Hedgehog通路对成骨细胞增殖和分化的作用

Hedgehog pathway is associated with the proliferation and differentiation of osteoblasts

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。

PURPOSE： The purpose of this study was to evaluate the effects of Hedgehog signaling pathway on osteoblast proliferation and differentiation. METHODS： Primary osteoblasts harvested from newborn rat ealvarial bone were cultured for 21 days. The temporary expression of Shh and Ihh was detected by RT-PCR. The culture medium was then treated with either a recombinant Shh N-terminals protein （N-Shh）or cyelopamine （cy）, a Hedgehog inhibitor. The proliferation of osteoblasts was quantified by MTF and FCM. The osteoblast differentiation and matrix mineralization were evaluated by ALP and Alizarin-Red staining. The expression of Ptch and Smo was quantified by real-time PCR.SAS 8.0 software package was used for statistical analysis. RESULTS： Osteoblast expressed both Shh and Ihh. The expression level of Shh decreased with culture time while the amount of Ihh expression increased. N-Shh treatment increased the cell population and the number of cells in S phase （P〈0.05）. N-Shh also promoted ALP activities （P〈0.05）and bone matrix calcification. Moreover, N-Shh application led to an increase in Ptch and Smo mRNA level （P〈0.05）. Treatment with cy, on the other side, inhibited osteoblast proliferation and differentiation （P〈0.05）. CONCLUSION： Heghehog signaling pathway is actively involved in regulating osteoblast proliferation and differentiation. Supported by Research Fund of Science and Technology Commission of Shanghai Municipality （Grant No.06QA14031 and 08DZ2271100） and Shanghai Leading Academic Discipline Project（Grant No.S30206）.