Effects Of Aflatoxin B₁ To Human Hepatoma Cell Line Smmc-7721

Objectives:1.To investigate the effects of aflatoxin B₁ to Human Hepatoma Cell Line SMMC-77212.To explore whether the aflatoxin B₁ activate the Ras-ERK signal transduction pathway in Human Hepatoma Cell Line SMMC-77213.The function of chlorophyllin on aflatoxinMethods:1.Firstly,apply different concentrations of aflatoxin B₁ 20ug/ml,10ug/ml, 5ug/ml,2ug/ml,1.0ug/ml,0.1ug/ml,0.01ug/ml to Human hepatoma cells Line SMMC-7721,and take MTT method to draw cell growth curve,observe the function of aflatoxin on SMMC-7721.Select the best concentration AFB₁ 1.0ug/ml as the further experimental intervention factor2.Observe the function of AFB₁1.0ug/ml on the cycle of SMMC-7721,apply AFB₁1.0ug/ml to hepatoma cells SMCC-7721 under the conditions of 37.0℃,5% CO2 and incubate for 24 hours,after that collect cells and detect them by flow cytometry.3.Observe whether the aflatoxin B₁ rapidly activate the receptor-type TPK-Ras-MAPK way,Apply AFB₁1.0ug/ml to cells,incubate them under the conditions of 37.0℃,5%CO2,deal with respectively 0,30,60,90,120 minutes,after that extract cell protein,select Ras,ERK1 protein antibodies for Western blot detection,observe whether express the Ras,ERK1 protein and change the expression quantity,and select proteinβ-catenin as the reference protein.4.Apply different concentrations of chlorophyllin 50ug/ml,30ug/ml,20ug/ml, 10ug/ml to hepatoma cell line SMMC-7721,observing the effect of chlorophyllin to SMMC-7721,select chlorophyllin 20ug/ml,10ug/ml as the further experimental intervention factor.5.Apply chlorophyllin 20ug/ml,10ug/ml and the AFB₁1.0ug/ml disposed hepatoma cell line SMMC-7721,incubate them under the conditions of 37.0℃,5% CO2,observe the function of chlorophyllin on cell growth and the AFB₁,select chlorophyllin 20ug/ml as the further experimental intervention factor which palys not significantly effect on the hepatoma cells and inhibits the function of AFB₁1.0ug/ml.6.Apply chlorophyllin 20ug/ml to the disposed AFB₁1.0ug/ml hepatoma cell line SMMC-7721,incubate them under the conditions of 37.0℃,5%CO2 for 24 hours, after that collect the cells and detect cell cycle by flow cytometry7.Apply chlorophyliin 20ug/ml to the disposed AFB₁1.0ug/ml hepatoma cell line SMMC-7721,incubate them under the conditions of 37.0℃,5%CO2,deal with respectively 0,30,60,90,120 minutes,after that extract cell protein,select Ras,ERK protein for Western blot detection and observe whether exist the changes of expression quantityResults:1.Different concentrations of AFB₁ played different effects on hepatoma cell line SMMC-7721,AFB₁20ug/ml,AFB₁10ug/ml,AFB₁5ug/ml,showed cytotoxicity, AFB₁1.0ug/ml,AFB₁0.1ug/ml,AFB₁0.01ug/ml promoted proliferation of hepatoma cell line SMMC-7721,and AFB₁1.0ug/ml roled significantly,the role of AFB₁0.1ug/ml,AFB₁10.01ug/ml gradually diminished.2.AFB₁1.0ug/ml disposed hepatoma cells which incubated under the conditions of 37.0℃,5%CO2 for 24 hours,S phase cells increased more than the control group3.Ras,ERK1 protein expressed in Human hepatoma cell Line SMMC-7721 4.AFB₁1.0ug/ml disposed hepatoma cells showed the more expression of Ras, ERK protein at 90,120-minute test piont than the control group5.Chlorophyllin disposed hepatoma cell line SMMC-7721 found chlorophyllin 30ug/ml can induce apoptosis of hepatoma cells.6.The AFB₁1.0ug/ml disposed hepatoma cell line SMMC-7721 treated with chlorophyllin showed chlorophyllin 20ug/ml can inhibit AFB₁ on SMMC-7721 proliferation,while not significantly effect on cell growth;chlorophyllin 10ug/ml not significant effect on AFB₁.7.AFB₁1.0ug/ml disposed hepatoma cell line SMMC-7721 treated with chlorophyllin which incubated under the conditions of 37.0℃,5%CO2 for 24 hours, S phase cells increased more than the group Of AFB₁1.0ug/ml.8.Chlorophyllin 20ug/ml treated AFB11.0ug/ml disposed hepatoma cells and incubated under the conditions of 37.0℃,5%CO2 for 24 hours,S phase cells showed less than the control group;the Ras,ERK1 protein expressed less than AFB₁1.0ug/ml disposed hepatoma cells at 90,120-minute test piont.Conclusions:1.Aflatoxin B₁ can promote the proliferation of Human Hepatoma Cell Line SMMC-7721.2 Aflatoxin B₁ can activate ras pathway,maybe interve cell cycle regulation through activating Ras pathway,and promote the proliferation of SMMC-7721,and play a role in the occurrence and the development of hepatocellular carcinoma.The mechanism needs more investigation.3.Chlorophyllin can inhibit the function of aflatoxin B₁,the mechanism needs further more research.Chlorophyllin can induce apoptosis of hepatoma cell line SMMC-7721 and probably play an anti-cancer role.The mechanism needs more investigation.