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目的研究全反式维甲酸(all-trans-retinoic acid,ATRA)与骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)对人骨肉瘤细胞增殖及分化的影响。方法利用四甲基偶氮唑蓝(MTT)法和流式细胞仪分析细胞增殖情况;蛋白质免疫印迹实验(Western blot)检测细胞增殖和成骨相关指标;半定量PCR及Western blot分析ATRA和ATRA与BMP9联用处理143B细胞株后对BMP9表达水平的影响。结果在人骨肉瘤143B细胞株中,与对照组相比,ATRA能明显抑制细胞增殖(P<0.05),并促使细胞周期阻滞在G1期(P<0.05),下调PCNA的蛋白水平,并呈浓度依赖性;ATRA能呈浓度依赖性增加晚期成骨指标OCN和OPN的蛋白水平(P<0.05);ATRA能上调BMP9的m RNA及蛋白水平,并呈浓度依赖性(P<0.05);单用BMP9并不能上调OCN和OPN表达,并促进其增殖(P<0.05),但与ATRA联用时,能够增强ATRA诱导的相关成骨指标表达和抗增殖作用(P<0.05)。结论ATRA对143B细胞的增殖具有抑制作用并诱导其成骨分化,并可能恢复BMP9的成骨诱导能力。
Objective To investigate the effects of all-trans-retinoic acid (ATRA) and bone morphogenetic proteins 9 (BMP9) on the proliferation and differentiation of human osteosarcoma cells. Methods MTT assay and flow cytometry were used to analyze cell proliferation. Western blotting was used to detect cell proliferation and osteogenesis-related indices. Semi-quantitative PCR and Western blot were used to analyze ATRA and ATRA Effect of BMP9 in combination with BMP9 on the expression of BMP9. Results In human osteosarcoma 143B cell line, ATRA inhibited cell proliferation significantly (P <0.05) and promoted cell cycle arrest in G1 phase (P <0.05), and down-regulated the protein level of PCNA compared with control group (P <0.05). ATRA could upregulate the mRNA and protein levels of BMP9 in a concentration-dependent manner (P <0.05). Single dose of ATRA could increase the expression of OCN and OPN in a concentration-dependent manner BMP9 could not up-regulate the expression of OCN and OPN, and promote the proliferation (P <0.05). However, when combined with ATRA, the expressions of osteoblast-related genes and anti-proliferative effects induced by ATRA were enhanced (P <0.05). Conclusion ATRA can inhibit the proliferation of 143B cells and induce its osteogenic differentiation, and may restore the osteogenic potential of BMP9.