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Objective: To investigate dehydrogenase Harmine (Harmine, HAM) in vitro anti-tumor effect, observed the different concentrations of harmine hepatoblastoma HepG2 cells in vitro proliferation inhibition and apoptosis induced clear its anticancer mechanism harmine to provide experimental evidence used in clinical anti-tumor therapy. Methods: 1.CCK-8 was detected harmine hepatoblastoma HepG2 cell proliferation inhibition: 0,0.625,1.25,2.50,5.00,10.00,20.00 μg / ml drug concentration in HepG2 cells 48 h, located DMSO control group, detection harmine hepatoblastoma HepG2 cell proliferation inhibition. Colony formation inhibition test: in hepatoblastoma HepG2 cells 0,0.625,1.25,2.50,5.00 μg / ml drug concentration, at the same time set the DMSO control group, after 7 days incubation, crystal violet staining, detection harmine alkali hepatoblastoma HepG2 cell colony formation. Morphological observation of apoptotic cells: 0,5.00 μg / ml drug concentration in hepatoblastoma HepG2 cells 48 h, at the same time set the DMSO control group to observe the morphological changes of apoptotic cells staining with Hoechest 33258. Asia twice the 4.PI single stain detection the hypodiploid rate and the cell cycle: 0,5.00,10.00,20.00 μg / ml drug concentration in hepatoblastoma HepG2 cells, at the same time set the DMSO control group, 48 h after detection body rate and cell cycle ratio. 5.Annexin V-PI double staining proportion of apoptotic cells was determined: in hepatoblastoma HepG2 cells 0,5.00,10.00,20.00 μg / ml drug concentration, at the same time set the DMSO control, detection of early apoptotic cells 48 h after rate. 6. Mitochondrial membrane potential detection: hepatoblastoma HepG2 cells 0,5.00 μg / ml drug concentration, at the same time set the DMSO control group, mitochondrial membrane potential changes detected after 48 h. Western-Blot Detection of Bcl-2 family proteins and Caspase family protein levels: 0,1.25,2.50,5.00 μg / ml drug concentration in hepatoblastoma HepG2 cells DMSO control group, and at the same time set up to detect the protein level. Results: 1. Harmine role in hepatoblastoma HepG2 cells after 48 h, significantly reduced cell viability, proliferation inhibition rate enhancement with increasing drug concentration has a significant (P lt; 0.05) harmine inhibits hepatoblastoma HepG2 cell proliferation IC50 of 9.80 gg / ml. 2. The harmine role hepatoblastoma HepG2 cells after 7 days, with the increase in drug concentration cloning gradually reduced, and gradually reduce the number of clones to produce significant colony formation inhibition. 3 the harmine role in the hepatoblastoma HepG2 cells after 48 h, the morphological structure of the control group normal intact HepG2 cell morphology visible in the drug group smaller nucleus pyknosis, the edge poly and cracking apoptotic body and the formation of apoptotic morphological changes. 4 the harmine role in the hepatoblastoma HepG2 cells after 48 h PI single dye flow cytometry detection subdiploid peak before the G1 phase of the cell cycle, harmine alkali to DMSO 0,5.00,10.00,20.00 μg / ml drug concentration in HepG2 cells apoptosis peak after 48h were 5.2%, 4.5%, 10.7%, 18.0%, 21.4%; detected significantly reduced the number of cells in G1 phase, S phase The number of cells and G2 phase cells significantly increased cell cycle arrest in the G2 phase. 5 the harmine role in the hepatoblastoma HepG2 cells 48h after Annexin V-PI double staining experiments testing the experimental group cells appear early apoptotic cell populations. 6. The harmine role hepatoblastoma HepG2 cells after 48h was observed in two-color filter, the control group for the high red high green, the experimental group for high green and low red Tip mitochondrial membrane potential was decreased. 7. Harmine role in hepatoblastoma HepG2 cells after 48 h, Western-Blot detected the Bcl-2 family members, Bcl-2, Mcl-1, Bcl-xl protein decreased Bax protein unchanged, Bcl the -2/Bax, Mcl-1/Bax, Mcl-1/Bax ratio decreased Caspase-3 and Caspase-9 activation. Conclusion: 1. Harmine can significantly inhibit the growth of hepatoblastoma HepG2 cells showed concentration-dependent 0-20μg/ml within; harmine hepatoblastoma HepG2 cells capable of inducing apoptosis. harmine hepatoblastoma HepG2 cells induced apoptosis through the mitochondrial pathway.
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