Preliminary Study Of Mechanism And Apoptosis Inducing By FB1 In Human Hepatocellular Carcinoma

Background and purpose: Fumonisins is a mycotoxin which was discovered by Gelderblom,-W-C in 1988. The toxins are produced by Fusarium moniliforme. Fumonisins have more than 10 kinds which are FA1,FA2,FB1,FB2,FB3,FB4,FC1,HFB1 etc. Fumonisin B1 is the main toxic factor. FB1 lies in many food for human and animal, it is harmful to human and animal, different animal and different organ have different response in which tissues were damaged and apoptosis caused by Fumonisin B1.Ross,-P-F concuded, the FB1 in food which concentration was 1mg/g～126mg/g could induce equine leukoencephalomalacia (ELEM), the concentration which was 1mg/g～330mg/g could induce porcine pulmonary edema syndrome (PPE); Schmelz,-E-M concluded 10mmol/L FB1 could induce HT29 cells apoptosis. Tolleson discovered FB1 could induce the HET-1A cells, keratinocytes,cv-1 cells apoptosis. Tsunoda,-M injected with fumonisin B1 subcutaneously at doses of 0,0.25, 0.75,2.25, and 6.75 mg/kg body weight daily for 5 days in five Groups male BALB/c mice, there were apoptosis in liver and kidney.Apoptosis is called programe cell death also, which is control by gene. Now it is known that the apoptosis is related to many life field, it is very important and necessary.It is the basic phenomena as same as the cell division, the basis of the life and development.Apoptosis is related to immune disease, tumor. The characters of apoptosis are the morphology change of nucleus and plsma, chromatin break regularly at special site. The mode can balance the tissue cells division; it is an important way to keep the creature alive. Now it has improved the apoptosis which is led by FB1, but the mechanism of apoptosis is unkown. To understand the mechanism which the FB1 induce apoptosis is important.Fas play an important role in the apoptosis, Fas lies in the membtana of cell which was called APO-1 or CD95 also. The expression vary within the different cells, such as thymic cells,active lymphocyte,virus infected cells,partial tumor cells. The rate of apoptosis rose with the expression of Fas mRNA when hydrogen peroxide (H2O2) induces the human umbilical vein endothelial cells apoptosis. Fas combine its ligand FasL can induce several kinds of cells apoptosis, such as Jurket cells,thymicolymphocyte. Our studies are based on the apoptosis in human hepatocellular carcinoma and observe the expression of Fas/FasL mRNA in vitro which was processed by different concentration FB1. Make the basis for the mechanism of the FB1 induce apoptosis in human hepatocellular carcinoma.Our study includes two parts:The first part: Objective Induce the human hepatocellular carcinoma apoptosis. Methods Culture the Human hepatocellular carcinoma in vitro.After the cells suspection , added the density of 4×10~5/well to 6-wells plate. When the density of the cells are 6×10~6, add the 1μmol/ml FB1 to the wells, the concentration are 5μmol/L, 15μmol/L, 20μmol/L, 30μmol/L, 40μmol/L in wells. After cells were cultured 24 hrs, The apoptosis was observed by Flow CytoMeter (FCM) detect and acridine orange dye Result 5μmol/L, 15μmol/L, 20μmol/L, 30μmol/L, 40μmol/L of FB1 can induce human hepatocellular carcinoma apoptosis.The second part: Objective Observe the effect of Fas/FasL mRNA expression Methods Human hepatocellular carcinoma were processed by 5μmol/L, 15μmol/L, 20μmol/L, 30μmol/L, 40μmol/L FB1 for 24 hours. Collecting cells, extracting total RNA, reversing transcription cDNA .Fluorescence quantitative PCR was used to examine the expression of Fas/FasL mRNA. Result The cells which were processed by 5μmol/L, 15μmol/L FB1, the expression of Fas mRNA are no difference compared with the control (p>0.05) . Meanwhile the cells were processed by 20μmol/L, 30μmol/L, 40μmol/L FB1, the expression of Fas mRNA is higher than the control group which the level is different with concentration (p<0.05). The expression of 30μmol/L FB1 processed is highest in 20μmol/L , 30μmol/L and 40μmol/L FB1 group, the difference is obviouse(p<0.05). The expression of FasL mRNA in every concentration processed groups are lower than control group, the difference is obviouse (p<0.05) .Conclusion The concentration of 5-40μmol/L FB1 can induce the human hepatocellular carcinoma apoptosis. The expression of Fas mRNA is raised with concentration when FB1 induce the human hepatocellular carcinoma apoptosis, but the expression of FasL mRNA is lower in every concentration FB1. FasL induce the apoptosis is not proved.