The Experimental Study Of The Inhibition Of A-amanitin On HBV Replication

Objective: To study the inhibitory effect ofα-amanitin on HBV in vitro, and provide an experimental basis for researching its effects on HBV replication in vivo.Methods: HBV genome permanently transfected HepG2.2.15 cell line was employed as the in vitro model in our study. Cell viability activity test of MTT assay was used to identify the nontoxic concentration ofα-amanitin to culture cells. Then different nontoxic concentration ofα-amanitin were added to HepG2.2.15 cells , and set up blank solution and positive control. Then culture medium contained the same concentration drug were changed by every 72 hours. And the culture supernate fluid were collected in 72 and 144 hours for determining the levels of HBsAg, HBeAg by time-resolved immunofluorometric assay and the quantitiy of HBV DNA by real-time fluorescent quantitation PCR.Results:(1)The result of viability activity test shows that TC50 on the third and sixth day is 10.19和8.47μg/ml respectively, and the concentration ofα-amanitin below 0.8μg/ml had no inhibition to growth of cells .(2)72 hours later, inhibition rate of 0.8 0.16μg/mlα-amanitin and 3TC to HBsAg were 19.88% 15.95% and 46.07%, but the other concentration had no effect; 144 hours later, inhibition rate of 0.8% 0.16 μg/mlα-amanitin and 3TC to HBsAg were 47.40% 29.30%和55.71 %, but the other concentration had no effect;(3) 72 hours later , inhibition rate of 0.8μg/mlα-amanitin and 3TC to HBeAg were 14.16% and 31.86%, but the other concentration had no effect;144 hours later, inhibition rate of 0.8 0.16 and 0.032μg/mlα-amanitin and 3TC to HBeAg were 51.52% 45.84% 29.09%和59.14 % . but the other concentration had no effect;(4) 72 hours later, inhibition rate of 0.8 0.16μg/mlα-amanitin and 3TC to HBV DNA were 29.25% 21.96%和33.30%, but the other concentration had no effect; 144 hours later, inhibition rate of 0.8 0.16 0.032μg/mlα-amanitin and 3TC to HBV DNA were 51.74% 42.12% 34.29 %和48.20%, but the other concentration had no effect.Conclusions:(1)The MTT results suggested thatα-amanitin had certain toxity to HepG2.2.15 cells, but the concentration ofα-amanitin below 0.8μg/ml had no inhibition to growth of cells;(2) At certain concentration below 0.8μg/mlα-amanitin could suppress HBsAg and HBeAg expression, and decrease the HBV DNA level cultured in HepG2.2.15 cell line. And within limited dose as the time and doses encreases,the inhibition rate becomes higher.