肾主骨理论指导下的后肾间充质细胞的成骨分化和促成骨作用研究

The development of the kidney is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. The pronephros and mesonephros degenerate, while the metanephros develops into adult kidneys eventually. The traditional Chinese medicine (TCM) believes that the kidneys store the essence, control the bone and produce the marrow. The metanephric mesenchymal cell (MMC) is a kind of renal stem cells and stores the essence as well. However, it remains unclear whether they can differentiate into osteoblasts and have the ability to promote osteogenesis or not. On the basis of the TCM, ①we plan to use the MMC, which is a patent of our research group, to induce its osteogenic differentiation; ②using the co-culture system, cytokine microarrays and antibody neutralizing technologies to investigate the effects of cytokines secreted by the MMC on the proliferation and osteogenesis of osteoblasts in vivo and to find out the effective cytokines and pathways; ③according to our previous works, we plan to investigate the promotive effects of naringin, an active ingredients of a kidney-tonifying medicine Rhizoma Drynariae, on the osteogenic differentiation of the MMC; ④ to investigate the effectiveness of the fluorescent protein labeled MMC and naringin on the osteonecrosis of femoral head (ONFH) of miniature pigs and to collect the morphological, pathological and molecular evidence. This study aims to enrich and develop scientific connotations of the theory of visceral manifestations and to provide new evidence for the theory of “Cong Shen Lun Zhi（从肾论治）”for bone diseases and the therapy of “Bu Shen Qiang Gu（补肾强骨）”.

哺乳动物肾脏的胚胎发育经历了前肾、中肾和后肾三个阶段，前肾和中肾在发育中退化，后肾发育为成体的肾脏。中医认为肾藏精、主骨、生髓。后肾间充质细胞是一种肾脏干细胞，具有肾精的属性，但能否分化为成骨细胞以及是否有促成骨作用，尚不清楚。本研究以中医理论为指导，①利用本课题组的专利产品-猪后肾间充质细胞，诱导分化为成骨细胞；②通过细胞共培养、细胞因子芯片和抗体中和技术，体外研究后肾间充质细胞对成骨细胞增殖和成骨作用的影响，揭示后肾间充质细胞分泌什么因子、经过什么通路促进成骨细胞成骨作用的分子机制；③在前期研究基础上，用补肾强骨中药骨碎补的有效成分柚皮苷强化后肾间充质细胞的成骨分化和促成骨作用；④体内观察荧光标记的猪后肾间充质细胞或柚皮苷或两者联合促进猪股骨头坏死的修复，并提供形态、结构和分子生物学证据。旨在丰富和发展肾主骨中医藏象理论新的现代科学内涵，为骨病的从肾论治和补肾强骨疗法提供新的科学依据。

中医认为“肾藏精、主骨、生髓”。后肾间充质细胞是一种胚胎肾脏干细胞，具有“肾精”的属性，但能否分化为成骨细胞以及是否有促成骨作用，尚不清楚。本项目以中医理论为指导，诱导猪后肾间充质细胞分化为成骨细胞，研究后肾间充质细胞对成骨细胞增殖和成骨作用的影响，用补肾强骨中药骨碎补的有效成分柚皮苷强化后肾间充质细胞的成骨分化和促成骨作用，旨在丰富和发展“肾主骨”中医藏象理论新的现代科学内涵，为骨病的从肾论治和补肾强骨疗法提供新的科学依据。.本项目的主要研究内容、重要结果、关键数据及其科学意义：①观察了后肾间充质细胞的成骨分化，结果发现后肾间充质细胞在成骨诱导培养基的作用下可以向成骨细胞分化；②研究了后肾间充质细胞上清液对成骨细胞增殖和成骨作用的影响，结果显示后肾间充质细胞上清液可提高成骨细胞的增殖活性，促进成骨细胞的成骨作用；上述①和②结果为中医“肾主骨”藏象理论提供了直接证据。③观察了补肾强骨中药骨碎补的有效成分柚皮苷对后肾间充质细胞成骨分化的影响和对成骨细胞的促成骨作用，结果显示柚皮苷可以促进后肾间充质细胞向成骨细胞分化，还可促进成骨细胞的成骨作用，为补肾强骨疗法提供了新的实验依据。④观察了后肾间充质细胞对糖皮质激素诱发的成骨细胞损伤的预防和治疗作用，创新性地发现后肾间充质细胞可以拮抗和逆转地塞米松对成骨细胞成骨作用的抑制，为骨病的从肾论治提供了新的科学依据。⑤ 进行了原代猪胚肾间充质细胞永生化及鉴定的系列工作，包括永生化基因的植入、干细胞特性鉴定及成骨分化和促成骨作用的验证等，最后获得具有自主知识产权的国家专利产品。⑥ 以第一发明人获得国家发明专利“后肾间充质细胞系及其制备方法与应用”授权1项，多次受邀作“肾主骨”相关的专题学术报告。