Regulation Of Apoptosis In Hepatoma Cells By Bcl-2 And Bax And Primary Study Of Hepatoma Apoptosis-related Monoclonal Antibody

Postgraduate for Doctor's degree: Lianjun YangSupervisor: Prof. Wenliang Wang Department of Pathology, Fourth Military Medical University, Xi 'an 710032To study the inducing effect of ethanol on apoptosis in hepatocytes and liver tumor cells is important and helpful to probe into the relationship between ethanol-induced apoptosis in hepatocytes and liver diseases. Currently, there are only few such reports as establishing the model of ethanol-induced apoptosis in hepatocellular carcinoma (HCC) cells. Bcl-2 and bax are respectively anti-apoptotic and proapoptotic genes, and both of them belong to bcl-2 gene family. There is no definite conclusion about the expression of bcl-2 and bax in HCC recently. There is no such report about the regulating effects of bax overexpression on apoptosis in HCC cells and bcl-2 overexpression on ethanol-induced apoptosis in HCC cells until now. Preparing anti-apoptotic cell monoclonal antibody (mAb) is helpful to investigate the change and production of apoptosis-related molecules, it may also be used in apoptosis detection, and it is a newly explored domain in the study of apoptosis There is no such report as preparing anti-apoptotic cell mAb as its antigen is unclear until now.Centering on ethanol-induced apoptosis in HCC cells and its expression of Bax and Bcl-2, the effect of bcl-2 and bax transfection on apoptosis in HCC cells and its expression of cell cycle proteins p21WAF1/cn>1 and p2710pl, and preparation of mAb against ethanol-induced apoptotic HCC cells, this study contains the following work:?The cytotoxicity of HCC cell line HCC-9204 treated with 20 ~ 100 mL / L ethanol for 6 h was detected by Methabenzthiazuron (MTT) assay. Then the cells were induced to undergo apoptosis with 60 mL / L for 6 h. The happening of apoptosis was detected by trypan blue staining, Haematine-eosin (HE) staining, May-Grunwald-Giemsa (MGG) staining, Hoechst 33258 staining, Acridine orange / Ethidium bromide (AO / EB) double staining, Transmission electronic microscope observation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and DNA contents assay in different cell cycles by flow cytometer. The change of Bax and Bcl-2 expression level in HCC-9204 cells before and after inducing apoptosis was detected by immunocytochemical ABC staining method and image-quantitative analysis. The results showed that, as the concentration of ethanol increased, the cytotoxicity of HCC-9204 cells increased gradually. Sixty mL / L for 6-6-h could make obviously morphologically apoptotic change in HCC-9204 cells, and most of the cells could be stained with trypan blue and were TUNEL positively-stained. There was significant sub-Gl apoptotic peak detected by flow cytometer. The Bax expression level in HCC-9204 cells after inducing apoptosis increased significantly, but there is still no detectable Bcl-2 both before and after inducing apoptosis.?Expression of Bcl-2 and Bax in HCC and normal liver tissues was detected by immunohistochemical ABC method, and its relationship with the pathological grades of HCC tissues was analyzed. Eukaryon expression vectors containing human bcl-2 and bax cDNA were respectively transfected into HCC-9204 cells, HCC cells that express Bax stably and highly were established, and an HCC cell strain that express Bcl-2 at a 100 % positive rate was obtained by cloning. The p21WAF1/CIP1 and p27Kipl expression level in the cells before and after transfected with bcl-2 and bax was detected by immunocytochemical ABC staining method and image-quantitative analysis. The happening of apoptosis in the cells before and after transfected with bcl-2 and bax was detected by MTT assay, TUNEL assay and flow cytometer. The results showed that, the positive rate of Bcl-2 in normal liver tissues was only 4.2 % (1 / 24), that in HCC tissues was 25.8 % (17 / 66), and there was significant difference between them (P < 0.05). The positive rate of Bax in normal liver tissues was 70.8 % (17 / 24), that in HCC tissues was 43.9 % (29 / 66), and there was...