基于血小板活化的血瘀模型生物学特征探讨及方证相关研究

Blood stasis syndrome which as a common traditional Chinese medicine syndrome is always concerned by the doctors and researchers, the animal model of the blood stasis syndrome is the major carrier of the research on the blood stasis syndromes and medicines for promoting blood circulation and removing blood stasis. But this animal model study is lack of systematic and deepen research for a long term. Though some new models have been tried to copy and explore constantly, the classical blood stasis syndrome model is still the major carrier for scientific research.This project is based on the study of the erythrocyte membrane biological characteristics on blood stasis syndrome models in earlier stage (the National Science Foundation 81173186),since the platelet activation and balance of fibrinolytic system are another major factors of the blood stasis formation, our project is aimed at the four associated process: platelet activation adhesion, aggregation, morphological change and release.We use the FCM(flow cytometry) and laser Confocal microtechnique to observe the platelet activation process of the blood stasis syndrome model constantly and dynamically.With the guidance of the research methodology for prescriptions and syndromes and making the classic prescriptions as the probe, we prove the biological characteristics of different blood stasis syndrome models and the promoting blood circulation and removing blood stasis function characters and superiorities in different prescriptions.And then we make this study link up and connect to our program in the National Science Foundation, hoping to set up a technology platform for the blood stasis biological characteristics to offer technical support for the research and development on traditional Chinese medicine blood stasis syndromes and medicines for promoting blood circulation and removing blood stasis.

血瘀证作为临床常见中医证型一直为医者和研究着所关注，然而作为血瘀证病证研究以及活血化瘀药物研究的主要载体--血瘀证动物模型研究却长期处于缺乏系统、深入研究的状态，虽然一些新的模型不断被尝试复制和探索，但经典的血瘀证模型仍旧是科学研究的主要载体。本项目旨在前期血瘀证模型红细胞膜生物学特征研究（国家自然基金资助81173186）的基础上，从瘀血形成的另外一个主要因素--血小板活化与纤溶系统平衡的角度，针对血小板活化粘附、聚集、形态改变及释放等四个相关联过程，运用流式细胞仪以及激光共聚焦技术连续、动态观察血瘀证模型血小板活化过程，并以经典方剂作为探针，在方证相应的研究方法学指导下，佐证不同血瘀模型的生物学特征以及不同方剂的活血化瘀作用特点和优势。进而，与在研国家自然科学基金项目相衔接和相结合，有望建立针对血瘀证生物学特征研究的技术平台，为中医血瘀证研究以及活血化瘀药物的研发提供技术支撑。

目的：复制寒凝血瘀证和气虚血瘀大鼠模型，分别给予补阳还五汤、少腹逐瘀汤、丹参饮三首方药。围绕血小板生物学指标，观察三首方药对寒凝血瘀和气虚血瘀模型大鼠的血小板生物学指标的影响。从血小板方向探讨大鼠寒凝血瘀和气虚血瘀形成机制及三首方药作用机制。方法：寒凝血瘀实验除空白组外，按照冰水浴加注射肾上腺素的造模方法连续造模15天；气虚血瘀实验采用一次性向气管内注射博莱霉素生理盐水溶液的方法复制气虚血瘀模型。两个血瘀模型实验分两批进行，每批选取SD大鼠为研究对象，80只大鼠按体重随机分成空白组和寒凝血瘀组。补阳还五汤、少腹逐瘀汤、丹参饮高低剂量共八组。并同时灌胃给药。末次给药后，禁食不禁水24h，第16天以乌拉坦1mg•kg-1麻醉，固定，颈总动脉取血，测与血小板相关的各项指标。结论：对于寒凝血瘀模型，1.三方均有改善血小板PLT、PDW、MPV含量的作用，并且能降低血液粘度。2.三方皆能降低TXB2含量，对6-keto-PGF1α、cAMP、cGMP含量变化有缓解作用。3.三方皆有升高β-TG、t-PA含量作用，改善PF4、PAI-1含量降低情况。对于气虚血瘀模型，1.气虚血瘀模型大鼠在全血黏度、血浆黏度、FIB等血液流变学指标方面呈现异常改变，符合血瘀证的客观诊断标准。2.补阳还五汤、少腹逐瘀汤、丹参饮三方均能不同程度降低气虚血瘀模型大鼠全血黏度、血浆粘度，改善血液的高黏状态。3.三方可均通过改善血小板聚集，抑制血小板释放亢进的状态，纠正血小板相关参数异常情况等方面来缓解气虚血瘀模型大鼠病理状态。4.在改善血小板粘附、血小板聚集、纤溶系统活性和抑制内源性凝血途径激活方面，补阳还五汤的作用优于其他两方。5.在改善血小板释放亢进状态及同时抑制内、外源性凝血途径激活方面，丹参饮作用优于其他两方。

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