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目的探讨鸦胆子素D对骨肉瘤细胞(143B HOS)增殖,骨肉瘤细胞凋亡与自噬的作用。方法用CCK8实验观察鸦胆子素D对143B HOS增殖的影响;用平板克隆实验观察鸦胆子素D对143B HOS细胞集落形成的影响;用流式细胞仪检测鸦胆子素D对143B HOS周期阻滞以及诱导凋亡的作用情况;用Western Blot检测143B HOS基础凋亡与自噬以及经鸦胆子素D诱导之后的凋亡与自噬情况;用荧光显微镜检测143B细胞基础LC3荧光鼬合蛋白表达以及经鸦胆子素D诱导之后的LC3荧光鼬合蛋白表达情况。结果(1)CCK8实验显示鸦胆子素D作用后143B HOS细胞的存活率下降,同时,也减少了143B HOS细胞克隆数的形成;(2)流式细胞仪检测细胞周期显示鸦胆子素D使143B HOS的G0/G1期细胞比例增高;(3)流式细胞仪检测细胞凋亡实验显示,鸦胆子素D使143B HOS的细胞凋亡比例增加;Western Blot检测143B HOS细胞Cleaved PARP的表达,结果显示,鸦胆子素D诱导Cleaved PARP表达增加;(4)Western Blot检测143B HOS细胞LC3B的表达,结果显示,鸦胆子素D诱导LC3B-II的表达增加;荧光显微镜检测143B细胞的LC3荧光鼬合蛋白实验,结果显示,鸦胆子素D诱导143B细胞绿色荧光斑点生成增多。结论 (1)鸦胆子素D抑制骨肉瘤细胞增殖;(2)鸦胆子素D诱导骨肉瘤细胞的凋亡;(3)鸦胆子素D诱导骨肉瘤细胞自噬。
Objective To investigate the effects of Brucein D on the proliferation of osteosarcoma cells (143B HOS), apoptosis of osteosarcoma cells and autophagy. Methods The effect of Brucein D on the proliferation of 143B HOS was observed by CCK8 assay. The effect of Brucein D on the colonization of 143B HOS cells was observed by plate clone assay. The cycle arrest of 143B HOS was detected by flow cytometry Western Blot was used to detect the basic apoptosis and autophagy of 143B HOS and the apoptosis and autophagy after induction with Brucein D. The fluorescence intensity of the basal LC3 was detected by fluorescence microscopy Expression of LC3 CAMP after induction with Brucein D. Results (1) The CCK8 experiment showed that the survival rate of 143B HOS cells decreased with the addition of Brucein D, and the number of 143B HOS cells was also reduced. (2) The cell cycle of FCB showed that Brucein D 143B HOS increased the proportion of G0 / G1 phase cells; (3) Flow cytometry apoptosis test showed that brucellin D 143H HOS apoptosis ratio increased; Western Blot 143B HOS cells Cleaved PARP expression, The results showed that javanin D induced the increase of Cleaved PARP expression; (4) The expression of LC3B in 143B HOS cells was detected by Western Blot. The results showed that the expression of LC3B-II increased with javanin D. The LC3 fluorescence of 143B cells was detected by fluorescence microscopy Protein experiments, the results showed that javanin D-induced 143B cells increased green fluorescent spots. Conclusion (1) Brucein D inhibits proliferation of osteosarcoma cells; (2) Brucein D induces osteosarcoma cell apoptosis; (3) Brucein D induces osteosarcoma cell autophagy.