肾虚椎间盘退变中盘源性干细胞的命运及调控机制研究

“lumbago due to kidney deficiency” is the theoretical foundation of Chinese medicine treatment of disc degeneration, but the exact mechanism is unclear. Lately researches show that “Deficiency of kidney” is mainly manifested as the changes of stem cells on proliferation, differentiation and microenvironment homeostasis in cellular and molecular level. Besides, the stem cells existing in intervertebral disc niches involve in disc degeneration repair. Our previous study found that Wnt/β-Catenin signaling pathway of disc cells played an significant role in disc degeneration. Thus, our hypothesis is that kidney deficiency induces the imbalance of microenvironment homeostasis, leading to the abnormal activity of Wnt/β-catenin signaling pathway in disc-derived stem cells. In addition, the change of Wnt/β-catenin signaling pathway affects their ability to differentiate into the intervertebral disc cells, resulting in the decreased ability to regenerate and repair disc degeneration. Our study will build the disc degeneration model caused by kidney deficiency with stem cell specific tdTomato transgenic mice and specific β-catenin gene knockout mice to definite the fate of disc-derived stem cells in the process of disc degeneration and the regulation mechanism on proliferation and differentiation by Wnt/β-catenin signaling pathway, and conduct counterevidence study with a classical fomula of reinforcing kidney, Liuweidihuang decoction. This study will reveal the mechanism of disc degeneration caused by kidney deficiency and provide the evidence of the modern interpretation of the “lumbago due to kidney deficiency” theory and clinical treatment of intervertebral disc degeneration.

“肾虚腰痛”是中医药治疗椎间盘退变的理论基础，但其具体机制尚不明确。最新研究表明“肾虚”主要体现为干细胞增殖分化等功能及其微环境稳态的变化；椎间盘干细胞龛中存在盘源性干细胞，参与椎间盘退变修复。我们前期研究发现椎间盘细胞Wnt/β-catenin信号通路在椎间盘退变过程中起关键作用。因此，我们假说肾虚可能诱发盘源性干细胞龛微环境稳态失衡，致使盘源性干细胞Wnt/β-catenin信号通路活性异常，影响其分化为椎间盘细胞的能力，导致椎间盘修复能力下降，出现椎间盘退变。本研究将以干细胞特异性tdTomato转基因小鼠和特异性β-catenin基因敲除小鼠为对象，研究盘源性干细胞在肾虚椎间盘退变过程中的命运以及Wnt/β-catenin通路对其增殖分化的调控机制，并用补肾经典方六味地黄汤进行佐证。研究结果将初步揭示肾虚椎间盘退变的发生机制，为“肾虚腰痛”理论的现代诠释和临床运用提供实验依据。

腰痛是引起患者丧失劳动能力的十大危险因素之一，全球50%至80%的成人都会在其一生中遭受至少一次的腰痛，而椎间盘退变（IVDD）是引起腰痛的主要原因之一。“肾虚腰痛”是中医药治疗IVDD的理论基础。最新研究表明“肾虚”主要体现为干细胞增殖迁移功能及其微环境稳态的变化；而椎间盘干细胞龛中存在盘源性干细胞，参与IVDD修复。我们前期研究发现椎间盘细胞Wnt/β-catenin信号通路在IVDD过程中起关键作用。为探究“肾虚腰痛”的物质基础及分子机制，我们首先构建了Gli1-CreER;tdTomato示踪工具小鼠，通过与间充质干细胞的各种特异性Marker进行免疫荧光共定位，发现Gli1+细胞和这些Marker的高度重叠（>80%），进而明确Gli1+细胞是一类存在于干细胞龛的盘源性干细胞。接着，利用体内定向动态追踪技术盘源性干细胞的发育生存模式，结果表明，发育过程中Gli1+细胞可逐步移行到椎间盘各个位置，包括纤维环、软骨终板和髓核。另外，通过力学失稳、去卵巢和全身振动等方式建立各种肾虚型IVDD小鼠模型，通过影像学和形态学染色对比筛选最优的IVDD造模方法（力学失稳法）进行后续示踪工具小鼠的IVDD造模。在对比分析IVDD模型中Gli1+细胞的生存模式变化表明，IVDD造模使椎间盘纤维环和髓核中Gli1+细胞数量显著降低；Wnt/β-catenin信号关键蛋白β-catenin的免疫荧光共定位结果表明，IVDD造模使Gli1+细胞中的β-catenin表达显著增加。上述结果表明，IVDD造模激活干细胞Wnt/β-catenin信号致使干细胞数量和迁移能力降低。进一步，通过给予IVDD模型小鼠以补肾经方六味地黄汤干预后，可以显著增加Gli1+细胞数量并降低其β-catenin表达。体外研究表明，六味地黄含药血清干预可以显著增加原代分离髓核细胞和髓核细胞系NP细胞的增殖迁移能力。通过本项目的实施，明确盘源干细胞在IVDD过程中的命运及Wnt/β-catenin信号通路对盘源性干细胞增殖分化中的作用，同时也揭示了补肾中药的干预机制，本研究是“肾虚腰痛”理论进行现代化医学诠释，为临床治疗肾虚IVDD提供重要的实验依据。