| Malignant tumor has been one of the main causations of death and about 7,000,000 people are killed by cancer annually all over the world. In China, hepatoma has a high incidence. 138,000 people are suffered by it every year which is approximately 43.7 percent of the 315,000 hepatoma sufferers all over the world. So far, hepatoma has been the main threat of people's health in our country and has the second death rate of the tumor cancers. However the current therapies of liver cancer can't serve the purpose very well, it becomes more and more important to research and develop new antihepatoma drugs. Recently, the orientation of research of antitumor drugs has changed from single cytotoxic drugs to diversified drugs. In this field, Chinese traditional medicine (CTM), as the heritage of thousands years, attracts more and more attention with its remarkable curative effect on tumor. At present, more and more evidence indicates that the lack of equilibrium among proliferation, differentiation and apoptosis is related to generation and evolution of cancer. As thus, using drugs to induce apoptosis of tumor cell is probably a promising way of tumor therapy and it also provides a new clew to the treatment of hepatoma and other cancers. Oxymatrine(OMT) is a kind of alkaloid which is the main product extracted from Chinese traditional medicine sophora flavescens ait. It plays an important role in anti-arrhythmic, antitumor, liver protection and immunity regulation. Experiments indicate that OMT can inhibit the apoptosis of human liver cells and rat liver cells induced by TNF-α, protect rat liver cells from damage induced by diethylnitrosamine(DEN) and delay the forming of hepatoma. Meanwhile, it is reported that OMT can inhibit the proliferation of human hepatoma cells (HCC) and ovary cancer cells in vitro. These suggest that OMT potentially have antitumor effect on hepatoma and its mechanism is probably related to inducing apoptosis of hepatoma cell. However, there are few detailed reports about apoptosis and its mechanism of cancer cells induced by OMT. Based on the methods of biochemistry and molecular biology, OMT's effect on the proliferation, cancer related gene expression and telomerase activity of human hepatoma cell line(SMMC-7721) were investigated in this thesis in order to elucidate the mechanism of HCC apoptosis induced by OMT. The main research methods, contents and conclusions are as follows: ①With MTT assay, the growth inhibition of human hepatoma cells line SMMC-7721 and human normal liver cells HL-7702 treated by various concentration of OMT after different time was observed. Results showed that OMT could inhibit the growth of SMMC-7721 cells and HL-7702 cells and it presented dose-and time-dependent. However the effect of OMT on SMMC-7721 cells was significantly strong than that of HL-7702 cells. ②Effect of cell cycle and apoptosis rate of SMMC-7721 cells was studied with flow cytometry(FCM), PI and AnnexinⅤfluorescent dyes. It showed that OMT could block the cell cycle at G2/M phase and S phase, prevent SMMC-7721 cells entering G0/G1 phase. It also showed that OMT could induce apoptosis of SMMC-7721 cells and the apoptosis rate was 61.12 percent after 48h treatment by 1.0mg/ml OMT. ③Expression of cancer related gene bcl-2 and p53 was measured by FCM and FITC marked antibody quantificationally. It was found that OMT could downregulate the expression of bcl-2 gene while upregulate the expression of p53 gene. ④Effect of OMT on the telomerease activity in SMMC-7721 cells was analyzed with TRAP-sliver stain assay. The result indicated that the telomerase activity of HCC was decreased obviously by OMT and the telomerase activity was undetectable when OMT concentration is higher than 1.0mg/ml.
|
PDF Full Text Request