用胚肾干细胞促进牙周膜干细胞成骨诠释"肾主骨"的新内涵

Traditional Chinese medicine acknowledges "Shen Zhu Gu". Modern medicine has also proved that kidney can activate vitamin D3, and regulate both calcium and phosphorus metabolism and bone growth and development. However, the connotation of "Shen Zhu Gu" is far more than that. This study aims to apply embryonic kidney stem cells in promoting periodontal ligament stem cells to differentiate into osteoblasts enriching and developing the new connotation of "Shen Zhu Gu", and to apply "Bu Shen Qiang Gu" traditional Chinese medicine in strengthening the above role of embryonic kidney stem cells providing a new strategy of "Cong Shen Lun Zhi" for the repair and regeneration treatment of bone tissue defects. First is, by means of the coculture system, to observe the role of embryonic kidney stem cells in promoting periodontal ligament stem cells to differentiate into osteoblasts, and to observe the strengthening effects of "Bu Shen Qiang Gu" traditional Chinese medicine total flavone of rhizoma drynaria as well as its main component naringin on the two types of stem cells. Second is, by means of omics technology, to screen and validate the functional molecules through which the embryonic kidney stem cells promote the osteogenesis of periodontal ligament stem cells, thereby revealing mechanisms of the molecules through both in vitro and in vivo intervention experiments. Finally, the collagen scaffold that contains both embryonic kidney stem cells and periodontal ligament stem cells marked with red fluorescent protein will be transplanted into the damaged alveolar cavity after tooth extraction of the miniature pig in order to observe the effects of embryonic kidney stem cells on osteogenesis of periodontal ligament stem cells in a particular in vivo environment, and to observe the effects on repair and regeneration of the damaged alveolar bone. At the same time, animal experiments will also be carried out to further observe the in vivo strengthening-effects of total flavone of rhizoma drynaria and naringin on embryonic kidney stem cells and periodontal ligament stem cells.

中医认为"肾主骨"，现代医学已证实肾脏活化维生素D3，调节钙磷代谢和骨的生长发育，但"肾主骨"的内涵远不仅如此。本研究旨在用胚肾干细胞促进牙周膜干细胞向成骨细胞分化，为"肾主骨"藏象理论增加新内涵；用补肾强骨中药强化这一作用，为骨组织缺损的修复再生提供"从肾论治"和"补肾强骨"的新策略。首先，通过细胞共培养观察胚肾干细胞促进牙周膜干细胞分化为成骨细胞以及补肾强骨中药骨碎补总黄酮或其主要成分柚皮苷对两种干细胞的强化作用。其次，利用组学技术筛选、验证胚肾干细胞促进牙周膜干细胞成骨作用的功能分子，通过体内外干预实验揭示该分子的作用机制。最后，将含有胚肾干细胞和红色荧光蛋白标志的牙周膜干细胞的胶原支架植入到小型猪被拔牙的受损牙槽内，观察在体内胚肾干细胞对牙周膜干细胞成骨作用的影响及其对受损牙槽骨的修复再生作用；同时通过动物实验进一步验证骨碎补总黄酮或柚皮苷对胚肾干细胞和牙周膜干细胞的体内强化作用。

中医认为“肾主骨”，现代医学已证实肾脏活化维生素D3，调节钙磷代谢和骨的生长发育，但“肾主骨”的内涵远不仅如此。本项目首先从妊娠的小型猪胚肾被膜下的生肾区，提取后肾间充质细胞，通过与牙周膜干细胞共培养，明确了后肾间充质细胞及其上清液可促进牙周膜干细胞的成骨分化，表现为成骨标志物碱性磷酸酶活性增高、RUNX2蛋白和OCN的mRNA表达量上调、矿化结节增多，为中医“肾主骨”藏象理论增添了新的科学内涵。然后，利用细胞因子芯片技术筛选后肾间充质细胞促进牙周膜干细胞成骨作用的功能分子VEGF-C，验证后通过正向和负向干预实验，发现VEGFR2受体阻滞剂KI8751和VEGFR3受体阻滞剂MAZ51，均可抑制VEGF-C对牙周膜干细胞成骨分化的促进作用；VEGF-C可时间依赖性地激活p38信号通路，p38的特异性阻断剂SB203580可以阻断VEGF-C对牙周膜干细胞的促成骨作用，揭示了后肾间充质细胞通过分泌VEGF-C经过VEGF2/ VEGF3受体激活p38通路促进牙周膜干细胞成骨分化的分子机制，为骨损伤的治疗提供了“从肾论治”的新策略。另外，研究了补肾强骨中药骨碎补总黄酮的主要成分柚皮苷对牙周膜干细胞的促成骨作用及其机制，明确了柚皮苷可以通过激活ERK1/2信号通路促进牙周膜干细胞成骨分化，ERK1/2通路的特异性阻断剂PD98059可以阻断柚皮苷对牙周膜干细胞的促成骨作用，为“补肾强骨”疗法提供了新的科学依据。同时，本项目通过实验培养了数名研究生，发表了标有本项目编号的论文14篇，其中SCI论文5篇，另有2篇在国际学术会议上交流，并受邀在全国学术年会上讲座。