从Caveolae介导的脂质转运探讨健脾化痰法调节脂代谢紊乱的机制

The previous researches pointed out that lipid transport barriers lead to lipid metabolism imbalance, and the basic pathogenesis is spleen deficiency. Lipid transport is the embodiment of spleen governing transportation and transformation for lipid to nourishing the whole body. Recent studies have shown that caveolae are cell surface plasma membrane invaginations, and caveolae-centered lipid transport is closely related to lipid metabolism. Therefore, based on previous work, golden hamster will be fed by high fat diet to establish the experimental lipid metabolism disorder model. After traditional Chinese prescription interventions, Real-time PCR and Western Blot will be used to detect the expression of caveolae-mediated lipid transporters in adipose and liver tissue of these golden hamster. Meanwhile, we will observe the localization of caveolae-mediated lipid transporters in adipose and liver tissue cells using immunofluorescence technique. The purpose is to clarify the close ties between spleen deficiency and caveolae-mediated lipid transport, and investigate the molecular mechanism of invigorating spleen to remove phlem regulating lipid metabolism disorders, and further enrich the scientific meaning of "spleen governing transportation and transformation".

本项目申请者团队前期研究发现脂质转运障碍导致脂代谢失衡，脾虚生痰是脂代谢紊乱的基本病机，因而认为脂质转运是“脾主运化”在水谷精微转输布散的具体体现之一。Caveolae是细胞表面参与脂质转运的特殊结构，其介导的脂质转运与脂代谢关系密切。本项目拟在前期研究基础上，以脂代谢紊乱金黄地鼠模型为对象，并对其进行健脾化痰的二陈汤干预后，观察脂代谢变化情况：（1）采用Real-time PCR和Western Blot技术分别检测地鼠脂肪和肝组织中Caveolae介导的脂质转运因子mRNA和蛋白的表达水平；（2）对地鼠脂肪和肝组织的冷冻切片进行油红染色和免疫荧光染色，观察二种组织细胞中脂肪的含量和Caveolae介导的脂质转运因子的定位。目的在于阐明脾虚生痰与Caveolae介导的脂质转运的密切联系，揭示健脾化痰法调节脂代谢紊乱的分子生物学机制，为进一步探究脾主运化在脂质代谢中的物质基础提供思路。

饮食不节、过逸少劳易损伤脾胃功能，导致脾失健运，使得膏脂运化输布失常。脂质转运是“脾主运化”在水谷精微转输布散的具体体现之一。Caveolae是细胞表面参与脂质转运的特殊结构，其介导的脂质转运与脂代谢关系密切。为了探讨Caveolae介导的脂质转运与“脾主运化”调节脂代谢的联系，本课题利用高脂饮食诱导建立脂代谢紊乱动物模型，并用健脾化痰的二陈汤以方测证，通过观察大鼠血脂含量及肝脏和脂肪组织中脂质的堆积变化情况，明确脂代谢紊乱的中医病理基础和基本病机。在此基础上，采用Real-time PCR、Western Blot和免疫组织化学等方法检测以Caveolae为中心的脂质胞外、跨膜和胞内转运因子的表达和定位。结果表明，脂代谢紊乱大鼠Caveolae介导的脂质转运因子表达异常，而二陈汤能够逆转这种异常状态。阐明脾虚生痰与Caveolae介导的脂质转运的密切联系，揭示了健脾化痰方—二陈汤通过调控Caveolae介导的脂质转运过程，进而起到调节脂代谢紊乱的作用，探明脾主运化在脂质代谢中的物质基础。此外，采用microRNA-seq的方法检测并筛选出高脂饮食诱导的脂代谢紊乱模型大鼠与正常大鼠肝脏中差异表达的microRNAs，为课题组后续从多靶点进一步探讨Caveolae介导的脂质转运在脾主运化中的内在机制提供思路。