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三结构域蛋白29(TRIM29)与HBV复制及聚乙二醇干扰素α-2b抗病毒作用的关联性分析

廖心 张宝芳

引用本文:
Citation:

三结构域蛋白29(TRIM29)与HBV复制及聚乙二醇干扰素α-2b抗病毒作用的关联性分析

DOI: 10.12449/JCH241111
基金项目: 

国家自然科学基金地区基金 (82060114);

贵州省卫健委2023年度科技基金项目 (gzwkj2023-042);

贵州省卫生健康委员会2024年科技基金项目 (gzwkj2024-010)

伦理学声明:本研究方案于2020年4月9日通过贵州医科大学附属医院伦理委员会审批,批号为2020208。所有研究对象入组前均签署了书面知情同意书。
利益冲突声明:本文不存在任何利益冲突。
作者贡献声明:廖心负责课题设计,实验实施,收据收集和撰写论文;张宝芳负责课题设计,指导撰写文章并最后定稿。
详细信息
    通信作者:

    张宝芳, 312368145@qq.com (ORCID: 0000-0002-5396-540X)

Association of TRIM29 with HBV replication and the antiviral effect of pegylated interferon α-2b

Research funding: 

National Natural Science Foundation of China (82060114);

Science & Technology Fund Project of Guizhou Health Commission in 2023 (gzwkj2023-042);

The Science and Technology Fund Project of Guizhou Health Commission in 2024 (gzwkj2024-010)

More Information
    Corresponding author: ZHANG Baofang, 312368145@qq.com (ORCID: 0000-0002-5396-540X)
  • 摘要:   目的  三结构域蛋白29(TRIM29)参与多种疾病的发生和发展,与部分DNA和RNA病毒复制密切相关,本研究对TRIM29与HBV复制及聚乙二醇干扰素(PEG-IFN)α-2b抗病毒作用之间的关系展开初步讨论。  方法  选取2021年10月—2022年6月在贵州医科大学附属医院感染内科门诊就诊的CHB患者64例,其中未治疗患者34例(CHB组),经PEG-IFN-α- 2b治疗患者30例,体检中心30例健康志愿者作为对照(健康对照组)。收集志愿者年龄、性别、ALT、AST、TBil、DBil、HBV DNA和外周血单个核细胞(PBMC)。采用HepG2和HepG2.2.15细胞作为细胞模型,将TRIM29特异性过表达质粒或siRNA和对照转染至细胞;PEG-IFN-α-2b(0、10、100、1 000和10 000 U/mL)处理HepG2细胞和Huh7细胞;TRIM29特异性si-RNA或阴性对照联合PEG-IFN-α-2b处理HepG2.2.15细胞。ELISA检测HBsAg和HBeAg的浓度,qRT-PCR检测TRIM29和HBV RNA相对表达水平,Western Blot检测STING、p-TBK1、TBK1、pIRF3、IRF3、MX1和IFIT1蛋白表达情况,免疫共沉淀检测TRIM29与STING蛋白相互作用关系。正态分布的计量资料两组间比较采用成组t检验,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。计数资料两组间比较采用χ2检验或Fisher检验。  结果  CHB组患者外周血TRIM29表达明显高于健康对照组(P<0.001)。在细胞实验中,HBsAg、HBeAg和HBV RNA的表达均随TRIM29表达上调而升高,随TRIM29表达下调而降低(P值均<0.05)。TRIM29与STING互相结合,并通过蛋白酶降解STING,与对照组相比,过表达TRIM29对TBK1和IRF3总蛋白无明显变化,STING、p-TBK1和p-IRF3蛋白表达水平均降低(P值均<0.05)。处理HepG2细胞和Huh7细胞的PEG-IFN-α-2b浓度越高,TRIM29蛋白和mRNA的表达水平越低(P值均<0.01)。CHB患者在PEG-IFN-α-2b治疗期间,TRIM29 mRNA表达水平逐渐降低,且早期应答组和无应答组间差异均有统计学意义(P值均<0.05)。在等量PEG-IFN-α-2b处理下,与对照相比,敲低TRIM29后HepG2.2.15细胞的MX1和IFIT1蛋白表达水平明显增高(P值均<0.05)。在PEG-IFN-α-2b治疗早期,CHB患者PBMC中TRIM29表达逐渐降低。  结论  TRIM29靶向并降解STING,通过抑制STING-TBK1-IRF3信号通路促进HBV复制。TRIM29干扰PEG-IFN-α-2b的抗病毒作用,CHB患者PBMC中TRIM29的表达水平可能作为预测患者对PEG-IFN-α-2b治疗应答的指标。

     

  • 图  1  TRIM29表达水平与HBV感染的关系

    注: a,CHB组与健康对照组TRIM29 mRNA表达差异;b,HepG2和HepG2.2.15细胞TRIM29蛋白表达差异。

    Figure  1.  The relationship between TRIM29 and HBV infection

    图  2  过表达TRIM29对HBV复制的影响

    注: a、c,Western Blot和qRT-PCR检测TRIM29转染效率;b、d,ELISA检测细胞培养基中HBsAg和HBeAg的浓度,qRT-PCR检测HBV RNA的相对表达水平。

    Figure  2.  Effect of TRIM29 overexpression on HBV replication and gene expression

    图  3  敲低TRIM29对HBV复制的影响

    注: a、c,Western Blot和qRT-PCR检测TRIM29特异性si-RNA转染效率;b、d,ELISA检测细胞培养基中HBsAg和HBeAg的浓度,qRT-PCR检测HBV RNA表达水平。

    Figure  3.  Effect of TRIM29 knockdown on HBV replication and gene expression

    图  4  TRIM29对STING-TBK1-IRF3信号通路的调控作用

    注: a,免疫共沉淀检测TRIM29和STING相互作用;b、c,qRT-PCR检测IFN-α mRNA表达水平;d、e,Western Blot检测STING、TBK1、IRF3、p-TBK1、p-IRF3蛋白水平。

    Figure  4.  Regulatory effect of TRIM29 on STING-TBK1-IRF3 signaling pathway

    图  5  PEG-IFN-α-2b对TRIM29表达的影响

    注: a、b HepG2细胞;c、d,Huh7细胞。a、c,Western Blot检测TRIM29蛋白表达水平;b、d,qRT-PCR检测TRIM29 mRNA水平。

    Figure  5.  Effect of PEG-IFN-α-2b on TRIM29 expression

    图  6  TRIM29表达对PEG-IFN-α-2b的抗病毒作用的影响

    Figure  6.  Effect of TRIM29 expression on the antiviral effect of PEG-IFN-α-2b

    表  1  引物序列

    Table  1.   List of primers

    引物 序列(5'-3')
    HBV RNA-F TCTTGCCTTACTTTTGGAAG
    HBV RNA-R AGTTCTTCTTCTAGGGGACC
    Hu-TRIM29-F TGCGAGCTGCATCTCAAGC
    Hu-TRIM29-R GGTGCTATGATTCTTGTGCTCC
    Hu-IFN-α-F TGACCTCAAAGCCTGTGTGATG
    Hu-IFN-α-R AAGTATTTCCTCACAGCCAGCAG
    Hu-GAPDH-F CGGATTTGGTCGTATTGGG
    Hu-GAPDH-R TCTCGCTCCTGGAAGATGG
    下载: 导出CSV

    表  2  健康对照组和CHB组患者的一般资料比较

    Table  2.   Comparison of clinical characteristics between healthy and CHB patients

    指标 健康对照组(n=30) CHB组(n=34) 统计值 P
    年龄(岁) 38.2±10.4 33.8±5.0 t=0.451 0.328
    男/女(例) 15/15 16/18 χ2=0.055 0.814
    汉族/其他(例) 23/7 24/10 χ2=0.302 0.583
    ALT(U/L) 19.4±4.8 17.4±4.9 t=1.702 0.094
    AST(U/L) 17.3±3.3 18.4±3.3 t=-1.293 0.201
    TBil(μmol/L) 6.2±3.7 8.6±2.7 t=-1.791 0.195
    DBil(μmol/L) 3.4±0.8 4.6±1.6 t=-2.219 0.138
    下载: 导出CSV

    表  3  一般临床资料

    Table  3.   Clinical characteristics of patients

    指标 早期应答组(n=11) 早期无应答组(n=19) t P
    女性(例) 7 12 >0.05
    年龄(岁) 26.8±4.49 29.3±7.20 -2.041 0.643
    HBV DNA (log10 IU/mL)
    0周 2.35±0.26 2.11±0.18 2.380 0.010
    12周 1.33±0.10 1.84±0.23 2.598 <0.001
    24周 1.09±0.16 1.56±0.27 -4.260 <0.001
    ALT(U/L)
    0周 23.64±3.70 24.09±4.00 -0.546 0.99
    12周 59.78±6.30 36.25±5.73 8.127 <0.001
    24周 33.66±6.74 45.68±4.96 -3.893 <0.001
    TRIM29 mRNA
    0周 1.57±0.43 1.32±0.75 2.07 0.047
    12周 1.22±0.26 0.72±0.34 3.83 <0.001
    24周 0.94±0.27 0.43±0.11 7.20 <0.001
    下载: 导出CSV
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