绝经后骨质疏松小鼠中Hedgehog信号对破骨细胞的作用

Osteoclast function of Hedgehog signaling in postmenopausal osteoporosis mice

目的探讨绝经后骨质疏松破骨细胞中Hedgehog信号的活性及其对破骨细胞的作用,了解雌激素与Hedgehog信号的关系。方法利用绝经后骨质疏松模型(OVX,Ovariotomized)小鼠3只与假手术组小鼠3只来源的破骨细胞进行培养,对比两组细胞Hedgehog信号的活性。将RAW264.7细胞用无酚红培养基进行破骨诱导培养,并随机分为6组:雌激素组,给予雌二醇以10-8 M浓度干预;雌激素+激动剂组,给予10-8 M浓度雌二醇和1μm浓度的Hedgehog信号激动剂Purmorphamine进行干预;雌激素+拮抗剂组,给予10-8 M浓度雌二醇和1μm浓度的Hedgehog信号拮抗剂Vismdegib进行干预;对照组,不给予任何干预;激动剂组,给予1μm浓度的Purmorphamine进行干预;拮抗剂组,给予1μm浓度的Vismdegib进行干预,定量PCR检测各组Hedgehog信号水平Gli1和Ptch1的表达。对雌激素组、拮抗剂组和对照组分别进行TRAP染色和F-actin染色,以检测破骨细胞数量。结果 (1)与正常小鼠来源的破骨细胞相比,OVX小鼠来源的破骨细胞中Ptch1 Gli1和Ptch1 Gli1表达量0.72400±0.04272、0.66794±0.07331水平更高于正常小鼠来源的量0.44196±0.06822、0.45229±0.05750,差异有统计学意义(P<0.05);(2)Gli1 Ptch1表达量0.4131±0.02511、0.54165±0.03931较对照组1.00000±0.11771、0.74160±0.07632低,差异有统计学意义(P<0.05);雌激素+激动剂组中Ptch1和Gli1表达水平0.38557±0.06785、1.00000±0.03138较激动剂组0.86403±0.05886、1.45856±0.05911低,差异有统计学意义(P<0.01);(3)与对照组相比,拮抗剂组与雌激素组破骨细胞数量均较低(P<0.05)。结论 (1)绝经后骨质疏松破骨细胞中Hedgehog信号活性提高;(2)破骨细胞中雌激素对Hedgehog信号有抑制作用;(3)体外培养时,抑制Hedgehog信号能降低破骨细胞的数量,改善雌激素缺乏时破骨细胞过多的情况。

Objective To investigate the activity of Hedgehog signaling and the relationship between estrogen and Hedgehog signaling in postmenopausal osteoporotic osteoclasts, and the effect of Hedgehog signaling on osteoporotic osteoclasts. Methods Culturing osteoclasts from postmenopausal ovariectomized osteoporosis （ OVX ） group mice （ n=3 ） and sham group mice （ n=3 ）, and compare the activity of Hedgehog signaling in different groups. RAW264.7 cells with osteoclastic inductive factors dissolved in phenol red free α-MEM culture medium. Cells were divide into 6 groups randomly： estrogen group, supplemented with 1 713-estradiol at the concentration of 10-8 M; estrogen ＋ agonist group, supplemented with 17β-estradiol at the concentration of 10-8 M and Hedgehog signaling agonist Purmorphamine at the concentration of 1 μm; estrogen ＋ antagonist group, supplemented with 17β-estradiol at the concentration of 10-8 M and Hedgehog signaling antagonist Vismdegib at the concentration of 1 μm; control group, no extra interference; agonist group, supplemented with Purmorphamine at the concentration of 1 μm; antagonist group, supplemented with Vismdegib at the concentration of 1 μm. Comparing the activity of Hedgehog signaling in different groups. Comparing the number of osteoclasts in estrogen group, antagonist group and control group by TRAP staining and F-actin staining. Results （ 1 ） Expression levels of Ptchl and Glil （ 0.72400±0.04272, 0.66794±0.07331 ） in osteoclasts from OVX group mice was higher than that of the sham group （ 0.44196±0.06822, 0.45229±0.05750 individually ）, which was statistically significant （ P〈0.05 ）. （ 2 ） Expression levels of Ptchl and Glil （ 0.4131±0.02511, 0.54165±0.03931 ） in osteoclasts from estrogen group was higher than that of the control group （ 1.00000±0.11771, 0.74160±0.07632 ）, which was statistically significant （ P〈0,05 ）. Expression level of Gli 1 and Ptchl （ 0.38557±0.06785, 1.00000±0.03138 ） in osteoclasts from estrogen ＋ agonist group was lower than that of the agonist group （ 0.86403±0.05886, 1.45856±0.05911 ）, which was statistically significant （P〈0.01）. （ 3 ） Compared with control group, less osteoclasts existed in estrogen group and antagonist group. Conclusions （ 1 ） Hedgehog signaling activity in postmenopausal osteoporotic osteoclasts was higher than the normal. （ 2 ） Estrogen inhibited Hedgehog signaling activity in osteoclast. （ 3 ） By inhibiting Hedgehog signaling, osteoclast number could be decreased thus over-activated osteoclast in estrogen deficient circumstance could be compromised in vitro.