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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 its effect on osteoclasts in osteoporotic osteoclasts of postmenopausal women and to understand the relationship between estrogen and Hedgehog signaling. Methods Three osteoblasts derived from ovariectomized osteoporosis (OVX, Ovariotomized) mice and three sham - operated mice were cultured, and the activity of Hedgehog signaling was compared between the two groups. RAW264.7 cells were cultured in osteoclast-free medium with phenol red free medium and randomly divided into 6 groups: estrogen group, estradiol group with 10-8 M concentration intervention; estrogen + agonist group, 10- 8 M concentration of estradiol and 1μm Hedgehog signal agonist Purmorphamine intervention; estrogen + antagonist group, given 10-8 M concentration of estradiol and 1μm Hedgehog signaling antagonist Vismdegib intervention; control group, without any The rats in the antagonist group were treated with Purmorphamine at a concentration of 1 μm. The antagonist group was treated with Vismdegib at a concentration of 1 μm. The levels of Gli1 and Ptch1 were detected by quantitative PCR. The estrogen group, the antagonist group and the control group were respectively subjected to TRAP staining and F-actin staining to detect the number of osteoclasts. Results (1) Compared with normal mouse-derived osteoclasts, the levels of Ptch1 Gli1 and Ptch1 Gli1 in OVX mouse-derived osteoclasts were 0.72400 ± 0.04272 and 0.66794 ± 0.07331, respectively, higher than that of normal mouse-derived cells (0.44196 ± 0.06822,0.45229 ± 0.05750, the difference was statistically significant (P <0.05); (2) The expression of Gli1 Ptch1 was 0.4131 ± 0.02511 and 0.54165 ± 0.03931, respectively, lower than the control group 1.00000 ± 0.11771,0.74160 ± 0.07632, the difference was statistically significant P <0.05). The expressions of Ptch1 and Gli1 in estrogen + agonist group were significantly lower than those in the agonist group (0.38557 ± 0.06785, 1.00000 ± 0.03138, 0.86403 ± 0.05886,1.45856 ± 0.05911, P <0.01) Compared with the control group, the number of osteoclasts in antagonist group and estrogen group was lower (P <0.05). Conclusions (1) Hedgehog signaling activity in osteoporotic osteoclasts after menopause is increased; (2) Estrogen inhibits Hedgehog signaling in osteoclasts; (3) Inhibition of Hedgehog signaling can reduce osteoclast Quantity, improve osteoclastogenesis when estrogen deficiency is excessive.