【作者】 杨茜；

【导师】 张伦理；

【作者基本信息】 南昌大学 ， 临床医学（专业学位）， 2018， 博士

【摘要】 研究背景和目的:干扰素α是目前广泛用于临床的一线抗乙肝病毒药物,它具有广谱性、高活性、间接性及种属特异性等特点的抗病毒效应。它是一种具有抗病毒作用的细胞因子,干扰素自身不能直接使病毒灭活,而往往是通过激活JAK-STAT信号转导通路发挥其抗病毒效应。研究表明,I型干扰素(IFN-α、IFN-β)通过与其特异性的干扰素受体(IFNAR)结合后,激活JAK激酶-JAK1、Tyk2,继而招募有SH2结构域的STAT1和STAT2(信号转导和转录活化因子),并使之磷酸化,激活的STAT1、2异源性二聚体与IRF-9(IFN调节因子9)结合,再结合上ISGF3γ组成ISGF3(IFN刺激基因因子3复合体),ISGF3从细胞质转位到细胞核与干扰素刺激应答元件(ISRE)结合,启动后者转录,激活蛋白激酶(PKR)等一系列调控抗病毒靶基因的转录,诱导多种抗病毒蛋白的表达,最主要的抗病毒蛋白有PKR与核糖核酸酶L(RNase L),它们分别通过影响病毒mRNA的翻译与降解RNA起抗病毒作用。干扰素α同时还具有抗肿瘤、免疫调节等作用。持续性的乙型肝炎病毒感染是引起原发性肝癌的主要原因。在HBV相关原发性肝癌的发生发展过程中乙肝病毒X蛋白(HBx)起十分关键的作用,它可以作为原癌基因或者共同作用因子促进肿瘤的发生。本研究目的是建立HBV相关的肝癌细胞模型,即转染携带HBx基因慢病毒的人肝癌细胞Huh-7细胞,观察干扰素α对该细胞模型细胞行为学和JAK-STAT信号通路相关分子表达的影响,从肝癌细胞生物学行为角度及干扰素α主要作用的JAK-STAT信号通路上重要分子(IFNAR1、IFNAR2、ISGF3、PKR、RNAse L)的表达水平来分析探讨干扰素α抑制HBV相关HCC的机制。同时设计使用干扰素α抗病毒治疗的慢性乙肝患者临床试验,检测各治疗节点有效组及无效组患者之间上述5个分子表达水平差异,以期寻找能早期判断干扰素α应答效果的因子。方法:1、构建携带乙肝病毒X基因的慢病毒载体,转染肝癌细胞Huh-7,采用免疫荧光进行鉴定;再以MTT法检测细胞增殖活性,划痕实验检测细胞迁移能力,侵袭实验检测细胞侵袭能力;之后将Huh-7和Huh-7-HBV X再加入干扰素α干预,观察干扰素α对上述细胞模型生物学行为的影响。2、同样选择肝癌细胞Huh-7为对照,转染携带HBV X基因的慢病毒载体形成Huh-7-HBx,之后将Huh-7和Huh-7-HBV X再加入干扰素α干预;自干扰素作用环节的始动环节起分别用RT-qPCR和Western-blot检测JAK-STAT通路重要相关分子即干扰素受体(IFNAR1、IFNAR2)干扰素刺激基因因子3(ISGF3)、RNA依赖蛋白激酶(PKR)、核糖核酸酶(RNase L)的mRNA和蛋白表达水平的变化。3、收集在南昌大学第一附属医院感染科就诊的符合干扰素α治疗指征的慢性乙型肝炎患者,入组患者给予聚乙二醇IFNα-2b,1.0μg/kg/周,每周1次,皮下注射,或普通IFN-α,500万U,隔日一次,皮下注射。疗程48周,治疗24周仍无应答者作无效病例停药。收集基线(治疗前)、治疗4周、8周、12周、24周时的PBMCs。所有患者在治疗24周疗程结束时判断疗效,依据疗效分为两组:(1)有效组:ALT正常,HBV-DNA<500IU/m L或下降幅度≥2log IU/m L,HBeAg发生或未发生血清学转换;(2)无效组:ALT正常或升高,HBV-DNA下降幅度<2log IU/mL,HBe Ag未发生血清学转换。分别采用RT-qPCR法和Western blot法检测各治疗时间点PBMCs中JAK-STAT通路各分子IFNAR1、IFNAR2、ISGF3、PKR、RNase-L的mRNA表达水平和蛋白表达水平。结果:1、Huh-7细胞经HBX修饰后迁移和侵袭能力部分增强,而经IFN-α干预后Huh-7细胞和Huh-7转染组细胞迁移和侵袭能力均有所减弱,差异均有统计学意义(P<0.05)。Huh-7经HBX修饰后增殖能力无明显影响,同时经IFN-α干预后Huh-7和Huh-7转染组细胞增殖能力亦无明显影响(P>0.5)。2、经慢病毒转染后Huh-7细胞中HBX基因显著高于转染前(P<0.05)。经IFN-α干预后Huh-7细胞IFNAR2、ISGF3、PKR、RNaseL基因表达均出现了上调,差异均有统计学意义(P<0.05)。而经IFN-α干扰后Huh-7-HBx转染组细胞IFNAR1、IFNAR2、ISGF3、PKR、RNase L基因表达亦均出现了上调,且上调水平高于Huh-7细胞组,差异均有统计学意义(P<0.05)。经慢病毒转染后Huh-7细胞HBX蛋白显著高于转染前(P<0.05)。经干扰素α干预后Huh-7细胞和Huh-7-HBx细胞上述5个分子的蛋白表达水平变化呈现与mRNA表达水平基本一致的结果,差异均有统计学意义(P<0.05)。3、共入选41例患者,包括30名男性及11名女性,18例患者接受PegIFNα-2b,1.5μg/Kg,每周1次;23名患者接受普通IFNα-2b,500万单位,每周3次,所有患者完成24周治疗。24周治疗结束,依据上述分组标准,在41名患者中25名患者为治疗有效组(A组),16名为治疗无效组(B组)。治疗基线、4周、8周、12周和24周时,IFNAR1、IFNAR2、ISGF3、PKR和RNASE-L的mRNA表达水平组间差异均有统计学意义,且均为A组>B组。治疗4周时,IFNAR2及PKR的mRNA水平治疗有效组显著高于无效组,在治疗8周时IFNR1及ISGF3的水平有效组显著高于无效组,而Western-blot检测显示的上述蛋白水平也呈现基本一致的结果。结论:1.HBx可增加肝癌细胞Huh-7的体外迁移和侵袭能力,但对增殖能力无明显影响;IFNα可抑制肝癌细胞Huh-7和Huh-7-HBx转染组细胞的迁移和侵袭能力,但对增殖能力亦无明显影响。2.干扰素α可上调肝癌细胞Huh-7中JAK-STAT通路分子IFNAR1、IFNAR2、ISGF3、PKR、RNase L基因以及蛋白的表达水平,而干扰素α干预后Huh-7-HBx转染组细胞IFNAR1、IFNAR2、ISGF3、PKR、RNaseL基因和蛋白表达亦均出现了上调,且上调水平高于Huh-7细胞组。提示干扰素α抗HBV相关性肝癌可能与其上调上述抗病毒蛋白有关。3.JAK-STAT通路分子IFNAR1、IFNAR2、ISGF3、PKR和RNAse-L为干扰素α抗病毒作用的重要分子,上述5个分子的mRNA以及蛋白的组间和时间点差异性表达可能是影响IFNα抗病毒效应的重要机制。从早期预测干扰素α治疗慢性乙肝应答价值的角度来看,IFNAR2及PKR有望成为早期预测干扰素应答的指标。更多还原

【Abstract】 Background and Objectives:Interferon alpha(IFN-α)is currently used as a first-line antiviral drug to treat chronic hepatitis B.Interferon alpha is an antiviral cytokine that has a broad spectrum of action,exhibits high activity and indirect and species specificity.IFN-α exerts its antiviral activity via activation of the Janus kinase/signal transducer and activator of transcription(JAK-STAT)signaling pathway.The binding of IFN-α to its receptor IFNAR1 activates Janus kinase-JAK1 and non-receptor tyrosine-protein kinase TYK2,which leads to the phosphorylation of signal transducer and activator of transcription(STAT)1 and STAT2.STAT1 and STAT2 then form a heterodimer and bind to interferon regulatory factor 9(IRF-9)to form ISGF3.ISGF3 translocates from the cytoplasm to the nucleus and binds to the IFN stimulated regulatory element to promote the transcription of antiviral genes,such as PKR and RNaseL.In addition,interferon alpha also has biological functions such as anti-tumor,immune regulation and so on.The occurrence of primary liver cancer in 50-55% is caused by persistent hepatitis B virus(HBV)infection.Hepatitis B X protein(HBx),encoded by HBV DNA,serves an important role during the development of chronic hepatitis B,liver cirrhosis and liver cancer.Therefore,the current study established a novel HBV-related liver cancer model by transfecting the hepatoma cell line Huh-7 with HBx-expressing lentivirus,and subsequently investigated the effect of IFN-α on the cellular behavior and the expression of JAK-STAT signal pathway related molecules of the cancer cells.In addition,our study observed the predictive value of JAK-STAT pathway related molecules on the response of IFN-α in the treatment of chronic hepatitis B.Methods:1.We produced HBx-expressing lentivirus and then transfected it into Huh-7 cells.Subsequently we used an MTT assay to detect the cell viability,cell scratch test to detect the cell migration ability and cell invasion test to detect the cell invasiveness.After IFN-α was added to Huh-7 and Huh-7-HBx,the effects of IFN-α on the biological behavior of the above cells were observed.2.We transfected HBx-expressing lentivirus into Huh-7 cells and then added IFN-α.Subsequently we used RT-qPCR and Western-blot to detect the changes of expression level of related molecules on JAK-STAT signaling pathway before and after IFN-α intervention in above cells.3.Patients with chronic hepatitis B who met the indications of interferon alpha treatment were collected.The patients in the group were given polyethylene glycol IFN alpha-2b,1 g/kg/ weeks,1 times a week,subcutaneous injection,or regular IFN alpha,5 million U,once every other day,subcutaneous injection.The basic course of treatment was 48 weeks,and the treatment of non responders was stopped at 24 weeks.We collected PBMCs at baseline(before treatment)and treatment for 4,8,12,and 24 weeks of these patients.The expression level of IFNAR1,IFNAR2,ISGF3,PKR and RNase-L in PBMCs were detected by RT-qPCR and Western blot respectively.Finally,the differences of the above molecular expression levels in PBMCs between the effective and ineffective groups were statistically analyzed.Results:1.The effect of IFN-α treatment alone on Huh-7 cell viability compared with the control group was not significant.Similarly,the difference in cell viability in the Huh-7-HBx and Huh-7-HBx+IFN-α treatment groups compared with the control was not significant.Cell migration was decreased(P<0.05)in the IFN-α treatment alone group and increased(P<0.05)in the Huh-7-HBx group compared with the control.There was a decrease(P<0.05)in cell migration in the Huh-7-HBx+IFN-α treatment group compared with the control,but not to the same extent as the decrease observed in the IFN-α treatment alone group.Huh-7 cell invasion was decreased(P<0.05)in the IFN-α treatment alone group and increased(P<0.05)in the Huh-7-HBx group compared with the control.There was a decrease(P<0.05)in cell invasion in the Huh-7-HBx+IFN-α treatment group compared with the control,but not to the same extent as the decrease observed in the IFN-α treatment alone group.2.RT-qPCR demonstrated that the expression of IFNAR1,IFNAR2,PKR,RNaseL and ISGF3 mRNA was significantly increased(P<0.05)in the IFN-α treatment only group compared to the control.mRNA levels of these antiviral genes were upregulated(P<0.05)in the Huh-7-HBx group compared with the control group however,not to the extent of the increase demonstrated in the IFN-α treatment only group.The most significant increase in expression of antiviral gene mRNA,compared with the control,was in the Huh-7-HBx+IFN-α treatment group.The protein expression of these antiviral genes was consistent with this.3.A total of 41 patients were enrolled,including 30 males and 11 females,18 patients were treated with Peg IFN alpha-2b,1.0μg/ kg,1 times a week;23 patients underwent conventional IFN alpha-2b,5 million units,3 times a week,all patients completed 24 weeks of treatment.At the end of the 24 week treatment,according to the above criteria,23 of the 36 patients were treated as effective group(group A),and 13 were treated as ineffective group(group B).At baseline,4 weeks,8 weeks,12 weeks and 24 weeks,mRNA expression level of IFNAR1,IFNAR2,ISGF3,PKR and RNASE-L were statistically significant in two groups,and all were A group >B group.At 4 weeks of treatment,the mRNA level of IFNR2 and PKR in the effective group was significantly higher than that in the ineffective group.At the 8 week treatment,the level of IFNR1 and ISGF3 in the effective group was significantly higher than that in the ineffective group,and the above protein levels detected by Western-blot showed the same results.Conclusions:1.HBx could increase the migration and invasion ability of Huh-7 cells in vitro,but had no significant effect on the viability,and IFN-α may inhibit cell migration and reduce cell invasion in HBV-related liver cancer.2.IFN-α can increase the expression of JAK-STAT pathway molecules IFNAR1,IFNAR2,ISGF3,PKR,RNase L in hepatocellular carcinoma cell Huh-7,and IFN-α can further increase the expression levels of the above molecules in Huh-7 cells transfected with HBx-expressing lentivirus.3.The differential expression of JAK-STAT pathway molecules IFNAR1,IFNAR2,IRF9,ISGF3 and RNASE-L genes and protein between groups and time points may be an important mechanism that affects the antiviral effect of IFN-α.From the point of view of the early predictive value of IFN-α in the response to chronic hepatitis B,IFNR2 and PKR are expected to be an early prediction of IFN-α response.更多还原