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目的:探讨上海地区丙型肝炎病毒(HCV)1b亚型慢性感染者的血清HCV非结构基因5A(NS5A)与干扰素(IFN)疗效的关系。方法:收集上海地区32例HCV1b亚型慢性感染者在干扰素治疗前后及随访过程中的血清标本,定量检测治疗血清HCV RNA,用逆转录-集合酶链反应方法扩增NS5A的干扰素敏感决定区(ISDR)基因并进行测序和分析。结果:治疗前血清HCV RNA的定量结果显示,持续应答组的病毒滴度(平均滴度4.50×104拷贝数.mL-1)明显低于复发组和无应答组(平均滴度1.82×107拷贝数.mL-1)。32例慢性丙型肝炎干扰素治疗前血清HCV的ISDR氨基酸序列与抗干扰素的HCV-J株比较,22例为野生型,10例为中间型,无突变型。12例完全应答者8例感染的是野生型株,4例感染的是中间型病毒株。5株HCV病毒的NS5A全长序列种系发生树显示,3种不同应答类型株在种系发生上分属3个组别,无应答株与抗干扰素的HCV-J株关系相近被归为1组。蛋白质二级结构预测显示,上述病毒株NSSA蛋白在二级结构方面基本相似,仅在2255—2289范围内有明显不同,这一区域与PKR结合域部分重叠。结论:HCVNS5A基因的ISDR区不能单独用于预测干扰素疗效,治疗前血清HCV RNA水平结合NS5A区的PKR结合域序列的分析,可能有助于预测干扰素疗效,指导临床用药。
Objective: To investigate the relationship between serum HCV non-structural gene 5A (NS5A) and interferon (IFN) in patients with chronic hepatitis C virus (HCV) 1b subtype infection in Shanghai. Methods: Serum samples of 32 patients with chronic HCV1b infection in Shanghai before and after interferon treatment and during follow-up were collected. Serum HCV RNA was detected quantitatively and interferon sensitivity of NS5A was amplified by reverse transcription-polymerase chain reaction Region (ISDR) genes and sequenced and analyzed. Results: Quantitative results of serum HCV RNA before treatment showed that the virus titer (average titer of 4.50 × 104 copies. ML-1) in the continuous response group was significantly lower than that of the recurrence group and the non-response group (mean titer of 1.82 × 10 7 copies Number.mL-1). The ISDR amino acid sequence of serum HCV of 32 patients with chronic hepatitis C was 22 wild type and 10 intermediate type with no mutation when compared with HCV-J strain of anti-interferon. Eight of the 12 complete responders were infected with the wild-type strain and the other 4 were infected with the intermediate-type strain. Five full-length NS5A sequences of five HCV strains showed that the three strains with different response types belonged to three groups and the non-responders had similar relationship with the anti-interferon HCV-J strain Group 1. Protein secondary structure prediction showed that the NSSA proteins of the above strains were similar in secondary structure, with obvious difference only in the range of 2255-2289, which partially overlap with the PKR binding domain. Conclusion: The ISDR region of HCVNS5A gene can not be used to predict the efficacy of interferon alone. The analysis of serum HCV RNA level and PKR binding domain sequence in NS5A region before treatment may be helpful in predicting the efficacy of interferon and guiding clinical application.