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慢性乙型肝炎患者干扰素-α治疗早期乙型肝炎病毒的准种进化
任玉莲
2014-05-15
导师谢尧
专业内科学
授予单位北京大学
授予地点北京地坛医院教学医院
学位硕士
关键词肝炎,乙型,慢性 干扰素-α 准种 适应性进化
其他题名Early evolution of hepatitis B virus quasispecies during IFN-α treatment in patients with chronic hepatitis B
分类号R512.6
摘要目的 应用生物信息学方法研究干扰素-α治疗早期乙型肝炎病毒(HBV)全基因组的准种变化与适应性进化及其与病毒学应答的关系。方法 对16例进入免疫清除期的慢性乙型肝炎患者进行干扰素-α抗病毒治疗,治疗12周有6例取得完全病毒学应答,3例部分病毒学应答,7例无应答。分别采集治疗前和治疗12周的血清,提取HBV DNA后扩增HBV全基因组,进行分子克隆并测序。对得到的序列进行以下生物信息学分析:(1)比较3组间相同基因位点上核苷酸的差异;(2)分别比较3组间4个开放读码框和全基因组准种复杂度和准种多样性的差异,并比较干扰素-α作用下HBV的进化速率;(3)应用最大似然法对治疗前和治疗第12周的HBV全基因组序列构建分子进化树;(4)对HBV基因组的4个开放读码框,分别用最大似然法检测治疗前以及在干扰素-α作用下的正选择位点。结果(1)比较3组间相同基因位点上的核苷酸差异,未发现具有统计学差异的位点(P>0.05)(2)除了乙肝病毒前C/C基因,在干扰素-α治疗早期无其他基因准种的动态变化(P>0.05)。治疗前完全应答组和部分应答组前C/C基因氨基酸水平的准种多样性高于无应答组(P<0.01);治疗12周部分应答组氨基酸水平准种多样性中的平均遗传距离仍高于无应答组(P<0.05)。部分应答组与无应答组在干扰素-α作用下病毒进化速率无显著差别(P>0.05)(3)用最大似然法分别检测治疗前后3组患者4个开放读码框的正选择位点,一共检测到28个密码子位点。治疗前无应答组检测到的正选择位点(16个)多于完全应答组(9个)和部分应答组(8个),治疗12周部分应答组和无应答组的正选择位点数量减少。对检测到的正选择位点进行抗原表位定位,发现28个正选择位点中有18个位于已知的抗原表位区。治疗后新出现的正选择位点S-ORF188位密码子位点、P-ORF 213密码子位点以及部分应答组和无应答组治疗12周都未消失的正选择位点X-ORF144位密码子位点突变可能与应答不良相关。结论 慢性乙型肝炎免疫清除期,前C/C基因氨基酸水平准种多样性高的患者在干扰素-α治疗早期更容易取得病毒学应答。慢性乙型肝炎免疫清除期各个基因都存在适应性进化,且检测到的正选择位点大部分位于免疫表位区。HBV基因组的正选择作用可能与氨基酸突变病毒逃逸宿主免疫应答有关,S-ORF188位密码子位点、P-ORF213位密码子位点以及X-ORF 144位密码子突变可能与干扰素-α治疗早期应答不佳相关。
英文摘要Objective: To investigate the dynamic change of hepatitis B virus (HBV) quasispeices and adaptive evloution within complete genome during the early stage of IFN-α treatment and its impact on virological response. Methods: Sixteen patients with chronic hepatitis B receiving IFN-α treatment were investigated, according to the HBV DNA levels at weeks 12, there were 6 responders, 3 partial responders and 7 non-responders,respectively. HBV DNA was extracted from serum sample at baseline and week 12. The complete genome of HBV was amplified, then cloned and sequenced. Bioinformatics analysis was conducted on all sequences as follows: (1) Compare the nucleotide differences between the 3 groups in the same nucleotide site. (2) The quasispecies heterogeneity of four ORFs and complete genome was depicted at baseline and week 12 in threes groups, the evolutionary rate of partial responders and non-responders was also calculated, too. (3) The molecular evolutionary tree was constructed to the sequences at beseline and at week 12, respectively. (4)The adaptive evolution was conducted using PAML at baseline and week 12 in threes groups. Results: (1) There were no differences between the 3 groups in the same nucleotide site (P>0.05). (2)The quasispecies heterogeneity of the genome except for C-ORF was comparable in three groups at baseline and week 12 (P>0.05). The quasispecies diversity at amino acid levels of responders within C-ORF were higher than that of non-responders at baseline (P<0.01). Furthermore, the mean genetic distance at amino acid levels of partial responders was significantly higher than that of the non-responders at week 12(P<0.05). The evolutionary rate was not different between non-responders and partial responders (P>0.05). (3) A total of 28 positive selection codons were resolved, and there were more positive selection sites in non-responders at baseline than the responders. Most of the positive selection codons were located at immune epitope. The positive selection codons 188 of S-ORF and 213 of P-ORF appeared after treatment of IFN-α. The positive selection codon of X144 codon did not disappear in partial responder group and non-responder group . Conclusions: In the immunoclearance phase, the patients who had greater viral quasispecies diversity within C-ORF at amino acid level had the more chance to obtain the early virological response during IFN-α treatment. The adaptive evolution was detected in all the four ORFs in the immunoclearance phase, the positive selection sites of HBV may be associated with immune escape caused by epitope mutation. The codons 188 of S-ORF, 213 of P-ORF, and 144 of X-ORF may be related to the poor response of IFN-α treatment.
语种中文
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/107982
专题北京地坛医院教学医院
作者单位北京地坛医院教学医院
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GB/T 7714
任玉莲. 慢性乙型肝炎患者干扰素-α治疗早期乙型肝炎病毒的准种进化[D]. 北京地坛医院教学医院. 北京大学,2014.
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