重组人白蛋白干扰素α-2b融合蛋白抗乙肝病毒的药效学及部分机制
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摘要
慢性乙型肝炎(Chronic Hepatitis B,CHB)是一种严重危害人类健康的病毒性传染病。我国是乙肝病毒(Hepatitis B Virus,HBV)高流行区,一般人群的乙型肝炎表面抗原(Hepatitis B surface Antigen,HBsAg)阳性率为9.09%,约有1.1～1.2亿为HBV携带者,其中部分有不同程度的肝细胞受损,并可能发展成肝硬化和肝癌。
干扰素(Interferon,IFN)是细胞和机体受到病毒感染,或者受核酸、细菌内毒素和促细胞分裂素等作用后,由淋巴细胞分泌的一种细胞因子,可分为α、β、γ三类。IFN-α具有抗病毒及免疫调节的双重作用,是临床常用的抗HBV药物。
目前用于临床的普通IFN-α半衰期只有4～6h,患者不得不接受频繁的皮下注射(疗程至少半年),但其长期疗效并不确切。重组人白蛋白融合干扰素α-2b融合蛋白( recombinant human serum albumin-interferon-α-2b fusion protein ,rHSA-IFNα-2b)是运用基因重组技术开发出来的一种新型长效干扰素,其在体内的半衰期显著长于IFNα-2b和聚乙二醇干扰素(Pegylated Interferon,PEG-IFN)。rHSA-IFNα-2b注射频率可以从IFNα-2b的每天一次或每周3次减少到每两周一次,这将极大地方便患者。同时长效干扰素所提供的较为稳定的血药浓度也将提高疗效,降低不良反应。
动物实验及临床试验研究已证明rHSA-IFNα-2b可发挥有效的抗丙肝病毒(Hepatitis C Virus,HCV)作用,但是否同样具有抗HBV作用还有待进一步研究。本研究拟通过探索rHSA-IFNα-2b对体外2.2.15细胞(HepG2 2.2.15 cell)HBsAg、HBeAg(Hepatitis B e Antigen)分泌、HBV DNA复制的影响,及对鸭乙型肝炎动物模型的肝功能影响和对鸭乙肝病毒(DHBV)DNA的抑制作用,来系统考察rHSA-IFNα-2b的抗HBV作用,为rHSA-IFNα-2b治疗CHB的临床试验提供依据。
IFN-α主要通过激动特异性细胞膜受体来发挥抗病毒作用。当IFN-α与靶细胞表面的特异性受体结合后,通过JAK-STAT信号通路,触发细胞内一系列酶活化,产生一组抗病毒蛋白,包括2'-5'-寡腺苷酸合成酶(2'-5'-oligoadenylate synthetase,2'-5'-OAS)、磷酸二脂酶(phosphodiesterase,PDE)及蛋白激酶(protein Kinase,PK)。OAS可催化2'-5'-寡核苷酸(2'-5'-oligoadenylate,2-5A)合成,激活内源性核酸内切酶,抑制病毒mRNA信息的传递,从而阻止病毒在宿主细胞内繁殖。本研究同时拟通过考察rHSA-IFNα-2b对抗病毒蛋白OAS的影响,及与Jak-Stat通路、p38-MAPK通路的关联,来阐述其发挥抗HBV作用的机制。
(一)体外药效学及机制研究:
本课题选用2.2.15细胞作乙型肝炎体外模型,首先采用MTT法考察rHSA-IFNα-2b对2.2.15细胞的毒性作用。选择含8个实验浓度梯度rHSA-IFNα-2b(500、250、125、62.5、31.2、15.6、7.8、3.9nmol/L)的培养液,培养2.2.15细胞9天后,加入含有400mg/L MTT的培养液孵育4 h,弃MTT液并加入DMSO,用酶标仪测定吸光度(absorbance,A)值,来考察药物对细胞的破坏程度。结果发现,rHSA-IFNα-2b对2.2.15细胞形态仅有轻微的损坏,且细胞破坏率与细胞形态变化无明显关系,TC50>500 nmol/L,远高于其有效剂量。
我们选用含三个不同rHSA-IFNα-2b浓度梯度(0.075,0.3,1.2 nmol/L)的培养液来培养2.2.15细胞,每3天更换含有药物的培养液,分别收集第3、6、9天上清液。细胞上清液中的HBsAg、HBeAg采用ELISA法检测,用酶标仪测定其A值,并根据标准品A值折算得出样品浓度。细胞上清液中的HBV DNA浓度通过实时荧光定量PCR测定,最后通过标准品的CT值来计算样品DNA的浓度。实验结果发现,rHSA-IFNα-2b明显抑制HBsAg、HBeAg分泌,其中浓度在0.075～1.2 nmol/L范围内时,其对HBsAg的抑制作用呈现浓度依赖性;rHSA-IFNα-2b能抑制培养上清液中HBV DNA复制,也呈明显浓度依赖性。IFNα-2b与rHSA-IFNα-2b作用无显著性差异。
随后,我们用rHSA-IFNα-2b(0.075、0.3、1.2 nmol/L)作用2.2.15细胞3天后,收集细胞并提取总RNA,用RT-PCR方法考察STAT1、ISGF3、2'-5'-OAS的变化;给予JAK抑制剂或p38抑制剂之后1h再给予rHSA-IFNα-2b(0.3 nmol/L),48h后收集培养上清液,用ELISA法检测培养上清液中的HBsAg,用实时荧光定量PCR检测上清液HBV-DNA,用Western Blot检测2.2.15细胞2'-5'-OAS蛋白的表达。
结果表明,rHSA-IFNα-2b作用3天后,2.2.15细胞的OAS表达明显增加,呈现一定的浓度依赖性;STAT1和ISGF3的表达也有所增加,但浓度依赖性不明显。
rHSA-IFNα-2b可抑制HBsAg分泌、HBV-DNA复制,增加2'-5'-OAS蛋白表达;p38抑制剂对rHSA-IFNα-2b作用产生轻微影响,JAK抑制剂可产生较大影响,两抑制剂合用可产生更明显影响,但并不能完全阻断rHSA-IFNα-2b的作用。这说明rHSA-IFNα-2b主要通过Jak-Stat通路发挥作用,p38-MAPK通路也起部分作用。
(二)体内药效学:
本课题选用感染了鸭乙型肝炎病毒的安徽麻鸭作动物模型,用PCR筛选DHBV阳性雏鸭,皮下注射rHSA-IFNα-2b(0.25、0.5、1.0nmol/kg),每周1次,共4周;选用IFNα-2b作阳性对照,0.2 nmol/kg,每周3次,共4周;停药1周后处死动物,取肝脏做病理切片。用试剂盒检测血清丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、血清总胆红素(total bilirubin,TBIL)的含量,用实时荧光定量PCR检测DHBV DNA拷贝数;对鸭肝脏进行病理检查;并用Western Blot检测肝脏2'-5'-OAS蛋白的表达。
结果表明,rHSA-IFNα-2b治疗4周后,鸭血清ALT、AST、TBIL水平明显降低,其中ALT对rHSA-IFNα-2b治疗更为敏感,表现出一定的剂量依赖性; IFNα-2b也能明显降低ALT、AST、TBIL,但ALT在停药7天后出现反跳,而rHSA-IFNα-2b三治疗组无明显反跳。rHSA-IFNα-2b可使鸭血清DHBV DNA拷贝数明显降低,治疗4周后显示明显剂量依赖性,停药1周后未见反跳;而IFNα-2b停药1周后有明显反跳。rHSA-IFNα-2b对DHBV DNA的抑制率明显高于IFNα-2b。鸭肝脏病理检查发现感染DHBV实验鸭肝细胞出现变性、坏死及炎性细胞浸润,而rHSA-IFNα-2b可使肝细胞变性明显减轻;肝脏组织学评分显示rHSA-IFNα-2b肝脏病理改善要明显强于IFNα-2b。rHSA-IFNα-2b还可增加肝脏细胞2'-5'-OAS蛋白表达,呈现剂量依赖性。
总之,rHSA-IFNα-2b能有效抑制2.2.15细胞和鸭乙肝动物模型中乙肝病毒的增殖,它主要是通过影响Jak-Stat通路,增加抗病毒蛋白OAS的分泌而实现的;p38-MAPK通路也发挥部分作用。
Chronic hepatitis B is a serious public health risk for human health problem. China is a high HBV endemic area, and the rate of hepatitis B surface antigen (HBsAg)-positive is about 9%, and the HBV carriers are about 110-120 million, some of which got varying degrees of liver cell damage and may develop into cirrhosis of the liver and liver cancer.
Currently, clinical anti-HBV drugs are: interferons, nucleoside/nucleoside analogues and immunomodulators. IFN is a cytokine secreted by lymphocytes after infected by virus or bacteria, and has a variety of functions such as anti-virus, anti-tumor and immune regulation. In accordance with immunogenicity the different molecular structures, IFNs can be divided intoα,β,γcategories, and the antiviral effect of IFN-αis strongest.
Interferons (IFNs) are a family of soluble glycoproteins produced by different cells and can be divided intoα,β,γcategories. IFN-αis one of anti-HBV drugs commonly used in clinic, and exhibits antiviral and immunomodulation functions.
Conventional IFN-αhas been used for the treatment of CHB for many decades. However, it also has some shortcomings, such as short half-life (4~6h), which makes patients have to accept frequent injections for at least half a year. In order to overcome these shortcomings, rHSA-IFNα-2b was developed, whose half-life is much longer than that of IFNα-2b, and even longer than that of PEG-IFNα. The reduction of injection frequency would greatly facilitate patient who need long period treatment. At the same time, long-acting interferon provided a more stable plasma concentration, which will also improve efficacy and reduce side effects.
Animal experiments and clinical trials have proved rHSA-IFN to be an effective anti-HCV drug. However, whether it can reach the same anti-HBV effect, will be further research. In this study, we are trying to investigate its anti-HBV effect and some mechanisms in vitro and in vivo, and to provide information for the clinical research of rHSA-IFNα-2b in CHB therapy.
IFN-αtriggers a series of signal transduction events induced by the binding of IFN-αto its cell surface receptors, and a series of enzymes are actived and produce a group of anti-viral protein, including 2'-5'-oligoadenylate synthetase (OAS), phosphodiesterase (PDE) and protein kinase. OAS can prevent virus reproduction in host cells by catalyzing oligonucleotide synthesis, activating endogenous endonuclease, and inhibiting the information transmission of virus mRNA. PDE could end virus tRNA degradation; and protein kinase can inhibit protein synthesis and viral replication. At the same time, in order to explore its anti-HBV mechanism, this study was to examine the effects of rHSA-IFNα-2b on antiviral protein OAS, and on the relationship of Jak-Stat pathway and p38-MAPK pathway.
(A) Pharmacodynamics in vitro
HepG2 2.2.15 cells were selected as the model of hepatitis B in vitro. Firstly, we study the toxicity of rHSA-IFNα-2b by MTT method. The cells were cultured for 9 days in different concentrations of rHSA-IFNα-2b (500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9 nmol/L), then MTT was added, and then DMSO. The absorbance (A) was measured by absorbance microplate reader to examine the degree of cell damage. The results showed that, 2.2.15 cells were slightly damaged by rHSA-IFNα-2b, but damage rate had no significant direct relationship with changes of morphology, and TC50>500 nmol/L, well above its effective dose.
Subsequently, the 2.2.15 cells were cultured in three different concentrations of rHSA-IFNα-2b (0.075, 0.3 and 1.2 nmol/L) for 9 days, and the culture medium was replaced every three days. HBsAg and HBeAg in cell supernatants were detected using ELISA kits, and the copies of HBV DNA were detected by real-time fluorescence quantitative PCR. The experimental results showed that, rHSA-IFNα-2b significantly inhibited secretion of HBsAg and HBeAg, and there was obvious dose-dependent relationship between the secretion of HBsAg with rHSA-IFNα-2b at concentrations from 0.075 to 1.2 nmol/L, but not with HBeAg. And the effect of rHSA-IFNα-2b was no significant difference with that of IFNα-2b.
Three days after administration of rHSA-IFNα-2b (0.075,0.3,1.2 nmol/L), the cells were collected and total RNA was extracted by Trizol.The expressions of signal transducers and transactivator 1 (STAT1), IFN-stimulated gene factor 3 (ISGF3) and 2'-5'-Oligoadenylate synthetase 1 (OAS1) in HepG2 2.2.15 cells were detected by RT-PCR. And then the changes of HBsAg, HBV DNA and 2'-5'-OAS were detected after the intervention of p38 inhibitor or JAK inhibitor.
The results showed that, three days after the treatment of rHSA-IFNα-2b, the expression of OAS1 in HepG2 2.2.15 cells was increased in a dose-dependent manner; STAT1 and ISGF3 expression also increased, but no dose-dependent manner.
rHSA-IFNα-2b could inhibit the secretion of HBsAg and HBV-DNA, increase the expression of 2'-5'-OAS protein. p38 inhibitor had a minor impact on the effects of rHSA-IFNα-2b, JAK inhibitors had great influence, the combination of two inhibitors could produce a more significant impact, but did not block the rHSA-IFNα-2b role completely. This indicated that rHSA-IFNα-2b produces a marked effect primarily through the Jak-Stat pathways, and partly through p38-MAPK pathway.
(B) The efficacy study in vivo
The DHBV-infected Anhui ducks were selected as the animal model in vivo. Firstly, the DHBV-positive ducklings were judged by PCR, then rHSA-IFNα-2b (0.25, 0.5, 1.0 nmol/kg) were injected subcutaneously, once a week for 4 weeks. IFNα-2b was the positive control, 0.2 nmol/kg, 3 times a week for 4 weeks. The animals were sacrificed one week after cessation of the treatment, and the livers were obtained immediately after collecting blood from the vena cruralis. The Changes of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum total bilirubin (TBIL) and DHBV DNA were explored, and the duck livers were examined by pathological method; and the expression of liver 2'-5'-OAS was detected by Western Blotting.
The results showed that, four weeks after treatment with rHSA-IFNα-2b, the major liver enzymes (ALT, AST and TBIL) were reduced significantly, and ALT was more significantly in a dose-dependent manner. IFNα-2b can significantly reduce the levels of ALT, AST, TBIL, and the ALT level had a rebound at day 35, but not obviously in rHSA-IFNα-2b-treated groups.
rHSA-IFNα-2b could markedly decrease the concentrations of DHBV DNA in the duckling model, and was dose-dependent after 4 weeks` treatment. All animals in the IFNα-2b-treated group showed a reduction in DHBV DNA levels over the course of treatment, with less potency than those treated with rHSA-IFNα-2b.
Histopathological profiles of the liver from the control group ducks revealed necrosis, inflammatory cell infiltration and massive ballooning degeneration of the hepatic cytoplasm. Administrations of rHSA-IFNα-2b to the experimental animals showed a dose-dependent improvement of the hepatocellular architecture over the control group. rHSA-IFNα-2b resulted in more obvious improvements than that IFNα-2b did.
rHSA-IFNα-2b could increase the expression of 2'-5'-OAS protein in liver cell remarkably, and showed significant dose-dependent manner.
In Conclusion, rHSA-IFNα-2b can effectively inhibit the proliferation of HBV in vitro and in vivo, and it is mainly through the Jak-Stat pathways, and partly through p38-MAPK pathway, to increase the secretion of OAS.
干扰素(Interferon,IFN)是细胞和机体受到病毒感染,或者受核酸、细菌内毒素和促细胞分裂素等作用后,由淋巴细胞分泌的一种细胞因子,可分为α、β、γ三类。IFN-α具有抗病毒及免疫调节的双重作用,是临床常用的抗HBV药物。
目前用于临床的普通IFN-α半衰期只有4～6h,患者不得不接受频繁的皮下注射(疗程至少半年),但其长期疗效并不确切。重组人白蛋白融合干扰素α-2b融合蛋白( recombinant human serum albumin-interferon-α-2b fusion protein ,rHSA-IFNα-2b)是运用基因重组技术开发出来的一种新型长效干扰素,其在体内的半衰期显著长于IFNα-2b和聚乙二醇干扰素(Pegylated Interferon,PEG-IFN)。rHSA-IFNα-2b注射频率可以从IFNα-2b的每天一次或每周3次减少到每两周一次,这将极大地方便患者。同时长效干扰素所提供的较为稳定的血药浓度也将提高疗效,降低不良反应。
动物实验及临床试验研究已证明rHSA-IFNα-2b可发挥有效的抗丙肝病毒(Hepatitis C Virus,HCV)作用,但是否同样具有抗HBV作用还有待进一步研究。本研究拟通过探索rHSA-IFNα-2b对体外2.2.15细胞(HepG2 2.2.15 cell)HBsAg、HBeAg(Hepatitis B e Antigen)分泌、HBV DNA复制的影响,及对鸭乙型肝炎动物模型的肝功能影响和对鸭乙肝病毒(DHBV)DNA的抑制作用,来系统考察rHSA-IFNα-2b的抗HBV作用,为rHSA-IFNα-2b治疗CHB的临床试验提供依据。
IFN-α主要通过激动特异性细胞膜受体来发挥抗病毒作用。当IFN-α与靶细胞表面的特异性受体结合后,通过JAK-STAT信号通路,触发细胞内一系列酶活化,产生一组抗病毒蛋白,包括2'-5'-寡腺苷酸合成酶(2'-5'-oligoadenylate synthetase,2'-5'-OAS)、磷酸二脂酶(phosphodiesterase,PDE)及蛋白激酶(protein Kinase,PK)。OAS可催化2'-5'-寡核苷酸(2'-5'-oligoadenylate,2-5A)合成,激活内源性核酸内切酶,抑制病毒mRNA信息的传递,从而阻止病毒在宿主细胞内繁殖。本研究同时拟通过考察rHSA-IFNα-2b对抗病毒蛋白OAS的影响,及与Jak-Stat通路、p38-MAPK通路的关联,来阐述其发挥抗HBV作用的机制。
(一)体外药效学及机制研究:
本课题选用2.2.15细胞作乙型肝炎体外模型,首先采用MTT法考察rHSA-IFNα-2b对2.2.15细胞的毒性作用。选择含8个实验浓度梯度rHSA-IFNα-2b(500、250、125、62.5、31.2、15.6、7.8、3.9nmol/L)的培养液,培养2.2.15细胞9天后,加入含有400mg/L MTT的培养液孵育4 h,弃MTT液并加入DMSO,用酶标仪测定吸光度(absorbance,A)值,来考察药物对细胞的破坏程度。结果发现,rHSA-IFNα-2b对2.2.15细胞形态仅有轻微的损坏,且细胞破坏率与细胞形态变化无明显关系,TC50>500 nmol/L,远高于其有效剂量。
我们选用含三个不同rHSA-IFNα-2b浓度梯度(0.075,0.3,1.2 nmol/L)的培养液来培养2.2.15细胞,每3天更换含有药物的培养液,分别收集第3、6、9天上清液。细胞上清液中的HBsAg、HBeAg采用ELISA法检测,用酶标仪测定其A值,并根据标准品A值折算得出样品浓度。细胞上清液中的HBV DNA浓度通过实时荧光定量PCR测定,最后通过标准品的CT值来计算样品DNA的浓度。实验结果发现,rHSA-IFNα-2b明显抑制HBsAg、HBeAg分泌,其中浓度在0.075～1.2 nmol/L范围内时,其对HBsAg的抑制作用呈现浓度依赖性;rHSA-IFNα-2b能抑制培养上清液中HBV DNA复制,也呈明显浓度依赖性。IFNα-2b与rHSA-IFNα-2b作用无显著性差异。
随后,我们用rHSA-IFNα-2b(0.075、0.3、1.2 nmol/L)作用2.2.15细胞3天后,收集细胞并提取总RNA,用RT-PCR方法考察STAT1、ISGF3、2'-5'-OAS的变化;给予JAK抑制剂或p38抑制剂之后1h再给予rHSA-IFNα-2b(0.3 nmol/L),48h后收集培养上清液,用ELISA法检测培养上清液中的HBsAg,用实时荧光定量PCR检测上清液HBV-DNA,用Western Blot检测2.2.15细胞2'-5'-OAS蛋白的表达。
结果表明,rHSA-IFNα-2b作用3天后,2.2.15细胞的OAS表达明显增加,呈现一定的浓度依赖性;STAT1和ISGF3的表达也有所增加,但浓度依赖性不明显。
rHSA-IFNα-2b可抑制HBsAg分泌、HBV-DNA复制,增加2'-5'-OAS蛋白表达;p38抑制剂对rHSA-IFNα-2b作用产生轻微影响,JAK抑制剂可产生较大影响,两抑制剂合用可产生更明显影响,但并不能完全阻断rHSA-IFNα-2b的作用。这说明rHSA-IFNα-2b主要通过Jak-Stat通路发挥作用,p38-MAPK通路也起部分作用。
(二)体内药效学:
本课题选用感染了鸭乙型肝炎病毒的安徽麻鸭作动物模型,用PCR筛选DHBV阳性雏鸭,皮下注射rHSA-IFNα-2b(0.25、0.5、1.0nmol/kg),每周1次,共4周;选用IFNα-2b作阳性对照,0.2 nmol/kg,每周3次,共4周;停药1周后处死动物,取肝脏做病理切片。用试剂盒检测血清丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、血清总胆红素(total bilirubin,TBIL)的含量,用实时荧光定量PCR检测DHBV DNA拷贝数;对鸭肝脏进行病理检查;并用Western Blot检测肝脏2'-5'-OAS蛋白的表达。
结果表明,rHSA-IFNα-2b治疗4周后,鸭血清ALT、AST、TBIL水平明显降低,其中ALT对rHSA-IFNα-2b治疗更为敏感,表现出一定的剂量依赖性; IFNα-2b也能明显降低ALT、AST、TBIL,但ALT在停药7天后出现反跳,而rHSA-IFNα-2b三治疗组无明显反跳。rHSA-IFNα-2b可使鸭血清DHBV DNA拷贝数明显降低,治疗4周后显示明显剂量依赖性,停药1周后未见反跳;而IFNα-2b停药1周后有明显反跳。rHSA-IFNα-2b对DHBV DNA的抑制率明显高于IFNα-2b。鸭肝脏病理检查发现感染DHBV实验鸭肝细胞出现变性、坏死及炎性细胞浸润,而rHSA-IFNα-2b可使肝细胞变性明显减轻;肝脏组织学评分显示rHSA-IFNα-2b肝脏病理改善要明显强于IFNα-2b。rHSA-IFNα-2b还可增加肝脏细胞2'-5'-OAS蛋白表达,呈现剂量依赖性。
总之,rHSA-IFNα-2b能有效抑制2.2.15细胞和鸭乙肝动物模型中乙肝病毒的增殖,它主要是通过影响Jak-Stat通路,增加抗病毒蛋白OAS的分泌而实现的;p38-MAPK通路也发挥部分作用。
Chronic hepatitis B is a serious public health risk for human health problem. China is a high HBV endemic area, and the rate of hepatitis B surface antigen (HBsAg)-positive is about 9%, and the HBV carriers are about 110-120 million, some of which got varying degrees of liver cell damage and may develop into cirrhosis of the liver and liver cancer.
Currently, clinical anti-HBV drugs are: interferons, nucleoside/nucleoside analogues and immunomodulators. IFN is a cytokine secreted by lymphocytes after infected by virus or bacteria, and has a variety of functions such as anti-virus, anti-tumor and immune regulation. In accordance with immunogenicity the different molecular structures, IFNs can be divided intoα,β,γcategories, and the antiviral effect of IFN-αis strongest.
Interferons (IFNs) are a family of soluble glycoproteins produced by different cells and can be divided intoα,β,γcategories. IFN-αis one of anti-HBV drugs commonly used in clinic, and exhibits antiviral and immunomodulation functions.
Conventional IFN-αhas been used for the treatment of CHB for many decades. However, it also has some shortcomings, such as short half-life (4~6h), which makes patients have to accept frequent injections for at least half a year. In order to overcome these shortcomings, rHSA-IFNα-2b was developed, whose half-life is much longer than that of IFNα-2b, and even longer than that of PEG-IFNα. The reduction of injection frequency would greatly facilitate patient who need long period treatment. At the same time, long-acting interferon provided a more stable plasma concentration, which will also improve efficacy and reduce side effects.
Animal experiments and clinical trials have proved rHSA-IFN to be an effective anti-HCV drug. However, whether it can reach the same anti-HBV effect, will be further research. In this study, we are trying to investigate its anti-HBV effect and some mechanisms in vitro and in vivo, and to provide information for the clinical research of rHSA-IFNα-2b in CHB therapy.
IFN-αtriggers a series of signal transduction events induced by the binding of IFN-αto its cell surface receptors, and a series of enzymes are actived and produce a group of anti-viral protein, including 2'-5'-oligoadenylate synthetase (OAS), phosphodiesterase (PDE) and protein kinase. OAS can prevent virus reproduction in host cells by catalyzing oligonucleotide synthesis, activating endogenous endonuclease, and inhibiting the information transmission of virus mRNA. PDE could end virus tRNA degradation; and protein kinase can inhibit protein synthesis and viral replication. At the same time, in order to explore its anti-HBV mechanism, this study was to examine the effects of rHSA-IFNα-2b on antiviral protein OAS, and on the relationship of Jak-Stat pathway and p38-MAPK pathway.
(A) Pharmacodynamics in vitro
HepG2 2.2.15 cells were selected as the model of hepatitis B in vitro. Firstly, we study the toxicity of rHSA-IFNα-2b by MTT method. The cells were cultured for 9 days in different concentrations of rHSA-IFNα-2b (500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9 nmol/L), then MTT was added, and then DMSO. The absorbance (A) was measured by absorbance microplate reader to examine the degree of cell damage. The results showed that, 2.2.15 cells were slightly damaged by rHSA-IFNα-2b, but damage rate had no significant direct relationship with changes of morphology, and TC50>500 nmol/L, well above its effective dose.
Subsequently, the 2.2.15 cells were cultured in three different concentrations of rHSA-IFNα-2b (0.075, 0.3 and 1.2 nmol/L) for 9 days, and the culture medium was replaced every three days. HBsAg and HBeAg in cell supernatants were detected using ELISA kits, and the copies of HBV DNA were detected by real-time fluorescence quantitative PCR. The experimental results showed that, rHSA-IFNα-2b significantly inhibited secretion of HBsAg and HBeAg, and there was obvious dose-dependent relationship between the secretion of HBsAg with rHSA-IFNα-2b at concentrations from 0.075 to 1.2 nmol/L, but not with HBeAg. And the effect of rHSA-IFNα-2b was no significant difference with that of IFNα-2b.
Three days after administration of rHSA-IFNα-2b (0.075,0.3,1.2 nmol/L), the cells were collected and total RNA was extracted by Trizol.The expressions of signal transducers and transactivator 1 (STAT1), IFN-stimulated gene factor 3 (ISGF3) and 2'-5'-Oligoadenylate synthetase 1 (OAS1) in HepG2 2.2.15 cells were detected by RT-PCR. And then the changes of HBsAg, HBV DNA and 2'-5'-OAS were detected after the intervention of p38 inhibitor or JAK inhibitor.
The results showed that, three days after the treatment of rHSA-IFNα-2b, the expression of OAS1 in HepG2 2.2.15 cells was increased in a dose-dependent manner; STAT1 and ISGF3 expression also increased, but no dose-dependent manner.
rHSA-IFNα-2b could inhibit the secretion of HBsAg and HBV-DNA, increase the expression of 2'-5'-OAS protein. p38 inhibitor had a minor impact on the effects of rHSA-IFNα-2b, JAK inhibitors had great influence, the combination of two inhibitors could produce a more significant impact, but did not block the rHSA-IFNα-2b role completely. This indicated that rHSA-IFNα-2b produces a marked effect primarily through the Jak-Stat pathways, and partly through p38-MAPK pathway.
(B) The efficacy study in vivo
The DHBV-infected Anhui ducks were selected as the animal model in vivo. Firstly, the DHBV-positive ducklings were judged by PCR, then rHSA-IFNα-2b (0.25, 0.5, 1.0 nmol/kg) were injected subcutaneously, once a week for 4 weeks. IFNα-2b was the positive control, 0.2 nmol/kg, 3 times a week for 4 weeks. The animals were sacrificed one week after cessation of the treatment, and the livers were obtained immediately after collecting blood from the vena cruralis. The Changes of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum total bilirubin (TBIL) and DHBV DNA were explored, and the duck livers were examined by pathological method; and the expression of liver 2'-5'-OAS was detected by Western Blotting.
The results showed that, four weeks after treatment with rHSA-IFNα-2b, the major liver enzymes (ALT, AST and TBIL) were reduced significantly, and ALT was more significantly in a dose-dependent manner. IFNα-2b can significantly reduce the levels of ALT, AST, TBIL, and the ALT level had a rebound at day 35, but not obviously in rHSA-IFNα-2b-treated groups.
rHSA-IFNα-2b could markedly decrease the concentrations of DHBV DNA in the duckling model, and was dose-dependent after 4 weeks` treatment. All animals in the IFNα-2b-treated group showed a reduction in DHBV DNA levels over the course of treatment, with less potency than those treated with rHSA-IFNα-2b.
Histopathological profiles of the liver from the control group ducks revealed necrosis, inflammatory cell infiltration and massive ballooning degeneration of the hepatic cytoplasm. Administrations of rHSA-IFNα-2b to the experimental animals showed a dose-dependent improvement of the hepatocellular architecture over the control group. rHSA-IFNα-2b resulted in more obvious improvements than that IFNα-2b did.
rHSA-IFNα-2b could increase the expression of 2'-5'-OAS protein in liver cell remarkably, and showed significant dose-dependent manner.
In Conclusion, rHSA-IFNα-2b can effectively inhibit the proliferation of HBV in vitro and in vivo, and it is mainly through the Jak-Stat pathways, and partly through p38-MAPK pathway, to increase the secretion of OAS.
引文
[1] Hayashi PH, Di Bisceglie AM. The progression of hepatitis B- and C-infections to chronic liver disease and hepatocellular carcinoma: presentation, diagnosis, screening, prevention, and treatment of hepatocellular carcinoma[J]. Med Clin North Am, 2005; 89(2):345-69
[2] Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures[J]. J Viral Hepat, 2004; 11(2):97-107
[3] Thompson JE, Cubbon RM, Cummings RT, Wicker LS, Frankshun R, Cunningham BR, Cameron PM, Meinke PT, Liverton N, Weng Y, DeMartino JA. Photochemical preparation of a pyridone containing tetracycle: a Jak protein kinase inhibitor[J]. Bioorg Med Chem Lett, 2002; 12(8):1219-23
[4] Korba BE, Milman G. A cell culture assay for compounds which inhibit hepatitis B virus replication[J]. Antiviral Res, 1991; 15(3):217-28
[5] Cooksley WG, Piratvisuth T, Lee SD, Mahachai V, Chao YC, Tanwandee T, Chutaputti A, Chang WY, Zahm FE, Pluck N. Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B e antigen-positive chronic hepatitis B[J]. J Viral Hepat, 2003; 10(4):298-305
[6] Hiscott J. Another detour on the Toll road to the interferon antiviral response[J]. Nat Struct Mol Biol, 2004; 11(11): 1028-30
[7]梁晓峰,陈园生,王晓军,贺雄,陈丽娟,王骏,林长缨,白呼群,严俊,崔钢,于竞进.中国3岁以上人群乙型肝炎血清流行病学研究[J].中华流行病学杂志, 2005; 26(9): 655-8
[8] Chen M, Sallberg M, Hughes J, Jones J, Guidotti LG, Chisari FV, Billaud JN, Milich DR. Immune tolerance split between hepatitis B virus precore and core proteins[J]. J Virol, 2005; 79(5):3016-27
[9]姜玉杰,丛雅琴.慢性乙型肝炎患者体内病毒免疫逃逸机制的研究进展[J].国外医学·临床生物化学与检验学分册, 2004; 25(3): 220-2
[10]段国荣,周永兴,王平忠,贾战生,聂青和. TH1/TH2在慢性乙型肝炎发病机制中的作用[J].西北国防医学杂志, 2002; 23(1): 34-5
[11] Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, King AS, Herberg J, Gilson R, Alisa A, Williams R, Vergani D, Naoumov NV, Ferrari C, Bertoletti A: The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection[J]. J Exp Med, 2000; 191(8):1269-80
[12] Liaw YF, Leung N, Guan R, Lau GK, Merican I, McCaughan G, Gane E, Kao JH, Omata M. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2005 update[J]. Liver Int, 2005; 25(3):472-89
[13] Loka S, McMahon BJ. Chronic hepatitis B[M]. Alexan dria(Va). American association for the Study of Liver Diseases, 2004
[14] el-Sayed MH, Shanab G, Karim AM, el-Tawil A, Black A, Dixon JS. Lamivudine facilitates optimal chemotherapy in hepatitis B virus-infected children with hematological malignancies: a preliminary report[J]. Pediatr Hematol Oncol, 2004; 21(2):145-56
[15] Telegdy L. [Treatment of hepatitis B] [J]. Orv Hetil, 2004; 145(45): 2293-6
[16] Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, Dienstag JL, Heathcote EJ, Little NR, Griffiths DA, Gardner SD, Castiglia M. Long-term safety of lamivudine treatment in patients with chronic hepatitis B[J]. Gastroenterology, 2003; 125(6):1714-22
[17] Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, Lu ZM, Piratvisuth T, Germanidis G, Yurdaydin C, Diago M, Gurel S, Lai MY, Button P, Pluck N: Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B[J]. N Engl J Med, 2004; 351(12):1206-17
[18] Zhang Q, Wang Y, Wei L, Jiang D, Wang JH, Rao HY, Zhu L, Chen H, Fei R, Cong X. Role of ISGF3 in modulating the anti-hepatitis B virus activity of interferon-alpha in vitro[J]. J Gastroenterol Hepatol 2008; 23(11):1747-61
[19] Tang TJ, Kwekkeboom J, Mancham S, Binda RS, de Man RA, Schalm SW, Kusters JG, Janssen HL. Intrahepatic CD8 + T lymphocyte response is important for t herapy-induced viral clearance in chronic hepatitis B infection[J]. J Hepatol, 2005; 43 (1): 45-52
[20] Carotenuto P, van Riel D , Artsen A , Bruijns S, Uytdehaag FG, Laman JD, van Nunen AB, Zondervan PE, De Man RA, Osterhaus AD, Pontesilli O. Antiviral treatment with alpha interferon up-regulates CD14 on liver macrophages and its soluble form in patients with chronic hepatitis B[J]. Antimicrob Agents Chemother, 2005; 49 (2): 590-599
[21] Craxi A, Di Bona D, Camma C. Interferon-alpha for HBeAg-positive chronic hepatitis B[J]. J Hepatol, 2003; 39 Suppl 1:S99-105
[22] Glue P, Fang JW, Rouzier-Panis R, Raffanel C, Sabo R, Gupta SK, Salfi M, Jacobs S. Pegylated interferon-alpha2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data. Hepatitis C Intervention Therapy Group[J]. Clin Pharmacol Ther, 2000; 68(5):556-67
[23] Osborn BL, Olsen HS, Nardelli B, Murray JH, Zhou JX, Garcia A, Moody G, Zaritskaya LS, Sung C. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys[J]. J Pharmacol Exp Ther, 2002; 303(2):540-8
[24] Sung C, Nardelli B, LaFleur DW, Blatter E, Corcoran M, Olsen HS, Birse CE, Pickeral OK, Zhang J, Shah D, Moody G, Gentz S, Beebe L, Moore PA. An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates[J]. J Interferon Cytokine Res, 2003; 23(1):25-36
[25] Wiegand J, Buggisch P, Boecher W, Zeuzem S, Gelbmann CM, Berg T, Kauffmann W, Kallinowski B, Cornberg M, Jaeckel E, Wedemeyer H, Manns MP. Early monotherapy with pegylated interferon alpha-2b for acute hepatitis C infection: the HEP-NET acute-HCV-II study[J]. Hepatology, 2006; 43(2):250-6
[26] Liu C, Zhu H, Subramanian GM, Moore PA, Xu Y, Nelson DR. Anti-hepatitis C virus activity of albinterferon alfa-2b in cell culture[J]. Hepatol Res, 2007; 37(11):941-7
[27] Zeuzem S, Yoshida EM, Benhamou Y, Pianko S, Bain VG, Shouval D, Flisiak R, Rehak V, Grigorescu M, Kaita K, Cronin PW, Pulkstenis E, Subramanian GM, McHutchison JG. Albinterferon alfa-2b dosed every two or four weeks in interferon-naive patients with genotype 1 chronic hepatitis C[J]. Hepatology, 2008; 48(2):407-17
[28] Balan V, Nelson DR, Sulkowski MS, Everson GT, Lambiase LR, Wiesner RH, Dickson RC, Post AB, Redfield RR, Davis GL, Neumann AU, Osborn BL, Freimuth WW, Subramanian GM. A Phase I/II study evaluating escalating doses of recombinant human albumin-interferon-alpha fusion protein in chronic hepatitis C patients who have failed previous interferon-alpha-based therapy[J]. Antivir Ther, 2006; 11(1):35-45
[29] Bain VG, Kaita KD, Yoshida EM, Swain MG, Heathcote EJ, Neumann AU, Fiscella M, Yu R, Osborn BL, Cronin PW, Freimuth WW, McHutchison JG, Subramanian GM. A phase 2 study to evaluate the antiviral activity, safety, and pharmacokinetics of recombinant human albumin-interferon alfa fusion protein in genotype 1 chronic hepatitis C patients[J]. J Hepatol, 2006; 44(4):671-8
[30] Su H, Wang H, Ji W, Zhao Y, Cai G. [In vitro evaluation on the effects of combined lamivudine (3TC) and Ara-AMP against hepatitis B virus] [J]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002; 16(1):16-9
[31] Hayashi Y, Koike K. Interferon inhibits hepatitis B virus replication in a stableexpression system of transfected viral DNA[J]. J Virol, 1989; 63(7):2936-40
[32]张士岭,王淑秋,李晓捷,庞伟.计算机图像分析与人工计数方法在癫痫脑组织GFAP免疫组化中的对比研究[J].中国体视学与图像分析, 2008; 13(3): 185-7
[33] Maximova OA, Taffs RE, Pomeroy KL, Piccardo P, Asher DM. Computerized morphometric analysis of pathological prion protein deposition in scrapie-infected hamster brain[J]. J Histochem Cytochem, 2006; 54(1):97-107
[34] Durbin JE, Fernandez-Sesma A, Lee CK, Rao TD, Frey AB, Moran TM, Vukmanovic S, Garcia-Sastre A, Levy DE. Type I IFN modulates innate and specific antiviral immunity[J]. J Immunol, 2000; 164(8):4220-8
[35] Bieche I, Olivi M, Champeme MH, Vidaud D, Lidereau R, Vidaud M. Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer[J]. Int J Cancer, 1998; 78(5):661-6
[36] Desmet VJ. Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW, Wollman J. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis [Hepatology 1981;1:431-435] [J]. J Hepatol, 2003; 38(4):382-6
[37] Ikawa H, Hayashi Y, Ninomiya T, Yano Y, Nakaji M, Nagano H, Seo Y, Kumon Y, Yoon S, Kasuga M, Itoh H, Ohbayashi C. Various scoring systems evaluating histologic features of chronic hepatitis C treated with interferon[J]. Hum Pathol, 2001; 32(9):910-7
[38] Lian Y, Tan J, Deng Z. [Experimental study of shuangcao granule no. 1 on duck hepatitis B virus in ducklings] [J]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 2000; 20(7):530-2
[39]李晖,田德英,许东,吴亮,倪明,马晓军.肝康栓抗鸭乙型肝炎病毒的实验研究[J].中西医结合肝病杂志, 2006; l6(5): 270-2
[40]阿特金森,主编;魏伟,主译.临床药理学原理(第二版)[M].北京,科学出版社, 2008
[41] Sells MA, Zelent AZ, Shvartsman M, Acs G. Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions[J]. J Virol, 1988; 62(8):2836-44
[42] Mason WS, Seal G, Summers J. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus[J]. J Virol, 1980; 36(3):829-36
[43] Heuss LT, Heim MH, Schultz U, Wissmann D, Offensperger WB, Staeheli P, Blum HE. Biological efficacy and signal transduction through STAT proteins of recombinant duck interferon in duck hepatitis B virus infection[J]. J Gen Virol, 1998; 79 ( Pt 8):2007-12
[44] Pestka S, Krause CD, Walter MR. Interferons, interferon-like cytokines, and their receptors[J]. Immunol Rev, 2004; 202:8-32
[45]罗红雨,杨旭,邹文,黄力. 2.2.15细胞上清HBVDNA定量检测方法的比较[J].中国现代医学杂志, 2005; 15(3): 383-4
[46]李芳芳.定量PCR与定性PCR及分子斑点杂交测定HBVDNA的比较[J].中国现代医学杂志. 1999; 9(11): 40
[47] Leemans WF, Flink HJ, Janssen HL, Niesters HG, Schalm SW, de Man RA. The effect of pegylated interferon-alpha on the treatment of lamivudine resistant chronic HBeAg positive hepatitis B virus infection[J]. J Hepatol, 2006; 44(3):507-11
[48] Hoofnagle JH. Alpha-interferon therapy of chronic hepatitis B. Current status and recommendations[J]. J Hepatol, 1990; 11 Suppl 1:S100-7
[49] Korenman J, Baker B, Waggoner J, Everhart JE, Di Bisceglie AM, Hoofnagle JH. Long-term remission of chronic hepatitis B after alpha-interferon therapy[J]. Ann Intern Med, 1991; 114(8):629-34
[50] Clemens MJ, Elia A. The double-stranded RNA-dependent protein kinase PKR: structure and function[J]. J Interferon Cytokine Res, 1997; 17(9):503-24
[51] Rebouillat D, Hovanessian AG. The human 2',5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties[J]. J Interferon Cytokine Res, 1999; 19(4):295-308
[52] Jagus R, Joshi B, Barber GN. PKR, apoptosis and cancer[J]. Int J Biochem Cell Biol, 1999; 31(1):123-38
[53] Ma XY, Wang H, Ding B, Zhong H, Ghosh S, Lengyel P. The interferon-inducible p202a protein modulates NF-kappaB activity by inhibiting the binding to DNA of p50/p65 heterodimers and p65 homodimers while enhancing the binding of p50 homodimers[J]. J Biol Chem, 2003; 278(25):23008-19
[54] Guo J, Sen GC. Characterization of the interaction between the interferon-induced protein p56 and the Int6 protein encoded by a locus of insertion of the mouse mammary tumor virus[J]. J Virol, 2000; 74(4): 1892-9
[55] Martensen PM, Justensen J. Small ISGs coming forward [J]. J Interferon Cytokine Res, 2004; 24(1): 1-19
[56] LeBon A, Tough DF. Links between innate and adaptive immunity via type I interferon[J]. Curr Opin Immunol, 2002; 14(4): 432-6
[57] Hovnanian A, Rebouillat D, Mattei MG, Levy ER, Marie I, Monaco AP, Hovanessian AG. The human 2',5'-oligoadenylate synthetase locus is composed of three distinct genes clustered on chromosome 12q24.2 encoding the 100-, 69-, and 40-kDa forms[J]. Genomics, 1998; 52(3):267-77
[58] Yakub I, Lillibridge KM, Moran A, Gonzalez OY, Belmont J, Gibbs RA, Tweardy DJ. Single nucleotide polymorphisms in genes for 2'-5'-oligoadenylate synthetase and RNase L inpatients hospitalized with West Nile virus infection[J]. J Infect Dis, 2005; 192(10):1741-8
[59] Hamano E, Hijikata M, Itoyama S, Quy T, Phi NC, Long HT, Ha le D, Ban VV, Matsushita I, Yanai H, Kirikae F, Kirikae T, Kuratsuji T, Sasazuki T, Keicho N. Polymorphisms of interferon-inducible genes OAS-1 and MxA associated withSARS in the Vietnamese population[J]. Biochem Biophys Res Commun, 2005; 329(4):1234-9
[60] He J, Feng D, de Vlas SJ, Wang H, Fontanet A, Zhang P, Plancoulaine S, Tang F, Zhan L, Yang H, Wang T, Richardus JH, Habbema JD, Cao W. Association of SARS susceptibility with single nucleic acid polymorphisms of OAS1 and MxA genes: a case-control study[J]. BMC Infect Dis, 2006; 6:106
[61] Haller O, Kochs G. Interferon-induced mx proteins.dynamin-like GTPases with antiviral activity[J]. Traffic, 2002; 3(10): 710-7
[62] Malathi K, Paranjape JM, Bulanova E, Shim M, Guenther-Johnson JM, Faber PW, Eling TE, Williams BR, Silverman RH. A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L[J]. Proc Natl Acad Sci USA, 2005; 102(41):14533-8
[63] Benech P, Merlin G, Revel M, Chebath J. 3' end structure of the human (2'-5') oligo A synthetase gene: prediction of two distinct proteins with cell type-specific expression[J]. Nucleic Acids Res, 1985; 13(4):1267-81
[64] Bonnevie-Nielsen V, Field LL, Lu S, Zheng DJ, Li M, Martensen PM, Nielsen TB, Beck-Nielsen H, Lau YL, Pociot F. Variation in antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the OAS1 gene[J]. Am J Hum Genet, 2005; 76(4):623-33
[65] Knapp S, Yee LJ, Frodsham AJ, Hennig BJ, Hellier S, Zhang L, Wright M, Chiaramonte M, Graves M, Thomas HC, Hill AV, Thursz MR. Polymorphisms in interferon-induced genes and the outcome of hepatitis C virus infection: roles of MxA, OAS-1 and PKR[J]. Genes Immun, 2003; 4(6):411-9
[66] Gil J, Esteban M. The interferon-induced protein kinase(PKR), triggers apoptosis through FADD-mediated activation of caspase 8 in a manner independent of Fas and TNF-alpha receptors[J]. Oncogene, 2000; 19(32): 3665-74
[67] Guan SH, Lu M, Grunewald P, Roggendorf M, Gerken G, Schlaak JF. Interferon-alpha response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment[J]. World J Gastroenterol, 2007; 13(2):228-35
[68] Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B, Davis RJ. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway[J]. Mol Cell Biol, 1996; 16(3):1247-55
[69] Ishida H, Ohkawa K, Hosui A, Hiramatsu N, Kanto T, Ueda K, Takehara T, Hayashi N. Involvement of p38 signaling pathway in interferon-alpha-mediated antiviral activity toward hepatitis C virus[J]. Biochem Biophys Res Commun, 2004; 321(3):722-7
[70] Guo H, Zhou T, Jiang D, Cuconati A, Xiao GH, Block TM, Guo JT. Regulation of hepatitis B virus replication by the phosphatidylinositol 3-kinase-akt signal transduction pathway[J]. J Virol, 2007; 81(18):10072-80
[71] Xiong W, Wang X, Liu X, Xiang L, Zheng L, Yuan Z. Interferon-inducible MyD88 protein inhibits hepatitis B virus replication[J]. Virology, 2004; 319(2):306-14
[72] Wu M, Xu Y, Lin S, Zhang X, Xiang L, Yuan Z. Hepatitis B virus polymerase inhibits the interferon-inducible MyD88 promoter by blocking nuclear translocation of Stat1[J]. J Gen Virol, 2007; 88(Pt 12):3260-9
[73] Nakao K, Nakata K, Yamashita M, Tamada Y, Hamasaki K, Ishikawa H, Kato Y, Eguchi K, Ishii N. p48 (ISGF-3gamma) is involved in interferon-alpha-induced suppression of hepatitis B virus enhancer-1 activity[J]. J Biol Chem, 1999; 274(40):28075-8
[74] Li Y, Sassano A, Majchrzak B, Deb DK, Levy DE, Gaestel M, Nebreda AR, Fish EN, Platanias LC. Role of p38alpha Map kinase in Type I interferon signaling[J]. J Biol Chem, 2004; 279(2):970-9
[75] Platanias LC. The p38 mitogen-activated protein kinase pathway and its role in interferon signaling[J]. Pharmacol Ther, 2003; 98(2):129-42
[1]刘新垣.干扰素研究及其重大突破性研究进展[J].中国处方药, 2004; 7(28): 33-5
[2]卢年芳,黄爱龙,唐霓,郑瑞强,林华,朱亚彬,吴莹,陶鹏.干扰素抗病毒活性的实验研究[J].中华肝脏病杂志, 2005; 13(12): 892-6
[3]张权,魏来,王燕. ISGF3:IFN-a抗乙肝病毒信号转导机制的重要因子[J].中华实验和临床病毒学杂志, 2005; 19(2): 110-113
[4] Nakao K, Nakata K, Yamashita M, Tamada Y, Hamasaki K, Ishikawa H, Kato Y, Eguchi K, Ishii N. p48 (ISGF-3gamma) is involved in interferon-alpha-induced suppression of hepatitis B virus enhancer-1 activity[J]. J Biol Chem, 1999; 274(40): 28075-8
[5] Samuel CE. Antiviral actions of interferons[J]. Clin Microbiol Rev, 2001; 14(4): 778-809
[6] Sarkar SN, Ghosh A, Wang HW, Sung SS, Sen GC. The nature of the catalytic domain of 2'-5'-oligoadenylate synthetases[J]. J Biol Chem, 1999; 274(36): 25535-42
[7] Rebouillat D, Hovanessian AG. The human 2',5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties[J]. J Interferon Cytokine Res, 1999; 19(4): 295-308
[8] Kubota K, Nakahara K, Ohtsuka T, Yoshida S, Kawaguchi J, Fujita Y, Ozeki Y, Hara A, Yoshimura C, Furukawa H, Haruyama H, Ichikawa K, Yamashita M, Matsuoka T, Iijima Y: Identification of 2'-phosphodiesterase, which plays a role in the 2-5A system regulated by interferon[J]. J Biol Chem, 2004; 279(36): 37832-41
[9] Malathi K, Paranjape JM, Bulanova E, Shim M, Guenther-Johnson JM, Faber PW, Eling TE, Williams BR, Silverman RH: A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L[J]. Proc Natl Acad Sci USA, 2005; 102(41):14533-8
[10] Clemens MJ, Elia A: The double-stranded RNA-dependent protein kinase PKR: structure and function[J]. J Interferon Cytokine Res, 1997; 17(9):503-24
[11] Gil J, Esteban M. The interferon-induced protein kinase(PKR), triggers apoptosis through FADD-mediated activation of caspase 8 in a manner independent of Fas and TNF-alpha receptors[J]. Oncogene, 2000; 19(32): 3665-74
[12] Kumar A, Yang Y L, Flati V, Der S, Kadereit S, Deb A, Haque J, Reis L, Weissmann C, Williams BR. Deficient cytokine signaling in mouse embryo fibroblasts with a targrted deletion in the PKR gene: role of IRF-1 and NF-kappaB[J]. EMBO J, 1997; 16(2): 406-16
[13]毛国强,许红梅,朱朝敏. MxA蛋白研究进展[J].国外医学病毒学分册, 2005; 12(4): 107-10
[14]吴金明,吴建胜,陈民新,林菊生.α干扰素于体外培养的2.2.15细胞中对MxA基因的诱导及抗HBV作用[J].中华实验和临床病毒学杂志, 2004; 18(4): 386-7
[15] Gordien E, Rosmorduc O, Peltekian C, Garreau F, Brechot C, Kremsdorf D. Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein[J]. J Virol, 2001; 75(6): 2684-91
[16] Kochs G, Haller O. GTP-bound human MxA protein interacts with the nucleocapsids of thogoto virus (orthomyxoviridae) [J]. J Biol Chem, 1999, 274(7): 43702-61
[17]管世鹤,杨东亮,陆蒙吉, Michael Roggendorf, Joerg F Schlaak.干扰素抗病毒蛋白MxA和2’,5’2OAS在肝胚瘤细胞株中的差异表达[J].中国感染与化疗杂志, 2006; 6(3): 149-153
[18] Guan SH, Lu M, Grünewald P, Roggendorf M, Gerken G, Schlaak JF. Interferon-αresponse in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment[J]. World J Gastroenterol, 2007; 13(2): 228-35
[19] Rosmorduc O, Sirma H, Soussan P, Gordien E, Lebon P, Horisberger M, BrechotC, Kremsdorf D. Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein[J]. J Gen Virol, 1999; 80(Pt 5): 1253-62
[20] Fernandez M, Quiroga JA, Carreno V. Hepatitis B virus downregulates the human interferon-inducible MxA promoter through direct interaction of precore/core proteins[J]. J Gen Virol, 2003; 84(Pt 8): 2073-82
[21] Raingeaud J, Whitmarsh AJ, Barrett T, Dérijard B, Davis RJ. MKK3 and MKK 6 regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway[J]. Mol Cell Biol, 1996; 16(3): 1247-55
[22]王抒,孙兰,杜冠华. p38-MAPK控制阿尔采末病进程的新靶点[J].中国药理学通报, 2004; 20(5): 595-8
[23] Jiang Y, Gram H, Zhao M, New L, Gu J, Feng L, Di Padova F, Ulevitch RJ, Han J. Characterization of the structure and function of the fourth member of p38 mitogen-activated protein kinases group, p38 delta[J]. J Biol Chem, 1997; 272(48): 30122–8
[24] Kuma Y, Sabio G, Bain J, Shpiro N, Marquez R, Cuenda A. BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo[J]. J Biol Chem, 2005; 280(20):19472-9
[25] Uddin S, Lekmine F, Sharma N, Majchrzak B, Mayer I, Young PR, Bokoch GM, Fish EN, Platanias LC. The Rac1/p38 mitogen-activated protein kinase pathwayis required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins[J]. J. Biol. Chem, 2000; 275: 27634-40
[26] Sassano A, Verma A, Platanias LC. Mitogen-activated protein kinase pathways in interferon signaling[J]. Methods Mol Med, 2005;116:135-50
[27] Uddin S, Majchrzak B, Woodson J, Arunkumar P, Alsayed Y, Pine R, Young PR, Fish EN, Platanias LC. Activation of the p38 mitogen-activated protein kinase by type I interferons[J]. J Biol Chem, 1999; 274(42):30127-31
[28] Uddin S, Majchrzak B, Wang PC, Modi S, Khan MK, Fish EN, Platanias LC. Interferon-dependent activation of the serine kinase PI 3'-kinase requiresengagement of the IRS pathway but not the Stat pathway[J]. Biochem Biophys Res Commun, 2000; 270(1):158-62
[29] Ishida H, Ohkawa K, Hosui A, Hiramatsu N, Kanto T, Ueda K, Takehara T, Hayashi N. Involvement of p38 signaling pathway in interferon-alpha-mediated antiviral activity toward hepatitis C virus[J]. Biochem Biophys Res Commun, 2004; 321(3): 722-7
[30] Fan H, Li H. Insulin receptor substrate and signals transduction[J]. Foreign Med Sci Sect Pathophysiol Clin Med, 2002; 22(5): 481-4
[31] Cantley LC. The phosphoinositide 3-kinase pathways[J]. Science, 2002, 296(5573): 1655-7
[32] Uddin S, Platanias LC. Mechanisms of type-I interferon signal transduction[J]. J Biochem Mol Biol, 2004; 37(6): 635-41
[33] Lekmine F, Uddin S, Sassano A, Parmar S, Brachmann SM, Majchrzak B, Sonenberg N, Hay N, Fish EN, Platanias LC. Activation of the p70 S6 Kinase and Phosphorylation of the 4E-BP1 Repressor of mRNA Translation by Type I Interferons[J]. J. Biol.Chem, 2003; 278(30): 27772–80
[34] Uddin S, Fish EN, Sher DA, Gardziola C, White MF, Platanias LC. Activation of the phosphatidylinositol 3'-kinase serine kinase by IFNα.[J]. J. Immunol.1997; 158(5): 2390-7
[35] Thyrell L, Hjortsberg L, Arulampalam V, Panaretakis T, Uhles S, Dagnell M, Zhivotovsky B, Leibiger I, Grander D, Pokrovskaja K. Interferon alpha-induced apoptosis in tumor cells is mediated through the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway[J]. J Biol Chem, 2004; 279(23): 24152-62
[36] Xiong W, Wang X, Liu XY, Xiang L, Zheng LJ, Liu JX, Yuan ZH. Analysis of gene expression in hepatitis B virus transfected cell line induced by interferon[J]. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003;35(12):1053-60
[37] Xiong W, Wang X, Liu X, Xiang L, Zheng L, Yuan Z. Interferon-inducible MyD88 protein inhibits hepatitis B virus replication[J]. Virology, 2004; 319 (2): 306-14
[38] Vlotides G, Sorensen AS, Kopp F, Zitzmann K, Cengic N, Brand S, Zachoval R, Auernhammer CJ. SOCS-1 and SOCS-3 inhibit IFN-alpha-induced expression of the antiviral proteins 2,5-OAS and MxA[J]. Biochem Biophys Res Commun, 2004; 320(3): 1007-14
[39] Brand S, Zitzmann K, Dambacher J, Beigel F, Olszak T, Vlotides G, Eichhorst ST, Goke B, Diepolder H, Auernhammer CJ. SOCS-1 inhibits expression of the antiviral proteins 2',5'-OAS and MxA induced by the novel interferon-lambdas IL-28A and IL-29[J]. Biochem Biophys Res Commun, 2005; 331(2):543-8
[40]范金水,庄辉,李远贵,朱晓洁,徐德忠,马为民,王跃民,陈雅洁,娄国强,马廷贤.我国8城市HBsAg阳性和阴性乙肝患者的病毒血清型和基因型分析[J].中华微生物和免疫学杂志, 1998; 18(1): 18-22
[41] Wai CT, Chu CJ, Hussain M, Lok AS. HBV genotype B is associated with better response to interferon therapy in HBeAg (+) chronic hepatitis than genotype C[J]. Hepatology, 2002; 36(6): 1425-30
[42]肖扬,胡操寒,周岳进,谢庆荣,朱冰星,胡侠,王开鉴,张文静.乙型肝炎病毒基因型对干扰素α-2b应答的影响[J].临床肝胆病杂志, 2006; 22(2): 105-6
[43] Xing TJ, Luo KX, Hou JL. Preliminary identification and analysis of point mutations correlated with response to interferon-alpha in hepatitis B virus post-transcriptional regulatory elements[J]. Chin Med J (Engl), 2005; 118(1):56-61
[44] Erhardt A, Reineke U, Blondin D, Gerlich WH, Adams O, Heintges T, Niederau C, Haussinger D. Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B[J]. Hepatology, 2000; 31(3): 716-25
[45] Wang Y, Wei L, Jiang D, Cong X, Fei R, Chen H, Xiao J, Wang Y. In vitroresistance to interferon-alpha of hepatitis B virus with basic core promoter double mutation[J]. Antiviral Res, 2007; 75(2): 139-45
[46] Zhang FK. Pegylated interferons in the treatment of chronic hepatitis C[J]. Chinese Medical Journal, 2003; 116(4): 495-8
[47] Cooksley WG, Piratvisuth T, Lee SD, Mahachai V, Chao YC, Tanwandee T, Chutaputti A, Chang WY, Zahm FE, Pluck N. Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B eantigen-positive chronic hepatitis B[J]. J Viral Hepat, 2003; 10(4): 298-30
[48] Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, Lu ZM, Piratvisuth T, Germanidis G, Yurdaydin C, Diago M, Gurel S, Lai MY, Button P, Pluck N. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J Med, 2004; 16;351(12):1206-17
[49] Vassiliadis T, Tziomalos K, Patsiaoura K, Zagris T, Giouleme O, Soufleris K, Grammatikos N, Theodoropoulos K, Mpoumponaris A, Dona K, Zezos P, Nikolaidis N, Orfanou-Koumerkeridou E, Balaska A, Eugenidis N.Lamivudine/pegylated interferon alfa-2b sequential combination therapy compared with lamivudine monotherapy in HBeAg-negative chronic hepatitis B[J]. J Gastroenterol Hepatol, 2007; 22(10):1582-8
[50] Monkarsh SP, Ma Y, Aglione A, et al. Positional isomers of monopegylated interferon alpha-2: isolatio, characterization, and biological activity[J]. Anal Biochem, 1997; 247 (2): 434-40
[51]崔建军,范平,蒋小玲,许诚,何清,周伯平.聚乙二醇干扰素α-2a治疗HBeAg阳性慢性乙型肝炎的疗效和安全性[J].中华实验和临床病毒学杂志, 2006; 20(4): 331-3
[52]赵鸿,斯崇文,魏来,万漠彬,尹有宽,侯金林,牛俊奇.聚乙二醇化干扰素a -2b与干扰素a -2b治疗e抗原阳性慢性乙型肝炎的疗效和安全性的随机对照多中心研究[J].中华肝脏病杂志, 2006; 14(5): 323-6
[53] Osborn BL, Olsen HS, Nardelli B, Murray JH, Zhou JX, Garcia A, Moody G, Zaritskaya LS, Sung C. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys[J]. J Pharmacol Exp Ther, 2002; 303(2):540-8
[54] Sung C, Nardelli B, LaFleur DW, Blatter E, Corcoran M, Olsen HS, Birse CE, Pickeral OK, Zhang J, Shah D, Moody G, Gentz S, Beebe L, Moore PA. An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates [J]. J Interferon Cytokine Res, 2003; 23(1): 25-36
[55]唱韶红,巩新,杨志愉,王同映,马国昌,马清钧,吴军.人血清白蛋白和人干扰素α2b的融合蛋白在毕赤酵母中的表达[J].生物工程学报, 2006; 22(2): 173-9
[56]张伍魁,范清林,邹文艺,仲皙,宋礼华.白蛋白融合α-2b干扰素在毕赤酵母中的表达[J].中国生物工程杂志, 2005; 25(12): 45-9
[57] Bain VG, Kaita KD, Yoshida EM, Swain MG, Heathcote EJ, Neumann AU, Fiscella M, Yu R, Osborn BL, Cronin PW, Freimuth WW, McHutchison JG, Subramanian GM. A phase 2 study to evaluate the antiviral activity, safety, and pharmacokinetics of recombinant human albumin-interferon alfa fusion protein in genotype 1 chronic hepatitis C patients[J]. J Hepatol, 2006; 44(4): 671-8
[58] Balan V, Nelson DR, Sulkowski MS, Everson GT, Lambiase LR, Wiesner RH, Dickson RC, Post AB, Redfield RR, Davis GL, Neumann AU, Osborn BL, Freimuth WW, Subramanian GM. A Phase I/II study evaluating escalating doses of recombinant human albumin-interferon-alpha fusion protein in chronic hepatitis C patients who have failed previous interferon-alpha-based therapy[J]. Antivir Ther, 2006; 11(1): 35-45
[59] Balan V, Nelson DR, Sulkowski MS, Everson GT, Lambiase LR, Wiesner RH,Dickson RC, Garcia A, Moore PA, Yu R, Subramanian GM. Modulation of interferon-specific gene expression by albumin-interferon-alpha in interferon-alpha-experienced patients with chronic hepatitis C[J]. Antivir Ther, 2006; 11(7): 901-8
[60]任进余,陈义,吴斌,崔红,张瑛,沈钟.病毒性肝炎的药物靶向治疗研究——抗HBV脂质体的研制及临床观察[J].中西医结合肝病杂志, 1997; 7(1):1-4
[61]黄耀芳,章廉,胡俊,王燕军,骆抗先.脂质体干扰素的体外抗乙型肝炎病毒效应[J].临床肝胆病杂志, 1997; 13(1): 38-39
[62]吕厚峰,孙迎基,邹文艺,张奉国,廖建民,沈子龙.基因重组干扰素a-2b脂质体的药代动力学研究[J].药物生物技术, 2005; 12(3): 186-9
[63]杜施霖,王吉耀,潘弘,陆伟跃,王剑.含有RGD序列环肽介导的干扰素脂质体对大鼠肝纤维化的治疗作用[J].中华医学杂志, 2005; 85(15): 1015-20
[64] Le Guerhier F, Thermet A, Guerret S, Chevallier M, Jamard C, Gibbs CS, Trepo C, Cova L, Zoulim F. Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model [J]. J Hepatol, 2003; 38(3): 328-334
[65] Addison WR, Wong WW, Fisher KP, Tyrrell DL. A quantitative competitive PCR assay for the covalently closed circular form of the duck hepatitis B virus [J]. Antiviral Res, 2000; 48(1): 27-37
[66] Deres K, Schroder CH, Paessens A, Goldmann S, Hacker HJ, Weber O, Kramer T, Niewohner U, Pleiss U, Stoltefuss J, Graef E, Koletzki D, Masantschek RN, Reimann A, Jaeger R, Gross R, Beckermann B, Schlemmer KH, Haebich D, Rubsamen-Waigmann H. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids[J]. Science, 2004; 299(5608): 893-6
[67] Hu J, Toft DO, Seeger C. Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids [J]. EMBO J, 1997; 16(1): 59-81
[68] Genovesi EV, Lamb L, Medina I, Taylor D, Seifer M, Innaimo S, Colonno RJ,Clark JM. Antiviral efficacy of lobucavir (BMS2180194), a cyclobutyl-guanosine nucleoside analogue, in the woodchuck (Marmota monax) model of chronic hepatitis B virus (HBV) infection [J]. Antiviral Res, 2000; 48(3):197-203
[69] Korba BE, Cote P, Hornbuckle W, Tennant BC, Gerin JL. Treatment of chronic woodchuck hepatitis virus infection in the eastern woodchuck (marmota monax) with nucleoside analogues is predictive of therapy for chronic hepatitis B virus infection in human [J]. Hepatology, 2000; 31(5): 1165-75
[70] Dahmen U, Li J, Dirsch O, Fiedler M, Lu M, Roggendorf M, Broelsch CE. A new model of hepatitis B virus reinfection: liver transplantation in the woodchuck[J]. Transplantation, 2002; 74 (3): 373-80
[71] Bruni R, D’Ugo E, Argentini C, Giuseppetti R, Rapicetta M. Scaffold attachment region located in a locus targeted by hepadnavirus integration in hepatocellular carcinoma [J]. Cancer Detect Prev, 2003; 27(3):175-81
[72] Wang Y, Cui F, Lv Y, Li C, Xu X, Deng C, Wang D, Sun Y, Hu G, Lang Z, Huang C, Yang X. HBsAg and HBx knocked into the p21 locus causes hepatocellular carcinoma in mice [J]. Hepatology, 2004; 39(2): 2318- 24
[73] Sell MA, Chen ML, ACs G. Production of hepatitis B virus padicles in HepG2 cells transfected with cloned hepatitis B virus DNA[J]. Pron Natl Acad Sci USA, 1987; 84: 1005-9
[74]劳国琴,王佳良,吴勇.干扰素治疗慢性乙型肝炎病毒感染的经济效益[J].中国药房, 2006; 17(16): 1223-5
[2] Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures[J]. J Viral Hepat, 2004; 11(2):97-107
[3] Thompson JE, Cubbon RM, Cummings RT, Wicker LS, Frankshun R, Cunningham BR, Cameron PM, Meinke PT, Liverton N, Weng Y, DeMartino JA. Photochemical preparation of a pyridone containing tetracycle: a Jak protein kinase inhibitor[J]. Bioorg Med Chem Lett, 2002; 12(8):1219-23
[4] Korba BE, Milman G. A cell culture assay for compounds which inhibit hepatitis B virus replication[J]. Antiviral Res, 1991; 15(3):217-28
[5] Cooksley WG, Piratvisuth T, Lee SD, Mahachai V, Chao YC, Tanwandee T, Chutaputti A, Chang WY, Zahm FE, Pluck N. Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B e antigen-positive chronic hepatitis B[J]. J Viral Hepat, 2003; 10(4):298-305
[6] Hiscott J. Another detour on the Toll road to the interferon antiviral response[J]. Nat Struct Mol Biol, 2004; 11(11): 1028-30
[7]梁晓峰,陈园生,王晓军,贺雄,陈丽娟,王骏,林长缨,白呼群,严俊,崔钢,于竞进.中国3岁以上人群乙型肝炎血清流行病学研究[J].中华流行病学杂志, 2005; 26(9): 655-8
[8] Chen M, Sallberg M, Hughes J, Jones J, Guidotti LG, Chisari FV, Billaud JN, Milich DR. Immune tolerance split between hepatitis B virus precore and core proteins[J]. J Virol, 2005; 79(5):3016-27
[9]姜玉杰,丛雅琴.慢性乙型肝炎患者体内病毒免疫逃逸机制的研究进展[J].国外医学·临床生物化学与检验学分册, 2004; 25(3): 220-2
[10]段国荣,周永兴,王平忠,贾战生,聂青和. TH1/TH2在慢性乙型肝炎发病机制中的作用[J].西北国防医学杂志, 2002; 23(1): 34-5
[11] Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, King AS, Herberg J, Gilson R, Alisa A, Williams R, Vergani D, Naoumov NV, Ferrari C, Bertoletti A: The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection[J]. J Exp Med, 2000; 191(8):1269-80
[12] Liaw YF, Leung N, Guan R, Lau GK, Merican I, McCaughan G, Gane E, Kao JH, Omata M. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2005 update[J]. Liver Int, 2005; 25(3):472-89
[13] Loka S, McMahon BJ. Chronic hepatitis B[M]. Alexan dria(Va). American association for the Study of Liver Diseases, 2004
[14] el-Sayed MH, Shanab G, Karim AM, el-Tawil A, Black A, Dixon JS. Lamivudine facilitates optimal chemotherapy in hepatitis B virus-infected children with hematological malignancies: a preliminary report[J]. Pediatr Hematol Oncol, 2004; 21(2):145-56
[15] Telegdy L. [Treatment of hepatitis B] [J]. Orv Hetil, 2004; 145(45): 2293-6
[16] Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, Dienstag JL, Heathcote EJ, Little NR, Griffiths DA, Gardner SD, Castiglia M. Long-term safety of lamivudine treatment in patients with chronic hepatitis B[J]. Gastroenterology, 2003; 125(6):1714-22
[17] Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, Lu ZM, Piratvisuth T, Germanidis G, Yurdaydin C, Diago M, Gurel S, Lai MY, Button P, Pluck N: Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B[J]. N Engl J Med, 2004; 351(12):1206-17
[18] Zhang Q, Wang Y, Wei L, Jiang D, Wang JH, Rao HY, Zhu L, Chen H, Fei R, Cong X. Role of ISGF3 in modulating the anti-hepatitis B virus activity of interferon-alpha in vitro[J]. J Gastroenterol Hepatol 2008; 23(11):1747-61
[19] Tang TJ, Kwekkeboom J, Mancham S, Binda RS, de Man RA, Schalm SW, Kusters JG, Janssen HL. Intrahepatic CD8 + T lymphocyte response is important for t herapy-induced viral clearance in chronic hepatitis B infection[J]. J Hepatol, 2005; 43 (1): 45-52
[20] Carotenuto P, van Riel D , Artsen A , Bruijns S, Uytdehaag FG, Laman JD, van Nunen AB, Zondervan PE, De Man RA, Osterhaus AD, Pontesilli O. Antiviral treatment with alpha interferon up-regulates CD14 on liver macrophages and its soluble form in patients with chronic hepatitis B[J]. Antimicrob Agents Chemother, 2005; 49 (2): 590-599
[21] Craxi A, Di Bona D, Camma C. Interferon-alpha for HBeAg-positive chronic hepatitis B[J]. J Hepatol, 2003; 39 Suppl 1:S99-105
[22] Glue P, Fang JW, Rouzier-Panis R, Raffanel C, Sabo R, Gupta SK, Salfi M, Jacobs S. Pegylated interferon-alpha2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data. Hepatitis C Intervention Therapy Group[J]. Clin Pharmacol Ther, 2000; 68(5):556-67
[23] Osborn BL, Olsen HS, Nardelli B, Murray JH, Zhou JX, Garcia A, Moody G, Zaritskaya LS, Sung C. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys[J]. J Pharmacol Exp Ther, 2002; 303(2):540-8
[24] Sung C, Nardelli B, LaFleur DW, Blatter E, Corcoran M, Olsen HS, Birse CE, Pickeral OK, Zhang J, Shah D, Moody G, Gentz S, Beebe L, Moore PA. An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates[J]. J Interferon Cytokine Res, 2003; 23(1):25-36
[25] Wiegand J, Buggisch P, Boecher W, Zeuzem S, Gelbmann CM, Berg T, Kauffmann W, Kallinowski B, Cornberg M, Jaeckel E, Wedemeyer H, Manns MP. Early monotherapy with pegylated interferon alpha-2b for acute hepatitis C infection: the HEP-NET acute-HCV-II study[J]. Hepatology, 2006; 43(2):250-6
[26] Liu C, Zhu H, Subramanian GM, Moore PA, Xu Y, Nelson DR. Anti-hepatitis C virus activity of albinterferon alfa-2b in cell culture[J]. Hepatol Res, 2007; 37(11):941-7
[27] Zeuzem S, Yoshida EM, Benhamou Y, Pianko S, Bain VG, Shouval D, Flisiak R, Rehak V, Grigorescu M, Kaita K, Cronin PW, Pulkstenis E, Subramanian GM, McHutchison JG. Albinterferon alfa-2b dosed every two or four weeks in interferon-naive patients with genotype 1 chronic hepatitis C[J]. Hepatology, 2008; 48(2):407-17
[28] Balan V, Nelson DR, Sulkowski MS, Everson GT, Lambiase LR, Wiesner RH, Dickson RC, Post AB, Redfield RR, Davis GL, Neumann AU, Osborn BL, Freimuth WW, Subramanian GM. A Phase I/II study evaluating escalating doses of recombinant human albumin-interferon-alpha fusion protein in chronic hepatitis C patients who have failed previous interferon-alpha-based therapy[J]. Antivir Ther, 2006; 11(1):35-45
[29] Bain VG, Kaita KD, Yoshida EM, Swain MG, Heathcote EJ, Neumann AU, Fiscella M, Yu R, Osborn BL, Cronin PW, Freimuth WW, McHutchison JG, Subramanian GM. A phase 2 study to evaluate the antiviral activity, safety, and pharmacokinetics of recombinant human albumin-interferon alfa fusion protein in genotype 1 chronic hepatitis C patients[J]. J Hepatol, 2006; 44(4):671-8
[30] Su H, Wang H, Ji W, Zhao Y, Cai G. [In vitro evaluation on the effects of combined lamivudine (3TC) and Ara-AMP against hepatitis B virus] [J]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002; 16(1):16-9
[31] Hayashi Y, Koike K. Interferon inhibits hepatitis B virus replication in a stableexpression system of transfected viral DNA[J]. J Virol, 1989; 63(7):2936-40
[32]张士岭,王淑秋,李晓捷,庞伟.计算机图像分析与人工计数方法在癫痫脑组织GFAP免疫组化中的对比研究[J].中国体视学与图像分析, 2008; 13(3): 185-7
[33] Maximova OA, Taffs RE, Pomeroy KL, Piccardo P, Asher DM. Computerized morphometric analysis of pathological prion protein deposition in scrapie-infected hamster brain[J]. J Histochem Cytochem, 2006; 54(1):97-107
[34] Durbin JE, Fernandez-Sesma A, Lee CK, Rao TD, Frey AB, Moran TM, Vukmanovic S, Garcia-Sastre A, Levy DE. Type I IFN modulates innate and specific antiviral immunity[J]. J Immunol, 2000; 164(8):4220-8
[35] Bieche I, Olivi M, Champeme MH, Vidaud D, Lidereau R, Vidaud M. Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer[J]. Int J Cancer, 1998; 78(5):661-6
[36] Desmet VJ. Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW, Wollman J. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis [Hepatology 1981;1:431-435] [J]. J Hepatol, 2003; 38(4):382-6
[37] Ikawa H, Hayashi Y, Ninomiya T, Yano Y, Nakaji M, Nagano H, Seo Y, Kumon Y, Yoon S, Kasuga M, Itoh H, Ohbayashi C. Various scoring systems evaluating histologic features of chronic hepatitis C treated with interferon[J]. Hum Pathol, 2001; 32(9):910-7
[38] Lian Y, Tan J, Deng Z. [Experimental study of shuangcao granule no. 1 on duck hepatitis B virus in ducklings] [J]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 2000; 20(7):530-2
[39]李晖,田德英,许东,吴亮,倪明,马晓军.肝康栓抗鸭乙型肝炎病毒的实验研究[J].中西医结合肝病杂志, 2006; l6(5): 270-2
[40]阿特金森,主编;魏伟,主译.临床药理学原理(第二版)[M].北京,科学出版社, 2008
[41] Sells MA, Zelent AZ, Shvartsman M, Acs G. Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions[J]. J Virol, 1988; 62(8):2836-44
[42] Mason WS, Seal G, Summers J. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus[J]. J Virol, 1980; 36(3):829-36
[43] Heuss LT, Heim MH, Schultz U, Wissmann D, Offensperger WB, Staeheli P, Blum HE. Biological efficacy and signal transduction through STAT proteins of recombinant duck interferon in duck hepatitis B virus infection[J]. J Gen Virol, 1998; 79 ( Pt 8):2007-12
[44] Pestka S, Krause CD, Walter MR. Interferons, interferon-like cytokines, and their receptors[J]. Immunol Rev, 2004; 202:8-32
[45]罗红雨,杨旭,邹文,黄力. 2.2.15细胞上清HBVDNA定量检测方法的比较[J].中国现代医学杂志, 2005; 15(3): 383-4
[46]李芳芳.定量PCR与定性PCR及分子斑点杂交测定HBVDNA的比较[J].中国现代医学杂志. 1999; 9(11): 40
[47] Leemans WF, Flink HJ, Janssen HL, Niesters HG, Schalm SW, de Man RA. The effect of pegylated interferon-alpha on the treatment of lamivudine resistant chronic HBeAg positive hepatitis B virus infection[J]. J Hepatol, 2006; 44(3):507-11
[48] Hoofnagle JH. Alpha-interferon therapy of chronic hepatitis B. Current status and recommendations[J]. J Hepatol, 1990; 11 Suppl 1:S100-7
[49] Korenman J, Baker B, Waggoner J, Everhart JE, Di Bisceglie AM, Hoofnagle JH. Long-term remission of chronic hepatitis B after alpha-interferon therapy[J]. Ann Intern Med, 1991; 114(8):629-34
[50] Clemens MJ, Elia A. The double-stranded RNA-dependent protein kinase PKR: structure and function[J]. J Interferon Cytokine Res, 1997; 17(9):503-24
[51] Rebouillat D, Hovanessian AG. The human 2',5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties[J]. J Interferon Cytokine Res, 1999; 19(4):295-308
[52] Jagus R, Joshi B, Barber GN. PKR, apoptosis and cancer[J]. Int J Biochem Cell Biol, 1999; 31(1):123-38
[53] Ma XY, Wang H, Ding B, Zhong H, Ghosh S, Lengyel P. The interferon-inducible p202a protein modulates NF-kappaB activity by inhibiting the binding to DNA of p50/p65 heterodimers and p65 homodimers while enhancing the binding of p50 homodimers[J]. J Biol Chem, 2003; 278(25):23008-19
[54] Guo J, Sen GC. Characterization of the interaction between the interferon-induced protein p56 and the Int6 protein encoded by a locus of insertion of the mouse mammary tumor virus[J]. J Virol, 2000; 74(4): 1892-9
[55] Martensen PM, Justensen J. Small ISGs coming forward [J]. J Interferon Cytokine Res, 2004; 24(1): 1-19
[56] LeBon A, Tough DF. Links between innate and adaptive immunity via type I interferon[J]. Curr Opin Immunol, 2002; 14(4): 432-6
[57] Hovnanian A, Rebouillat D, Mattei MG, Levy ER, Marie I, Monaco AP, Hovanessian AG. The human 2',5'-oligoadenylate synthetase locus is composed of three distinct genes clustered on chromosome 12q24.2 encoding the 100-, 69-, and 40-kDa forms[J]. Genomics, 1998; 52(3):267-77
[58] Yakub I, Lillibridge KM, Moran A, Gonzalez OY, Belmont J, Gibbs RA, Tweardy DJ. Single nucleotide polymorphisms in genes for 2'-5'-oligoadenylate synthetase and RNase L inpatients hospitalized with West Nile virus infection[J]. J Infect Dis, 2005; 192(10):1741-8
[59] Hamano E, Hijikata M, Itoyama S, Quy T, Phi NC, Long HT, Ha le D, Ban VV, Matsushita I, Yanai H, Kirikae F, Kirikae T, Kuratsuji T, Sasazuki T, Keicho N. Polymorphisms of interferon-inducible genes OAS-1 and MxA associated withSARS in the Vietnamese population[J]. Biochem Biophys Res Commun, 2005; 329(4):1234-9
[60] He J, Feng D, de Vlas SJ, Wang H, Fontanet A, Zhang P, Plancoulaine S, Tang F, Zhan L, Yang H, Wang T, Richardus JH, Habbema JD, Cao W. Association of SARS susceptibility with single nucleic acid polymorphisms of OAS1 and MxA genes: a case-control study[J]. BMC Infect Dis, 2006; 6:106
[61] Haller O, Kochs G. Interferon-induced mx proteins.dynamin-like GTPases with antiviral activity[J]. Traffic, 2002; 3(10): 710-7
[62] Malathi K, Paranjape JM, Bulanova E, Shim M, Guenther-Johnson JM, Faber PW, Eling TE, Williams BR, Silverman RH. A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L[J]. Proc Natl Acad Sci USA, 2005; 102(41):14533-8
[63] Benech P, Merlin G, Revel M, Chebath J. 3' end structure of the human (2'-5') oligo A synthetase gene: prediction of two distinct proteins with cell type-specific expression[J]. Nucleic Acids Res, 1985; 13(4):1267-81
[64] Bonnevie-Nielsen V, Field LL, Lu S, Zheng DJ, Li M, Martensen PM, Nielsen TB, Beck-Nielsen H, Lau YL, Pociot F. Variation in antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the OAS1 gene[J]. Am J Hum Genet, 2005; 76(4):623-33
[65] Knapp S, Yee LJ, Frodsham AJ, Hennig BJ, Hellier S, Zhang L, Wright M, Chiaramonte M, Graves M, Thomas HC, Hill AV, Thursz MR. Polymorphisms in interferon-induced genes and the outcome of hepatitis C virus infection: roles of MxA, OAS-1 and PKR[J]. Genes Immun, 2003; 4(6):411-9
[66] Gil J, Esteban M. The interferon-induced protein kinase(PKR), triggers apoptosis through FADD-mediated activation of caspase 8 in a manner independent of Fas and TNF-alpha receptors[J]. Oncogene, 2000; 19(32): 3665-74
[67] Guan SH, Lu M, Grunewald P, Roggendorf M, Gerken G, Schlaak JF. Interferon-alpha response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment[J]. World J Gastroenterol, 2007; 13(2):228-35
[68] Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B, Davis RJ. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway[J]. Mol Cell Biol, 1996; 16(3):1247-55
[69] Ishida H, Ohkawa K, Hosui A, Hiramatsu N, Kanto T, Ueda K, Takehara T, Hayashi N. Involvement of p38 signaling pathway in interferon-alpha-mediated antiviral activity toward hepatitis C virus[J]. Biochem Biophys Res Commun, 2004; 321(3):722-7
[70] Guo H, Zhou T, Jiang D, Cuconati A, Xiao GH, Block TM, Guo JT. Regulation of hepatitis B virus replication by the phosphatidylinositol 3-kinase-akt signal transduction pathway[J]. J Virol, 2007; 81(18):10072-80
[71] Xiong W, Wang X, Liu X, Xiang L, Zheng L, Yuan Z. Interferon-inducible MyD88 protein inhibits hepatitis B virus replication[J]. Virology, 2004; 319(2):306-14
[72] Wu M, Xu Y, Lin S, Zhang X, Xiang L, Yuan Z. Hepatitis B virus polymerase inhibits the interferon-inducible MyD88 promoter by blocking nuclear translocation of Stat1[J]. J Gen Virol, 2007; 88(Pt 12):3260-9
[73] Nakao K, Nakata K, Yamashita M, Tamada Y, Hamasaki K, Ishikawa H, Kato Y, Eguchi K, Ishii N. p48 (ISGF-3gamma) is involved in interferon-alpha-induced suppression of hepatitis B virus enhancer-1 activity[J]. J Biol Chem, 1999; 274(40):28075-8
[74] Li Y, Sassano A, Majchrzak B, Deb DK, Levy DE, Gaestel M, Nebreda AR, Fish EN, Platanias LC. Role of p38alpha Map kinase in Type I interferon signaling[J]. J Biol Chem, 2004; 279(2):970-9
[75] Platanias LC. The p38 mitogen-activated protein kinase pathway and its role in interferon signaling[J]. Pharmacol Ther, 2003; 98(2):129-42
[1]刘新垣.干扰素研究及其重大突破性研究进展[J].中国处方药, 2004; 7(28): 33-5
[2]卢年芳,黄爱龙,唐霓,郑瑞强,林华,朱亚彬,吴莹,陶鹏.干扰素抗病毒活性的实验研究[J].中华肝脏病杂志, 2005; 13(12): 892-6
[3]张权,魏来,王燕. ISGF3:IFN-a抗乙肝病毒信号转导机制的重要因子[J].中华实验和临床病毒学杂志, 2005; 19(2): 110-113
[4] Nakao K, Nakata K, Yamashita M, Tamada Y, Hamasaki K, Ishikawa H, Kato Y, Eguchi K, Ishii N. p48 (ISGF-3gamma) is involved in interferon-alpha-induced suppression of hepatitis B virus enhancer-1 activity[J]. J Biol Chem, 1999; 274(40): 28075-8
[5] Samuel CE. Antiviral actions of interferons[J]. Clin Microbiol Rev, 2001; 14(4): 778-809
[6] Sarkar SN, Ghosh A, Wang HW, Sung SS, Sen GC. The nature of the catalytic domain of 2'-5'-oligoadenylate synthetases[J]. J Biol Chem, 1999; 274(36): 25535-42
[7] Rebouillat D, Hovanessian AG. The human 2',5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties[J]. J Interferon Cytokine Res, 1999; 19(4): 295-308
[8] Kubota K, Nakahara K, Ohtsuka T, Yoshida S, Kawaguchi J, Fujita Y, Ozeki Y, Hara A, Yoshimura C, Furukawa H, Haruyama H, Ichikawa K, Yamashita M, Matsuoka T, Iijima Y: Identification of 2'-phosphodiesterase, which plays a role in the 2-5A system regulated by interferon[J]. J Biol Chem, 2004; 279(36): 37832-41
[9] Malathi K, Paranjape JM, Bulanova E, Shim M, Guenther-Johnson JM, Faber PW, Eling TE, Williams BR, Silverman RH: A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L[J]. Proc Natl Acad Sci USA, 2005; 102(41):14533-8
[10] Clemens MJ, Elia A: The double-stranded RNA-dependent protein kinase PKR: structure and function[J]. J Interferon Cytokine Res, 1997; 17(9):503-24
[11] Gil J, Esteban M. The interferon-induced protein kinase(PKR), triggers apoptosis through FADD-mediated activation of caspase 8 in a manner independent of Fas and TNF-alpha receptors[J]. Oncogene, 2000; 19(32): 3665-74
[12] Kumar A, Yang Y L, Flati V, Der S, Kadereit S, Deb A, Haque J, Reis L, Weissmann C, Williams BR. Deficient cytokine signaling in mouse embryo fibroblasts with a targrted deletion in the PKR gene: role of IRF-1 and NF-kappaB[J]. EMBO J, 1997; 16(2): 406-16
[13]毛国强,许红梅,朱朝敏. MxA蛋白研究进展[J].国外医学病毒学分册, 2005; 12(4): 107-10
[14]吴金明,吴建胜,陈民新,林菊生.α干扰素于体外培养的2.2.15细胞中对MxA基因的诱导及抗HBV作用[J].中华实验和临床病毒学杂志, 2004; 18(4): 386-7
[15] Gordien E, Rosmorduc O, Peltekian C, Garreau F, Brechot C, Kremsdorf D. Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein[J]. J Virol, 2001; 75(6): 2684-91
[16] Kochs G, Haller O. GTP-bound human MxA protein interacts with the nucleocapsids of thogoto virus (orthomyxoviridae) [J]. J Biol Chem, 1999, 274(7): 43702-61
[17]管世鹤,杨东亮,陆蒙吉, Michael Roggendorf, Joerg F Schlaak.干扰素抗病毒蛋白MxA和2’,5’2OAS在肝胚瘤细胞株中的差异表达[J].中国感染与化疗杂志, 2006; 6(3): 149-153
[18] Guan SH, Lu M, Grünewald P, Roggendorf M, Gerken G, Schlaak JF. Interferon-αresponse in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment[J]. World J Gastroenterol, 2007; 13(2): 228-35
[19] Rosmorduc O, Sirma H, Soussan P, Gordien E, Lebon P, Horisberger M, BrechotC, Kremsdorf D. Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein[J]. J Gen Virol, 1999; 80(Pt 5): 1253-62
[20] Fernandez M, Quiroga JA, Carreno V. Hepatitis B virus downregulates the human interferon-inducible MxA promoter through direct interaction of precore/core proteins[J]. J Gen Virol, 2003; 84(Pt 8): 2073-82
[21] Raingeaud J, Whitmarsh AJ, Barrett T, Dérijard B, Davis RJ. MKK3 and MKK 6 regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway[J]. Mol Cell Biol, 1996; 16(3): 1247-55
[22]王抒,孙兰,杜冠华. p38-MAPK控制阿尔采末病进程的新靶点[J].中国药理学通报, 2004; 20(5): 595-8
[23] Jiang Y, Gram H, Zhao M, New L, Gu J, Feng L, Di Padova F, Ulevitch RJ, Han J. Characterization of the structure and function of the fourth member of p38 mitogen-activated protein kinases group, p38 delta[J]. J Biol Chem, 1997; 272(48): 30122–8
[24] Kuma Y, Sabio G, Bain J, Shpiro N, Marquez R, Cuenda A. BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo[J]. J Biol Chem, 2005; 280(20):19472-9
[25] Uddin S, Lekmine F, Sharma N, Majchrzak B, Mayer I, Young PR, Bokoch GM, Fish EN, Platanias LC. The Rac1/p38 mitogen-activated protein kinase pathwayis required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins[J]. J. Biol. Chem, 2000; 275: 27634-40
[26] Sassano A, Verma A, Platanias LC. Mitogen-activated protein kinase pathways in interferon signaling[J]. Methods Mol Med, 2005;116:135-50
[27] Uddin S, Majchrzak B, Woodson J, Arunkumar P, Alsayed Y, Pine R, Young PR, Fish EN, Platanias LC. Activation of the p38 mitogen-activated protein kinase by type I interferons[J]. J Biol Chem, 1999; 274(42):30127-31
[28] Uddin S, Majchrzak B, Wang PC, Modi S, Khan MK, Fish EN, Platanias LC. Interferon-dependent activation of the serine kinase PI 3'-kinase requiresengagement of the IRS pathway but not the Stat pathway[J]. Biochem Biophys Res Commun, 2000; 270(1):158-62
[29] Ishida H, Ohkawa K, Hosui A, Hiramatsu N, Kanto T, Ueda K, Takehara T, Hayashi N. Involvement of p38 signaling pathway in interferon-alpha-mediated antiviral activity toward hepatitis C virus[J]. Biochem Biophys Res Commun, 2004; 321(3): 722-7
[30] Fan H, Li H. Insulin receptor substrate and signals transduction[J]. Foreign Med Sci Sect Pathophysiol Clin Med, 2002; 22(5): 481-4
[31] Cantley LC. The phosphoinositide 3-kinase pathways[J]. Science, 2002, 296(5573): 1655-7
[32] Uddin S, Platanias LC. Mechanisms of type-I interferon signal transduction[J]. J Biochem Mol Biol, 2004; 37(6): 635-41
[33] Lekmine F, Uddin S, Sassano A, Parmar S, Brachmann SM, Majchrzak B, Sonenberg N, Hay N, Fish EN, Platanias LC. Activation of the p70 S6 Kinase and Phosphorylation of the 4E-BP1 Repressor of mRNA Translation by Type I Interferons[J]. J. Biol.Chem, 2003; 278(30): 27772–80
[34] Uddin S, Fish EN, Sher DA, Gardziola C, White MF, Platanias LC. Activation of the phosphatidylinositol 3'-kinase serine kinase by IFNα.[J]. J. Immunol.1997; 158(5): 2390-7
[35] Thyrell L, Hjortsberg L, Arulampalam V, Panaretakis T, Uhles S, Dagnell M, Zhivotovsky B, Leibiger I, Grander D, Pokrovskaja K. Interferon alpha-induced apoptosis in tumor cells is mediated through the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway[J]. J Biol Chem, 2004; 279(23): 24152-62
[36] Xiong W, Wang X, Liu XY, Xiang L, Zheng LJ, Liu JX, Yuan ZH. Analysis of gene expression in hepatitis B virus transfected cell line induced by interferon[J]. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003;35(12):1053-60
[37] Xiong W, Wang X, Liu X, Xiang L, Zheng L, Yuan Z. Interferon-inducible MyD88 protein inhibits hepatitis B virus replication[J]. Virology, 2004; 319 (2): 306-14
[38] Vlotides G, Sorensen AS, Kopp F, Zitzmann K, Cengic N, Brand S, Zachoval R, Auernhammer CJ. SOCS-1 and SOCS-3 inhibit IFN-alpha-induced expression of the antiviral proteins 2,5-OAS and MxA[J]. Biochem Biophys Res Commun, 2004; 320(3): 1007-14
[39] Brand S, Zitzmann K, Dambacher J, Beigel F, Olszak T, Vlotides G, Eichhorst ST, Goke B, Diepolder H, Auernhammer CJ. SOCS-1 inhibits expression of the antiviral proteins 2',5'-OAS and MxA induced by the novel interferon-lambdas IL-28A and IL-29[J]. Biochem Biophys Res Commun, 2005; 331(2):543-8
[40]范金水,庄辉,李远贵,朱晓洁,徐德忠,马为民,王跃民,陈雅洁,娄国强,马廷贤.我国8城市HBsAg阳性和阴性乙肝患者的病毒血清型和基因型分析[J].中华微生物和免疫学杂志, 1998; 18(1): 18-22
[41] Wai CT, Chu CJ, Hussain M, Lok AS. HBV genotype B is associated with better response to interferon therapy in HBeAg (+) chronic hepatitis than genotype C[J]. Hepatology, 2002; 36(6): 1425-30
[42]肖扬,胡操寒,周岳进,谢庆荣,朱冰星,胡侠,王开鉴,张文静.乙型肝炎病毒基因型对干扰素α-2b应答的影响[J].临床肝胆病杂志, 2006; 22(2): 105-6
[43] Xing TJ, Luo KX, Hou JL. Preliminary identification and analysis of point mutations correlated with response to interferon-alpha in hepatitis B virus post-transcriptional regulatory elements[J]. Chin Med J (Engl), 2005; 118(1):56-61
[44] Erhardt A, Reineke U, Blondin D, Gerlich WH, Adams O, Heintges T, Niederau C, Haussinger D. Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B[J]. Hepatology, 2000; 31(3): 716-25
[45] Wang Y, Wei L, Jiang D, Cong X, Fei R, Chen H, Xiao J, Wang Y. In vitroresistance to interferon-alpha of hepatitis B virus with basic core promoter double mutation[J]. Antiviral Res, 2007; 75(2): 139-45
[46] Zhang FK. Pegylated interferons in the treatment of chronic hepatitis C[J]. Chinese Medical Journal, 2003; 116(4): 495-8
[47] Cooksley WG, Piratvisuth T, Lee SD, Mahachai V, Chao YC, Tanwandee T, Chutaputti A, Chang WY, Zahm FE, Pluck N. Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B eantigen-positive chronic hepatitis B[J]. J Viral Hepat, 2003; 10(4): 298-30
[48] Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, Lu ZM, Piratvisuth T, Germanidis G, Yurdaydin C, Diago M, Gurel S, Lai MY, Button P, Pluck N. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J Med, 2004; 16;351(12):1206-17
[49] Vassiliadis T, Tziomalos K, Patsiaoura K, Zagris T, Giouleme O, Soufleris K, Grammatikos N, Theodoropoulos K, Mpoumponaris A, Dona K, Zezos P, Nikolaidis N, Orfanou-Koumerkeridou E, Balaska A, Eugenidis N.Lamivudine/pegylated interferon alfa-2b sequential combination therapy compared with lamivudine monotherapy in HBeAg-negative chronic hepatitis B[J]. J Gastroenterol Hepatol, 2007; 22(10):1582-8
[50] Monkarsh SP, Ma Y, Aglione A, et al. Positional isomers of monopegylated interferon alpha-2: isolatio, characterization, and biological activity[J]. Anal Biochem, 1997; 247 (2): 434-40
[51]崔建军,范平,蒋小玲,许诚,何清,周伯平.聚乙二醇干扰素α-2a治疗HBeAg阳性慢性乙型肝炎的疗效和安全性[J].中华实验和临床病毒学杂志, 2006; 20(4): 331-3
[52]赵鸿,斯崇文,魏来,万漠彬,尹有宽,侯金林,牛俊奇.聚乙二醇化干扰素a -2b与干扰素a -2b治疗e抗原阳性慢性乙型肝炎的疗效和安全性的随机对照多中心研究[J].中华肝脏病杂志, 2006; 14(5): 323-6
[53] Osborn BL, Olsen HS, Nardelli B, Murray JH, Zhou JX, Garcia A, Moody G, Zaritskaya LS, Sung C. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys[J]. J Pharmacol Exp Ther, 2002; 303(2):540-8
[54] Sung C, Nardelli B, LaFleur DW, Blatter E, Corcoran M, Olsen HS, Birse CE, Pickeral OK, Zhang J, Shah D, Moody G, Gentz S, Beebe L, Moore PA. An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates [J]. J Interferon Cytokine Res, 2003; 23(1): 25-36
[55]唱韶红,巩新,杨志愉,王同映,马国昌,马清钧,吴军.人血清白蛋白和人干扰素α2b的融合蛋白在毕赤酵母中的表达[J].生物工程学报, 2006; 22(2): 173-9
[56]张伍魁,范清林,邹文艺,仲皙,宋礼华.白蛋白融合α-2b干扰素在毕赤酵母中的表达[J].中国生物工程杂志, 2005; 25(12): 45-9
[57] Bain VG, Kaita KD, Yoshida EM, Swain MG, Heathcote EJ, Neumann AU, Fiscella M, Yu R, Osborn BL, Cronin PW, Freimuth WW, McHutchison JG, Subramanian GM. A phase 2 study to evaluate the antiviral activity, safety, and pharmacokinetics of recombinant human albumin-interferon alfa fusion protein in genotype 1 chronic hepatitis C patients[J]. J Hepatol, 2006; 44(4): 671-8
[58] Balan V, Nelson DR, Sulkowski MS, Everson GT, Lambiase LR, Wiesner RH, Dickson RC, Post AB, Redfield RR, Davis GL, Neumann AU, Osborn BL, Freimuth WW, Subramanian GM. A Phase I/II study evaluating escalating doses of recombinant human albumin-interferon-alpha fusion protein in chronic hepatitis C patients who have failed previous interferon-alpha-based therapy[J]. Antivir Ther, 2006; 11(1): 35-45
[59] Balan V, Nelson DR, Sulkowski MS, Everson GT, Lambiase LR, Wiesner RH,Dickson RC, Garcia A, Moore PA, Yu R, Subramanian GM. Modulation of interferon-specific gene expression by albumin-interferon-alpha in interferon-alpha-experienced patients with chronic hepatitis C[J]. Antivir Ther, 2006; 11(7): 901-8
[60]任进余,陈义,吴斌,崔红,张瑛,沈钟.病毒性肝炎的药物靶向治疗研究——抗HBV脂质体的研制及临床观察[J].中西医结合肝病杂志, 1997; 7(1):1-4
[61]黄耀芳,章廉,胡俊,王燕军,骆抗先.脂质体干扰素的体外抗乙型肝炎病毒效应[J].临床肝胆病杂志, 1997; 13(1): 38-39
[62]吕厚峰,孙迎基,邹文艺,张奉国,廖建民,沈子龙.基因重组干扰素a-2b脂质体的药代动力学研究[J].药物生物技术, 2005; 12(3): 186-9
[63]杜施霖,王吉耀,潘弘,陆伟跃,王剑.含有RGD序列环肽介导的干扰素脂质体对大鼠肝纤维化的治疗作用[J].中华医学杂志, 2005; 85(15): 1015-20
[64] Le Guerhier F, Thermet A, Guerret S, Chevallier M, Jamard C, Gibbs CS, Trepo C, Cova L, Zoulim F. Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model [J]. J Hepatol, 2003; 38(3): 328-334
[65] Addison WR, Wong WW, Fisher KP, Tyrrell DL. A quantitative competitive PCR assay for the covalently closed circular form of the duck hepatitis B virus [J]. Antiviral Res, 2000; 48(1): 27-37
[66] Deres K, Schroder CH, Paessens A, Goldmann S, Hacker HJ, Weber O, Kramer T, Niewohner U, Pleiss U, Stoltefuss J, Graef E, Koletzki D, Masantschek RN, Reimann A, Jaeger R, Gross R, Beckermann B, Schlemmer KH, Haebich D, Rubsamen-Waigmann H. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids[J]. Science, 2004; 299(5608): 893-6
[67] Hu J, Toft DO, Seeger C. Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids [J]. EMBO J, 1997; 16(1): 59-81
[68] Genovesi EV, Lamb L, Medina I, Taylor D, Seifer M, Innaimo S, Colonno RJ,Clark JM. Antiviral efficacy of lobucavir (BMS2180194), a cyclobutyl-guanosine nucleoside analogue, in the woodchuck (Marmota monax) model of chronic hepatitis B virus (HBV) infection [J]. Antiviral Res, 2000; 48(3):197-203
[69] Korba BE, Cote P, Hornbuckle W, Tennant BC, Gerin JL. Treatment of chronic woodchuck hepatitis virus infection in the eastern woodchuck (marmota monax) with nucleoside analogues is predictive of therapy for chronic hepatitis B virus infection in human [J]. Hepatology, 2000; 31(5): 1165-75
[70] Dahmen U, Li J, Dirsch O, Fiedler M, Lu M, Roggendorf M, Broelsch CE. A new model of hepatitis B virus reinfection: liver transplantation in the woodchuck[J]. Transplantation, 2002; 74 (3): 373-80
[71] Bruni R, D’Ugo E, Argentini C, Giuseppetti R, Rapicetta M. Scaffold attachment region located in a locus targeted by hepadnavirus integration in hepatocellular carcinoma [J]. Cancer Detect Prev, 2003; 27(3):175-81
[72] Wang Y, Cui F, Lv Y, Li C, Xu X, Deng C, Wang D, Sun Y, Hu G, Lang Z, Huang C, Yang X. HBsAg and HBx knocked into the p21 locus causes hepatocellular carcinoma in mice [J]. Hepatology, 2004; 39(2): 2318- 24
[73] Sell MA, Chen ML, ACs G. Production of hepatitis B virus padicles in HepG2 cells transfected with cloned hepatitis B virus DNA[J]. Pron Natl Acad Sci USA, 1987; 84: 1005-9
[74]劳国琴,王佳良,吴勇.干扰素治疗慢性乙型肝炎病毒感染的经济效益[J].中国药房, 2006; 17(16): 1223-5