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半枝莲碱对人骨肉瘤细胞增殖、侵袭与迁移的抑制作用及其机制
作者:李国湘1  方业汉2  吴彬1 
单位:1. 定安县人民医院 骨科, 海南 定安 571200;
2. 海南省人民医院 骨科, 海南 海口 570311
关键词:半枝莲碱 骨肉瘤 Wnt/β-连环蛋白 增殖 侵袭 迁移 
分类号:R738.1
出版年·卷·期(页码):2021·40·第四期(456-462)
摘要:

目的:分析和确认半枝莲碱(SBT)是否对骨肉瘤细胞具有一定的抑制作用,并对SBT抑制骨肉瘤细胞的相关分子机制进行初步解析。方法:四甲基偶氮唑盐(MTT)实验和克隆形成实验检测细胞增殖,Horchest 33258染色检测细胞凋亡,划痕实验检测细胞迁移能力,细胞侵袭迁移实验检测细胞侵袭能力;蛋白质印迹法检测凋亡相关分子B淋巴细胞瘤-2基因相关启动子(BAD)、B淋巴细胞瘤-2基因(BCL2)和活化的半胱氨酸蛋白酶3(cleaved caspase 3)的蛋白水平,β-连环蛋白(β-catenin)、原癌基因(C-myc)、G1/S-特异性周期蛋白-D1(Cyclin D1)、糖原合成酶激酶-3β(GSK3β)的蛋白水平及GSK3β第9位丝氨酸(Ser9)的磷酸化水平;荧光素酶报告基因实验检测TopLuc荧光素酶活性。结果:SBT可抑制骨肉瘤细胞的增殖,减弱细胞迁移和侵袭;促进骨肉瘤细胞发生凋亡,抑制BCL蛋白水平但上调BAD和cleaved caspase 3蛋白水平;抑制TopLuc荧光素酶活性、Ser9的磷酸化,下调β-catenin及其靶分子C-myc和Cyclin D1的蛋白水平,最终使Wnt/β-catenin信号失活。结论:SBT可抑制人骨肉瘤细胞的增殖、迁移和侵袭,促进骨肉瘤细胞发生凋亡,这可能与SBT抑制Wnt/β-catenin信号途径有关。

Objective: To analyze and confirm whether scutebarbatine(SBT) has a certain inhibitory effect on osteosarcoma cells, and to preliminarily analyze its molecular mechanism in inhibiting osteosarcoma cells. Methods: Cell proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay and colony formation assay. Apoptosis, migration and invasion ability of the cells were respectively determined by Hoechst 33258 staining, scratch test and by cell invasion and migration assays. Western blot was used to analyze the protein levels of apoptosis related molecules, including BAD, BCL2 and cleaved caspase 3. The luciferase activity of TopLuc was detected by luciferase reporter gene assay. Western blot was used to detect the expression of β-Catenin, proto-oncogene(C-myc), G1/s-specific cyclin D1, glycogen synthase kinase-3β(GSK3β) and the phosphorylation of serine 9(Ser9) of GSK3 β. Results: SBT could inhibit the proliferation, migration and invasion of osteosarcoma cells, promote apoptosis of osteosarcoma cells and inhibit BCL protein level, but it upregulated BAD and cleaved caspase 3 protein levels. SBT inhibited the activity of TopLuc luciferase and Ser9, down-regulated the protein levels of β-catenin and its target molecules, C-myc and cyclin D1, which ultimately led to Wnt/β-catenin signal inactivated. Conclusion: SBT can inhibit the proliferation, migration and invasion of human osteosarcoma cells, and promote the apoptosis of osteosarcoma cells, which may be related to the inhibition of Wnt/β-Catenin signaling pathway by SBT.

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