Inhibition of hepatitis B virus by a novel L-nucleoside,β-L-D4A and related analogues  

作　　者：Jin-MingWu Ju-ShengLin NaXie Kuo-HuanLiang 

机构地区：[1]DepartmentofDigestiveMedicine,theFirstAffiliatedHospital,WenzhouMedicalCollege,Wenzhou325000,ZhejiangProvince,China [2]Instituteofliverdiseases,TongjiMedicalCollege,HuazhongUniversityofScienceandTeclmologv,Wuhan430030,China

出　　处：《World Journal of Gastroenterology》2003年第8期1840-1843,共4页世界胃肠病学杂志（英文版）

基　　金：National Natural Science Foundation of China,No.39970858

摘　　要：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 5×10^4 per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. In the end,5μl 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 HindⅢ. Both DNAs were subjected to Southern blot,hybridized with a ^32P-labeled HBV probe and autoradiophed.Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED50 was calculated. Then Hybond-N membrane was washed and rehybridized with a ^32P-labeled mtDNA-specific probe, and 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 different concentrations was examined by MTT method. ID50 was calculated. Structure-activity relationships between D2A and D4A were also studied as above.RESULTS: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNAwas inhibited in a dose-dependent manner. ED50 was 0.2μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID50 at 200 μM.CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxidty 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 5×10~4 per well in 12-well tissue culture plates,and treated with various 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 HindⅢ.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 ED_(50) was calculated.Then Hybond-N membrane was washed and rehybridized with a ^(32)P-labeled mtDNA-specific probe,and 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 different concentrations was examined by MTT method.ID_(50) was calculated.Structure- activity relationships between D2A and D4A were also studied 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 experiment 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.

关 键 词：乙型肝炎病毒 L-核苷 Β-L-D4A 细胞毒性 线粒体 

分 类 号：R373.21[医药卫生—病原生物学]