补骨脂素通过调控CD4^+T细胞分化抑制RAW264.7向破骨细胞分化和骨吸收

Psoralen inhibits RAW264. 7 differentiation into osteoclasts and bone resorption by regulating CD4^+T cell differentiation

探讨补骨脂素（psoralen）是否通过调控CIM^＋T细胞分化抑制RANKL诱导RAW264．7向破骨细胞分化及骨吸收作用，初步阐明补骨脂素抗骨质疏松作用的骨免疫学机制。采用免疫磁珠分选法从Balb／c小鼠脾脏细胞分离制备CD4^＋T细胞，分为空白组和补骨脂素组接种于74孑L板，加入补骨脂素诱导培养3d，第4天收集各组细胞用于共培养实验。共培养实验分为RAW264．7细胞组（a组）、RAW264．7细胞^＋psoralen-^＋CD4^＋T细胞组（b组）、RAW264．7细胞＋补骨脂素组（终浓度为10μmol·L^-1，C组）、RAW264．7细胞＋psoralen^＋CIM^＋T细胞组（d组）等4组，共培养5d后，TRAP染色检测破骨细胞数目，共培养8d后，骨片用甲苯胺蓝染色评价骨吸收情况，RT．PCR检测CIM^＋T细胞RORTt，Foxp3，IL-17，TNF-α，TGF-β和IL-10等基因的表达，以及破骨细胞相关基因MMP-9，TRAP和Cat-K的表达，ELlSA试剂盒检测1L-17，TNF-α，TGF-β和IL-10等细胞因子的水平。结果显示，补骨脂素显著促进CD4^＋T细胞的Foxp3，TGF-β和IL-10的表达，抑制CIM’T细胞的RORTt，IL-17和TNF-α的表达。psoralen-CD4＋T细胞能显著促进RANKL诱导RAW264．7向破骨细胞分化，psoralen＋CIM＋T细胞能显著抑制RANKL诱导RAW264．7向破骨细胞分化及骨吸收作用。补骨脂素通过促进CD4^＋T细胞中CD4^＋CD25’Treg／Thl7平衡向CD4^＋CD25^＋Treg发展，从而抑制RANKL诱导RAW264．7向破骨细胞分化及骨吸收作用。

This paper aimed to investigate whether psoralen inhibits the differentiation and bone resorption by regulating CD4^＋ T cell differentiation in RANKL-induced osteoclastogenesis in RAW264. 7 cells, and elucidate its mechanism for osteoporosis. CD4^＋ T cells were isolated from spleen cells of Balb/c mice by immunomagnetic separation method. The ceils were divided into blank control group and psoral.en group. Tile cells were cultured in 24-well plates and cultured for 3 days, and then they were collected for co-culture ex- periments after 4 days. Co-culture experiments were divided into RAW264. 7 cell group, psoralen ＋ RAW264. 7 cell group, without psoralen treatment of CD4^＋ T cells ＋ RAW264. 7 cell group, psoralen treatment of CD4^＋ T cells ＋ RAW264. 7 cell group. After 5 days of co-culture, TRAP staining was used to detect the number of osteoclasts, and after 8 days of co-culture, bone resorption was evaluated by toluidine blue staining. The expressions of RORTt, Foxp3, IL-17, TNF-α, TGF-β and IL-10 in CD4^＋ T cells and osteoclast differ- entiation-related genes MMP-9, TRAP and Cat-K were detected by Real-time polymerase chain reaction （RT-PCR） ; ELISA kit was used to detect IL-17, TNF-α, TGF-β and IL-10 and other cytokines levels. Our data confirmed that the psoralen significantly promoted the expression of Foxp3, TGF-α and IL-10 in CD4^＋T, and inhibited the expression of RORTt, IL-17 and TNF-α in CD^4＋ T, the CD4^＋ T cells without treatment by psoralen can significantly promote RANKL-induced differentiation of RAW264. 7 to osteoclasts, and psoralen treatment of CD^4＋ T can significantly inhibit RANKL-induced RAW264. 7 osteoclast differentiation and bone resorption. Taken togeth- er, psoralen inhibits the differentiation and bone resorption of RAW264. 7 into osteoclasts by promoting the development of CD^4＋ CD25^＋ Treg/Thl7 balance in CD^4＋T cells to CD^4＋ CD25^＋ T.