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研究莲房原花青素(LSPCs)诱导肝癌Hep G2细胞凋亡的机制。利用MTT法检测LSPCs对肝癌Hep G2细胞的生长抑制作用,Hochest 33258染色观察凋亡细胞的核形态,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡水平,彗星实验用来检测细胞DNA损伤,JC-1染色用来检测线粒体膜电位,Western blotting法检测细胞凋亡蛋白水平的表达。LSPCs作用细胞后,显著抑制Hep G2细胞的增殖;细胞凋亡征象明显,核染色体高度凝聚,细胞核碎裂,核固缩,染色质凝聚的细胞数从3.86%增至42.76%(p<0.01);活细胞率急剧减少,而凋亡率从5.32%增至67.05%(p<0.01);LSPCs诱发细胞产生DNA损伤,线粒体膜电位下降,导致Cytochromec、Caspase-3和Caspase-9蛋白的表达量增加,Bax蛋白的表达量上调,Bcl-2蛋白的表达量下调,Bax/Bcl-2的比值上升。而凋亡抑制剂Z-VAD能够削弱LSPCs对Hep G2细胞增殖的抑制作用,显著下调细胞核聚缩,抑制线粒体损伤及Caspase相关蛋白的表达量,最终阻断LSPCs的致凋亡作用。由此得出结论,LSPCs可通过线粒体介导的内源性Caspase途径诱导人肝癌细胞凋亡。
To investigate the mechanism of lotus proanthocyanidins (LSPCs) inducing apoptosis in Hep G2 cells. The inhibitory effects of LSPCs on Hep G2 cells were detected by MTT assay. The morphology of apoptotic cells was observed by Hochest 33258 staining. Cell apoptosis was detected by Annexin V-FITC / PI double staining flow cytometry. DNA damage, JC-1 staining was used to detect mitochondrial membrane potential, and Western blotting was used to detect the expression of apoptosis protein. LSPCs significantly inhibited the proliferation of Hep G2 cells. The apoptotic signs were obvious, the nuclear chromosomes were highly aggregated, and the number of nucleus fragmentation and nuclear pyknosis increased from 3.86% to 42.76% (p <0.01) (P <0.01). The DNA damage induced by LSPCs and the decrease of mitochondrial membrane potential resulted in the expression of Cytochrome c, Caspase-3 and Caspase-9 protein Increased, the expression of Bax protein increased, the expression of Bcl-2 protein decreased, Bax / Bcl-2 ratio increased. The apoptotic inhibitor Z-VAD attenuated the inhibitory effect of LSPCs on the proliferation of Hep G2 cells, significantly down-regulated the nuclear condensation, inhibited the mitochondrial damage and the expression of Caspase-related proteins, and ultimately blocked the apoptotic effects of LSPCs. It was concluded that LSPCs could induce apoptosis in human hepatoma cells through mitochondria-mediated endogenous Caspase pathway.