白介素-21与肝炎病毒感染的关系以及肝损伤相关小分子抑制剂的筛选研究
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摘要
流行病学的调查发现乙肝病毒(Hepatitis B Virus, HBV)和丙肝病毒(Hepatitis C Virus, HCV)的感染,黄曲霉毒素(Aflatoxin)和过量酒精的摄入是肝细胞癌(Hepatocellular Carcinoma, HCC)发生的主要危险因素,其中HBV和HCV慢性感染造成的HCC占了HCC总数的80%以上。从HBV或HCV造成的慢性感染到肝癌的发生,一般需要30年以上的时间。作为一种最常见的恶性肿瘤,HCC预后极差,存活率只有3%-5%。因此,如何阻止疾病的进展,降低肝硬化、肝癌的发生率和死亡率,成为医生和患者关注的焦点问题。
第一部分白介素-21与肝炎病毒感染的关系
目的：
白介素21(IL-21)是近期研究的一个热点,已有学者对艾滋病病人以及自身免疫性肝炎患者外周血中IL-21及IL-21R进行了检测和探讨,但迄今为止没有关于HBV和HCV患者外周血中IL-21水平的报道。IL-21参与了多种自身免疫性疾病的发生,而HBV和HCV也与机体的免疫功能有着不可分割的联系。为此,本研究拟通过检测HBV和HCV感染者血清IL-21水平,同时检测其它肝功能指标、病毒载量、抗核抗体等,探讨IL-21与HBV、HCV感染的相关性。
方法：
采用ELISA法定量测定IL-21和抗核抗体(ANA)。肝功能指标包括谷丙转氨酶(ALT)、谷草转氨酶(AST)、总蛋白(TP)、白蛋白(ALB)、球蛋白(GLO)、总胆红素(TBIL)、直接胆红素(DBIL)均采用全自动生化分析仪检测。ELISA方法检测HBV血清标志物即HBsAg、HBsAb、HBeAg、HBeAb、HBcAb。HBVDNA采用荧光定量PCR法。
结果：
1、抗核抗体ANA和白介素21对HCV患者具有保护作用。
ALT<40 U/L的HCV感染者中ANA阳性率为22.4%,而ALT>40 U/L的患者阳性率为15.0%,反映ANA阳性患者大部分肝功能较好。表明自身免疫抗体的产生对肝细胞损伤有一定的保护作用,导致ALT降低,有助于控制HCV病毒的发展。高ALT组、低ALT组及对照组间AST均值与ALT均值有同步变化,部分患者的ALT较高可能也与ANA抗体水平较低有一定的关系。ALT>40U/L的HCV感染组ALT/AST比率>1,原因可能一方面是肝细胞损伤较厉害,另一方面是纤维化程度较高。本试验中ALT<40 U/L感染组、ALT>40 U/L感染组的IL-21水平均低于对照组。但相比之下,低ALT感染组的水平更高一些。可见,HCV的感染会导致IL-21表达水平降低,且IL-21水平的高低与肝细胞受损程度呈负相关,可能与HCV感染后引起的细胞免疫有牵连。证实了IL-21的存在有助于控制HCV病毒的发展,降低肝细胞损伤。本研究提示ANA的表达与IL-21的存在均能降低HCV感染者的肝细胞损伤,且ANA阳性HCV感染者的IL-21水平低于ANA阴性HCV感染者,进一步对ANA、IL-21、ALT、AST、ALB这五个变量两两间进行相关分析,结果都没有相关性。ANA与IL-21对肝细胞的保护作用应该是与其他因素共同作用的结果。
2、HBsAg阳性和HBeAg阳性病毒携带者组以及肝硬化组的IL-21水平显著低于对照组,而HBsAg阳性病毒携带者组和慢乙肝组的IL-21水平却显著高于对照组。
在HBV与IL-21相互关系的研究中,我们将收集的血清分为五组,分别为：健康对照组(control)、HBsAg阳性病毒携带者组(AsCs)、HBsAg阳性和HBeAg阳性病毒携带者组(AsCse)、慢乙肝组(CHB)和肝硬化组(Cir)。control、AsCs、AsCse、CHB、Cir组ALT均值依次为16.75±1.11、24.44±2.09、23.68±2.01、201.47±36.51、59.28±12.97 U/L,具有显著性差异(F=12.368,ν=122.158,P=0.000)。AST均值依次为22.24±0.72、28.23±1.27、27.53±1.37、150.82±27.40、63.83±8.98U/L,具有显著性差异(F=15.680,v=122.362,P=0.000)。ALB均值依次为47.03±0.46、45.79±0.54、46.12±0.59、43.02±0.66、39.57±0.94g/L,具有显著性差异(F=16.367,v=116.789,P=0.000)。GLO均值依次为25.57±0.62、26.61±0.61、25.94±0.65、27.96±0.48、30.70±0.65g/L,具有显著性差异(F=10.395,v=109.138,P=0.000)。TBIL均值依次为15.20±1.44、14.13±0.80、14.25±1.73、36.17±11.00、41.96±7.27μmol/L,具有显著性差异(F=4.557,ν=114.548,P=0.002)。DBIL均值依次为4.10±0.26、4.25±0.25、4.04±0.38、17.84±7.04、20.69±5.16μmol/L,具有显著性差异(F=3.518,ν=121.135,P=0.009)。而TP均值没有显著性差异(F=1.608,ν=122.766,P=0.177)。另外,AsCs、AsCse、CHB、Cir组的HBV DNA滴度(logDNA)均值依次为3.76±0.19、6.80±0.28、5.41±0.18、3.64±0.14,具有显著性差异(F=47.324, v=97.334, P=0.000)。Control、AsCs、AsCse、CHB、Cir组的IL-21均值依次为67.22±5.87、117.17±23.92、47.90±7.46、112.73±18.54、50.18±5.87pg/ml,具有显著性差异(F=5.096,ν=120.252,P=0.001)。将所有标本按照各抗原、抗体的阴、阳性分别进行分组,用t检验比较IL-21在阴阳性组间的差别。结果表明,HBsAb阴性组的IL-21水平显著高于阳性组,依次为79.55±5.73和62.30±6.35pg/ml (t=-2.016, P=0.046);HBeAg阴性组的IL-21水平显著高于阳性组,依次为90.49±10.76和66.92±5.07pg/ml(t=-1.982,P=0.049); HBeAb阳性组的IL-21水平显著高于阴性组,依次为90.06±9.94和66.50±3.93pg/ml (t=2.205, P=0.029)。将所有标本按照男性、女性分成两组,比较IL-21在两组间的差别。结果显示,女性组IL-21均值略高于男性组,但没有显著性差异(t=-0.375,P=0.708)。对所有标本的ALT和AST作双变量相关性分析,结果相关系数r=0.959,P=0.000,表明两者显著性相关,且相关关系非常密切。对所有标本的ALB和TP作双变量相关性分析,结果相关系数r=0.689,P=0.000,表明两者显著性相关。对所有标本的DBIL和TBIL作双变量相关性分析,结果相关系数r=0.988,P=0.000,表明两者显著性相关,且相关关系非常密切。对所有标本的ALB和GLO作双变量相关性分析,结果相关系数r=-0.413,P=0.000,表明两者显著性负相关。其它变量之间也有一些较弱的相关性存在,例如LogDNA与ALT之间的r=0.328,P=0.000;LogDNA与AST之间的r=0.302,P=0.000;GLO与TP之间的r=0.363,P=0.000;ALB与DBIL之间的r=0.333,P=0.000;ALB与TBIL之间的r=0.323,P=0.000等。但是,IL-21与其它变量之间不存在显著相关性。
结论：
1、ALT<40U/L的HCV感染者中ANA阳性率高于ALT>40 U/L的感染者,表明自身免疫抗体的产生对肝细胞损伤有一定的保护作用。
2、ALT<40 U/L的HCV感染组和ALT>40 U/L感染组的IL-21水平均低于对照组,表明HCV的感染会导致IL-21表达水平降低,反过来也说明IL-21的产生有助于控制HCV病毒发展,降低肝细胞损伤。
3、HBsAg阳性和HBeAg阳性病毒携带者组以及肝硬化组的IL-21水平显著低于对照组,而HBsAg阳性病毒携带者组和慢乙肝组的IL-21水平却显著高于对照组。说明HBeAg阳性乙肝患者的预后较差,也验证了IL-21的积极作用。
4、所有HBV标本的ALT和AST显著密切相关(r=0.959,P=0.000),ALB和TP显著相关(r=0.689,P=0.000),DBIL和TBIL显著密切相关(r=0.988,P=0.000),ALB和GLO显著负相关(r=-0.413,P=0.000)。
第二部分肝损伤相关小分子抑制剂的筛选研究
目的：
利用HBV DNA指标在细胞水平筛选抗HBV药物,并研究药物对HBV复制中间体的抑制效果。从胞外、胞内两个角度深入研究化合物与DNA的结合方式、对DNA的断裂作用及其抗肝癌活性,从多方面探讨其作用机制。
方法：
采用WST-8法检测细胞毒性,按照CCK-8试剂盒进行操作。采用HBV DNA提取及扩增荧光检测试剂盒检测细胞内外HBV DNA。采用Western blot法检测HBcAg。采用时间分辨免疫荧光(TRFIA)法定量检测HBeAg和HBsAg水平。采用紫外吸收光谱、荧光光谱、荧光Scatchard图及黏度测定实验研究镍配合物与小牛胸腺DNA (CT DNA)的作用,琼脂糖凝胶电泳分析镍配合物与pBR322质粒DNA的作用。通过MTT法进行镍配合物体外抗肝癌活性的评估,DAPI荧光染色分析及流式细胞术检测镍配合物对细胞凋亡及细胞周期的影响。
结果：
1、化合物XLWG-54-52抑制HepAD38细胞内HBV DNA的IC50为11.87±3.94μM,抑制胞外HBV DNA的IC50为10.84±0.60μM。
通过本研究确立了一个合适的细胞模型HepAD38,该模型能较稳定地表达HBsAg和HBeAg抗原,DNA复制也较高,并确定细胞培养时间为7天；建立了一套筛选抗HBV药物的方法,并筛选出了XLWG-54-52、XLWG-61-3A、XLWG-61-7、XLWG-48-6、XLWG-48-11五个能有效抑制HBV DNA的化合物；重点对其中的XLWG-54-52进行了研究,XLWG-54-52对胞内外HBV DNA均有较强的抑制作用,胞内IC50为11.87±3.94μM,胞外IC50为10.84±0.60μM；XLWG-54-52对HepAD38的半数致死浓度CC50为0.339mM;XLWG-54-52对HBeAg、HBsAg和HBcAg三种抗原均没有抑制作用。XLWG-54-52对HBV所致乙型肝炎的治疗作用可能要结合其类似物双环醇的作用机理,从它对肝损伤的保护作用入手。
2、NiL配合物与小牛胸腺DNA (CT DNA)有良好的结合,且结合方式为插入模式。它还能将质粒pBR322 DNA由超螺旋形式切割成开环形式,切割效果呈剂量依赖型,切割机理为自由基机理。
紫外吸收光谱法测得配合物的吸收峰伴随有明显的减色,减色率为18%,推测NiL配合物与DNA可能是以插入模式结合,计算得出结合常数Kb为2.18×105(L·mol-1)；荧光光谱实验表明随着配合物浓度的不断增加,EB-DNA复合物的荧光明显发生淬灭,验证了配合物的吡咯环插入到DNA双链之间与之结合；荧光Scatchard图进一步显示,随着DNA溶液中配合物浓度的增大,k值逐渐减小,而n值在0.190附近变化不大,说明配合物竞争性地抑制EB与DNA的结合,即以插入方式与DNA结合；黏度实验中,配合物与CT DNA发生相互作用后,随着配合物浓度的增大,DNA黏度值逐渐增加,由此可确定配合物是以插入方式与DNA作用的；DNA琼脂糖凝胶电泳的结果是,配合物能有效断裂超螺旋DNA,且随着配合物浓度的不断增大,超螺旋DNA的量逐渐减少,缺刻产物的量逐渐增多,呈明显的量效关系,配合物切割DNA的最佳反应温度为50℃；加入自由基捕捉剂后,镍配合物对pBR322 DNA的切割效果明显减弱,说明镍配合物可能是以自由基机理断裂DNA的。
3、NiL配合物能有效抑制HepG2肿瘤细胞增殖,抑制效果呈剂量依赖性,抑制机理为促进细胞凋亡
MTT法结果表明镍配合物对HepG2细胞有明显的抑制作用,且存在浓度依赖性,计算得出,24h、48h和72h对应的IC50分别为328.68±66.31μM、389.81±52.56μM和202.60±38.01μM,相应地,阳性对照药物cisplatin在24h、48h和72h的IC50分别为70.87±9.60μM、28.77±5.18μM和39.97±3.66μM,而5FU的IC50分别为10.35±5.78mM、5.57±0.60mM、0.54±0.21mM相比之下,镍配合物对HepG2肝癌细胞的体外抑制活性强于5-FU,但比Cisplatin弱；DAPI染色的结果显示,镍配合物处理细胞后,细胞核呈致密浓染,染色质固缩,染色体凝聚,还有些细胞核破裂形成碎片,核解体,体现了细胞凋亡的典型特征,与阳性对照药物cisplatin类似；流式细胞仪检测结果表明,随着镍配合物浓度的增加,凋亡细胞所占的百分比也逐渐增大,进一步证实镍配合物能够促进HepG2细胞的凋亡,就细胞周期而言,镍配合物处理细胞后,与空白对照组相比,S期细胞百分比明显增加,G1和G2期细胞百分比减少,且药物浓度越高,S期比例越高。表明镍配合物能阻止HepG2细胞由S期向G2期移行,使细胞阻滞于S期,且类似于顺铂的作用效果。
4、四种新的磺胺类药物均能通过凋亡途径有效抑制肿瘤细胞HepG2增殖。
对四种新的磺胺类药物抗肝癌的作用进行了研究,用MTT法得到四种化合物对HepG2细胞的IC50分别为31.48±1.49μM、13.19±2.15μM.20.11±1.48μM和13.05±1.69μM。用DAPI染色后,在荧光显微镜下明显观察到四种化合物促进HepG2细胞凋亡形成的碎片状细胞核及浓缩的染色质。
结论：
1、化合物XLWG-54-52对HepAD38细胞内外的HBV DNA都有较强的抑制效果,其IC50分别为11.87±3.94μM和10.84±0.60μM。
2、紫外吸收光谱、荧光光谱实验和黏度实验证实NiL配合物与小牛胸腺DNA(CT DNA)有良好的结合,且结合方式为插入模式。DNA琼脂糖凝胶电泳实验表明NiL配合物能将质粒pBR322 DNA由超螺旋形式切割成开环形式,切割效果呈剂量依赖型,切割机理为自由基机理。
3、NiL配合物能有效抑制HepG2肿瘤细胞增殖,24h、48h和72h对应的IC50分别为328.68±66.31μM.389.81±52.56μM和202.60±38.01μM。DAPI染色结果表明NiL配合物通过促进细胞凋亡抑制HepG2细胞增殖。流式细胞术实验表明NiL配合物使HepG2细胞周期阻滞于S期。
4、四种新的磺胺类药物均能通过凋亡途径有效抑制肿瘤细胞HepG2增殖。对应的IC50分别为31.48±1.49μM、13.19±2.15μM、M.20.11±1.48μM和13.05±1.69μM。
Epidemiological survey found that infection of HBV, HCV, Aflatoxin and excessive drink are the most dangerous factors of hepatocellular carcinoma (HCC). Among them, HCC caused by chronic HBV and HCV occupied more than 80%. It usually takes more than thirty years from chronic infection to HCC. As one of the most popular malignant tumors, the prognosis of HCC is extremely bad and the survival rate is only 3%-5%. How to prevent the progress of the disease and decrease the morbidity and mortality of hepatocirrhosis and HCC has become hot issues of both doctors and scientists.
PartⅠ. Relationship of interleukin 21 with HBV and HCV infection
OBJECTIVES:
Interleukin 21(IL-21) has become a hot topic recently. Some scholars have examined and discussed the IL-21 and IL-21R levels in peripheral blood of AIDS patients and autoimmune hepatitis patients. However, there is no report about IL-21 level in peripheral blood of HBV and HCV patients so far. In fact, IL-21 participates in a variety of autoimmune diseases. At the same time, there is indiscerptible relation between HBV, HCV progress and immune function of the body. This study was designed to explore the relationship between IL-21 level and HBV, HCV infection by testing IL-21 level, liver functional indices, viral load and anti-nuclear antibody (ANA) of the patients.
METHODS:
IL-21 and ANA levels were detected by enzyme linked immunosorbent assay (ELISA). Liver functional indices including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLO), total bilirubin (TBIL) and direct bilirubin (DBIL) were measured by auto-biochemical analyzer. ELISA method was also used to examine HBV serum markers (HBsAg、HBsAb、HBeAg、HBeAb、HBcAb). HBV DNA was detected by FQ-PCR.
RESULTS:
1. The ANA and IL-21 were found to be protective for HCV patients.
The positive rate of ANA in HCV positive patients with low ALT (ALT<40 U/L) was 22.4%, while that of high ALT (ALT>40 U/L) group was 15.0%, indicating that most ANA positive HCV patients had better liver function. Autoimmune antibody showed certain protective effect of hepatocyte injury and helped to control the development of HCV with the result of lower ALT. Synchronous changes were found between means of ALT and AST among high ALT, low ALT and control groups. The higher ALT level of some patients maybe correlated with the lower ANA level. The average ALT/AST ratio of high ALT (ALT>40U/L) group was greater than 1, probably because of the seriously damaged liver cells on one hand and the high fibrosis extent on the other. In this study, the average IL-21 levels of both low ALT (ALT<40 U/L) HCV patients and high ALT (ALT>40 U/L) group were lower than that of control group, and comparatively that of low ALT group was a bit higher. It was concluded that HCV infection might result in decreased IL-21 level, which was negatively correlated with the degree of hepatocellular damage, indicating HCV infection can induce cellular immune response. It was confirmed that IL-21 helped to control virus development and decrease liver cell damage. This study suggested that the expression of both ANA and IL-21 can decrease hepatocellular damage of HCV patients and the IL-21 level of ANA positive HCV patients was lower than the negative ones. Correlation analysis among ANA、IL-21、ALT、AST and ALB, showed no correlation between these parameters. So the protection effect of ANA and IL-21 should be a result of joint function by other factors.
2. The IL-21 levels of both HBV carriers with HBsAg and HBeAg and HBV-related hepatocirrhosis patients were significantly lower than healthy control, but those of HBV carriers with HBsAg alone and chronic HBV patients were significantly higher.
HBV patients and healthy control were separated into five groups. The average ALT of control, AsCs, AsCse, CHB and Cir group were 16.75±1.11,24.44±2.09, 23.68±2.01,201.47±36.51 and 59.28±12.97 U/L, respectively. The difference was significant (F=12.368, v=122.158, P=0.000). The mean AST of each group was 22.24±0.72,28.23±1.27,27.53±1.37,150.82±27.40 and 63.83±8.98 U/L, respectively. The difference was also significant (F=15.680, v=122.362, P=0.000). The mean ALB of each group was 47.03±0.46,45.79±0.54,46.12±0.59,43.02±0.66 and 39.57±0.94 g/L, respectively. The difference was significant (F=16.367, v=116.789, P=0.000). The mean GLO of each group was 25.57±0.62,26.61±0.61,25.94±0.65,27.96±0.48 and 30.70±0.65 g/L, respectively. Similarly, the difference was significant (F=10.395, v=109.138, P=0.000). The mean TBIL of each group was 15.20±1.44,14.13±0.80, 14.25±1.73,36.17±11.00 and 41.96±7.27μmol/L, respectively. The difference was significant (F=4.557, v=114.548, P=0.002). The mean DBIL of each group was 4.10±0.26,4.25±0.25,4.04±0.38,17.84±7.04 and 20.69±5.16μmol/L, respectively. The difference was significant (F=3.518, v=121.135, P=0.009). There was no significant difference among the mean TP level. And the average logDNA of AsCs, AsCse, CHB and Cir group was 3.76±0.19,6.80±0.28、5.41±0.18 and 3.64±0.14, respectively. The difference was significant (F=47.324, v=97.334, P=0.000). The IL-21 level of control, AsCs, AsCse, CHB and Cir group was 67.22±5.87, 117.17±23.92,47.90±7.46,112.73±18.54 and 50.18±5.87 pg/ml, respectively. The difference was significant (F=5.096, v=120.252, P=0.001). When regrouping all samples according to the status of antigen or antibody, we found that IL-21 level of HBsAb negative group (79.55±5.73 pg/ml) was notably higher than that of HBsAb positive one (62.30±6.35 pg/ml) (t=-2.016, P=0.046). IL-21 level of HBeAg negative group (90.49±10.76 pg/ml) was also notably higher than that of HBeAg positive group (66.92±5.07 pg/ml)(t=-1.982, P=0.049), and IL-21 level of HBeAb positive group (90.06±9.94 pg/ml) was notably higher than that of HBeAb negative group (66.50±3.93 pg/ml)(t=2.205,P=0.029). IL-21 value of female group was a bit higher than that of male group if all specimens regrouped by gender (t=-0.375, P=0.708). Bivariate correlation analysis of all samples showed significant correlations between ALT and AST (r=0.959, P=0.000, closely related), ALB and TP (r=0.689, P=0.000), DBIL and TBIL (r=0.988, P=0.000, closely related), and negative correlation between ALB and GLO (r=-0.413, P=0.000). There were also some weak correlations between other variables such as logDNA and ALT (r=0.328, P=0.000), logDNA and AST (r=0.302, P=0.000), GLO and TP (r=0.363, P=0.000), ALB and DBIL (r=0.333, P=0.000), ALB and TBIL (r=0.323, P=0.000) and so on. But there was no significant correlation between IL-21 and others.
CONCLUSIONS:
1. The positive rate of ANA in HCV positive patients with low ALT (ALT<40 U/L) was high than that of high ALT (ALT>40 U/L) group, indicating that the production of ANA was protective to liver injry.
2. The average IL-21 levels of both low ALT (ALT<40 U/L) HCV patients and high ALT (ALT>40 U/L) group were lower than that of control group, indicating that HCV infection could result in decreased IL-21 level. And on the contrary, the production of IL-21 helped to control HCV development and reduce hepatocellular injury.
3. The IL-21 levels of both HBV carriers with HBsAg and HBeAg and HBV-related hepatocirrhosis patients were significantly lower than healthy control, but those of HBV carriers with HBsAg alone and chronic HBV patients were significantly higher. It was concluded that HBeAg positive HBV patients had bad prognosis. The active effect of IL-21 was also validated.
4. Bivariate correlation analysis of all HBV samples showed that ALT was closely related to AST (r=0.959, P=0.000), ALB was related to TP (r=0.689, P=0.000), DBIL was closely related to TBIL (r=0.988, P=0.000), and that ALB was negatively related to GLO (r=-0.413,P=0.000).
PartⅡ. The screening of small molecular inhibitors related to liver injury.
OBJECTIVE:
To screen anti-HBV drugs in cells by HBV DNA level, and to study the inhibitory activities of drugs on HBV replication intermediates. To study the binding pattern of the compound with DNA, the cleavage effect of the compound on DNA, and the anti-tumor activity within and without cells, Futhermore, the mechanism of action was also investigated in various ways.
METHODS:
WST-8 method was adopted to measure cytotoxicity using CCK-8 kit. HBV DNA inside and outside cells was detected by Fluorescence Quantitative PCR Diagnostic Kit for Hepatitis B Virus DNA. Western blot was adoptive in testing HBcAg. While HBeAg and HBsAg were measured using TRFIA technique. The function of nickel complex on CT DNA was investigated by uv absorption spectrum、fluorescence spectrum、Scatchard plot and viscosity analysis. The action of nickel complex on pBR322 DNA is explored by agarose gel electrophoresis. MTT test was used to evaluate the anti-tumor activity of nickel complex in vitro. The influences of nickel complex on cell apoptosis and cell cycle were analyzed using DAPI fluorescent dying and flow cytometry.
RESULTS:
1. Compound XLWG-54-52 was found to be effective in reducing HBV DNA both inside cells and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively.
HepAD38 is a cell line which can express HBsAg and HBeAg stably and replicate DNA at a high level. The cultivation time was fixed on seven days. We also screen anti-HBV drugs from a small compound library and found five compounds which inhibited HBV DNA effectively. They were XLWG-54-52, XLWG-61-3A, XLWG-61-7, XLWG-48-6 and XLWG-48-11. Among them, XLWG-54-52 was the most effective and studied further. This compound suppressed HBV DNA both inside and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively. The CC50 of XLWG-54-52 on HepAD38 was 0.339 mM. It did not inhibit HBeAg, HBsAg and HBcAg. XLWG-54-52 is an analogue of bicyclol. Therfore, this compiound may protect liver damage by inhibiting the HBV DNA replication.
2. Nickel complex exhibited excellent performance in binding with CT DNA by insertion mode. And it could break pBR322 DNA from supercoiled form to open circular form dose-dependently through free radical mechanism.
In UV absorption spectrum test, the absorption peak of nickel complex was accompanied with hypochromic effect to 18%. The binding pattern of nickel complex to DNA was presumed to be insertion. The binding constant Kb was calculated to be 2.18×105(L·mol-1). In the fluorescence spectrum experiment, the fluorescence of EB-DNA complex quenched distinctly as the concentration of nickel complex increased, thus validating that the pyrrole ring inserted into DNA double chain for binding. Fluorescent Scatchard plot further suggested the k value reduce when the concentration of nickel complex increased in the mixture. But the n value almost maintained at 0.190, indicating nickel complex inhibited the association of EB and DNA competitively. From another viewpoint, the complex inserted into DNA. When nickel complex interacted with CT DNA, the viscosity augmented with the increase of complex. Therefore, it was ascertainable that nickel complex acted with DNA through insertion. Nickel complex was found to promote the cleavage of pBR322 DNA dose-dependently from supercoiled form to open circular form by agarose gel electrophoresis. The optimal reaction temperature was 50℃. While free radical capture agent was added to the reaction system, the cleavage effect was remarkably weakened. These results indicated that nickel complex can cleave DNA chain through free radical mechanism.
3. Nickel complex could inhibit HepG2 cell proliferation effectively and dose-dependently through apoptosis pathway.
Nickel complex was found to inhibit HepG2 cell proliferation effectively and dose-dependently. The IC50 values of 24h,48h and 72h were calculated to be 328.68±66.31μM,389.81±52.56μM and 202.60±38.01μM, respectively. Accordingly, of the IC50S of positive control drug, cisplatin, were 70.87±9.60μM, 28.77±5.18μM and 39.97±3.66μM, respectively. Another positive control drug, 5-FU, has an IC50 of 10.35±5.78 mM、5.57±0.60 mM and 0.54±0.21 mM, respectively. Therefore, the in vitro inhibition activity of nickel complex on hepatoma cells HepG2 was better than 5-FU but weaker than cisplatin. DAPI staining showed that nickel complex treated cells displayed chromatin condensation, chromosome aggregation and nuclear fragmentation, indicating a typical morphological changes of apoptosis, which was similar to cisplatin.. The apoptosis of HepG2 cells was confirmed by FACS examination. The rate of apoptotic cells enhanced as the concentration of nickel complex increased. The cells in S phase were increased and those in G1 and G2 phases were decreased after nickel complex treatment. The higher the concentration of nickel complex, the lareger the percentage of cells in S phase. It suggested that nickel complex could prevent HepG2 cells proliferation by blocking the cell cycle shift from S phase to G2 phase, thus arresting them at S phase. This effect was similar to cisplatin.
4. Sulpha compounds could inhibit HepG2 proliferation through apoptosis pathway.
The anti-tumor effects of four sulpha compounds were also investigated. The IC50 of them against HepG2 cells proliferation was obtained by MTT, which was 31.48±1.49μM,13.19±2.15μM,20.11±1.48μM and 13.05±1.69μM, respectively. After DAPI staining, the concentrated chromatin and fragmented nucleus was seen in all of the four active compounds treated HepG2 cells under fluorescent-microscope, indictating that they inhibited the growth of HepG2 cells through apoptosis pathway.
CONCLUSIONS:
1. Compound XLWG-54-52 was found to be effective in reducing HBV DNA both inside cells and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively.
2. The results of the UV absorption spectrum test, the fluorescence spectrum experiment and the viscosity examination all indicated the excellent character of nickel complex in binding with CT DNA by insertion mode. DNA agarose gel electrophoresis technique validated that nickel comple could break pBR322 DNA from supercoiled form to open circular form dose-dependently through free radical mechanism.
3. Nickel complex could inhibit HepG2 cell proliferation effectively and dose-dependently. The IC50 at 24h,48h and 72h was 328.68±66.31μM,389.81±52.56μM, and 202.60±38.01μM, respectively. DAPI staining results showed the apoptosis mechanism. FACS examination validated that the cell cycle was blocked at S phase.
4. Four sulpha compounds could inhibit HepG2 proliferation effectively through apoptosis pathway. The IC50 was 31.48±1.49μM,13.19±2.15μM,20.11±1.48μM and 13.05±1.69μM, respectively.
第一部分白介素-21与肝炎病毒感染的关系
目的：
白介素21(IL-21)是近期研究的一个热点,已有学者对艾滋病病人以及自身免疫性肝炎患者外周血中IL-21及IL-21R进行了检测和探讨,但迄今为止没有关于HBV和HCV患者外周血中IL-21水平的报道。IL-21参与了多种自身免疫性疾病的发生,而HBV和HCV也与机体的免疫功能有着不可分割的联系。为此,本研究拟通过检测HBV和HCV感染者血清IL-21水平,同时检测其它肝功能指标、病毒载量、抗核抗体等,探讨IL-21与HBV、HCV感染的相关性。
方法：
采用ELISA法定量测定IL-21和抗核抗体(ANA)。肝功能指标包括谷丙转氨酶(ALT)、谷草转氨酶(AST)、总蛋白(TP)、白蛋白(ALB)、球蛋白(GLO)、总胆红素(TBIL)、直接胆红素(DBIL)均采用全自动生化分析仪检测。ELISA方法检测HBV血清标志物即HBsAg、HBsAb、HBeAg、HBeAb、HBcAb。HBVDNA采用荧光定量PCR法。
结果：
1、抗核抗体ANA和白介素21对HCV患者具有保护作用。
ALT<40 U/L的HCV感染者中ANA阳性率为22.4%,而ALT>40 U/L的患者阳性率为15.0%,反映ANA阳性患者大部分肝功能较好。表明自身免疫抗体的产生对肝细胞损伤有一定的保护作用,导致ALT降低,有助于控制HCV病毒的发展。高ALT组、低ALT组及对照组间AST均值与ALT均值有同步变化,部分患者的ALT较高可能也与ANA抗体水平较低有一定的关系。ALT>40U/L的HCV感染组ALT/AST比率>1,原因可能一方面是肝细胞损伤较厉害,另一方面是纤维化程度较高。本试验中ALT<40 U/L感染组、ALT>40 U/L感染组的IL-21水平均低于对照组。但相比之下,低ALT感染组的水平更高一些。可见,HCV的感染会导致IL-21表达水平降低,且IL-21水平的高低与肝细胞受损程度呈负相关,可能与HCV感染后引起的细胞免疫有牵连。证实了IL-21的存在有助于控制HCV病毒的发展,降低肝细胞损伤。本研究提示ANA的表达与IL-21的存在均能降低HCV感染者的肝细胞损伤,且ANA阳性HCV感染者的IL-21水平低于ANA阴性HCV感染者,进一步对ANA、IL-21、ALT、AST、ALB这五个变量两两间进行相关分析,结果都没有相关性。ANA与IL-21对肝细胞的保护作用应该是与其他因素共同作用的结果。
2、HBsAg阳性和HBeAg阳性病毒携带者组以及肝硬化组的IL-21水平显著低于对照组,而HBsAg阳性病毒携带者组和慢乙肝组的IL-21水平却显著高于对照组。
在HBV与IL-21相互关系的研究中,我们将收集的血清分为五组,分别为：健康对照组(control)、HBsAg阳性病毒携带者组(AsCs)、HBsAg阳性和HBeAg阳性病毒携带者组(AsCse)、慢乙肝组(CHB)和肝硬化组(Cir)。control、AsCs、AsCse、CHB、Cir组ALT均值依次为16.75±1.11、24.44±2.09、23.68±2.01、201.47±36.51、59.28±12.97 U/L,具有显著性差异(F=12.368,ν=122.158,P=0.000)。AST均值依次为22.24±0.72、28.23±1.27、27.53±1.37、150.82±27.40、63.83±8.98U/L,具有显著性差异(F=15.680,v=122.362,P=0.000)。ALB均值依次为47.03±0.46、45.79±0.54、46.12±0.59、43.02±0.66、39.57±0.94g/L,具有显著性差异(F=16.367,v=116.789,P=0.000)。GLO均值依次为25.57±0.62、26.61±0.61、25.94±0.65、27.96±0.48、30.70±0.65g/L,具有显著性差异(F=10.395,v=109.138,P=0.000)。TBIL均值依次为15.20±1.44、14.13±0.80、14.25±1.73、36.17±11.00、41.96±7.27μmol/L,具有显著性差异(F=4.557,ν=114.548,P=0.002)。DBIL均值依次为4.10±0.26、4.25±0.25、4.04±0.38、17.84±7.04、20.69±5.16μmol/L,具有显著性差异(F=3.518,ν=121.135,P=0.009)。而TP均值没有显著性差异(F=1.608,ν=122.766,P=0.177)。另外,AsCs、AsCse、CHB、Cir组的HBV DNA滴度(logDNA)均值依次为3.76±0.19、6.80±0.28、5.41±0.18、3.64±0.14,具有显著性差异(F=47.324, v=97.334, P=0.000)。Control、AsCs、AsCse、CHB、Cir组的IL-21均值依次为67.22±5.87、117.17±23.92、47.90±7.46、112.73±18.54、50.18±5.87pg/ml,具有显著性差异(F=5.096,ν=120.252,P=0.001)。将所有标本按照各抗原、抗体的阴、阳性分别进行分组,用t检验比较IL-21在阴阳性组间的差别。结果表明,HBsAb阴性组的IL-21水平显著高于阳性组,依次为79.55±5.73和62.30±6.35pg/ml (t=-2.016, P=0.046);HBeAg阴性组的IL-21水平显著高于阳性组,依次为90.49±10.76和66.92±5.07pg/ml(t=-1.982,P=0.049); HBeAb阳性组的IL-21水平显著高于阴性组,依次为90.06±9.94和66.50±3.93pg/ml (t=2.205, P=0.029)。将所有标本按照男性、女性分成两组,比较IL-21在两组间的差别。结果显示,女性组IL-21均值略高于男性组,但没有显著性差异(t=-0.375,P=0.708)。对所有标本的ALT和AST作双变量相关性分析,结果相关系数r=0.959,P=0.000,表明两者显著性相关,且相关关系非常密切。对所有标本的ALB和TP作双变量相关性分析,结果相关系数r=0.689,P=0.000,表明两者显著性相关。对所有标本的DBIL和TBIL作双变量相关性分析,结果相关系数r=0.988,P=0.000,表明两者显著性相关,且相关关系非常密切。对所有标本的ALB和GLO作双变量相关性分析,结果相关系数r=-0.413,P=0.000,表明两者显著性负相关。其它变量之间也有一些较弱的相关性存在,例如LogDNA与ALT之间的r=0.328,P=0.000;LogDNA与AST之间的r=0.302,P=0.000;GLO与TP之间的r=0.363,P=0.000;ALB与DBIL之间的r=0.333,P=0.000;ALB与TBIL之间的r=0.323,P=0.000等。但是,IL-21与其它变量之间不存在显著相关性。
结论：
1、ALT<40U/L的HCV感染者中ANA阳性率高于ALT>40 U/L的感染者,表明自身免疫抗体的产生对肝细胞损伤有一定的保护作用。
2、ALT<40 U/L的HCV感染组和ALT>40 U/L感染组的IL-21水平均低于对照组,表明HCV的感染会导致IL-21表达水平降低,反过来也说明IL-21的产生有助于控制HCV病毒发展,降低肝细胞损伤。
3、HBsAg阳性和HBeAg阳性病毒携带者组以及肝硬化组的IL-21水平显著低于对照组,而HBsAg阳性病毒携带者组和慢乙肝组的IL-21水平却显著高于对照组。说明HBeAg阳性乙肝患者的预后较差,也验证了IL-21的积极作用。
4、所有HBV标本的ALT和AST显著密切相关(r=0.959,P=0.000),ALB和TP显著相关(r=0.689,P=0.000),DBIL和TBIL显著密切相关(r=0.988,P=0.000),ALB和GLO显著负相关(r=-0.413,P=0.000)。
第二部分肝损伤相关小分子抑制剂的筛选研究
目的：
利用HBV DNA指标在细胞水平筛选抗HBV药物,并研究药物对HBV复制中间体的抑制效果。从胞外、胞内两个角度深入研究化合物与DNA的结合方式、对DNA的断裂作用及其抗肝癌活性,从多方面探讨其作用机制。
方法：
采用WST-8法检测细胞毒性,按照CCK-8试剂盒进行操作。采用HBV DNA提取及扩增荧光检测试剂盒检测细胞内外HBV DNA。采用Western blot法检测HBcAg。采用时间分辨免疫荧光(TRFIA)法定量检测HBeAg和HBsAg水平。采用紫外吸收光谱、荧光光谱、荧光Scatchard图及黏度测定实验研究镍配合物与小牛胸腺DNA (CT DNA)的作用,琼脂糖凝胶电泳分析镍配合物与pBR322质粒DNA的作用。通过MTT法进行镍配合物体外抗肝癌活性的评估,DAPI荧光染色分析及流式细胞术检测镍配合物对细胞凋亡及细胞周期的影响。
结果：
1、化合物XLWG-54-52抑制HepAD38细胞内HBV DNA的IC50为11.87±3.94μM,抑制胞外HBV DNA的IC50为10.84±0.60μM。
通过本研究确立了一个合适的细胞模型HepAD38,该模型能较稳定地表达HBsAg和HBeAg抗原,DNA复制也较高,并确定细胞培养时间为7天；建立了一套筛选抗HBV药物的方法,并筛选出了XLWG-54-52、XLWG-61-3A、XLWG-61-7、XLWG-48-6、XLWG-48-11五个能有效抑制HBV DNA的化合物；重点对其中的XLWG-54-52进行了研究,XLWG-54-52对胞内外HBV DNA均有较强的抑制作用,胞内IC50为11.87±3.94μM,胞外IC50为10.84±0.60μM；XLWG-54-52对HepAD38的半数致死浓度CC50为0.339mM;XLWG-54-52对HBeAg、HBsAg和HBcAg三种抗原均没有抑制作用。XLWG-54-52对HBV所致乙型肝炎的治疗作用可能要结合其类似物双环醇的作用机理,从它对肝损伤的保护作用入手。
2、NiL配合物与小牛胸腺DNA (CT DNA)有良好的结合,且结合方式为插入模式。它还能将质粒pBR322 DNA由超螺旋形式切割成开环形式,切割效果呈剂量依赖型,切割机理为自由基机理。
紫外吸收光谱法测得配合物的吸收峰伴随有明显的减色,减色率为18%,推测NiL配合物与DNA可能是以插入模式结合,计算得出结合常数Kb为2.18×105(L·mol-1)；荧光光谱实验表明随着配合物浓度的不断增加,EB-DNA复合物的荧光明显发生淬灭,验证了配合物的吡咯环插入到DNA双链之间与之结合；荧光Scatchard图进一步显示,随着DNA溶液中配合物浓度的增大,k值逐渐减小,而n值在0.190附近变化不大,说明配合物竞争性地抑制EB与DNA的结合,即以插入方式与DNA结合；黏度实验中,配合物与CT DNA发生相互作用后,随着配合物浓度的增大,DNA黏度值逐渐增加,由此可确定配合物是以插入方式与DNA作用的；DNA琼脂糖凝胶电泳的结果是,配合物能有效断裂超螺旋DNA,且随着配合物浓度的不断增大,超螺旋DNA的量逐渐减少,缺刻产物的量逐渐增多,呈明显的量效关系,配合物切割DNA的最佳反应温度为50℃；加入自由基捕捉剂后,镍配合物对pBR322 DNA的切割效果明显减弱,说明镍配合物可能是以自由基机理断裂DNA的。
3、NiL配合物能有效抑制HepG2肿瘤细胞增殖,抑制效果呈剂量依赖性,抑制机理为促进细胞凋亡
MTT法结果表明镍配合物对HepG2细胞有明显的抑制作用,且存在浓度依赖性,计算得出,24h、48h和72h对应的IC50分别为328.68±66.31μM、389.81±52.56μM和202.60±38.01μM,相应地,阳性对照药物cisplatin在24h、48h和72h的IC50分别为70.87±9.60μM、28.77±5.18μM和39.97±3.66μM,而5FU的IC50分别为10.35±5.78mM、5.57±0.60mM、0.54±0.21mM相比之下,镍配合物对HepG2肝癌细胞的体外抑制活性强于5-FU,但比Cisplatin弱；DAPI染色的结果显示,镍配合物处理细胞后,细胞核呈致密浓染,染色质固缩,染色体凝聚,还有些细胞核破裂形成碎片,核解体,体现了细胞凋亡的典型特征,与阳性对照药物cisplatin类似；流式细胞仪检测结果表明,随着镍配合物浓度的增加,凋亡细胞所占的百分比也逐渐增大,进一步证实镍配合物能够促进HepG2细胞的凋亡,就细胞周期而言,镍配合物处理细胞后,与空白对照组相比,S期细胞百分比明显增加,G1和G2期细胞百分比减少,且药物浓度越高,S期比例越高。表明镍配合物能阻止HepG2细胞由S期向G2期移行,使细胞阻滞于S期,且类似于顺铂的作用效果。
4、四种新的磺胺类药物均能通过凋亡途径有效抑制肿瘤细胞HepG2增殖。
对四种新的磺胺类药物抗肝癌的作用进行了研究,用MTT法得到四种化合物对HepG2细胞的IC50分别为31.48±1.49μM、13.19±2.15μM.20.11±1.48μM和13.05±1.69μM。用DAPI染色后,在荧光显微镜下明显观察到四种化合物促进HepG2细胞凋亡形成的碎片状细胞核及浓缩的染色质。
结论：
1、化合物XLWG-54-52对HepAD38细胞内外的HBV DNA都有较强的抑制效果,其IC50分别为11.87±3.94μM和10.84±0.60μM。
2、紫外吸收光谱、荧光光谱实验和黏度实验证实NiL配合物与小牛胸腺DNA(CT DNA)有良好的结合,且结合方式为插入模式。DNA琼脂糖凝胶电泳实验表明NiL配合物能将质粒pBR322 DNA由超螺旋形式切割成开环形式,切割效果呈剂量依赖型,切割机理为自由基机理。
3、NiL配合物能有效抑制HepG2肿瘤细胞增殖,24h、48h和72h对应的IC50分别为328.68±66.31μM.389.81±52.56μM和202.60±38.01μM。DAPI染色结果表明NiL配合物通过促进细胞凋亡抑制HepG2细胞增殖。流式细胞术实验表明NiL配合物使HepG2细胞周期阻滞于S期。
4、四种新的磺胺类药物均能通过凋亡途径有效抑制肿瘤细胞HepG2增殖。对应的IC50分别为31.48±1.49μM、13.19±2.15μM、M.20.11±1.48μM和13.05±1.69μM。
Epidemiological survey found that infection of HBV, HCV, Aflatoxin and excessive drink are the most dangerous factors of hepatocellular carcinoma (HCC). Among them, HCC caused by chronic HBV and HCV occupied more than 80%. It usually takes more than thirty years from chronic infection to HCC. As one of the most popular malignant tumors, the prognosis of HCC is extremely bad and the survival rate is only 3%-5%. How to prevent the progress of the disease and decrease the morbidity and mortality of hepatocirrhosis and HCC has become hot issues of both doctors and scientists.
PartⅠ. Relationship of interleukin 21 with HBV and HCV infection
OBJECTIVES:
Interleukin 21(IL-21) has become a hot topic recently. Some scholars have examined and discussed the IL-21 and IL-21R levels in peripheral blood of AIDS patients and autoimmune hepatitis patients. However, there is no report about IL-21 level in peripheral blood of HBV and HCV patients so far. In fact, IL-21 participates in a variety of autoimmune diseases. At the same time, there is indiscerptible relation between HBV, HCV progress and immune function of the body. This study was designed to explore the relationship between IL-21 level and HBV, HCV infection by testing IL-21 level, liver functional indices, viral load and anti-nuclear antibody (ANA) of the patients.
METHODS:
IL-21 and ANA levels were detected by enzyme linked immunosorbent assay (ELISA). Liver functional indices including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLO), total bilirubin (TBIL) and direct bilirubin (DBIL) were measured by auto-biochemical analyzer. ELISA method was also used to examine HBV serum markers (HBsAg、HBsAb、HBeAg、HBeAb、HBcAb). HBV DNA was detected by FQ-PCR.
RESULTS:
1. The ANA and IL-21 were found to be protective for HCV patients.
The positive rate of ANA in HCV positive patients with low ALT (ALT<40 U/L) was 22.4%, while that of high ALT (ALT>40 U/L) group was 15.0%, indicating that most ANA positive HCV patients had better liver function. Autoimmune antibody showed certain protective effect of hepatocyte injury and helped to control the development of HCV with the result of lower ALT. Synchronous changes were found between means of ALT and AST among high ALT, low ALT and control groups. The higher ALT level of some patients maybe correlated with the lower ANA level. The average ALT/AST ratio of high ALT (ALT>40U/L) group was greater than 1, probably because of the seriously damaged liver cells on one hand and the high fibrosis extent on the other. In this study, the average IL-21 levels of both low ALT (ALT<40 U/L) HCV patients and high ALT (ALT>40 U/L) group were lower than that of control group, and comparatively that of low ALT group was a bit higher. It was concluded that HCV infection might result in decreased IL-21 level, which was negatively correlated with the degree of hepatocellular damage, indicating HCV infection can induce cellular immune response. It was confirmed that IL-21 helped to control virus development and decrease liver cell damage. This study suggested that the expression of both ANA and IL-21 can decrease hepatocellular damage of HCV patients and the IL-21 level of ANA positive HCV patients was lower than the negative ones. Correlation analysis among ANA、IL-21、ALT、AST and ALB, showed no correlation between these parameters. So the protection effect of ANA and IL-21 should be a result of joint function by other factors.
2. The IL-21 levels of both HBV carriers with HBsAg and HBeAg and HBV-related hepatocirrhosis patients were significantly lower than healthy control, but those of HBV carriers with HBsAg alone and chronic HBV patients were significantly higher.
HBV patients and healthy control were separated into five groups. The average ALT of control, AsCs, AsCse, CHB and Cir group were 16.75±1.11,24.44±2.09, 23.68±2.01,201.47±36.51 and 59.28±12.97 U/L, respectively. The difference was significant (F=12.368, v=122.158, P=0.000). The mean AST of each group was 22.24±0.72,28.23±1.27,27.53±1.37,150.82±27.40 and 63.83±8.98 U/L, respectively. The difference was also significant (F=15.680, v=122.362, P=0.000). The mean ALB of each group was 47.03±0.46,45.79±0.54,46.12±0.59,43.02±0.66 and 39.57±0.94 g/L, respectively. The difference was significant (F=16.367, v=116.789, P=0.000). The mean GLO of each group was 25.57±0.62,26.61±0.61,25.94±0.65,27.96±0.48 and 30.70±0.65 g/L, respectively. Similarly, the difference was significant (F=10.395, v=109.138, P=0.000). The mean TBIL of each group was 15.20±1.44,14.13±0.80, 14.25±1.73,36.17±11.00 and 41.96±7.27μmol/L, respectively. The difference was significant (F=4.557, v=114.548, P=0.002). The mean DBIL of each group was 4.10±0.26,4.25±0.25,4.04±0.38,17.84±7.04 and 20.69±5.16μmol/L, respectively. The difference was significant (F=3.518, v=121.135, P=0.009). There was no significant difference among the mean TP level. And the average logDNA of AsCs, AsCse, CHB and Cir group was 3.76±0.19,6.80±0.28、5.41±0.18 and 3.64±0.14, respectively. The difference was significant (F=47.324, v=97.334, P=0.000). The IL-21 level of control, AsCs, AsCse, CHB and Cir group was 67.22±5.87, 117.17±23.92,47.90±7.46,112.73±18.54 and 50.18±5.87 pg/ml, respectively. The difference was significant (F=5.096, v=120.252, P=0.001). When regrouping all samples according to the status of antigen or antibody, we found that IL-21 level of HBsAb negative group (79.55±5.73 pg/ml) was notably higher than that of HBsAb positive one (62.30±6.35 pg/ml) (t=-2.016, P=0.046). IL-21 level of HBeAg negative group (90.49±10.76 pg/ml) was also notably higher than that of HBeAg positive group (66.92±5.07 pg/ml)(t=-1.982, P=0.049), and IL-21 level of HBeAb positive group (90.06±9.94 pg/ml) was notably higher than that of HBeAb negative group (66.50±3.93 pg/ml)(t=2.205,P=0.029). IL-21 value of female group was a bit higher than that of male group if all specimens regrouped by gender (t=-0.375, P=0.708). Bivariate correlation analysis of all samples showed significant correlations between ALT and AST (r=0.959, P=0.000, closely related), ALB and TP (r=0.689, P=0.000), DBIL and TBIL (r=0.988, P=0.000, closely related), and negative correlation between ALB and GLO (r=-0.413, P=0.000). There were also some weak correlations between other variables such as logDNA and ALT (r=0.328, P=0.000), logDNA and AST (r=0.302, P=0.000), GLO and TP (r=0.363, P=0.000), ALB and DBIL (r=0.333, P=0.000), ALB and TBIL (r=0.323, P=0.000) and so on. But there was no significant correlation between IL-21 and others.
CONCLUSIONS:
1. The positive rate of ANA in HCV positive patients with low ALT (ALT<40 U/L) was high than that of high ALT (ALT>40 U/L) group, indicating that the production of ANA was protective to liver injry.
2. The average IL-21 levels of both low ALT (ALT<40 U/L) HCV patients and high ALT (ALT>40 U/L) group were lower than that of control group, indicating that HCV infection could result in decreased IL-21 level. And on the contrary, the production of IL-21 helped to control HCV development and reduce hepatocellular injury.
3. The IL-21 levels of both HBV carriers with HBsAg and HBeAg and HBV-related hepatocirrhosis patients were significantly lower than healthy control, but those of HBV carriers with HBsAg alone and chronic HBV patients were significantly higher. It was concluded that HBeAg positive HBV patients had bad prognosis. The active effect of IL-21 was also validated.
4. Bivariate correlation analysis of all HBV samples showed that ALT was closely related to AST (r=0.959, P=0.000), ALB was related to TP (r=0.689, P=0.000), DBIL was closely related to TBIL (r=0.988, P=0.000), and that ALB was negatively related to GLO (r=-0.413,P=0.000).
PartⅡ. The screening of small molecular inhibitors related to liver injury.
OBJECTIVE:
To screen anti-HBV drugs in cells by HBV DNA level, and to study the inhibitory activities of drugs on HBV replication intermediates. To study the binding pattern of the compound with DNA, the cleavage effect of the compound on DNA, and the anti-tumor activity within and without cells, Futhermore, the mechanism of action was also investigated in various ways.
METHODS:
WST-8 method was adopted to measure cytotoxicity using CCK-8 kit. HBV DNA inside and outside cells was detected by Fluorescence Quantitative PCR Diagnostic Kit for Hepatitis B Virus DNA. Western blot was adoptive in testing HBcAg. While HBeAg and HBsAg were measured using TRFIA technique. The function of nickel complex on CT DNA was investigated by uv absorption spectrum、fluorescence spectrum、Scatchard plot and viscosity analysis. The action of nickel complex on pBR322 DNA is explored by agarose gel electrophoresis. MTT test was used to evaluate the anti-tumor activity of nickel complex in vitro. The influences of nickel complex on cell apoptosis and cell cycle were analyzed using DAPI fluorescent dying and flow cytometry.
RESULTS:
1. Compound XLWG-54-52 was found to be effective in reducing HBV DNA both inside cells and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively.
HepAD38 is a cell line which can express HBsAg and HBeAg stably and replicate DNA at a high level. The cultivation time was fixed on seven days. We also screen anti-HBV drugs from a small compound library and found five compounds which inhibited HBV DNA effectively. They were XLWG-54-52, XLWG-61-3A, XLWG-61-7, XLWG-48-6 and XLWG-48-11. Among them, XLWG-54-52 was the most effective and studied further. This compound suppressed HBV DNA both inside and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively. The CC50 of XLWG-54-52 on HepAD38 was 0.339 mM. It did not inhibit HBeAg, HBsAg and HBcAg. XLWG-54-52 is an analogue of bicyclol. Therfore, this compiound may protect liver damage by inhibiting the HBV DNA replication.
2. Nickel complex exhibited excellent performance in binding with CT DNA by insertion mode. And it could break pBR322 DNA from supercoiled form to open circular form dose-dependently through free radical mechanism.
In UV absorption spectrum test, the absorption peak of nickel complex was accompanied with hypochromic effect to 18%. The binding pattern of nickel complex to DNA was presumed to be insertion. The binding constant Kb was calculated to be 2.18×105(L·mol-1). In the fluorescence spectrum experiment, the fluorescence of EB-DNA complex quenched distinctly as the concentration of nickel complex increased, thus validating that the pyrrole ring inserted into DNA double chain for binding. Fluorescent Scatchard plot further suggested the k value reduce when the concentration of nickel complex increased in the mixture. But the n value almost maintained at 0.190, indicating nickel complex inhibited the association of EB and DNA competitively. From another viewpoint, the complex inserted into DNA. When nickel complex interacted with CT DNA, the viscosity augmented with the increase of complex. Therefore, it was ascertainable that nickel complex acted with DNA through insertion. Nickel complex was found to promote the cleavage of pBR322 DNA dose-dependently from supercoiled form to open circular form by agarose gel electrophoresis. The optimal reaction temperature was 50℃. While free radical capture agent was added to the reaction system, the cleavage effect was remarkably weakened. These results indicated that nickel complex can cleave DNA chain through free radical mechanism.
3. Nickel complex could inhibit HepG2 cell proliferation effectively and dose-dependently through apoptosis pathway.
Nickel complex was found to inhibit HepG2 cell proliferation effectively and dose-dependently. The IC50 values of 24h,48h and 72h were calculated to be 328.68±66.31μM,389.81±52.56μM and 202.60±38.01μM, respectively. Accordingly, of the IC50S of positive control drug, cisplatin, were 70.87±9.60μM, 28.77±5.18μM and 39.97±3.66μM, respectively. Another positive control drug, 5-FU, has an IC50 of 10.35±5.78 mM、5.57±0.60 mM and 0.54±0.21 mM, respectively. Therefore, the in vitro inhibition activity of nickel complex on hepatoma cells HepG2 was better than 5-FU but weaker than cisplatin. DAPI staining showed that nickel complex treated cells displayed chromatin condensation, chromosome aggregation and nuclear fragmentation, indicating a typical morphological changes of apoptosis, which was similar to cisplatin.. The apoptosis of HepG2 cells was confirmed by FACS examination. The rate of apoptotic cells enhanced as the concentration of nickel complex increased. The cells in S phase were increased and those in G1 and G2 phases were decreased after nickel complex treatment. The higher the concentration of nickel complex, the lareger the percentage of cells in S phase. It suggested that nickel complex could prevent HepG2 cells proliferation by blocking the cell cycle shift from S phase to G2 phase, thus arresting them at S phase. This effect was similar to cisplatin.
4. Sulpha compounds could inhibit HepG2 proliferation through apoptosis pathway.
The anti-tumor effects of four sulpha compounds were also investigated. The IC50 of them against HepG2 cells proliferation was obtained by MTT, which was 31.48±1.49μM,13.19±2.15μM,20.11±1.48μM and 13.05±1.69μM, respectively. After DAPI staining, the concentrated chromatin and fragmented nucleus was seen in all of the four active compounds treated HepG2 cells under fluorescent-microscope, indictating that they inhibited the growth of HepG2 cells through apoptosis pathway.
CONCLUSIONS:
1. Compound XLWG-54-52 was found to be effective in reducing HBV DNA both inside cells and outside cells with the IC50 of 11.87±3.94μM and 10.84±0.60μM, respectively.
2. The results of the UV absorption spectrum test, the fluorescence spectrum experiment and the viscosity examination all indicated the excellent character of nickel complex in binding with CT DNA by insertion mode. DNA agarose gel electrophoresis technique validated that nickel comple could break pBR322 DNA from supercoiled form to open circular form dose-dependently through free radical mechanism.
3. Nickel complex could inhibit HepG2 cell proliferation effectively and dose-dependently. The IC50 at 24h,48h and 72h was 328.68±66.31μM,389.81±52.56μM, and 202.60±38.01μM, respectively. DAPI staining results showed the apoptosis mechanism. FACS examination validated that the cell cycle was blocked at S phase.
4. Four sulpha compounds could inhibit HepG2 proliferation effectively through apoptosis pathway. The IC50 was 31.48±1.49μM,13.19±2.15μM,20.11±1.48μM and 13.05±1.69μM, respectively.
引文
[1]Parrish NJ, Dillon SR, Nelson A et al. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function[J]. Nature,2000,408:57-63.
[2]Spolski, R., Leonard, W. J. Interleukin-21:basic biology and implications for cancer and autoimmunity[J]. Annu. Rev. Immunol,2008,26,57-79.
[3]Onoda, T., Rahman, M., Nara, H., et al. Human CD4+ central and effector memory T cells produce IL-21:effect on cytokine-driven proliferation of CD4+ T cell subsets[J]. Int. Immunol,2007,19:1191-1199.
[4]Ozaki, K., Kikly, K., Michalovich, D. et al. Cloning of a type Ⅰ cytokine receptor most related to the IL-2 receptor beta chain[J]. Proc. Natl. Acad. Sci. USA,2000,97:11439-11444.
[5]Asao, H., Okuyama, C., Kumaki, S., et al. Cutting edge:the common gamma-chain is an indispensable subunit of the IL-21 receptor complex[J]. J. Immunol,2001,167:1-5.
[6]Habib, T., Senadheera, S., Weinberg, K. et al. The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3[J]. Biochemistry,2002,41: 8725-8731.
[7]Yi, J. S., Du, M., Zajac, A. J. A vital role for interleukin-21 in the control of a chronic viral infection[J]. Science,2009,324:1572-1576.
[8]Zeng, R., Spolski, R., Finkelstein, S. E., et al. Synergy of IL-21 and IL-15 in regulating CD8+T cell expansion and function[J]. J. Exp. Med.,2005,201: 139-148.
[9]Ozaki, K. A critical role for IL-21 in regulating immunoglobulin production[J]. Science,2002,298:1630-1634.
[10]Ozaki, K., Spolski, R. Ettinger, H., et al. Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6[J]. J. Immunol.,2004,173:5361-5371.
[11]Jin, H., Carrio, R., Yu, A., et al. Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis[J]. J. Immunol.,2004,173:657-665.
[12]Pene, J., J. F. Gauchat, S. Lecart, et al. Cutting edge:IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells[J]. J. Immunol.,2004, 172:5154-5157.
[13]Avery, D. T., V. L. Bryant, C. S. Ma, et al. IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4[J]. J. Immunol.,2008,181:1767-1779.
[14]Sivori, S. et al. IL-21 induces both rapid maturation of human CD34+cell precursors towards NK cells and acquisition of surface killer Ig-like receptors[J]. Eur. J. Immunol.,2003,33:3439-3447.
[15]Toomey, J. A., Gays, F., Foster, D., et al. Cytokine requirements for the growth and development of mouse NK cells in vitro[J]. J.Leukoc.Biol.,2003, 74:233-242.
[16]Vinuesa, C. G., Cook, M. C., Angelucci, C., et al. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity[J]. Nature,2005,35:52-458.
[17]Herber, D., Brown, T. P., Liang, S., et al. IL-21 has a pathogenic role in a lupus-prone mouse model and its blockade with IL-21 R.Fc reduces disease progression[J]. J. Immunol.,2007,178:3822-3830.
[18]King, C., Ilic, A., Koelsch, K., et al. Homeostatic expansion of T cells during immune insufficiency generates autoimmunity[J]. Cell,2004,117:265-277.
[19]Warren J. Leonard, Rosanne Spolski. Interleukin 21:a modulator of lymphoid proliferation, apoptosis and differentiation[J]. Nature,2005,5:688-698.
[20]Wang, G., Tschoi, M., Spolski, R., et al. In vivo antitumor activity of interleukin 21 mediated by natural killer cells[J]. Cancer Res.,2003,63: 9016-9022.
[21]Heidi Elsaesser, Karsten Sauer, David G. Brooks. IL-21 Is Required to Control Chronic Viral Infection[J]. Science,2009,324:1569-1572.
[22]Anja Frohlich, Jan Kisielow, Iwana Schmitz, et al. IL-21R on T Cells Is Critical for Sustained Functionality and Control of Chronic Viral Infection[J]. Science,2009,324:1576-1580.
[23]Alexandre I, Cecile T, Jean PR et al. Decreased levels of circulating IL-21 in HIV-Infected AIDS patients:correlation with CD4+ T-cell counts[J]. Viral Immunol.,2008,21(3):385-388.
[24]Bolesta E, Kowalczyk A, Wierzbicki A et al. Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery[J]. J. Immunol.,2006,177(1):177-191.
[25]Tsui LV, Guidotti LG, Ishikawa T, et al. Posttranscriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyte-activated hepatocytes[J]. Proc Natl Acad Sci USA.,1995,92:12398-12402.
[26]Maini MK, Boni C, Lee CK, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection[J]. J Exp Med.,2000,191:1269-1280.
[27]Thimme R, Wieland S, Steiger C, et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection[J]. J Virol.,2003,77:68-76.
[28]Chisari F.V., Isogawa M., Wieland S.F.. Pathogenesis of hepatitis B virus infection[J]. Pathol Biol (Paris),2010, doi:10.1016/j.patbio.2009.11.001.
[29]Asabe S, Wieland SF, Chattopadhyay PK, et al. The size of the viral inoculum contributes to the outcome of hepatitis B virus infection[J]. J Virol,2009,83: 9652-9662.
[30]Steinman RM, Inaba K, Turley S, et al. Antigen capture, processing, and presentation by dendritic cells:recent cell biological studies[J]. Hum Immunol, 1999,60:562-567.
[31]Isogawa M, Furuichi Y, Chisari FV. Oscillating CD8(+) T cell effector functions after antigen recognition in the liver[J]. Immunity,2005,23:53-63.
[32]Roncarolo MG, Levings MK. The role of different subsets of T regulatory cells in controlling autoimmunity[J]. Curr Opin Immunol,2000,12:676-683.
[33]Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science,2003,299:1057-1061.
[34]Guilin Yang, Ailian Liu, Qing Xie, et al. Association of CD4+CD25+Foxp3+ regulatory T cells with chronic activity and viral clearance in patients with hepatitis B[J]. International Immunology,19(2):133-140.
[35]Shoukry NA, Grakoui A, Houghton M, et al. Memory CD8+T cells are required for protection from persistent hepatitis C virus infection[J]. J Exp Med, 2003,197:1645-1655.
[36]Cox AL, Mosbruger T, Lauer GM, et al. Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C[J]. Hepatology,2005,42:104-112.
[37]Thimme R, Oldach D, Chang KM, et al. Determinants of viral clearance and persistence during acute hepatitis C virus infection [J]. J Exp Med,2001,194: 1395-1406.
[38]Christoph NH, Hans CS, Hubert EB, et al. Host and viral factors contributing to CD8+T cell failure in hepatitis C virus infection[J]. World J Gastroenterol, 2007,13(36):4839-4847.
[39]Nasser Semmo, Paul Klenerman. CD4+T cell responses in hepatitis C virus infection[J]. World J Gastroenterol,2007,13(36):4831-4838.
[40]Tsai SL, Liaw YF, Chen MH, et al. Detection of type 2-like T-helper cells in hepatitis C virus infection:implications for hepatitis C virus chronicity[J]. Hepatology,1997,25:449-458.
[41]L. J. Yee, P. Kelleher, R. D. Goldin, et al. Antinuclear antibodies (ANA) in chronic hepatitis C virus infection:correlates of positivity and clinical relevance[J]. Journal of Viral Hepatitis,2004,11:459-464.
[42]Whitacre CC. Sex differences in autoimmune disease[J]. Nat Immunol,2001, 2:777-780.
[43]Williams MJ, Lawson A, Neal K.R., et al. Autoantibodies in chronic hepatitis C virus infection and their association with disease profile[J]. J Viral Hepat,2008,16,325-331.
[44]Blumberg BS, Alter HJ, Visnick S. A 'new' antigen in leukemia sera[J]. JAMA,1965,191:541-6.
[45]Christine N, Yu Wei, Marie AB. Mechanisms of HBV-related hepatocarcinogenesis[J]. Journal of hepatology,2010,52:594-604.
[46]Raso G M, Pacilio M, Esposito E, et al. Leptin potentiates IFN-gamma-induced expression of nitric oxide synthase and cyclo-oxygenase-2 in murine macrophage J774A. I[J]. Br JPharmacol,2002.137:799-804.
[47]Hassan MM, Hwang LY, Hatten CJ, et al. Risk factors for hepatocellular carcinoma:synergism of alcohol with viral hepatitis and diabetes mellitus[J]. Hepatology,2002,36:1206-1213.
[48]Scott K. Fung, Anna S.F. Lok. Management of patients with hepatitis B virus-induced cirrhosis[J]. Journal of hepatology,2005,42:S54-S64.
[49]Chemin I., Zoulim F.. Hepatitis B virus induced hepatocellular carcinoma[J]. Cancer Letters,2009,286:52-59.
[50]Kaito M, Ishida S, Tanaka H, et al. Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy[J]. Med Mol Morphol,2006,39(2):63-71
[51]Oberhaus SM, Newbold JE. Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay[J]. J. Virol,1993,67:6558-6566.
[52]Colgrove R, Simon G, Ganem D. Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication[J]. J. Virol.,1989,63:4019-4026.
[53]Arauz-Ruiz, P. Genotype H:a new Amerindian genotype of hepatitis B virus revealed in Central America[J]. J Gen Virol,2002,83(Pt 8):2059-2073.
[54]Stoll-Becker S, Repp R, Glebe D et al. Transcription of hepatitis B virus in peripheral blood mononuclear cells from persistently infected patients[J]. J. Virol.,1997,71:5399-5407.
[55]Ganem D, Schneider R. Hepadnaviridae:the viruses and their replication[J]. Virology,2001,2(4):2923-2970.
[56]Rehermann, B.& Nascimbeni, M. Immunology of hepatitis B virus and hepatitis C virus infection[J]. Nature reviews,2005,5:215-229.
[57]Massimo L, Teresa P, Jorg P, et al. Control of cccDNA function in hepatitis B virus infection[J]. Journal of hepatology,2009,51:581-592.
[58]Geoffrey F, Suzanne K, Johan N, et al.Antiviral treatment of chronic hepatitis B virus infections:the past, the present and the future[J]. Rev Med Virol,2008, 18:19-34.
[59]Chang CN, Skalski V, Zhou JH, et al. Biochemical pharmacology of (+)-and (-)-2',3'-dideoxy-3'-thiacytidine as anti-hepatitis B virus agents[J]. J Biol Chem, 1992,267:22414-22420.
[60]Wang HP, Li Y. Protective effect of bicyclol on acute hepatic failure induced by lipopolysaccharide and D-galactosamine in mice[J]. Eur J Pharmacol,2006, 534:194-201.
[61]Rosenberg B, Camp L V, Trosko J E, et al. Platinum Compounds:a New Class of Potent Antitumour Agents[J]. Nature,1969,222:385-386.
[62]Wong E, Giandomenico C M. Current status of platinum-based antitumor drugs[J]. Chem Rev,1999,99(9):2451-2466.
[63]Liao Y J, Tan G H, Jin Y P, Study of toxic efects on hearing, kidney and liver of mice induced by anticancer agent of cis-platin and their mechanisms[J]. Chin Pharmacol Bull,2004,20(1):82-85.
[64]Perez J M, Fuertes M A, Alonso C, et al. Current status of the development of trans plantinum antitumor drugs[J]. Crit Rev Oncol Hematol,2000,35: 109-120.
[65]Faggioni R, Feingold KR, Grunfeld C. Leptin regulation of the immune response and the immunodeficiency of malnutrition[J]. FASEB J,2000,14: 2565-2571.
[66]Zou Min, Liu Shuwen, Zhou Chunqiong. Interaction of nickel complex of pyrrole-amide and CT DNA and antitumor activity against liver-cancer cell[J].Acta Chimica Sinica,2010,68(6):481-486.
[1]Geoffrey F, Suzanne K, Johan N, et al.Antiviral treatment of chronic hepatitis B virus infections:the past, the present and the future[J]. Rev Med Virol,2008,18:19-34.
[2]Glebe D & Urban S. Viral and cellular determinants involved in hepadnaviral entry[J]. World J Gastroenterol,2007,13(1):22-38.
[3]Philippe Gripon, Isabelle Cannie and Stephan Urban. Efficient Inhibition of Hepatitis B Virus Infection by Acylated Peptides Derived from the Large Viral Surface Protein[J]. J Virol,2005, 79(3):1613-1622.
[4]Chunxiao Ying, Ying Li, Chung-Hang Leung, et al. Unique antiviral mechanism discovered in anti-hepatitis B virus research with a natural product analogue[J]. PNAS,2007,104(20): 8526-8531.
[5]Li Y, Fu L, Yeo H, et al. Inhibition of hepatitis B virus gene expression and replication by helioxanthin and its derivative. Antiviral Chem Chemother,2005,16(3):193-201.
[6]Hacker HJ, Deres K, Mildenberger M, et al. Antivirals interacting with hepatitis B virus core ptotein and core mutations may misdirect capsid assembly in a similar fashion[J]. Biochem Pharmacol,2003,66(12):2273-2279.
[7]Chang CN, Skalski V, Zhou JH, Cheng YC. Biochemical pharmacology of (+)-and (-)-2',3'-dideoxy-3'-thiacytidine as anti-hepatitis B virus agents[J]. J Biol Chem,1992,267: 22414-22420.
[8]Fung SK, Wong F, Hussain M, Lok ASF. Sustained response after a 2-year course of lamivudine treatment of hepatitis B e antigen-negative chronic hepatitis B[J]. J Viral Hepat, 2004,11:432-438.
[9]Lok ASF, Lai CL, Leung N, et al. Long-term safety of lamivudine treatment in patients with chronic hepatitis B[J]. Gastroenterology,2003,125:1714-1722.
[10]Werle-Lapostolle B, Bowden S, Locarnini S, et al. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy[J]. Gastroenterology,2004,126(7):1750-1758.
[11]Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B[J]. N Engl J Med,2005,352:2673-2681.
[12]Zoulim F. Entecavir:a new treatment option for chronic hepatitis B[J]. J Clin Virol,2006,36: 8-12.
[13]Chang TT, Gish RG, de Man R, et al. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B[J]. N Engl J Med,2006,354:1001-1010.
[14]Lai CL, Lim SG, Brown NA, et al. A dose-finding study of once-daily oral telbivudine in HBeAgpositive patients with chronic hepatitis B virus infection[J]. Hepatology,2004,40: 719-726.
[15]Gane E, Lai CL, Liaw YE, et al. Phase Ⅲ comparison of telbivudine vs lamivudine in HBeAg-positive patients with chronic hepatitis B:efficacy, safety, and predictors of response at 1 year[J]. J Hepatol,2006,44(2):183-184.
[16]Chong Y, Chu CK. Understanding the unique mechanism of L-FMAU (clevudine) against hepatitis B virus:molecular dynamics studies[J]. Bioorg Med Chem Lett,2002,12:3459-3462.
[2]Spolski, R., Leonard, W. J. Interleukin-21:basic biology and implications for cancer and autoimmunity[J]. Annu. Rev. Immunol,2008,26,57-79.
[3]Onoda, T., Rahman, M., Nara, H., et al. Human CD4+ central and effector memory T cells produce IL-21:effect on cytokine-driven proliferation of CD4+ T cell subsets[J]. Int. Immunol,2007,19:1191-1199.
[4]Ozaki, K., Kikly, K., Michalovich, D. et al. Cloning of a type Ⅰ cytokine receptor most related to the IL-2 receptor beta chain[J]. Proc. Natl. Acad. Sci. USA,2000,97:11439-11444.
[5]Asao, H., Okuyama, C., Kumaki, S., et al. Cutting edge:the common gamma-chain is an indispensable subunit of the IL-21 receptor complex[J]. J. Immunol,2001,167:1-5.
[6]Habib, T., Senadheera, S., Weinberg, K. et al. The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3[J]. Biochemistry,2002,41: 8725-8731.
[7]Yi, J. S., Du, M., Zajac, A. J. A vital role for interleukin-21 in the control of a chronic viral infection[J]. Science,2009,324:1572-1576.
[8]Zeng, R., Spolski, R., Finkelstein, S. E., et al. Synergy of IL-21 and IL-15 in regulating CD8+T cell expansion and function[J]. J. Exp. Med.,2005,201: 139-148.
[9]Ozaki, K. A critical role for IL-21 in regulating immunoglobulin production[J]. Science,2002,298:1630-1634.
[10]Ozaki, K., Spolski, R. Ettinger, H., et al. Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6[J]. J. Immunol.,2004,173:5361-5371.
[11]Jin, H., Carrio, R., Yu, A., et al. Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis[J]. J. Immunol.,2004,173:657-665.
[12]Pene, J., J. F. Gauchat, S. Lecart, et al. Cutting edge:IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells[J]. J. Immunol.,2004, 172:5154-5157.
[13]Avery, D. T., V. L. Bryant, C. S. Ma, et al. IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4[J]. J. Immunol.,2008,181:1767-1779.
[14]Sivori, S. et al. IL-21 induces both rapid maturation of human CD34+cell precursors towards NK cells and acquisition of surface killer Ig-like receptors[J]. Eur. J. Immunol.,2003,33:3439-3447.
[15]Toomey, J. A., Gays, F., Foster, D., et al. Cytokine requirements for the growth and development of mouse NK cells in vitro[J]. J.Leukoc.Biol.,2003, 74:233-242.
[16]Vinuesa, C. G., Cook, M. C., Angelucci, C., et al. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity[J]. Nature,2005,35:52-458.
[17]Herber, D., Brown, T. P., Liang, S., et al. IL-21 has a pathogenic role in a lupus-prone mouse model and its blockade with IL-21 R.Fc reduces disease progression[J]. J. Immunol.,2007,178:3822-3830.
[18]King, C., Ilic, A., Koelsch, K., et al. Homeostatic expansion of T cells during immune insufficiency generates autoimmunity[J]. Cell,2004,117:265-277.
[19]Warren J. Leonard, Rosanne Spolski. Interleukin 21:a modulator of lymphoid proliferation, apoptosis and differentiation[J]. Nature,2005,5:688-698.
[20]Wang, G., Tschoi, M., Spolski, R., et al. In vivo antitumor activity of interleukin 21 mediated by natural killer cells[J]. Cancer Res.,2003,63: 9016-9022.
[21]Heidi Elsaesser, Karsten Sauer, David G. Brooks. IL-21 Is Required to Control Chronic Viral Infection[J]. Science,2009,324:1569-1572.
[22]Anja Frohlich, Jan Kisielow, Iwana Schmitz, et al. IL-21R on T Cells Is Critical for Sustained Functionality and Control of Chronic Viral Infection[J]. Science,2009,324:1576-1580.
[23]Alexandre I, Cecile T, Jean PR et al. Decreased levels of circulating IL-21 in HIV-Infected AIDS patients:correlation with CD4+ T-cell counts[J]. Viral Immunol.,2008,21(3):385-388.
[24]Bolesta E, Kowalczyk A, Wierzbicki A et al. Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery[J]. J. Immunol.,2006,177(1):177-191.
[25]Tsui LV, Guidotti LG, Ishikawa T, et al. Posttranscriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyte-activated hepatocytes[J]. Proc Natl Acad Sci USA.,1995,92:12398-12402.
[26]Maini MK, Boni C, Lee CK, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection[J]. J Exp Med.,2000,191:1269-1280.
[27]Thimme R, Wieland S, Steiger C, et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection[J]. J Virol.,2003,77:68-76.
[28]Chisari F.V., Isogawa M., Wieland S.F.. Pathogenesis of hepatitis B virus infection[J]. Pathol Biol (Paris),2010, doi:10.1016/j.patbio.2009.11.001.
[29]Asabe S, Wieland SF, Chattopadhyay PK, et al. The size of the viral inoculum contributes to the outcome of hepatitis B virus infection[J]. J Virol,2009,83: 9652-9662.
[30]Steinman RM, Inaba K, Turley S, et al. Antigen capture, processing, and presentation by dendritic cells:recent cell biological studies[J]. Hum Immunol, 1999,60:562-567.
[31]Isogawa M, Furuichi Y, Chisari FV. Oscillating CD8(+) T cell effector functions after antigen recognition in the liver[J]. Immunity,2005,23:53-63.
[32]Roncarolo MG, Levings MK. The role of different subsets of T regulatory cells in controlling autoimmunity[J]. Curr Opin Immunol,2000,12:676-683.
[33]Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science,2003,299:1057-1061.
[34]Guilin Yang, Ailian Liu, Qing Xie, et al. Association of CD4+CD25+Foxp3+ regulatory T cells with chronic activity and viral clearance in patients with hepatitis B[J]. International Immunology,19(2):133-140.
[35]Shoukry NA, Grakoui A, Houghton M, et al. Memory CD8+T cells are required for protection from persistent hepatitis C virus infection[J]. J Exp Med, 2003,197:1645-1655.
[36]Cox AL, Mosbruger T, Lauer GM, et al. Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C[J]. Hepatology,2005,42:104-112.
[37]Thimme R, Oldach D, Chang KM, et al. Determinants of viral clearance and persistence during acute hepatitis C virus infection [J]. J Exp Med,2001,194: 1395-1406.
[38]Christoph NH, Hans CS, Hubert EB, et al. Host and viral factors contributing to CD8+T cell failure in hepatitis C virus infection[J]. World J Gastroenterol, 2007,13(36):4839-4847.
[39]Nasser Semmo, Paul Klenerman. CD4+T cell responses in hepatitis C virus infection[J]. World J Gastroenterol,2007,13(36):4831-4838.
[40]Tsai SL, Liaw YF, Chen MH, et al. Detection of type 2-like T-helper cells in hepatitis C virus infection:implications for hepatitis C virus chronicity[J]. Hepatology,1997,25:449-458.
[41]L. J. Yee, P. Kelleher, R. D. Goldin, et al. Antinuclear antibodies (ANA) in chronic hepatitis C virus infection:correlates of positivity and clinical relevance[J]. Journal of Viral Hepatitis,2004,11:459-464.
[42]Whitacre CC. Sex differences in autoimmune disease[J]. Nat Immunol,2001, 2:777-780.
[43]Williams MJ, Lawson A, Neal K.R., et al. Autoantibodies in chronic hepatitis C virus infection and their association with disease profile[J]. J Viral Hepat,2008,16,325-331.
[44]Blumberg BS, Alter HJ, Visnick S. A 'new' antigen in leukemia sera[J]. JAMA,1965,191:541-6.
[45]Christine N, Yu Wei, Marie AB. Mechanisms of HBV-related hepatocarcinogenesis[J]. Journal of hepatology,2010,52:594-604.
[46]Raso G M, Pacilio M, Esposito E, et al. Leptin potentiates IFN-gamma-induced expression of nitric oxide synthase and cyclo-oxygenase-2 in murine macrophage J774A. I[J]. Br JPharmacol,2002.137:799-804.
[47]Hassan MM, Hwang LY, Hatten CJ, et al. Risk factors for hepatocellular carcinoma:synergism of alcohol with viral hepatitis and diabetes mellitus[J]. Hepatology,2002,36:1206-1213.
[48]Scott K. Fung, Anna S.F. Lok. Management of patients with hepatitis B virus-induced cirrhosis[J]. Journal of hepatology,2005,42:S54-S64.
[49]Chemin I., Zoulim F.. Hepatitis B virus induced hepatocellular carcinoma[J]. Cancer Letters,2009,286:52-59.
[50]Kaito M, Ishida S, Tanaka H, et al. Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy[J]. Med Mol Morphol,2006,39(2):63-71
[51]Oberhaus SM, Newbold JE. Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay[J]. J. Virol,1993,67:6558-6566.
[52]Colgrove R, Simon G, Ganem D. Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication[J]. J. Virol.,1989,63:4019-4026.
[53]Arauz-Ruiz, P. Genotype H:a new Amerindian genotype of hepatitis B virus revealed in Central America[J]. J Gen Virol,2002,83(Pt 8):2059-2073.
[54]Stoll-Becker S, Repp R, Glebe D et al. Transcription of hepatitis B virus in peripheral blood mononuclear cells from persistently infected patients[J]. J. Virol.,1997,71:5399-5407.
[55]Ganem D, Schneider R. Hepadnaviridae:the viruses and their replication[J]. Virology,2001,2(4):2923-2970.
[56]Rehermann, B.& Nascimbeni, M. Immunology of hepatitis B virus and hepatitis C virus infection[J]. Nature reviews,2005,5:215-229.
[57]Massimo L, Teresa P, Jorg P, et al. Control of cccDNA function in hepatitis B virus infection[J]. Journal of hepatology,2009,51:581-592.
[58]Geoffrey F, Suzanne K, Johan N, et al.Antiviral treatment of chronic hepatitis B virus infections:the past, the present and the future[J]. Rev Med Virol,2008, 18:19-34.
[59]Chang CN, Skalski V, Zhou JH, et al. Biochemical pharmacology of (+)-and (-)-2',3'-dideoxy-3'-thiacytidine as anti-hepatitis B virus agents[J]. J Biol Chem, 1992,267:22414-22420.
[60]Wang HP, Li Y. Protective effect of bicyclol on acute hepatic failure induced by lipopolysaccharide and D-galactosamine in mice[J]. Eur J Pharmacol,2006, 534:194-201.
[61]Rosenberg B, Camp L V, Trosko J E, et al. Platinum Compounds:a New Class of Potent Antitumour Agents[J]. Nature,1969,222:385-386.
[62]Wong E, Giandomenico C M. Current status of platinum-based antitumor drugs[J]. Chem Rev,1999,99(9):2451-2466.
[63]Liao Y J, Tan G H, Jin Y P, Study of toxic efects on hearing, kidney and liver of mice induced by anticancer agent of cis-platin and their mechanisms[J]. Chin Pharmacol Bull,2004,20(1):82-85.
[64]Perez J M, Fuertes M A, Alonso C, et al. Current status of the development of trans plantinum antitumor drugs[J]. Crit Rev Oncol Hematol,2000,35: 109-120.
[65]Faggioni R, Feingold KR, Grunfeld C. Leptin regulation of the immune response and the immunodeficiency of malnutrition[J]. FASEB J,2000,14: 2565-2571.
[66]Zou Min, Liu Shuwen, Zhou Chunqiong. Interaction of nickel complex of pyrrole-amide and CT DNA and antitumor activity against liver-cancer cell[J].Acta Chimica Sinica,2010,68(6):481-486.
[1]Geoffrey F, Suzanne K, Johan N, et al.Antiviral treatment of chronic hepatitis B virus infections:the past, the present and the future[J]. Rev Med Virol,2008,18:19-34.
[2]Glebe D & Urban S. Viral and cellular determinants involved in hepadnaviral entry[J]. World J Gastroenterol,2007,13(1):22-38.
[3]Philippe Gripon, Isabelle Cannie and Stephan Urban. Efficient Inhibition of Hepatitis B Virus Infection by Acylated Peptides Derived from the Large Viral Surface Protein[J]. J Virol,2005, 79(3):1613-1622.
[4]Chunxiao Ying, Ying Li, Chung-Hang Leung, et al. Unique antiviral mechanism discovered in anti-hepatitis B virus research with a natural product analogue[J]. PNAS,2007,104(20): 8526-8531.
[5]Li Y, Fu L, Yeo H, et al. Inhibition of hepatitis B virus gene expression and replication by helioxanthin and its derivative. Antiviral Chem Chemother,2005,16(3):193-201.
[6]Hacker HJ, Deres K, Mildenberger M, et al. Antivirals interacting with hepatitis B virus core ptotein and core mutations may misdirect capsid assembly in a similar fashion[J]. Biochem Pharmacol,2003,66(12):2273-2279.
[7]Chang CN, Skalski V, Zhou JH, Cheng YC. Biochemical pharmacology of (+)-and (-)-2',3'-dideoxy-3'-thiacytidine as anti-hepatitis B virus agents[J]. J Biol Chem,1992,267: 22414-22420.
[8]Fung SK, Wong F, Hussain M, Lok ASF. Sustained response after a 2-year course of lamivudine treatment of hepatitis B e antigen-negative chronic hepatitis B[J]. J Viral Hepat, 2004,11:432-438.
[9]Lok ASF, Lai CL, Leung N, et al. Long-term safety of lamivudine treatment in patients with chronic hepatitis B[J]. Gastroenterology,2003,125:1714-1722.
[10]Werle-Lapostolle B, Bowden S, Locarnini S, et al. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy[J]. Gastroenterology,2004,126(7):1750-1758.
[11]Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B[J]. N Engl J Med,2005,352:2673-2681.
[12]Zoulim F. Entecavir:a new treatment option for chronic hepatitis B[J]. J Clin Virol,2006,36: 8-12.
[13]Chang TT, Gish RG, de Man R, et al. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B[J]. N Engl J Med,2006,354:1001-1010.
[14]Lai CL, Lim SG, Brown NA, et al. A dose-finding study of once-daily oral telbivudine in HBeAgpositive patients with chronic hepatitis B virus infection[J]. Hepatology,2004,40: 719-726.
[15]Gane E, Lai CL, Liaw YE, et al. Phase Ⅲ comparison of telbivudine vs lamivudine in HBeAg-positive patients with chronic hepatitis B:efficacy, safety, and predictors of response at 1 year[J]. J Hepatol,2006,44(2):183-184.
[16]Chong Y, Chu CK. Understanding the unique mechanism of L-FMAU (clevudine) against hepatitis B virus:molecular dynamics studies[J]. Bioorg Med Chem Lett,2002,12:3459-3462.