The Empirical Study Of Arsenic Compound Of Nanometer Inducing Hepatoma Cells To Apoptosis

Objective: As the effects of As₂S₃ inducing the hepatoma cells to be apoptotic inhibitive and proliferative, the arsenic compound of nanometer is composition by man-made As₂S₃ (Diameter<100nm), which provides a way to the new treatment to hepatoma.Methods:1 The cells of the experiment were divided to three groups: As₂S₃ group, As₂O₃ group (applied triple-distilled water to dilute the As₂O₃ powder to suitable density), control group. Detect the inhibition of As₂S₃ to the in vitro multiplication of SMMC-7721 by MTT. Four kinds of density of As₂S₃ were selected, including 1,2,4,8μmol/L. After intervening cells 24,48,72 hours, we chose the best density and the best time. The treatment of As₂O₃ group was the same as As₂S₃ group, and the culture medium was added to control group. While cells entered to the logarithm phase, the supernatant of adherent cell was thrown away. Then the cells were washed 2 times by PBS buffer solution, and digested by 0.25% trypase. Adjust the density of cells to 1×105 cells/ml by PRMI1640, seed them in cultivatble board with 96 holes, 1×104/100μL per hole. After the cells were raised 24 hours, the medicines in different density were added to different group, and add 100μL medium to control group for 5 duplicate holes in each group, put the experimental instrument to incubaton of 37℃and 5% CO₂ and then cultivante 24h,48h,72h, and then cultivation 4h after adding 20ul MTT in each holes, throw away supernatant, add DMSO 150μL then oscillate to dissolve for 10min, shade selection after Formosan dissolve completely, then use enzyme scale instrument detect the Optical Density (OD), and the wave length is 570nm, compute inhibition ratio of medicine affect on tumor cell. The equation is: inhibition ratio (%) = (1-group of experiment average OD/ control group average OD)×100%.2 Applying Transmission Electron Microscope (TEM) to observe the morphological changes and apoptosis of As₂S₃ cells affect on liver cancer SMMC-7721. according to the MTT result, add As₂S₃ and As₂O₃ of final density 8μmol/L to asepsis cell culture bottle, in the bottle is suspension of SMMC-7721 (5×106/ml), add medium to control group , put it to the incubator at 37℃, 5% CO₂, gather the cells after 72h, fore fix by glutaraldehyde of 2.5%, and postfix by 1% osmic acid. Alcoholic dehydration step by step, embedded by the balsam and makes it be ultramicrocut, double staining by Uranyl Acetate and Lead Citrate, then review the apoptosis of cells with the TEM (HITACH H-7500)3 Detection the cell cycle and the cell apoptotic ratio of the SMMC-7721 be action by As₂S₃ by FCM. According to the MTT result, add As₂S₃ and As₂O₃ of final density 8μmol/L, group of negative add medium action the SMMC-7721, after 72 hours'treatment, centrifuge and gather the cell, washout 2 times by PBS, infuse the alcohol 70%, centrifuge and throw away supernatant, add 200μl RNA enzyme at 37℃for 1h, and then add 800μL PI, put it to the refrigerator at 4℃for 30min, then detection the cell cycle and the cell apoptotic ratio by FCM.Results:1 The result of MTT method showed that 2-8μmol/L As₂S₃ have a notable inhibitory effect against the ectogenetic SMMC-7721, and 1μmol/L As₂S₃ not to have affected. The density and time of the best inhibition is 8μmol/L and 72h, the same to As₂O₃. They all rely the concentration and the time, the inhibition proportional to the concentration and the time, the inhibition of As₂S₃ surpass group of As₂O₃ and control group(P<0.05).2 The result of TEM showed that after 72h when As₂S₃ roles in the SMMC-7721, the mostly mitochondrial cristae inosculated or disappear and become blurring, there was vary size vacuoles in the cytoplast, compare to As₂S₃, As₂O₃ applied group have less vacuolus, and control group didn't change.3 The result of FCM showed that after 72h when As₂S₃ roles in the SMMC-7721, it was obvious encourage the apoptosis, the apoptotic ratio is 18.4%. At the same process, apply As₂O₃ to the cells, the ratio is 14.4%, and the control group is 2.2%, the difference between the experiment group and the other groups have statistical significance (P<0.01). Compare the cell's cycle of three groups, As₂S₃ role to the SMMC-7721 for 72h, the cell increase in the G₂/M phase, decrease in the S and G₀/G₁ phase, that indicated the cell's cycle hold-up at G₂/M phase.Conclusions:The results of the experiment have shown that the inhibition As₂S₃ to the in vitro multiplication of SMMC-7721 is powerful, and the apoptotic changes of SMMC-7721 in morphology can be observed. The function of As₂S₃ inducing the hepatoma cell to be apoptotic and causing the cell cycle to be blocked is better than that of As₂O₃.