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目的:采用多种方法探究天然植物抗菌液(PAMs)对人肝癌细胞HEPG-2的杀伤及初步分子作用机理。方法:运用形态学观察、MTT法测定PAMs对HEPG-2的抑制作用,AO/EB细胞双染、Annexin V-FITC/PI流式细胞术检测PAMs作用于HEPG-2细胞后细胞的凋亡情况,再通过qRT-PCR与western blot检测HEPG-2中相关基因在m RNA水平和蛋白水平的表达量变化。最后利用划痕实验和transwell小室检测PAMs对HEPG-2细胞迁移能力的抑制。结果:PAMs可以抑制和杀伤HEPG-2细胞,作用效果呈浓度和时间依赖性,且与细胞的凋亡途径密切相关。PAMs可下调HEPG-2细胞中foxm1、survivin、bcl-2的m RNA表达水平,同时在蛋白水平下调foxm1的表达量,上调p53的表达量。除此之外,PAMs还能够有效地抑制HEPG-2细胞的体外迁移。结论:PAMs杀伤HEPG-2细胞的途径之一是通过下调foxm1、上调p53基因的表达量,从而抑制或减少其下游靶基因survivin和bcl-2的转录表达,促进细胞凋亡。本研究为基于PAMs抗癌创新民族医药的研发提供了重要的前期工作基础。
OBJECTIVE: To explore the killing effect of natural plant antibacterial solution (PAMs) on human hepatocellular carcinoma cell line HEPG-2 by a variety of methods and the preliminary molecular mechanism. Methods: Morphological observation was used to determine the inhibitory effect of PAMs on HEPG-2 by MTT assay. AO / EB double staining and Annexin V-FITC / PI were used to detect the apoptosis of HEPG-2 cells The mRNA expression level of HEPG-2 at mRNA level and protein level was detected by qRT-PCR and western blot. Finally, scratch test and transwell chamber were used to detect the inhibitory effect of PAMs on the migration ability of HEPG-2 cells. Results: PAMs could inhibit and kill HEPG-2 cells in a concentration-and time-dependent manner, which was closely related to the apoptotic pathway. PAMs could down-regulate the m RNA expression levels of foxm1, survivin and bcl-2 in HEPG-2 cells, and down-regulate the expression of foxm1 and up-regulate the expression of p53 in the protein level. In addition, PAMs can effectively inhibit the in vitro migration of HEPG-2 cells. CONCLUSION: One of the pathways of killing HEPG-2 cells by PAMs is down-regulating foxm1 and upregulating the expression of p53 gene, thereby inhibiting or reducing the transcriptional expression of survivin and bcl-2 downstream target genes and promoting cell apoptosis. This study provides an important foundation for the preliminary work of R & D based on PAMs anti-cancer innovation and national medicine.