| Objective To investigate the cellulotoxic effect of PSP on HepG2 2.2.15 cell line, the inhibition to HBeAg , HBsAg and HBV-DNA cultured in 2.2.15 cell line , and the influence on the expression of IFN-a R mRNA in the cell line.Methods HepG 2 2.2.15 cells were added into 96 well plate by 10% blood serum of foetus cattle MEM culture media, which concentration was 3x105Per- ml.In every well, 200 ul was added in and fluid was changed after 48 hours. PSP was diluted by MEM culture media,which contains 2% blood serum of foetus cattle, till the end dilution were 3200 ug/ml,1600ug/ml,800 ug/ml 400ug/ml and 200ug/ml. The cells were placed 37 C and with 5% CO2 in air for 72 hours.MTT assay and counting of alive cells were used to observe the cytotoxicity of PSP to the HepG 2 2.2.15 cells; The cells were then planted in 24-well culture plate, Four concentrations-400ug/ml,200ug/ml,100ug/ml and 50ug/ml of PSP ( lml) were added into each well. At the different time,The inhibition to HBeAg and HBsAg were tested by ELISA(added standard sample); The quantity of HBV-DNA was detected by real-time fluorescence quantitative PCR(FQ-PCR). The expression of IFN-a R mRNA was observed by in situ hybridization;Results (1)PSP has little cellulotoxicity,TC50>3200ug/ml, TC0=600+28ug/ml (2)24hours later, inhibition rate of PSP(400ug/ ml) to HBsAg secretion was 22.35+3.06% (P<0.05) , but the others concentration of PSP had no effect on HBsAg secretion; Respectively, 48hours later, inhibition rate of PSP (400ug/ml, 200ug/ml) were 29.19+5.09% (P<0.01) and 21.47+2.26% (P<0.05) , the other two concentration of PSP had no effect; Respectively, 72hours later, inhibition rate of different concentration PSP were 34.67+4.66% (P<0.01) , 26.86+3.00 % (P<0.01) ,21.70+3.05% (P<0.01) and 15.74+1.61 (P<0.05); Respectively, 144hours later, inhibition rate were 6.50+2.41 % (P<0.01),35.83+1.10% (P<0.01) , 29.27+1.68 % (P<0.01) and 23.83+1.94 % (P<0.01) . (3) 24hours later, In each concentration of PSP, there were no significant difference between the inhibition rate of experimental group and that of control group to HBeAg secretion; Respectively,48hours later, inhibition rate of PSP (400ug/ml, 200ug/ml) were 43.23+1.05% (P<0.01) and 38.46+4.63(P<0.05); Respectively, 72 hours later, inhibition rate of PSP were 47.87+3.58%(P<0.01), 39.84+4.45%(P<0.01), 23.88+2.08%(P<0.01) and 16.81+0.33% (P<0.05); Respectively, 144hours later, inhibition rate of PSP were 40.99+6.35% (p<0.01), 38.47+2.73% (P<0.01), 23.52+0.90% (P<0.05)and 20.46+0.91% (P<0.05) ; (4)Respectively, 72hours later, inhibition rate of PSP to HBV-DNA synthesis were33.94+0.36%(P<0.),23.87+6.03% (P<0.01) ,16.72+0.51% (P<0.05)andl3.64+0.13% (P<0.05) ; Respectively, 144 hours later, inhibition rate of PSP were 36.79+4.14%(P<0.01), 29.22+3.79% (P<0.01), 22.38+6.87% (P<0.01)and 19.32+5.49% (P<0.05); (5) In situ hybridization results showed that there were significant difference between the disposal bundle and control bundle, Respectively,72 hours later, The positive rate of IFN-a R mRNA were 49.17+2.56 % (P<0.01), 41.33+1.76% (P<0.01), 31.83+4.67% (P<0.05) and23.67+2.75% (P<0.05) in disposal bundles, but was 11.00+2.29 % in control bundle; 144 hours later, the positive rate were 81.67+3.25% (P<0.01), 71.33+2.08% (P<0.01), 57.83 + 3.51% (P<0.05)and 38.67+5.53% (P<0.05) in disposal bundles, but it was 17.83+4.93% in control bundle.Conclusions These results suggested that PSP at non-cellulotoxic concentration could suppress HBeAg and HBsAg expression and decrease the HBV-DNA level cultured in HepG2 2.2.15 cell line, furthermore, It can also significantly facilitate transcription of IFN-aRmRNA.Its showed that PSP can not only reinforce the level of cellular immunity,But also inhibit the secretion of HBV-Antigen and the duplication of HBV-DNA. The study provided therical and experimental evidence for the further research of the pharmacological mechanism and the application as anti-HBV medicine of PSP.
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