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AIM:To explore the inhibition of β-L-D4A on hepatitis B virus(HBV) in 2.2.15 cells derived from HepG2 cells transfectedwith HBV genome.METHODS:2.2.15 cells were plated at a density of 5×10~4per well in 12-well tissue culture plates,and treated withvarious concentrations of β-L-D4A for 6 days.In the end,5 μl of medium was used for the estimation of HBsAg andHBeAg,the other medium was processed to obtain virionsby a polyethlene glycol precipitation method.At the sametime,intracellular DNA was also extracted and digestedwith HindⅢ.Both DNAs were subjected to Southern blot,hybridized with a ~(32)p-labeled HBV probe and autoradiographed.Intensity of the autoradiographic bands was quantitatedby densitometric scans of computer and ED_(50) wascalculated.Then Hybond-N membrane was washed andrehybridized with a ~(32)P-labeled mtDNA-specific probe,andeffect of β-L-D4A on mitochondrial DNA was studied.2.2.15 cells were also seeded in 24-well tissue culture plates,and cytotoxicity with different concentrations wasexamined by MTT method.ID_(50) was calculated.Structure-activity relationships between D2A and D4A were alsostudied as above.RESULTS:Autoradiographic bands were similar betweensupernatant and intracellular HBV DNA.Episomal HBV DNAwas inhibited in a dose-dependent manner.ED_(50) was 0.2μM.HBsAg or HBeAg was not apparently decreased,andinhibition of mitochondrial DNA was not obvious.Theexperiment of cytotoxicity gained ID_(50) at 200 μM.CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxicity and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.
AIM: To explore the inhibition of β-L-D4A on hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome. METHODS: 2.2.15 cells were plated at a density of 5 × 10-4per well in 12-well tissue culture plates, and treated withvarious concentrations of β-L-D4A for 6 days.In the end, 5 μl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both DNAs were subjected to Southern blot, hybridized with a ~ (32) p-labeled HBV probe and autoradiographed. Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and (50) wascalculated. Hybond-N membrane was washed and replaced with a -32 (P-labeled mtDNA-specific probe, an effect of β-L-D4A on mitochondrial DNA was studied.2.2.15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with diffe rent concentrations wasexamined by MTT method. ID (50) was calculated. Structure-activity relationships between D2A and D4A were alsostudied as above .RESULTS: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose- dependent manner. ED_ (50) was 0.2 μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The activity of cytotoxicity gained ID_ (50) at 200 μM. CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxicity and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.