通络救脑注射液对大鼠海马神经元的保护作用

【作者】 李小靓；

【导师】 华茜；

【作者基本信息】 北京中医药大学 ， 微生物与生化药学， 2009， 硕士

【摘要】 现代医学在对脑病的研究中,尤其是近年分子基因技术的发展,逐渐认识到脑病(无论是脑缺血,还是神经退行性疾病)发生发展与脑微环境息息相关,“毒损脑络”病机理论提出痰浊停聚和脉络瘀阻后化生的“内生之毒”为脑病发病过程中的基本病理环节,毒损脑络,则神机失用。用于急性期中风治疗的通络救脑注射液具有清热解毒、化痰通络、醒神开窍的功效,三七活血通络和栀子清热解毒在不同病理环节上的生物效应相辅相成。前期研究提示,通络救脑注射液对拟阿尔茨海默症模型大鼠学习记忆障碍具有显著的改善作用,可以抑制β分泌酶的酶活性,减少淀粉样蛋白的生成,同时,抑制由氧糖剥夺引起的炎症反应。基于上述理论,我们推测通络救脑注射液能有效地实现对氧糖剥夺造成的脑微血管内皮细胞损伤的保护以及对淀粉样蛋白造成海马神经元损伤的保护作用。本部分工作以氧糖剥夺及淀粉样蛋白的大鼠海马神经细胞为模型,观察通络救脑注射液及其不同组分在脑缺血期是否能够阻抑脑缺血后血管破损导致的神经损伤和神经细胞修复,观察能否抑制淀粉样蛋白造成的神经细胞毒,从而发挥神经保护作用。具体包括以下两部分内容及相关实验:第一部分,以脑微血管内皮细胞为着眼点,观察通络救脑注射液对氧糖剥夺的脑微血管内皮细胞以及神经细胞的影响,探讨通络救脑注射液对缺血性中风后神经系统损伤与修复过程的干预趋势。第二部分,以淀粉样蛋白模拟老年痴呆的细胞模型,深入研究通络救脑注射液及其不同组分的神经保护作用与它们对淀粉样蛋白代谢的干预作用。各部分实验结果如下:1、使用免疫细胞化学鉴定分离培养大鼠原代脑微血管内皮细胞和海马神经细胞。采用氧糖剥夺6小时模拟脑缺血再灌模型,采用LDH方法证明,与模型组比较,通络救脑注射液预处理后能够不同程度地抑制氧糖剥夺造成的损伤。2、收集通络救脑注射液作用于不同时期氧糖剥夺的脑微血管内皮细胞条件培养基,观察药物间接作用于神经细胞存活率的影响,可知,正常条件和复糖复氧加药组均明显提高细胞存活率,说明通络救脑注射液在该环节上体现出了阻抑脑血管内皮细胞和神经细胞损伤的效应。3、采用培养基观察药物间接作用于神经细胞突触生长情况的影响,可知,各个环节均能不同程度的促进突触延伸,提示药物作用于脑微血管内皮细胞后,能促进其分泌神经营养物质,促使突触生长。4、在线预测通络救脑注射液不同药物成分的功效,提示具有神经保护作用。采用淀粉样蛋白作用于神经细胞模拟阿尔茨海默病细胞模型。细胞存活率统计和免疫组织化学结果显示,通络救脑注射液保护了细胞的存活,防止了突触损伤。其中栀子苷在细胞存活的保护效果更明显,三七总皂苷在其突触损伤保护作用更明显。本文采用计算机预测药物活性与实验验证相结合的方法,融合神经生物学等学科知识,从细胞水平对脑微血管内皮细胞与海马神经元相互关系进行了初步研究,初步阐明了药物不同成分对大鼠海马神经细胞的影响及作用方式和作用时间窗,为临床用药提供依据。更多还原

【Abstract】 In the modern medical study of encephalopathy,especially in the development of molecular genetic technology,an increasing recognition that the relationship between encephalopathy and brain micro-environment(whether cerebral ischemia or neurodegenerative diseases) is much closed."Toxic hurts brain collaterals" is the key point in its pathogenesis,and remarkable clinical curative effect has obtained with the treatment targeting on the functional characteristic of collateral. TongLuoJiuNao(TLJN) is a herb extract that mainly contains ginsenoside Rg1 and geniposide which are clinically used for treating ischemic damages in the brain, however,the mechanism of this protection is unclear yet.Futhermore,under the instruction of "collateral theory" of Chinese medicine and focus the target on the Brain Microvascular Endothelial Cells(BMEC).We further studied the regulatory influence of rat brain microvascular endothelial cells on the activity of cortical neurons.The research focus on the neuroprotective effects which by using the cell model of oxygen glucose deprivation and amyloid protein in hippocampal neurons,the objective is to observe TLJN injection and its different components whether can rescue the nerve injury of cerebral ischemia andβ-amyloid neurotoxicity.The following two parts are related experiments:The first part,to observe TLJN injection can or cannot rescue neurons by cerebral oxygen glucose deprivation through brain microvascular endothelial cells,and to explore TLJN injection’s intervention effects on cerebral ischemic injury after stroke.The second part,to deeply study the neuroprotective effect of TLJN injection and its different components in cell model can or cannot rescue the neurotoxicity of amyloid protein.1,Identificated isolated primary cultured rat brain microvascular endothelial cells and hippocampal neurons by using immunocytochemical.By the method of six hours of oxygen glucose deprivation to model the cerebral ischemia-reperfusion, and using LDH methods to observe the neuroprotective effect in oxygen glucose deprivation injury by compared with the TLJN injection and model group. 2,Collected conditioned medium of brain microvascular endothelial cells during the different periods of oxygen deprivation after TLJN injection treatment,and observed the indirect effects of drugs on the neuronal survival.We find that the normal conditions and reoxygenation group can markedly improved cell survival.3,Observed the indirect effects of drugs on the synapses growth of neurons in condition medium.We find that TLJN injection can promote synaptic extension in different levels of conditioned medium.It prompted that the drug can effect on brain microvascular endothelial cells and then promote them to secret some neurotrophic substances to promote synaptic growth.4,Online predict the TLJN injection and its different compounds,suggesting that they has neuroprotective effect.Adopted amyloid protein in neuronal cell model to model the Alzheimer’s disease.The results showed that TLJN injection can protect the survival and can prevent the synaptic damage by using cell survival statistics and immunohistochemical.Geniposide’s protective effect is more obvious while the Ginsenoside can obviously present the synaptic damage.In this study,we investigated whether TLJN can protect neurons against damages by ischemia in brain vasculature,and the effects was evaluated with cell viability and neurite outgrowth before or after OGD.We found that TLJN could protected both cultured primary rat hippocampal neurons and BMECs from cell death under both normal and oxygen/glucose-deprivation(OGD) conditions.Moreover,under the same conditions,BMECs-conditioned media pretreated by TNJN could also promote neuron viability and neurite outgrowth,indicating that TLJN stimulated BMECs to secret some neuroprotective/ neurotrophic factors.These findings suggest that TLJN has a marked neuroprotective and neurotrophic roles by either direct or indirect operation,and provide insight into the mechanism of clinical efficacy of this drug against stroke and AD.更多还原