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目的:分析Wnt-β-catenin信号通路在骨肉瘤发展中的作用和对化疗效果的影响。方法:采用免疫组织化学、实时定量PCR与Western blotting比较人成骨细胞(human fetal osteoblasts,h FOB)和骨肉瘤(human OS,Saos2)细胞及人骨肉瘤细胞样本中Wnt-β-catenin信号通路相关分子的表达,比较h FOB和Saos2细胞的表达差异。采用萤光素酶实验观察Wnt-β-catenin、Notch、Hh信号通路对氨甲喋呤(methotrexate,MTX)疗效的调控。结果:同h FOB细胞相比该通路的主要分子包括:Wnt3(5.5倍)、β-catenin(5.3倍)、LEF1(7.6倍),在Saos2细胞中表达明显上调。Western blotting分析表明总β-catenin以及活化β-catenin的表达都升高。MTX处理后诱导了Saos2细胞凋亡和坏死。对Wnt-β-catenin、Notch、Hh信号通路的化学抑制也能够诱导细胞死亡,Wnt-β-catenin抑制剂更为明显。结论:采用小分子/化合物来抑制Wnt-β-catenin和Notch信号,并同目前常用的OS药物化疗联合使用,对于复发和转移的患者,有望改善患者的生存期。
OBJECTIVE: To analyze the role of Wnt-β-catenin signaling in the development of osteosarcoma and its effect on the efficacy of chemotherapy. Methods: The Wnt-β-catenin signaling pathway was compared between human fetal osteoblasts (h FOB) and human OS (Saos2) cells and human osteosarcoma cell lines by immunohistochemistry, real-time quantitative PCR and Western blotting Molecule expression, h FOB and Saos2 cells compared expression differences. The luciferase assay was used to observe the effects of Wnt-β-catenin, Notch and Hh signaling pathway on the efficacy of methotrexate (MTX). Results: The main molecules of this pathway were Wnt3 (5.5-fold), β-catenin (5.3-fold) and LEF1 (7.6-fold), which were significantly up-regulated in Saos2 cells compared with h FOB cells. Western blotting analysis showed that the expression of total β-catenin and activated β-catenin increased. MTX treatment induced Saos2 cell apoptosis and necrosis. The chemical inhibition of Wnt-|Â-catenin, Notch, Hh signaling pathway can induce cell death, Wnt-|Â-catenin inhibitor is more obvious. CONCLUSIONS: The use of small molecules / compounds to inhibit Wnt-|Â-catenin and Notch signaling, in combination with the currently used OS drug chemotherapy, is expected to improve patient survival in patients with recurrence and metastasis.