【作者】 徐丙发；

【导师】 宋礼华； 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2009， 博士

【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。更多还原

【Abstract】 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 vitroHepG2 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 vivoThe 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.更多还原