从CD4+CD25+Tregs／Th17平衡角度探讨壮骨止痛方抗绝经后骨质疏松的骨免疫学机理

Zhuangguzhitong formula could prevent women from post-menopausal osteoporosis, but the mechanisms keep unveil. This subject proposed the hypothesis that Zhuangguzhitongfang could keep the balance between CD4+CD25+ regulatary T cells(CD4+CD25+Tregs) and Th17 in bone . Then four sub-hypothesis have been proposed to the mechanisms on keeping the balance of CD4+CD25+Tregs and Th17. OVX and ERKO were used to identify the hypothesis. Then co-culture of CD2+CD2+Tregs、T17、OB and OC will be found to study the inter-reaction between them. At last, the signalling path will be screened with the protein chip,and the RT-PCR and western blot will be used to identify the signalling path. Not only will the mechanisms be explained in the project, but also the direction to the clinic will be gotten, what`s more, the data to osteoimmunology of TCM will be gotten.

在已获得壮骨止痛方防治绝经后骨质疏松有效部位和主要有效成分的基础上，本项目从骨免疫学角度推论其通过调节CD4+CD25+Tregs/Th17平衡实现抗骨质疏松的作用机制假说，综合利用去势雌鼠模型和雌激素受体敲除鼠病理模型验证假说，建立CD4+CD25+Tregs、Th17、OC的细胞培养体系及共培养体系，从细胞水平进一步验证假说，探寻其调节CD4+CD25+Tregs／Th17平衡的作用环节，最后，利用蛋白芯片筛选其调节细胞信号传导通路，并利用RT-PCR、Western blot技术验证蛋白芯片实验结果，然后根据信号传导实验结果反推其细胞因子或激素。本项目不仅阐明壮骨止痛方防治绝经后骨质疏松的骨免疫学机制，更深入、更精细地阐明壮骨止痛方多环节、多途径、多靶点的作用特点，而且能为临床更好应用壮骨止痛方提供指导，并为构建中西医结合骨免疫学提供宝贵实验资料，促进中西医结合骨免疫学的发展。

本课题阐明了壮骨止痛方从雌激素／雌激素受体途径、雌激素相关受体途径抗绝经后骨质疏松作用，从细胞水平、蛋白水平和基因水平验证了壮骨止痛方通过调节CD4+CD25+Tregs/Th17平衡实现抗骨质疏松的作用机制研究假说。通过脾淋巴细胞混和培养，从细胞水平进一步证实壮骨止痛方含药血清具有使CD4+CD25+Tregs/Th17平衡向CD4+CD25+Tregs发展的作用。从脾淋巴细胞分离CD4+淋巴细胞与破骨细胞共培养，结果表明壮骨止痛方含药血清能通过CD4+淋巴细胞抑制破骨细胞的分化和活性，其作用机理主要调节CD4+CD25+Tregs/Th17平衡向CD4+CD25+Tregs发展。从脾淋巴细胞中分离的CD4+CD25+Tregs与破骨细胞共培养实验表明，壮骨止痛方含药血清能增强CD4+CD25+Tregs对破骨细胞分化和活性的抑制作用，其作用主要通过增强CD4+CD25+Tregs分泌IL-10、TGFβ 等细胞因子，抑制破骨细胞TNFR、IL-17R的表达。该研究还发现壮骨止痛方可通过ER／miRNA-503通路抑制破骨细胞分化和溶骨活性。