| Objective:Our research team had selected mushroom strains C91-3 and had studied the lentinus edodes for many years. We found that fermentation of mycelium extract protein LFP91-3 not only inhibited tumor growth in vivo role also has significant in vitro anti-tumor cells directly. Preliminary studies show that the LFP91-3 on mouse ascites sarcoma cells (S180) inhibited the growth of the test by MTT method validation LFP91-3 in human cervical carcinoma cell (Hela) in vitro also has direct cytotoxic effect, and further discussed shiitake mushroom broth C91.3 protein on human cervical carcinoma cell (Hela) and mouse ascites sarcoma cells (S180) the process of growth inhibition induced Caspase-3, Caspase-8, Caspase-9 protein activity changes, flow cytometry detection of Caspase inhibitor apoptosis in synergy to verify the changes.Methods:(1) LFP91-3 protein preparation; (2) Hela,S180 cells subcultured. (3) LFP91-3 inhibited the growth of human cervical carcinoma cell (Hela) in vitro by the test of MTT method validation LFP91-3 (4) LFP91-3 protein 5μg/ml effect on the logarithmic growth phase Hela and S180 cells, Caspase inhibitor Z-VAD-FMK synergy control samples24h,48h,72h,and then the cells were collected,the apoptosis was analyzed by flow cytometry;(4) LFP91-3 effect on Hela and S180 cells 24h,48h,72h,and then the cells were collected,detected by Caspase-3, Caspase-8, Caspase-9 kits Results:Fermentative liquor of lentinus edodes C91-3 proteins effect on Hela cells 24h,48h,72h, concentration were 1μg/ml,5μng/ml,10μg/ml, the lowest rate of 18.621 percent growth inhibition, the maximum growth inhibition rate of 51.583percent, with the the role of prolonged growth inhibition was significantly increased, P<0.05. That its inhibitory effect on Hela cells in a dose-dependent with time.Flow cytometry analysis, and Caspase inhibitor Z-VAD-FMK collabo-rative intervention of cells compared with C91-3 protein in the role of prolonged fermentation, Hela early apoptosis rate measured value 24h 5.55%,48h the value increased by 6.80% and 72h to 11.55%, and Caspase inhibitor Z-VAD-FMK intervention of early apoptotic cells in the corresponding time points was 1.16%,1.32%,2.41%, the difference was significant (P<0.01); the corresponding start time start-type cysteine aspartase Caspase-8, Caspase-9 with Fermentative liquor of lentinus edodes C91-3 protein in the role of prolonged fermentation, protein activity increased by reagent kits (Beyotime) activity was measured by 24h protein Activity of the value was 13.545±1.181,11.750±0.800 up to 48h the value was 22.636±1.500,13.250±1.550, the 72h value was 35.364±1.455,17.250±1.350. Fermentative liquor of lentinus edodes C91-3 protein acts on cell fermentation 24h, a death by Caspase-3 was determined by the kit protein 6.043±0.086, effect on 48h protein activity assay value 8.217±0.173, to test value 72h protein activity reached 35.364±0.304, and apoptosis showed the same trend growth rate of expression. Compared with the negative of the photo, mushroom broth C91-3 protein induction in Hela cells, protein expression was increased, indicated the difference was significant (P<0.01).Fermentative liquor of lentinus edodes C91-3 protein effect on tumor cells S180 in vitro, the early apoptosis rate measured 24h to 48h increased by 5.86% to 7.51%,72h to 8.47%, Caspase inhibitor Z-VAD-FMK cell early intervention of withered death rate of the corresponding time points was 1.22%,2.08%,2.30%. Thus, with the role of prolonged protein, tumor cell apoptosis and death increased significantly, the early apoptosis rate was significantly higher than by the Caspase inhibitor Z-VAD-FMK intervention of tumor cells, significant difference between them (P<0.01). Kit (Beyotime) detection Caspase-3, Caspase-8, Caspase-9, C91-3 found in shiitake mushroom broth protein of time from 24h to 48h, and then to 72h, Caspase-9 from the 1.25±0.350 up to 4.25±0.15,72 h to 7.25±0.550, and Caspase-8 protein activity in 24h is reached 53.091±2.331, Caspase-3 increased from 8.652±0.086 9.956±0.210,72 h supreme almost doubled to 16.478±1.087. Compared with the negative of the photo, Fermentative liquor of lentinus edodes C91-3 protein induction in S180 cells, protein expression was increased, the difference was significant (P<0.01)Conclusion:Fermentative liquor of lentinus edodes C91-3 protein significantly inhibited the growth of tumor cells Hela (P<0.01), with the role of time, the growth inhibition rate in a dose-dependent. LFP91-3 5μg/ml effect on Hela, S180, flow cytometry analysis with the extended role of early apoptosis rate and mortality rate was significantly increased compared with the control group, Caspase inhibitor Z-VAD-FMK intervention of Apoptosis by death reduction, the difference was significant (P<0.01);It was found that Caspase-3, Caspase-8, Caspase-9 protein activity was significantly increased by kit detected,the anti-tumor effect of Fermentative liquor of lentinus edodes C91-3 protein were by influencing the expression and the activity of Caspase-3, Caspase-8, Caspase-9 protein that induced tumor cell apoptosis.
|
PDF Full Text Request