【摘要】：目的以FTY720-P作用于成骨細(xì)胞后,分析相關(guān)蛋白和基因表達(dá)的變化,明確FTY720-P對成骨細(xì)胞的作用機(jī)制。方法取Wistar大鼠乳鼠顱蓋骨進(jìn)行成骨細(xì)胞原代培養(yǎng)。將不同濃度的FTY720-P(0、200、300、400 ng/m L)作用于成骨細(xì)胞,MTT試驗(yàn)和流式細(xì)胞術(shù)進(jìn)行生長速率和細(xì)胞周期的檢測,堿性磷酸酶(ALP)染色和鈣化結(jié)節(jié)染色對比FTY720-P用藥前后對細(xì)胞形態(tài)學(xué)功能的影響,并分別采用ELISA、Western blot和Real Time RT-PCR檢測相關(guān)蛋白和基因的表達(dá)。結(jié)果 MTT顯示FTY720-P對細(xì)胞生長影響不大。流式細(xì)胞術(shù)結(jié)果提示,細(xì)胞阻滯在G_2期,對周期影響不大。ALP染色顯示對照組藍(lán)紫色顆粒更加明顯;茜素紅染色可見數(shù)量較多和更加明顯的鈣化結(jié)節(jié)。PCR結(jié)果中ALP的表達(dá)是下調(diào)的,骨鈣素(OCN)和胰島素樣生長因子(IGF)的表達(dá)是上調(diào)的;而在ELISA實(shí)驗(yàn)中,OCN和IGF的分泌是增加的,Western blot檢測中,胞外膠原蛋白I型分泌也增加,而在ALP活性實(shí)驗(yàn)中,胞內(nèi)ALP活性是降低的。結(jié)論 FTY720-P可以促進(jìn)成骨細(xì)胞細(xì)胞外基質(zhì)的成熟和礦化,但對細(xì)胞增殖和周期沒影響。
[Abstract]:Objective to investigate the effect of FTY720-P on osteoblasts by analyzing the changes of protein and gene expression in osteoblasts. Methods the calvaria of Wistar rats was cultured in primary culture. Osteoblasts were treated with different concentrations of FTY720-P (0200300400 ng/m L). The growth rate and cell cycle were measured by MTT test and flow cytometry. Alkaline phosphatase (ALP) staining and calcified nodule staining were used to compare the effects of FTY720-P on cellular morphology, and ELISA,Western blot and Real Time RT-PCR were used to detect the expression of related proteins and genes. Results MTT showed that FTY720-P had little effect on cell growth. The results of flow cytometry showed that the cells were blocked in the second phase of Gal and had little effect on the cycle. ALP staining showed that the blue purple granules were more obvious in the control group. The expression of ALP was down-regulated and the expression of osteocalcin (OCN) and insulin-like growth factor (IGF) was up-regulated in PCR. In ELISA assay, the secretion of OCN and IGF was increased in, Western blot assay, and the type I secretion of extracellular collagen was also increased. In ALP activity test, the intracellular ALP activity was decreased. Conclusion FTY720-P can promote the maturation and mineralization of extracellular matrix of osteoblasts, but has no effect on cell proliferation and cell cycle.
【作者單位】： 廣東醫(yī)科大學(xué)研究生學(xué)院;廣東省第二人民醫(yī)院創(chuàng)傷骨科;南方醫(yī)科大學(xué)研究生學(xué)院;
【基金】：廣東省科技計(jì)劃項(xiàng)目(編號:2016A020215124)
【分類號】：R68

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