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目的:研究Hedgehog通路因子Shh和Ihh对成骨细胞增殖和分化的影响。方法:体外分离和培养新生大鼠颅顶骨成骨细胞,RT-PCR检测Shh和Ihh信号的表达;用HedgehogN端重组蛋白(N-Shh)及Hedgehog通道抑制剂Cyclopamine(cy)对成骨细胞进行干预,分别采用MTT比色法、流式细胞仪、碱性磷酸酶(ALP)定性定量、茜素红染色检测成骨细胞的增殖、分化及基质钙化情况;实时定量PCR检测Hedgehog通路相关基因Ptch和Smo的表达。采用SAS8.0软件包对数据进行t检验。结果:在成骨细胞体外生长过程中,Shh的表达逐渐减弱,而Ihh的表达逐渐增强;N-Shh促进成骨细胞的增殖(P<0.05)和S期细胞比例增加(P<0.05),促进ALP的活性并促进Ptch和Smo表达(P<0.05);cy则抑制成骨细胞的增殖和分化(P<0.05)。结论:Hedgehog通路参与对成骨细胞增殖和分化的调节。
Objective: To study the effects of Hedgehog pathway factors Shh and Ihh on the proliferation and differentiation of osteoblasts. Methods: The osteoblasts were isolated and cultured in vitro. The expression of Shh and Ihh was detected by RT-PCR. The osteoblasts were treated with Hedgehog N-terminal recombinant protein (N-Shh) and Hedgehog channel inhibitor Cyclopamine (cy) The cell proliferation, differentiation and matrix calcification of osteoblasts were detected by MTT colorimetric assay, flow cytometry, alkaline phosphatase (ALP) qualitative analysis and Alizarin red staining respectively. The expression of Hedgehog pathway-related gene Ptch And Smo expression. SAS8.0 software package for t-test data. Results: During the in vitro growth of osteoblasts, the expression of Shh decreased and the expression of Ihh gradually increased. N-Shh promoted the proliferation of osteoblasts (P <0.05) and the proportion of cells in S phase (P <0.05) Promote the activity of ALP and promote the expression of Ptch and Smo (P <0.05); cy inhibited the proliferation and differentiation of osteoblasts (P <0.05). Conclusion: The Hedgehog pathway is involved in the regulation of osteoblast proliferation and differentiation.