| Objective:Primary hepatocellular carcinoma(HCC)is one of the most popular malignant tumors in the world.It is characterized by early detection and high mortality in the late stage.In recent years,great progress has been made in the diagnosis and treatment of hepatocellular carcinoma in China.However,because of the large population base and a considerable number of HBV\HCV infection or carriers,liver cancer will still be the most important of cancer prevention and diagnosis in China.Therefore,it is very important to explore the mechanism of action related to the apoptosis and development of hepatocellular carcinoma.The purpose of this study is to explore the preliminary leguminous plant stem mechanism of total flavonoids from Caulis Spatholobi extract on two classical hepatocellular carcinoma cell line apoptosis,is expected to provide a new pathway for the role of caspase-9,so as to regulate the apoptosis mechanism.Methods:1.cytotoxicity test.In HepG2 and HL7702 cell lines,CCK8 was performed after different drug concentrations were treated with 48h cells,and their IC50 values were measured.2.Cell morphological examination:HepG2 and HL-7702 cells,different concentrations of traditional Chinese medicine treatment of 48h,DAPI staining,room temperature lOmin,PBS cleaning,fluorescence microscope photography.3.,The drug concentration with a half inhibitory rate is the highest concentration(such as lOug/ml),set low two concentrations(such as 5ug/ml,lug/ml),and blank Blank group,that is,each cell has 4 groups.After the action of 48h,DAPI is stained and photographed.4.The 4 groups were detected by flow cytometry(Annexin V-FITC/PI).The significant difference between the number of apoptotic cells after treatment with different concentrations of drugs was compared.5.,The above 4 groups,after extracting the total protein,used Western Blotting to repeat three times to detect the expression of Caspase-3,Caspase-8 and caspase-9 3 proteins in two cell lines.Results:1.By calculating the cell inhibition rate found spatholobus IC50 16.937 g/ml,according to the IC50 data to determine the concentration of subsequent experiments as follows:0,5,10,17 g/ml.2.FCM results showed that in the HL7702 group,the difference between the control group and the 5ug drug group,5ug drug group and lOug drug group was very significant,and the difference between 10ug drug group and 17ug drug group was significant.In the HepG2 group,the difference between the control group and the 5ug drug group,the 10ug drug group and the 17ug drug group was very significant,and the difference between the 5ug drug group and the 10ug group was significant.3.Detection of target protein by Westren Blotting revealed that caspase-9 expression in HepG2 cell lines was statistically significant;caspase-3 and caspase-8 had no significant difference between HL7702 and HepG2.Conclusion:1.Total flavonoids from Caulis Spatholobi substances can promote apoptosis in HepG2 cells.2.Total flavonoids from Caulis Spatholobi substances promotes HepG2 cell apoptosis play a role in liver cancer cells by regulating caspase-9.3,With the comparison of the blank group,total flavonoids from Caulis Spatholobi were different degrees of inhibition of the proliferation of hepatocellular carcinoma cells.4.Total flavonoids from Caulis Spatholobi containing related groups showed both dose-dependent inhibitory effect on the proliferation of hepatoma cells.4.The results of DAPI staining showed that with the increase of drug concentration,the number of early apoptotic cells and late apoptotic cells increased gradually,and nuclear fragmentation and nuclear condensation appeared more frequently. |
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