【作者】 王淑娟；

【导师】 李德新；

【作者基本信息】 辽宁中医学院 ， 中医基础理论， 2002， 博士

【摘要】 衰老是一种复杂的生物学过程，是机体随着年龄的增长，各脏腑器官功能全面地逐渐降低的过程。人口老龄化是当今世界各国面临的社会问题，也是我国面临的社会问题之一。科学要为健康老龄化服务，防止早衰，延缓衰老，延长人类寿命，提高人们的生命质量，已成为当代世界性的重大研究课题。 抗衰老研究在我国具有悠久的历史，属于中医药学的优势领域。本项研究，根据中医学关于衰老的脏腑衰竭说，从人以水谷为本，脾胃为水谷之海，气血生化之源，后天之本，故脾胃为养生之本立论，在李德新教授领导的课题组既往研究成果的基础上，观察了补益脾胃法（包括健脾益气法、温运脾阳法、滋补脾阴法）对脾虚老龄大鼠海马神经元PKC活性与bcl-2和bax蛋白表达及超微结构的影响。 根据中医学的病因发病学理论，本项研究采用饮食不节、疲劳过度和药物损伤等综合因素，成功地建立了脾气虚、脾阳虚和脾阴虚三种证候老龄大鼠模型，分别应用导师李德新教授拟定的健脾益气、温运脾阳和滋补脾阴方药灌胃治疗两个月，以改良的Takai法检测其海马组织的PKC活性，以免疫印迹法（Western—blotting）检测其海马组织中PKCα、β₁、γ亚型的表达，以免疫组织化学SABC法检测了脾阳虚证大鼠海马组织bcl-2和bax蛋白的表达，并用透射电镜观察了脾阴虚证大鼠海马超微结构。 结果表明： 1．补益脾胃方药具有调控脾虚老龄大鼠海马神经元蛋白激酶C活性的作用。 脾虚老龄大鼠海马神经元PKC活性的变化： 海马神经元细胞膜PKC活性：与青年正常对照组相比，老龄大鼠及青年各模型组大鼠海马神经元细胞膜PKC活性不同程度升高，但老龄各模型组之间无显著性差异，青年各模型组间有显著性差异，以脾阳虚证组升高最为明显。老龄各模型组海马神经元细胞膜PKC活性升高幅度较青年组小。 海马神经元细胞浆PKC活性：与正常对照组相比，各模型组海马神经元细胞浆PKC活性明显降低，脾气虚证与脾阳虚证、脾阴虚证模型组之间有显著性差异，老龄组大鼠海马神经元细胞浆PKC活性明显高于青年组；老龄各模型组海马神经元细胞浆PKC活性下降幅度较青年组大。 补益脾胃方药可以降低各脾虚治疗组大鼠海马神经元细胞膜PKC的活性，其中对脾气虚证大鼠治疗效果最为明显。老龄组与青年组之间有显著性差异，老龄组海马神经元细胞膜PKC活性明显高于青年组：但年龄因素对治疗效果无影响。 补益脾胃方药可以提高各脾虚治疗组大鼠海马神经元细胞浆PKC活性，老龄组与青年组之间有显著性差异，老龄组海马神经元细胞浆PKC活性明显高于青年组，但年龄因素对治疗效果无影响。说明在调控海马神经元PKC活性方面，补益脾胃方药对老龄大鼠的疗效与青年组一致。 2．补益脾胃方药具有调控脾虚老龄大鼠海马神经元蛋白激酶C亚型表达的作用 在所检测的PKCα、β₁、γ三个亚型中，大鼠海马神经元细胞膜与细胞浆均以α亚型表达最明显，但各证候模型大鼠、治疗大鼠与正常大鼠之间无明显差异。 各组PKCβ₁、γ亚型的变化与PKC活性的变化基本一致，老龄及青年各证候脾虚模型大鼠海马神经元细胞膜PKCβ₁、γ亚型表达均增强，老龄及青年补益脾胃方药治疗大鼠β₁、 博 士 学 位 论 文 Y亚型表达减弱。 老龄及青年各证候脾虚模型大鼠海马神经元细胞浆PKC e；、Y亚型表达减弱，而补益脾 胃方药治疗大鼠p；亚型表达增强；Y亚型除脾阴虚治疗老龄大鼠与模型大鼠比较无显著性差 异外，脾气虚、脾阳虚治疗老龄大鼠Y亚型表达增强。但青年各模型大鼠与治疗大鼠之间无 明显差异。 3．温补脾阳方药具有调节脾阳虚大鼠海马神经元bclZ及bax基因蛋白表达的作用 与青年正常组大鼠比较，老龄大鼠海马神经元be12蛋白表达减弱，bax蛋白的表达明 显增强；老龄脾阳虚模型组大鼠表现更为明显，几乎未检测到bC12蛋白的阳性表达。 青年脾阳虚模型大鼠海马神经元仇 12蛋白表达减弱，而 bax蛋白表达明显增强，与青 年正常对照组比较有明显差异。 青年脾阳虚治疗组大鼠海马神经元bclZ蛋白表达增强，bax蛋白表达明显减弱；与正 常对照组比较，无明显性差异。老龄脾阳虚治疗组大鼠海马神经元仇－2蛋白表达有所增强， bax蛋白表达减弱，但无统计学意义。 4．滋补脾阴方药具有改善脾阴虚证大鼠海马超微结构的作用 老龄脾阴虚证大鼠海马神经细胞有典型的细胞凋亡特征，一些细胞核浓缩呈斑点状荧光， 有些在核周边呈环状或颗粒状荧光浓聚，细胞膜呈泡状改变。老龄脾阴虚治疗组大鼠变化不 明显。 青年脾阴虚证大鼠海马神经元胞体内细胞器形态不规则，线粒体出现肿胀变形，峭断裂 和空泡样改变；细胞核出现核膜内稻断裂：个别细胞还可见到内含条纹状结构的溶酶体。青 年脾阴虚治疗组大鼠虽可见到核膜内招及线粒体肿胀现象，但程度较模型组明显减轻。更多还原

【Abstract】 Objective:Aging is a complex procedure that the functions of various visceras and organs of the body decline gradually with the increasing of the age. With the increasing of aging tendency of population, it has become a world wide important medical topic to delay and prevent aging.Anti-aging research has a long history and belongs to the TCM field. On the basis of former findings achieved by the project group led by Prof. Li Dexin and based on the doctrine of Zangfu organs exhaustion in TCM, this study set forth its views from " the cereal essence is the origin of the body, the spleen-stomach is the source of Qi-blood and the origin of the acquired constitution so as to the fundamental of life-cultivation " . The effect of method for replenishing spleen and stomach (including methods of nourishing qi to invigorate spleen, warming up spleen yang and reinforcing spleen yin) on the PKC activities of hippocampus neuron and bcl-2, bax protein expression and ultrastructure in rats with spleen deficiency syndromes was observed.Based on the theory of etiology in TCM, the models of old rats with three types of spleen deficiency syndromes (spleen qi deficiency, spleen yang deficiency and spleen yin deficiency) were set up successfully by applying multiply factors, such as abnormal diet, overstrain and damage from abuse administration drugs. The models were fed with the prescriptions for nourishing qi to invigorate spleen, warming up spleen yang and reinforcing spleen yin for about two months separately. After that, the PKC activity of hippocampus neuron was detected by improved Takai method, PKC subtype of hippocampus neuron was tested by immunological ink mist method, and the bcl-2, bax genie protein expression of hippocampuswas also detected by immunochemistry tissue method (SABC method). The changes of hippocampus neuron ultrastructure were observed under electron microscope as well. The results showed that:1. A series changes of the activity of protein kinase C of hippocampus neuron cells in rats with different spleen deficiency syndromes have happened and the prescriptions for replenishing spleen can effectively regulate them.1. 1 The changes of PKC activity of hippocampus neuron in different model groups were different.PKC activities in hippocampus neuron cytomembrane in the control group of old healthy rats and each of model groups increased with different degree. The order is as follows: old group with spleen Yang deficiency syndrome) old group with spleen Yin deficiency syndrome) old group with spleen Qi deficiency syndrome) old normal group) young group with spleen Yang deficiency syndrome) young group with spleen Yin deficiency syndrome) young group with spleen Qi deficiency syndrome; there were obvious difference between the young and old groups, PKC activities in hippocampus neuron cytomembrane of old groups were higher than that of young groups significantly and there was the alternative effect between the age and the syndrome types. The increasing range of PKC activities in hippocampus neuron cytomembrane in the young groups with spleen Yang and spleen Yin deficiency syndromes is bigger than that of the old groups.1.2 Chinese herbs for replenishing spleen and stomach can effectively regulate PKC activities.The treatment method for replenishing spleen and stomach can effectively regulate PKC activities of hippocampus neuron cells and reduce PKC activities in hippocampus neuron cytomembrane in rats of treatment groups, meanwhile, it can increase PKC activities in cytoplasm. But there is obvious difference between the young groups and the old groups, PKC activities in hippocampus neuron cytomembrane of the old groups are higher than that of the young groups; but there is no alternative effect between the age and the curative effect.PKC activities in hippocampus neuron cytoplasm: there is significant difference between the old groups and the young groups, PKC activities in hippocampus neuron cytoplasm in the old groups are higher than that of the young groups obviously; and there i更多还原