右归饮诱导造血干细胞分化抗破骨细胞骨溶解的机制研究

Youguiyin was used to treat bone metabolic diseases such as avascular necrosis of femoral head, and it had obtained satisfactory curative effect. Previous studies had showed that Youguiyin could prevent prosthetic appliance loosening by inhibiting osteoclast(OC) and decreasing periprosthetic osteolysis (PO). With OC activating as its key point, PO was caused in the bone marrow. It had been revealed that OC activation could stimulate the release of the hematopoietic stem cell (HSC) which could then differentiate into OC. Youguiyin may regulate hematopoietic stem cell(HSC) to differentiate into hematopoietic cell, not into OC, and inhibite PO. So the researches on the correlation between HSC and OC have turned to be a new chapter, which has become a new research field after the coupled reaction between OC and osteoblast (OB). Based on the animal model of osteolysis caused by polythene, by transfecting lentivirus containing green fluorescent protein into HSC, marking it and tracking its differentiation, this task is to study the mechanism of the PO stimulated by the abnormal differentiation of the HSC by OC activation. We aimed at revealing the function of Youguiyin in inhibiting the differentiation of HSC into OC in endosteal bone marrow HSC niche, inhibiting osteoclast-induced bone osteolysis，remolding the microenvironment. This could provide more theory and mechanism for the clinical treating of the bone absorbing diseases.

临床上右归饮治疗股骨头坏死等骨代谢疾病疗效确切，前期动物实验表明右归饮可抑制破骨细胞(osteoclast, OC)性骨吸收、抗假体周围骨溶解(periprosthetic osteolysis, PO) 防止关节松动。OC活化是PO发病的关键，骨髓微环境内OC活化刺激造血干细胞(hematopoietic stem cell, HSC)大量释放并向OC分化。右归饮可能通过诱导HSC向造血细胞分化，抑制向OC分化，而发挥作用。HSC与OC相关研究是继破骨-成骨细胞偶联后又一研究方向。本课题以聚乙烯颗粒诱导骨溶解模型为基础，应用携带绿色荧光蛋白的慢病毒转染标识HSC并追踪其分化情况，揭示OC活化致HSC分化异常，加速PO的可能机制，探究体内外右归饮抑制HSC在骨内膜下细胞龛分化为OC，诱导其向造血细胞分化，抗OC性骨溶解，重塑骨髓微环境的机制，为右归饮治疗骨溶解疾病提供理论依据。

研究背景：骨髓破骨细胞（osteoclast, OC）活化是假体周围骨溶解（periprosthetic osteolysis, PO）发生的关键，与造血干细胞（hematopoietic stem cell, HSC）的分化刺激有关。前期研究表明，右归饮能调节骨代谢，发挥抑制OC性骨吸收的作用。本项目以聚乙烯颗粒诱导骨溶解模型为基础，追踪HSC分化情况以及右归饮的调节作用。.研究内容：（1）建立小鼠HSC体外培养体系；（2）建立HSC向OC前体细胞分化的培养体系，观察右归饮含药血清体外抑制HSC向OC前体细胞分化的作用；（3）制备HSC向OC前体细胞体内分化小鼠模型；（4）制备PO小鼠模型，明确右归饮抑制HSC向OC分化抗PO内在机制。.重要结果：（1）成功采用c-kit抗体藕联磁珠通过阳性分选的方法，从BABL/c小鼠骨髓细胞中分选高纯度具有造血干/祖细胞多向分化的HSC，建立了HSC体外分选、培养体系；通过ERFP慢病毒成功转染HSC，检测显示成功转染的HSC仍可保持多向分化的能力；（2）采用OC前体细胞集落形成单位（CFU-OC）半固体形成实验、HSC液体培养实验，发现右归饮含药血清体外抑制小鼠骨髓HSC细胞向OC前体细胞分化的作用显著，能减少OC前体细胞生成数量，其机制可能是通过抑制OC特异分化因子RANKL，下调OC分化相关蛋白PU.1的表达；（3）致死剂量60Co-γ射线照射小鼠，出现骨密度下降，提示辐射后体内HSC向OC前体细胞分化，出现大量活化OC致骨吸收活跃，成功建立HSC体内向OC前体细胞分化小鼠模型；（4）经右归饮治疗后，促成骨相关因子AKP上调，小鼠骨髓成骨前体细胞CFU-F数量、OPG mRNA表达水平均有不同程度提高，可见右归饮促PO小鼠C-kit+细胞向OB前体细胞分化，且优于密固达，同时下调OC骨吸收标志酶TRAP和ATP6i mRNA表达水平，可见PO小鼠骨吸收能力受到抑制。此外，右归饮治疗后外周血炎症因子IFN、TNF、IL-6和IL-17A含量均有所下降，甚至接近正常组水平，明显改善了PO小鼠骨髓OC前体细胞分化的内环境。.关键数据及其科学意义：本项目研究揭示了PO的发生机制，从体内外明确了右归饮抑制HSC向OC前体细胞分化、抗OC性骨溶解、重塑骨髓OC前体细胞微环境的作用机制，为右归饮治疗骨溶解疾病提供科学依据。