靶向调控AKT/mTOR信号通路对骨肉瘤细胞MG63增殖、凋亡、自噬及成骨分化的影响

Effects of Targeting Regulation of AKT/mTOR Signaling Pathway on Proliferation, Apoptosis, Autophagy and Osteogenic Differentiation of Osteosarcoma Cell Line MG63

该文探究靶向调控AKT/mTOR信号通路后,对人骨肉瘤细胞株(MG63)增殖、凋亡、自噬及成骨分化的影响并探讨其机制。RT-PCR检测在不同恶性程度骨肉瘤细胞中AKT、mTOR基因表达的情况;选择靶向mTOR信号通路的抑制剂雷帕霉素和激活剂3-苄基-5-(2-硝基苯氧甲基)-γ-丁内酯(3-BDO),分别用CCK-8检测细胞增殖;DAPI染色、Annexin V-FITC/PI双染法检测凋亡;碱性磷酸酶(ALP)染色检测早期成骨能力;茜素红染色检测中晚期成骨能力;Western blot技术检测自噬相关蛋白及分化抑制因子(Id1)表达。结果显示,AKT/mTOR表达情况与骨肉瘤恶性程度有关;通过靶向抑制AKT/mTOR信号通路后,可抑制骨肉瘤细胞MG63增殖,促进凋亡,上调自噬水平,抑制其早、晚期成骨分化;靶向激活AKT/mTOR信号通路后,对骨肉瘤细胞MG63增殖、凋亡无明显影响,下调自噬水平,但可促进其早、晚期成骨分化。该研究表明,靶向调控AKT/mTOR信号通路与分化抑制因子(Id1)表达有关,可进一步阐明骨肉瘤发病机制,为诱导分化治疗提供理论依据。

The aim of this paper was to investigate the effects of targeted regulation of AKT/mTOR signaling pathway on the proliferation, apoptosis, autophagy and osteogenic differentiation of human osteosarcoma cell line（MG63）, and to explore its mechanism. RT-PCR was used to detect m RNA expression of AKT and m TOR in osteosarcoma cells with different degrees of malignancy. Rapamycin was used as a targeted inhibitor of the mtor signaling pathway, and 3-BDO was used as a targeted activator of the mtor signaling pathway. Cell proliferation activity was detected by CCK-8 assay, apoptosis was detected by DAPI staining and Annexin V-FITC/PI double staining; alkaline phosphatase（ALP） staining was used to detect cell early osteogenic differentiation ability. Alizarin red staining was used to detect cell middle-late osteogenic differentiation ability. Autophagy and Id1 was detected by Western blot. The results showed that expression of AKT/mTOR was related to the degree of malignancy of osteosarcoma. After targeted inhibition of AKT/mTOR signaling pathway, inhibitor of m TOR signaling pathway could inhibit the proliferation of osteosarcoma cell MG63 and promote apoptosis, upregulate autophagy, but inhibite early and late osteogenic differentiation. After targeted activation of AKT/mTOR signaling pathway, there was no significant effect on the proliferation and apoptosis of osteosarcoma cell MG63, down-regulated autophagy, but promoted its early and late osteogenic differentiation. This study indicated that targeted regulation of AKT/mTOR signaling pathway was related to the expression of differentiation suppressor factor（Id1）, which could further elucidate the pathogenesis of osteosarcoma and provide theoretical basis for inducing differentiation therapy.