A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma

AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo.METHODS: GE7,a 16-peptide specific to EGFR, and HA20,a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA.BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured.RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1.The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4 % and 58.5 % respectively. RT-PCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4 injections of AFP-enhancing 4-element complex containing 0.2 μg DNA, the diameter of the tumor was 0.995 cm±0.35,which was significantly smaller than that of the control groups (2.215 cm±025, P<0.05).CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo.     

Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication, a smooth muscle actin expression and typeⅠcollagen synthesis in vitro

AIM: To study the effect of oxymatrine-baicalin combination (OB) against HBV replication in 2.2.15 cells and a smooth muscle actin (αSMA) expression, typeⅠ, collagen synthesis in HSC-T6 cells. METHODS: The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to p actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by p actin. RESULTS: In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group (HBsAg, P=0.043; HBeAg, P=0.026; respectively); HBV DNA level in the OB group was significantly lower than that in the oxymatrine group (P = 0.041). In HSC-T6 cells the mRNA and protein expression levels of a SMA in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P = 0.013; protein, P-0.042; respectively); The mRNA and protein expression levels of typeⅠcollagen in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P<0.01; protein, P<0.01; respectively). CONCLUSION: OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against a SMA expression and typeⅠcollagen synthesis in HSC-T6 cells than oxymatrine in vitro.     

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

AIM： To explore the inhibitory effects of pokeweed antiviral protein seed （PAP-S） and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus （HBV） replication in vitro. METHODS： HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold overlength plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS： The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION： Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.     

乙型肝炎免疫球蛋白聚氰基丙烯酸正丁酯纳米粒抑制HBsAg、HBV DNA分泌的体外实验

Objective To investigate the inhibitive activities of hepatitis B immunoglobulin(HBIG)poly(butylcynaoacrylate)nanoparticles(HBIG-PBCA-NP)to hepatitis B surface antigen(HBsAg)and hepatitis B virus(HBV)DNA secretions using HBV infected cell model in vitro.Methods HepG 2.2.15 cells were cultured with media containing HBIG-PBCA-NP or HBIG for several days,or cultured with HBIG-PBC-NP and HBIG for 2 days and without HBIG-PBCA-NP and HBIG from day 3.The supernatants at different time points were collected for quantitative detection of HBsAg and HBV DNA.The comparisons between groups were done by variance analysis.Resalts Secretions of HBsAg and HBV DNA in supernatants of HepG2.2.15 cultured with 0.1-10.0 IU/mL of HBIG-PBCA-NP and HBIG were inhibited significantly compared with control group.HBsAg titers and HBV DNA levels in supernatants of HBIG-PBCA-NP group and HBIG group cultured with media without HBIG-PBCA-NP and HBIG kept decreasing at day 5 and 7,then rebounded at day 9 and 11.HBsAg titera in supernatants of 0.1,1.0,5.0 IU/mL HBIG-PBCA-NP group were all significantly different from those in HBIG group at day 9[(31.31±1.98)μg/L vs(40.62±2.99)μg/L,(23.79±1.31)μg/L vs(36.51±2.12)μg/L,(19.91±1.74)μg/L vs(33.03±1.65)μg/L;F=412.24,P＜0.01].Couclusion HBIG-PBCA-NP can inhibit secretions of HgsAg and HBV DNA in vitro,which is more effective than HBIG.     

乙型肝炎免疫球蛋白聚氰基丙烯酸正丁酯纳米粒抑制HBsAg、HBV DNA分泌的体外实验

Objective To investigate the inhibitive activities of hepatitis B immunoglobulin(HBIG)poly(butylcynaoacrylate)nanoparticles(HBIG-PBCA-NP)to hepatitis B surface antigen(HBsAg)and hepatitis B virus(HBV)DNA secretions using HBV infected cell model in vitro.Methods HepG 2.2.15 cells were cultured with media containing HBIG-PBCA-NP or HBIG for several days,or cultured with HBIG-PBC-NP and HBIG for 2 days and without HBIG-PBCA-NP and HBIG from day 3.The supernatants at different time points were collected for quantitative detection of HBsAg and HBV DNA.The comparisons between groups were done by variance analysis.Resalts Secretions of HBsAg and HBV DNA in supernatants of HepG2.2.15 cultured with 0.1-10.0 IU/mL of HBIG-PBCA-NP and HBIG were inhibited significantly compared with control group.HBsAg titers and HBV DNA levels in supernatants of HBIG-PBCA-NP group and HBIG group cultured with media without HBIG-PBCA-NP and HBIG kept decreasing at day 5 and 7,then rebounded at day 9 and 11.HBsAg titera in supernatants of 0.1,1.0,5.0 IU/mL HBIG-PBCA-NP group were all significantly different from those in HBIG group at day 9[(31.31±1.98)μg/L vs(40.62±2.99)μg/L,(23.79±1.31)μg/L vs(36.51±2.12)μg/L,(19.91±1.74)μg/L vs(33.03±1.65)μg/L;F=412.24,P＜0.01].Couclusion HBIG-PBCA-NP can inhibit secretions of HgsAg and HBV DNA in vitro,which is more effective than HBIG.     

乙型肝炎免疫球蛋白聚氰基丙烯酸正丁酯纳米粒抑制HBsAg、HBV DNA分泌的体外实验

Objective To investigate the inhibitive activities of hepatitis B immunoglobulin(HBIG)poly(butylcynaoacrylate)nanoparticles(HBIG-PBCA-NP)to hepatitis B surface antigen(HBsAg)and hepatitis B virus(HBV)DNA secretions using HBV infected cell model in vitro.Methods HepG 2.2.15 cells were cultured with media containing HBIG-PBCA-NP or HBIG for several days,or cultured with HBIG-PBC-NP and HBIG for 2 days and without HBIG-PBCA-NP and HBIG from day 3.The supernatants at different time points were collected for quantitative detection of HBsAg and HBV DNA.The comparisons between groups were done by variance analysis.Resalts Secretions of HBsAg and HBV DNA in supernatants of HepG2.2.15 cultured with 0.1-10.0 IU/mL of HBIG-PBCA-NP and HBIG were inhibited significantly compared with control group.HBsAg titers and HBV DNA levels in supernatants of HBIG-PBCA-NP group and HBIG group cultured with media without HBIG-PBCA-NP and HBIG kept decreasing at day 5 and 7,then rebounded at day 9 and 11.HBsAg titera in supernatants of 0.1,1.0,5.0 IU/mL HBIG-PBCA-NP group were all significantly different from those in HBIG group at day 9[(31.31±1.98)μg/L vs(40.62±2.99)μg/L,(23.79±1.31)μg/L vs(36.51±2.12)μg/L,(19.91±1.74)μg/L vs(33.03±1.65)μg/L;F=412.24,P＜0.01].Couclusion HBIG-PBCA-NP can inhibit secretions of HgsAg and HBV DNA in vitro,which is more effective than HBIG.     

乙型肝炎免疫球蛋白聚氰基丙烯酸正丁酯纳米粒抑制HBsAg、HBV DNA分泌的体外实验

Objective To investigate the inhibitive activities of hepatitis B immunoglobulin(HBIG)poly(butylcynaoacrylate)nanoparticles(HBIG-PBCA-NP)to hepatitis B surface antigen(HBsAg)and hepatitis B virus(HBV)DNA secretions using HBV infected cell model in vitro.Methods HepG 2.2.15 cells were cultured with media containing HBIG-PBCA-NP or HBIG for several days,or cultured with HBIG-PBC-NP and HBIG for 2 days and without HBIG-PBCA-NP and HBIG from day 3.The supernatants at different time points were collected for quantitative detection of HBsAg and HBV DNA.The comparisons between groups were done by variance analysis.Resalts Secretions of HBsAg and HBV DNA in supernatants of HepG2.2.15 cultured with 0.1-10.0 IU/mL of HBIG-PBCA-NP and HBIG were inhibited significantly compared with control group.HBsAg titers and HBV DNA levels in supernatants of HBIG-PBCA-NP group and HBIG group cultured with media without HBIG-PBCA-NP and HBIG kept decreasing at day 5 and 7,then rebounded at day 9 and 11.HBsAg titera in supernatants of 0.1,1.0,5.0 IU/mL HBIG-PBCA-NP group were all significantly different from those in HBIG group at day 9[(31.31±1.98)μg/L vs(40.62±2.99)μg/L,(23.79±1.31)μg/L vs(36.51±2.12)μg/L,(19.91±1.74)μg/L vs(33.03±1.65)μg/L;F=412.24,P＜0.01].Couclusion HBIG-PBCA-NP can inhibit secretions of HgsAg and HBV DNA in vitro,which is more effective than HBIG.     

Inhibition of hepatitis B virus gene expression and replication by artificial microRNA

AIM： To investigate the inhibitory effects of hepatitis B virus （HBV） replication and expression by transfecting artificial microRNA （amiRNA） into HepG2.2.15 cells. METHODS： Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays （TRFIA）. HBV DNA replication was examined by ？ uorescence quantitative PCR, and the level of HBV S mRNA was measured by semi- quantitative RT-PCR. RESULTS： The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection （P 〈 0.01 for all）. The secretion of HBsAg and HBeAg into the supernatant was inhibited by 49.8% ± 4.7% and 39.9% ± 6.7%, respectively, at 72 h in amiRNA- HBV-S608 plasmid transfection group. The copy of HBV DNA within culture supernatant was also significantly decreased at 72 h and 96 h after transfection （P 〈0.01 for all）. In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups （P 〈 0.01 for all, vs negative control）. The copies of HBV DNA were inhibited by 33.4% ± 3.0%, 60.8% ± 2.3% and 70.1% ± 3.3%, respectively. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression could be inhibited by artif icial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.     

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with In vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log 10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.     

乙型肝炎免疫球蛋白聚氰基丙烯酸正丁酯纳米粒抑制HBsAg、HBV DNA分泌的体外实验

Objective To investigate the inhibitive activities of hepatitis B immunoglobulin(HBIG)poly(butylcynaoacrylate)nanoparticles(HBIG-PBCA-NP)to hepatitis B surface antigen(HBsAg)and hepatitis B virus(HBV)DNA secretions using HBV infected cell model in vitro.Methods HepG 2.2.15 cells were cultured with media containing HBIG-PBCA-NP or HBIG for several days,or cultured with HBIG-PBC-NP and HBIG for 2 days and without HBIG-PBCA-NP and HBIG from day 3.The supernatants at different time points were collected for quantitative detection of HBsAg and HBV DNA.The comparisons between groups were done by variance analysis.Resalts Secretions of HBsAg and HBV DNA in supernatants of HepG2.2.15 cultured with 0.1-10.0 IU/mL of HBIG-PBCA-NP and HBIG were inhibited significantly compared with control group.HBsAg titers and HBV DNA levels in supernatants of HBIG-PBCA-NP group and HBIG group cultured with media without HBIG-PBCA-NP and HBIG kept decreasing at day 5 and 7,then rebounded at day 9 and 11.HBsAg titera in supernatants of 0.1,1.0,5.0 IU/mL HBIG-PBCA-NP group were all significantly different from those in HBIG group at day 9[(31.31±1.98)μg/L vs(40.62±2.99)μg/L,(23.79±1.31)μg/L vs(36.51±2.12)μg/L,(19.91±1.74)μg/L vs(33.03±1.65)μg/L;F=412.24,P＜0.01].Couclusion HBIG-PBCA-NP can inhibit secretions of HgsAg and HBV DNA in vitro,which is more effective than HBIG.     

反义锁核酸与拉米夫定在HepG2.2.15细胞中抗HBV作用的比较

目的:应用反义锁核酸与拉米夫定作用HepG2.2.15细胞,对他们抗乙型肝炎病毒(hepatitis B virus,HBV)效果进行比较.方法:设计针对HBV翻译起始区S基因mRNA的反义寡核苷酸,并进行锁核酸修饰,以阳离子脂质体介导反义锁核酸转染HepG2.2.15细胞;拉米夫定组直接作用HepG2.2.15细胞;分别于用药后第2、4、6、8、10天收集细胞培养上清液.用ELISA法和FQ-PCR法检测收集上清液HBsAg、HBeAg和HBVDNA的含量.MTT法分别检测反义锁核酸与拉米夫定对细胞存活率的影响.结果:拉米夫定对HBVDNA具有明显抑制作用,最高可达46.52%,但对HBsAg、HBeAg影响较小;反义锁核酸对HBsAg、HBeAg及HBVDNA均有较强抑制作用,对HBsAg、HBeAg和HBVDNA的最高抑制率分别达67.69%、59.71%和62.96%(P<0.05),且抑制随时间呈增高趋势.反义锁核酸与拉米夫定对细胞代谢均无明显影响.结论:反义锁核酸抗HBV作用机制与拉米夫定不同,反义锁核酸抗HBV作用明显优于拉米夫定.     

三螺旋形成寡核苷酸抑制乙型肝炎病毒复制及抗原合成的实验研究

目的 探讨三螺旋形成寡核苷酸抗乙型肝炎病毒（ＨＢＶ）作用。方法 针对ＨＢＶ核心启动子ＳＰ１位点,合成２１ｍｅｒ硫代磷酸三螺旋形成寡核苷酸及２１ｍｅｒ无关对照寡核苷酸。采用ＬＥＩＳＡ,斑点杂交法分别检测了经寡核苷酸处理的ＨｅｐＧ２．２．１５细胞及空白对照组细胞培养上清ＨＢｓＡｇ,ＨＢｅＡｇ及ＨＢＶ ＤＮＡ水平。结果 ＴＦＯ２１组２．２．１５细胞ＨＢｓＡｇ及ＨＢＶ ＤＮＡ分泌量明显低于空白对照组。ＴＦ     

甲胎蛋白启动子介导反义乙型肝炎病毒X基因在肝癌细胞中的表达及其作用

目的 构建含甲胎蛋白(AFP)启动子和增强子的反义乙型肝炎病毒X基因(HBX)真核表达载体，研究其特异性和有效性，为开发肝癌细胞特异性HBX反义RNA基因治疗乙型肝炎病毒(HBV)奠定基础。方法 聚合酶链反应(PCR)扩增HBX(1370—1872nt)基因，克隆至EB病毒表达载体，双轮PCR筛选、鉴定基因插入方向。脂质体转染肝癌细胞和ECV304细胞，Northernblot检测HBX mRNA的表达，酶联免疫试验(ELISA)检测HBV抗原，荧光定量PCR检测HBV DNA。结果 成功构建正、反义RNA表达载体pEBAF—s—HBX、pEBAF—as—HBX。Northernblot证实反义RNA仅在AFP阳性的肝癌细胞中表达。pEBAF—as—HBX转染3d后，可显著抑制2．2．15细胞HBV复制和抗原表达，其HBsAg、HBeAg抗原表达较正义对照分别下降37．9％和36．8％，HBV DNA降低25％。结论 反义RNA表达载体pEBAF—as—HBX仅在肝癌细胞中特异表达、并可有效抑制HBV，有良好的开发应用前景。     

靶向S区和C区基因的M1GS RNA核酶共同抑制HBV的表达

目的:研究靶向HBVS区和C区基因的M1GSRNA核酶共同作用对HBV基因表达的影响.方法:选择HBVayw亚型S区基因294nt和C区基因2333nt为切割位点,以含有编码M1RNA的DNA序列的质粒pTK117为模板,通过PCR扩增得到M1GSRNA核酶的DNA模板,并将其克隆至真核表达载体pEGFP-C1得到重组质粒pEGFP-GSS和pEGFP-GSC.将2个重组质粒共转染HepG2.2.15细胞,转染后ELISA法测细胞培养液中的HBsAg和HBeAg,RT-PCR检测HBVmRNA.结果:成功构建了分别靶向HBVS区基因和C区基因的真核表达载体.共转染HepG2.2.15细胞后,HBsAg和HBeAg的表达分别被抑制了33.2%和39.1%,HBVCmRNA和SmRNA分别被抑制了32.5%和29.7%,而HepG2.2.15细胞的增殖无明显变化.结论:靶向HBVS区和C区基因的M1GSRNA核酶共同作用可特异性抑制HBVS区和C区基因的表达.     

乙型肝炎病毒(HBV)反基因与HBV的结合及对HBV复制的影响

目的 研究三螺旋形成寡核苷酸与HBV基因的结合情况及其对HBV复制的影响。方法 全盛一段能与HBV核心启动子位点（1734-1753nt)形成三螺旋的寡核苷酸（TF020）并标记上生物素分别用脂质体-TF020及裸TF020转染HepG2.2.15细胞,用链酶亲和素-生物素法（SABC）检测其转染效率及其与HepG2.2.15细胞中HBVDNA的结合情况;并采用ELISA、半定量逆转录（RT）-PCR及荧光定量PCR法分别检测经寡核苷酸处理的HepG2.2.15细胞及空白对照细胞上清HBsAg、HBeAg、HBV DNA及细胞中HBV RNA的水平。结果 脂质体-TF020转染HepG2.2.15细胞的效率可达80%,而裸TFO20转染率最高为40%;TF020主要定位于细胞核及胞浆中,TF020处理组细胞上清中HBsAg、HBeAg含量分别较空白对照组降低37.0%及78.2%,HBV DNA水平较空白对照组明显降低;而细胞中2.4kb/2.1kb RNA、3.5kb/3.4kbRNA亦分别降低31.6%及70.2Z%。结论 TF020通过脂质体包裹后可以很好地进入细胞并与HBV DNA结合;TF020能够有效地抑制HBV复制,发挥抗病毒作用。     

Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells

AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression by combination of siRNA and lamivudine in HepG2.2.15 cells. METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. The transfected cells were cultured in lamivudine-containing medium (0.05 μmol/L) and harvested at 48, 72 and 96 h. The concentration of HBeAg and HBsAg was determined using ELISA. HBV DNA replication was examined by real- time PCR and the level of HBV mRNA was measured by RT-PCR. RESULTS: In HepG2.2.15 cells treated with combination of siRNA and lamivudine, the secretion of HBeAg and HBsAg into the supernatant was found to be inhibited by 91.80% and 82.40% (2.89 ± 0.48 vs 11.73 ± 0.38, P < 0.05; 4.59 ± 0.57 vs 16.25 ± 0.48, P < 0.05) at 96 h, respectively; the number of HBV DNA copies within culture medium was also significantly decreased at 96 h (1.04 ± 0.26 vs 8.35 ± 0.33, P < 0.05). Moreover, mRNA concentration in HepG2.2.15 cells treated with combination of siRNA and lamivudine was obviously lower compared to those treated either with siRNA or lamivudine (19.44 ± 0.17 vs 33.27 ± 0.21 or 79.9 ± 0.13, P < 0.05). CONCLUSION: Combination of siRNA and lamivudine is more effective in inhibiting HBV replication as compared to the single use of siRNA or lamivudine in HepG2.2.15 cells.     

RNA干扰技术用于抗乙型肝炎病毒的实验研究

目的 构建针对乙型肝炎病毒表面抗原(HBsAg)和核心抗原的小干扰RNA(siRNA)表达载体pSuper-C，观察其对HepG2 2．2．15细胞(简称2、2．15细胞)中HBV DNA转录和翻译相应蛋白的影响。方法 根据RNA干扰(RNAi)作用原理设计针对HBV核心区的相应序列，再将其克隆入含聚合酶ⅢH1-RNA启动子的真核表达载体pSuper，将此重组质粒以电转染法转入2．2．15细胞中，用酶联免疫吸附法(Abbott试剂)检测培养上清液中HBsAg和e抗原(HBeAg)的表达。结果 经酶切鉴定、电泳和测序分析证明，成功构建了含作用序列的重组质粒pSuper-C；但以电穿孔法转染 2．2．15细胞后末能发现其对2．2．15细胞培养上清液中的HBsAg和HBeAg的表达有影响。结论 RNAi在2．2．15细胞中的作用还需进一步的实验来证实。