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目的研究初级纤毛对蛇床子素促进成骨细胞成骨性分化的影响。方法取新生SD大鼠颅骨,多次酶消化法获得成骨细胞,免疫荧光染色观察成骨细表面初级纤毛,RNA干扰法抑制鞭毛运输系统蛋白(IFT88)的表达,从而抑制成骨细胞初级纤毛的产生。以浓度为1×10-6 mol×L~(-1)蛇床子素分别作用于RNA干扰成骨细胞,3,6 d后检测胞内碱性磷酸酶(ALP)活性;药物处理24 h后,Real-time PCR检测1型胶原(collagen 1,COL-1)、RUNX-2、ALP mRNA的表达量;Western blot检测COL-1、RUNX-2蛋白表达量。结果超过70%的成骨细胞表面具有长度约为5μm的初级纤毛,RNA干扰法显著抑制IFT88基因和蛋白的表达,并抑制了初级纤毛的发生。1×10-6 mol×L~(-1)蛇床子素能够显著地促进胞内ALP的活性,成骨性分化相关的因子COL-1、RUNX-2和ALP基因的表达也相应增高,同时COL-1、RUNX-2蛋白表达量显著增加。当成骨细胞初级纤毛干扰后,药物促进ALP活性升高的作用消失,COL-1、Runx-2基因和蛋白的表达量也相应的降低。结论成骨细胞初级纤毛的去除,能够显著性地抑制蛇床子素促进成骨细胞成骨性分化的能力。
Objective To study the effect of primary cilia on osteoblast differentiation induced by osthole. Methods Osteoblasts were obtained from the skulls of newborn SD rats by multiple enzymatic digestion. The primary cilia on the surface of osteoblasts were observed by immunofluorescence staining. The RNA interference method inhibited the expression of IFT88 and inhibited the primary cilia of osteoblasts The production. Osteoblasts were treated with RNA at a concentration of 1 × 10-6 mol × L ~ (-1) osthole respectively, and alkaline phosphatase (ALP) activity was detected 3 and 6 days later. After 24 h Real-time PCR was used to detect the expression of collagen 1 (COL-1), RUNX-2 and ALP mRNA. Western blot was used to detect the expression of COL-1 and RUNX-2. RESULTS: More than 70% of the primary cilia on the osteoblast surface had a length of about 5μm. RNA interference significantly inhibited the expression of IFT88 gene and protein and inhibited the occurrence of primary cilia. 1 × 10-6 mol × L -1 osthole could significantly promote intracellular ALP activity, and the expression of osteogenic differentiation-related factors COL-1, RUNX-2 and ALP gene also increased correspondingly COL-1, RUNX-2 protein expression increased significantly. When the primary cilia of osteoblasts interfered, the role of drugs in promoting the increase of ALP activity disappeared, and the expression of COL-1 and Runx-2 genes and proteins also decreased correspondingly. Conclusion The removal of primary cilia of osteoblasts significantly inhibits the ability of osthole to promote the osteogenic differentiation of osteoblasts.