提供了理论依据。3.本文首次对两种植物源原小檗碱类化合物小檗碱和巴马汀的氢化产物——四氢小檗碱和四氢巴马汀的NGF诱导的PC12细胞轴突生长的作用进行了研究,首次发现四氢小檗碱和四氢巴马汀具有促NGF依赖的PC12细胞轴突生长活性。通过同一浓度下四种化合物分别对PC12细胞p-Trk A和p-ERK作用的比较,发现氢化修饰能显著提高原小檗碱类化合物的生物学活性。本文为含有小檗碱成分的植物的有效利用及新药开发提供理论基础。
【Abstract】 Nerve growth factor (NGF), a neurotrophic polypeptide, is an indispensable pleiotrophicfactor in human and animals. NGF plays a crucial role in neuronal survival, differentiation,growth, axon growth, neuro-transmitter synthesis and cell apoptosis. The potential of NGF asa therapeutic agent for several diseases including neurodegenerative disorders, diabeticneuropathy, nerve injury, inflammation and certain types of cancers has been indicated byseveral investigators. It involves with the genesis and development of a variety of diseases,and the changes in level of NGF itself or the receptor and in their functions have an importantinfluence on the pathophysiology of certain diseases. Despite the amazing therapeuticpotential, NGF is not considered an ideal drug candidate. In fact, they show a poorpharmacokinetic behavior, short half-lives, instability in vivo, unable to cross the blood-brainbarrier to reach target tissues. And NGF binds two receptors (Trks and p75NTR) which cancommand opposite actions, activate various signal pathways and produce different responses.Moreover, the NGF protein is relatively expensive to produce for medicinal applications, so itis difficult to large-scale application in neurodegenerative diseases, diabetic neuropathy andnerve injury. Therefore, efforts have been devoted to search for NGF mimics, NGF inducor orNGF agonist/antagonist activity compound with small molecule, better pharmacokinetics andless side effects.In order to find small molecules that can promote neuronal differentiation, we detectedthe NGF-dependent neurite outgrowth activity of a series of compounds isolated from thefruiting bodies of Sarcodon scabrosus (Fr.) karst, using PC-12cells as model. On the basis ofmorphological observation and statistical analysis of the cell differentiation, we chosecyathane-type diterpenoids and two protoberberines and their hydrogenation products asobject to study. Through morphological observation, cell counting, ELISA, Western blotting,we studied the structure-activity relationships and explained the differences on a molecularlevel. The main results were as follows:1. This is the first time to detect the NGF-induced PC12cell neurite outgrowth of20smallmolecules (including six new compounds and a synthetic compound). We reported firstlysarcodonin G and sarcodonin A have the NGF-dependent neurite outgrowth-promotingactivity in vitro. On the basis of the biological results such as detection of p-Trk A and p-ERK, statistics results of cell morphology and the structure of compounds, we initiallyclarified the structure-activity relationship on a molecular and mechanistic level and foundsome functional groups important for the NGF-dependent neurite outgrowth-promotingactivity. At the same time, we also confirmed they promoted the NGF-induced neuriteoutgrowth through NGF/Trk A and ERK1/2MAPK signal pathway in does-dependentmanner. The results provided the experimental base for further utilization of themushroom Sarcodon scabrosus (Fr.) karst and theoretical foundation for design of NGFagonist on the basis of discovery of bioactive lead compounds.2. We reported firstly scabronine M, a new compound has a suppressive effect onNGF-induced neuronal differentiation in vitro. We confirmed that it interacted with Trk A,partilly influenced the ERK1/2MAPK pathway to suppress the cell differentiationprocesses. The results provided the experimental base for further utilization of themushroom Sarcodon scabrosus (Fr.) karst and theoretical foundation for design of NGFagonist on the basis of discovery of bioactive lead compounds.3. We firstly reported tetrahydroberberine and tetrahydropalmatine, two hydrogenationproducts of plant origin protoberberines: bererine and palmatine, have the NGF-dependentneurite outgrowth-promoting activity in vitro. We compared the NGF-dependent neuriteoutgrowth-promoting activity of these four compounds through the lever of p-Trk A andp-ERK, and concluded that derivatives had better activity than their parent compoundsrespectively, the hydrogenation of protoberberines had a significant effect on their activity.The results provided a theoretical basis for the effective use of berberine-containing plantsand development of new drugs.
【Key words】 NGF; cyathane-type diterpenoids; PC12cell; protoberberine; signal transduction;