FTY720-P对成骨细胞分化成熟的作用

FTY720-P promotes the differentiation and maturation of osteoblasts

目的以FTY720-P作用于成骨细胞后,分析相关蛋白和基因表达的变化,明确FTY720-P对成骨细胞的作用机制。方法取Wistar大鼠乳鼠颅盖骨进行成骨细胞原代培养。将不同浓度的FTY720-P(0、200、300、400 ng/m L)作用于成骨细胞,MTT试验和流式细胞术进行生长速率和细胞周期的检测,碱性磷酸酶(ALP)染色和钙化结节染色对比FTY720-P用药前后对细胞形态学功能的影响,并分别采用ELISA、Western blot和Real Time RT-PCR检测相关蛋白和基因的表达。结果 MTT显示FTY720-P对细胞生长影响不大。流式细胞术结果提示,细胞阻滞在G_2期,对周期影响不大。ALP染色显示对照组蓝紫色颗粒更加明显;茜素红染色可见数量较多和更加明显的钙化结节。PCR结果中ALP的表达是下调的,骨钙素(OCN)和胰岛素样生长因子(IGF)的表达是上调的;而在ELISA实验中,OCN和IGF的分泌是增加的,Western blot检测中,胞外胶原蛋白I型分泌也增加,而在ALP活性实验中,胞内ALP活性是降低的。结论 FTY720-P可以促进成骨细胞细胞外基质的成熟和矿化,但对细胞增殖和周期没影响。

Objective To clarify the mechanism of FTY720 - P on osteoblast. Methods Primary osteoblasts were obtained from the skull of Wistar neonatal rats. The growth rate and cell cycle were detected by MTT assay and flow cytometry. Alkaline phosphatase assay and calcified nodule staining were used to compare the morphological changes of FTY720 - P cells before and after treatment with FTY720 - P. The expression of related proteins and genes were detected by ELISA, Western blot and real time RT - PCR. Results MTT showed that FTY720 - P had little effect on cell growth. Flow cytometry showed that the cells were arrested at G2 phase and cell cycle was little affected. Compared with the con- trol group, alizarin red staining showed increased and distinct calcified nodules were observed in FTY720 - P treatment group. The blue -violet granules were more prominent in control group than the experimental group in the alkaline phos- phatase staining. The expression of OCN and IGF was up - regulated, while the expression of ALP was down - regulated in experimental group. The secretion of OCN and IGF were increased in experimental group, and so was the secretion of ex- tracellular collagen type I. Intracellular ALP activity was reduced in experimental group. Conclusion FTY720 - P can promote the maturation and mineralization of extracellular matrix of osteoblasts, but has no effect on cell growth and cycle.