The Study Of Effect Of Artemisitene In Tumor-bearing Mice And In Hepatocellular Carcinoma And The Research Of Its Mechanism

Objective1 The first step of this experiment is to investigate the inhibitory effect on tumor-bearing by Artemisitene.2 The second step is to investigate the potential molecular mechanism of Artemisitene through DNA lesion the lead to apoptosis of hepatocellular carxinoma cells.Method1 In vivo experiments1.1 The effect of volume of xenograft liver neoplasms in NODSCID mice by Artemisitene.Through subcutaneous respectively inoculated to NODSCID mice with hepatoma cell lines of Hep3B2.1-7 and QGY-7703,we established xenograft tumor model.Then subcutaneous injection of different doses of Artemisitene,we observed the effect of Artemisitene to the volume on mice bearing xenografts.1.2 The effect of Artemisitene on tumor bearing mice to their livers and kidneys.After mices sacrificed,the tumor bearing mice s ' livers and kidneys were fixed in formaldehyde,and then dealt with dehydration,embedding,HE staining.Finally we did the pathological sections and observed the effects of Artemisitene in the body after absorption to the livers and kidneys.2 In vitro experiments2.1 The effect of the growth in HCCs by ArtemisiteneIn HCCs of Hep3B2.1-7?QGY-7703 Huh7 and sk-hepl,we detected the effect of Artemisitene to the cells growth in a dose-manner by MTT assays.We preliminary cleared concentration range of the effective of Artemisitene.2.2 The effect of the growth in normal cells by ArtemisiteneIn normal cells,including liver fiber cells(liver fiberblast),normal liver cells(L02),human renal tubular epithelial cells(HK2),human bronchial epithelial cells(BEAS-2B),we detected the effect of Artemi sitene to the cells growth in a dose-manner by MTT assays.And preliminary proved the inhibition on tumor cells of Artemisitene non toxicity.2.3 The effect of Artemisitene on HCCs apoptosis and the apoptosis related proteins PARP,Caspase3,caspase9In hepatoma cells of Hep3B2.1-7 and QGY-7703,we detected cell apoptosis by flow cytometry in which cells were dyed by anne xin V/PI in a dose-dependent manner.Then we initially identified the effective dose of Artemisitene;We used Western blot method to explore the expression of apoptosis related protein PARP,Caspase3 and caspase9 in a dose-dependent manner.Further validated the pro apoptotic effect of the Artemisitene.2.4 The effect of Artemisitene on HCCs of the DNA damage and phosphorylation of H2AXIn hepatoma cells Hep3B2.1-7 and QGY-7703,we used Western blot to detect the H2AX protein phosphorylation of Artemisitene in a dose-dependent manner.We cleared the sites of DNA damage caused by Artemisitene of in hepatoma cells.2.5 The effect of phosphorylation of ATM in HCCs by ArtemisiteneWe detected the phosphprylation of ATM protein expression in different time points by western blot method in order to get the range of the does that'the activation of Artemisitene.Results1 In vivo experiments1.1 Artemisitene inhibited the volume of the tumor tissues in a dose-dependent manner on mice bearing xenografts.1.2 Artemisitene has no obvious injury to the livers and kidneys of NODSCID mices.2 In vitro experiments2.1 Artemisitene inhibited the cell growth in HCCs in a dose-dependent manner2.2 Artemisitene has no obvious effect to the normal cells2.3 Artemisitene could induced the apoptosis of HCCs in a dose-dependent manner,and up-regulated the expression of the apoptosis related protein including PARP?caspase3 and caspase9 in a dose-dependent manner2.4 Artemisitene could cause DNA lesion and increased phosphorylation of? H2AX in HCCs in a dose-dependent manner.2.5 Artemisitene increased phosphorylation of ATM in HCCs in a dose-dependent manner.Conclusion1 Through the establishment of subcutaneous transplantation tumor models of hepatocellular carcinoma,confirmed the Artemisitene e by the body's circulatory absorption can inhibit tumor growth.Meanwhile,with the advantages of traditional Chinese Medicine---little toxic effect on organs.Mice liver,kidney pathological section showed that Artemisitene was no significant damage to organ.2 Inhibition of hepatocellular carcinoma cells growth activity in vitro experimental results show Artemisitene can obviously inhibit the growth activity of human hepatoma cell lines,but on significant effect on normal cell lines.Artemisitene can cause DNA damage in hepatocellular carcinoma cells,and activate the phosphorylation of ATM,which cause the apoptosis of hepatoma cells.DNA damage is the main factors to induced apoptosis of hepatomacells,but the mechanism of how the Artemisitene to induce DNA damage in hepatoma cells need to be confirmed in further experiments.3 Finally,the experimental results are consistent in vivo and in vitro.We can come up with the conclusion that Artemisitene can cause DNA lesion in hepatoma cells.