绝经后骨质疏松症肾阴虚证关联基因CLCF1对OPG/RANKL/RANK信号系统的骨免疫调控

For nearly 112 million patients with osteoporosis in China, it is of great significance for preventing and treating by clearly understanding the molecular mechanism of kidney deficiency. Thus, the research group has demonstrated in the earlier research that CLCF1 is an associated gene that can regulate JAK2/STAT3 signal pathway and impact bone metabolism for kidney yin deficiency of postmenopausal osteoporosis (PMOP). To make clear understanding of the direct-acting mechanism of CLCF1 for bone metabolism, this study intends to: ①observe impacts of low expression of CLCF1 upon immunities in mice and OPG/RANKL/RANK signal system using the technology of adenovirus associated virus. ②explore impacts of over-expression and silencing of CLCF1 on B lymphocytes by culcuturing the cells together with osteoblasts. ③ analyze the impacts of treating kidney yin deficiency of PMOP by Liuwei Dihuang pill upon immunities and OPG/RANKL/RANK system, and discuss the mechanism of regulating bone metabolism by CLCF1 by OPG/RANKL/RANK system via the bridge between immune system and bone metabolism, so as to demonstrate if the hypothesis of this study that “the molecular osteoimmunological mechanism of kidney yin deficiency of postmenopausal osteoporosis (PMOP) is possibly closely related to the impacts of CLCF1 regulation of OPG/RANKL/RANK signal system on bone metabolism” is right or not.

对于我国现有约1.12亿骨质疏松症患者来说，弄清其主要病因肾虚的分子机制对其防治具有重大的意义。为此，课题组前期研究已证实CLCF1是绝经后骨质疏松症（PMOP）肾阴虚证的关联基因，可调控JAK2/STAT3信号通路，影响骨代谢。为厘清CLCF1对骨代谢的直接作用机制，本课题拟：①采用腺相关病毒敲低技术，观察CLCF1低表达对小鼠免疫及OPG/RANKL/RANK信号系统的影响；②B淋巴细胞和成骨细胞共培养，研究B淋巴细胞中CLCF1基因过表达和沉默对OPG/RANKL系统的影响；③分析六味地黄丸治疗PMOP肾阴虚证对免疫和OPG/RANKL/RANK系统的影响；从免疫系统与骨代谢的桥梁--OPG/RANKL/RANK信号系统探讨CLCF1调控骨代谢的机制，从而论证课题假说：“PMOP肾阴虚证的骨免疫机制可能与CLCF1调控OPG/RANKL/RANK信号系统影响骨代谢密切相关”的正确性。

随着社会的老年化，骨质疏松防治形势日趋严峻。中医药治疗骨质疏松症（OP）具有较好的疗效，探讨其治疗机制具有重要意义。本次研究围绕课题组提出的“绝经后骨质疏松症（PMOP）肾阴虚证的骨免疫机制可能与心肌营养蛋白样细胞因子1（CLCF1）调控OPG/RANKL/RANK信号系统影响骨代谢密切相关”假说，通过基因敲除动物实验、细胞实验和临床试验，结合之前国基的研究数据，探讨PMOP肾阴虚证关联基因CLCF1的调控机制和六味地黄丸的治疗机理。结果：①绝经后妇女外周血CLCF1表达降低与骨密度降低、骨质疏松的发生密切相关；在PMOP肾阴虚患者中，CLCF1和OPG蛋白表达降低，六味地黄丸治疗后表达提高。②PMOP肾阴虚组血红蛋白、骨代谢PINP高于无骨质疏松对照组，单核细胞数低于对照组，但免疫指标和骨代谢S-CTX组间比较无显著差异，数据分析证实绝经后妇女OP组的HGB、血细胞计数均高于无OP组，随着骨密度的降低，HGB、血细胞计数反而增高。③绝经后妇女在骨量减少阶段的中医证候表现为肾虚、肝虚为主，心虚、肺虚次之。④细胞实验证实CLCF1过表达能够上调STAT3表达和p-JAK2、p-STAT3水平，CLCF1敲除能够下调p-STAT3和OPG、RANKL表达，说明CLCF1与OPG/RANKL信号通路有密切联系。⑤基因条件性敲除实验说明：12月龄CLCF1-Cd19-Cre雌性小鼠OPG mRNA表达高于野生对照组，RANKL mRNA低于对照组，但组间CLCF1、JAK2、STAT3、OPG、RANK、Rankl蛋白表达无明显差异；CLCF1基因条件性敲除对3月龄、12月龄CLCF1-Cd19-Cre、CLCF1-Cd4-Cre小鼠的骨密度、体重无明显影响，对12月龄基因敲除小鼠的免疫指标、胸腺和脾重量、Micro-CT检测的骨密度也无显著影响。.课题组认为：①CLCF1可能是影响骨质疏松发病的一个重要基因；②上调CLCF1和OPG蛋白表达是六味地黄丸治疗PMOP肾阴虚证的分子机制之一；③在骨量减少阶段，可根据中医证候提早进行中医药干预治疗。对于CLCF1与OPG/RANKL/RANK信号系统的关系，由于实验仅观察3月、6月龄的小鼠情况，观察时间窗不够全面，仍有待后续研究证实。上述成果将为防治OP提供了一个新的基因，将对防治骨质疏松和推动中医药发展产生积极的作用。