Study On The Regulatory Effect Of Fuhebeihua Formulaon Liver Cancer Stem Cells

Objective:Fuhebeihua formula is a famous Chinese medicine Professor Rong yuanming prescription for the treatment of liver cancer.The purpose of this study was to select CD133 + Hep G2 hepatoma stem cells by immunomagnetic bead method.The expression of CD133,the transcription factors Sox2,Nanog and Oct4 related to the differentiation of hepatoma stem cells,and the m RNA and protein levels of CD133,the surface marker of hepatoma stem cells,were detected by applying and preparing the drug containing serum co culture.The results showed that the serum of Fuhe Peihua formula could induce the differentiation of liver cancer stem cells by regulating the expression of CD133,transcription factors Sox2,Nanog and Oct4,which provided theoretical and experimental basis for the prevention and treatment of liver cancer by traditional Chinese medicine.Methods:Methods: 1.Isolation and identification of CD133 + Hep G2 liver cancer stem cells: CD133 + Hep G2 liver cancer stem cells were selected by immunomagnetic bead method,and the positive rate of CD133 + Hep G2 liver cancer stem cells identified by flow cytometry was more than 90%,which was used as the cells for subsequent experiments.2.CD133 + Hep G2 liver cancer stem cells were treated with the serum containing 2%,4%,8%,10%,12% and 16% respectively.The CCK-8method was used to determine the cell inhibition rate,and the best serum concentration was selected.The cells were divided into positive control group(DMSO group),application and preparation group,serum control group,blank control group,DMSO group with 0.5% DMSO intervention,application and preparation group with application and preparation containing serum intervention,serum control group with normal rat serum intervention,3.Flow cytometry was used to detect the percentage of CD133 + Hep G2 liver cancer stem cells,4.Q-PCR was used to detect transcription factors Sox2,Nanog and Oct4 M RNA level.5.Western blot was used to detect the protein levels of transcription factors Sox2,Nanog and Oct4.Results: 1.isolation and identification of CD133 + Hep G2 hepatoma stem cells: Hep G2 hepatoma cells were cultured,and CD133 + Hep G2 hepatoma stem cells were selected by immunomagnetic bead method.The positive rate of CD133 + Hep G2 cells identified by flow cytometry was 99.6%.The cell structure was complete and the growth condition was good,which could be used for follow-up test.2.CCK-8 method:application and preparation of the drug serum on CD133 + Hep G2 cell proliferation inhibition,and time,concentration related,16% application and preparation of the drug serum intervention 72 hours most obvious inhibition(P<0.05).In the follow-up test,16% application and preparation formula containing serum were used.3.The percentage of CD133 + Hep G2 cells detected by flow cytometry was 94.1% in serum control group,94.9% in blank control group,90.8% in DMSO group and84.2% in Fuhebeihua group.The percentage of CD133 + Hep G2 cells in both the application and preparation group and DMSO group was decreased(P<0.05),and the effect of the application and preparation group was more obvious(P<0.05).4.The m RNA levels of Sox2,Nanog and Oct4 were detected by Q-PCR.Compared with the blank control group,the expression of Sox2,Nanog and Oct4 could be down regulated in the Fuhebeihua group and DMSO group(P<0.05),and the effect of application and preparation group was more significant(P<0.05).5.Western blot method was used to detect the protein and phosphorylation level of transcription factors Sox2,Nanog and Oct4: compared with the blank control group,the protein levels of Sox2,Nanog and Oct4 in the Fuhebeihua group and DMSO group could be down regulated(P<0.05).The effect of Fuhebeihua group on the protein level of Nanog and Oct4 was better(P<0.05).The protein level of Sox2 was better in the application and DMSO group(P< 0.05).Conclusion: 1.Application and preparation of drug serum can effectively reduce the expression level of CD133,a marker on the surface of liver cancer stem cells,and reduce the malignant degree of liver cancer stem cells.2.The expression of Sox2,Nanog and Oct4 may be decreased by the prescription and preparation and Differentiation of hepatoma stem cells was induced.