• 中国科技论文统计源期刊
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Volume 47 Issue 4
May  2022
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Article Navigation >  Journal of Bengbu Medical College  > 2022,  47 > 4: (529-532)  

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Study on the relationships between the genotyping and disease severity, expression of immunocompetent cells in patients with hepatitis C

  • 1.

    Department of Laboratory, Qinghai Fourth People's Hospital, Xining Qinghai 810000, China

  • 2.

    Department of Liver Disease, Qinghai Fourth People's Hospital, Xining Qinghai 810000, China

  • Received Date: 2019-11-26
    Accepted Date: 2020-05-23
  • ObjectiveTo detect the relationships between the genotype of hepatitis C virus(HCV) and severity of the disease, expression of immunocompetent cells in patients with chronic hepatitis C(CHC).MethodsFrom June 2017 to July 2018, 169 Tibetan CHC patients were selected as the study objects(CHC group), and 170 Tibetan healthly examinees in Qinghai were selected as the control group.The levels of HCV-RNA in all cases were detected using PCR fluorescence probe, the PCR reverse hybridization was used to detect the HCV genotyping, and the levels of serum interferon-γ(INF-γ) and interleukin-4(IL-4) were measured by enzyme-linked immunosorbent assay(ELISA).The relationships between the genotype distribution, and disease severity, serum levels of INF-γ and IL-4;and between disease severity, and levels of INF-γ and IL-4 were analyzed.ResultsCompared with the control group, the positive rate of HCV-RNA and serum level of IL-4 increased, and the serum level of INF-γ decreased in CHC group(P < 0.01).The positive rates of HCV-RNA in severe chronic hepatitis group and acute hepatitis group were higher than that in mild and moderate chronic hepatitis group(P<0.05).The differences of the positive rates of HCV-RNA among different genotypes and different severity of CHC were statistically significant(P < 0.01).Compared with the HCV genotype 1a, the serum levels of INF-γ in patients with genotype 2a and 3b increased(P < 0.01).Compared with the HCV genotype 1b, the serum level of INF-γ increased(P < 0.01), and the serum level of IL-4 level decreased in genotype 2a(P < 0.01).The serum level of INF-γ in genotype 3b decreased compared with the genotype 2a(P<0.05).The differences of the serum levels of INF-γ and IL-4 among different severity of CHC were statistically significant(P < 0.01).There was no statistical significance in the levels of INF-γ between mild and moderate group, and between moderate and severe group(P>0.05).The serum level of INF-γ in the mild group was higher than that in severe group, it decreased gradually in moderate group, severe group and acute group, and the differences of which were statistically significant(P < 0.01).The serum level of IL-4 increased gradually in the mild group, moderate group, severe group and acute group, and the differences of which were statistically significant(P < 0.01).ConclusionsFour subtypes of HCV(1a, 1b, 2a and 3b) are detected in Tibetan patients with hepatitis C in Qinghai area, the proportions of 1b and 2a are high.The genotype 1b is closely related to the severity of the disease, the level of INF-γ decreases, and the level of IL-4 increases.
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  • [1] 夏幼辰, 陆伦根. 上海地区人群丙型肝炎病毒1b型进化树及其影响因素分析[J]. 国际消化病杂志, 2019, 39(3): 189. doi: 10.3969/j.issn.1673-534X.2019.03.011
    [2] 孙宏. 慢性丙型肝炎患者HCV核心抗原和HCV RNA检测价值研究[J]. 检验医学与临床, 2018, 15(4): 556. doi: 10.3969/j.issn.1672-9455.2018.04.042
    [3] 常凤霞, 董力, 闫金南. HCV基因分型在丙肝诊断和治疗中的应用探讨[J]. 青海医药杂志, 2016, 46(6): 53.
    [4] 中华医学会肝病学分会, 中华医学会感染病学分会. 丙型肝炎防治指南(2015更新版)[J]. 肝脏, 2015, 33(12): 933. doi: 10.3969/j.issn.1008-1704.2015.12.002
    [5] SAITO Y, IMAMURA M, UCHIDA T, et al. Ribavirin induces hepatitis C virus genome mutations in chronic hepatitis patients who failed to respond to prior daclatasvir plus asunaprevir therapy[J]. J Med Virol, 2019, 10(1002): 25602.
    [6] 纪伟, 张勇, 周吉坤, 等. 献血感染丙型肝炎病毒人群的T细胞免疫及与肝损伤的相关性研究[J]. 病毒学报, 2019, 35(3): 357.
    [7] 马玉秀, 于国英, 杨秀兰, 等. 青海地区206例藏、汉族丙型肝炎患者基因分型及临床特征分析[J]. 高原医学杂志, 2018, 108(1): 18.
    [8] 徐辉, 杨伟国, 李永红. 甘肃地区藏族丙型肝炎患者的HCV基因分型研究[J]. 国际检验医学杂志, 2012, 33(17): 2081.
    [9] 张彦芳, 赵冬梅, 张彦敏, 等. 邢台地区HCV基因型分布特征及其与疾病进展和持续病毒学应答的关系[J]. 临床肝胆病杂志, 2015, 31(11): 111.
    [10] KOMOLMIT P, CHAROENSUK K, THANAPIROM K, et al. Correction of vitamin D deficiency facilitated suppression of IP-10 and DPP Ⅳ levels in patients with chronic hepatitis C: A randomised double-blinded, placebo-control trial[J]. PLoS One, 2017, 12(4): e0174608. doi: 10.1371/journal.pone.0174608
    [11] 崔彭华, 李志艳, 张玉娟, 等. 卵巢癌患者外周血Treg细胞和Th1、Th2细胞因子的变化及其与临床病理特征的关系[J]. 江苏大学学报(医学版), 2019, 29(2): 66.
    [12] CACHEM FC, DIAS AS, MONTEIRO C, et al. The proportion of different IL-17-producing T cell subsets is associated with liver fibrosis in chronic hepatitis C[J]. Immunology, 2017, 151(2): 167. doi: 10.1111/imm.12720
    [13] GHONIM MA, JEFFREY W, IBBA SV, et al. Sulfated non-anticoagulant heparin blocks Th2-induced asthma by modulating the IL-4/signal transducer and activator of transcription 6/Janus kinase 1 pathway[J]. J Trans Med, 2018, 16(1): 243.
    [14] 杨丽, 吕晓东, 刘妍彤, 等. 参龙煎剂干预肺纤维化大鼠血清IFN-γ、IL-4表达水平影响的实验研究[J]. 中华中医药学刊, 2017, 36(6): 84
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Study on the relationships between the genotyping and disease severity, expression of immunocompetent cells in patients with hepatitis C

  • 1. Department of Laboratory, Qinghai Fourth People's Hospital, Xining Qinghai 810000, China
  • 2. Department of Liver Disease, Qinghai Fourth People's Hospital, Xining Qinghai 810000, China
  • Received Date: 2019-11-26
  • Accepted Date: 2020-05-23
  • Available Online: 2022-04-15

Abstract: ObjectiveTo detect the relationships between the genotype of hepatitis C virus(HCV) and severity of the disease, expression of immunocompetent cells in patients with chronic hepatitis C(CHC).MethodsFrom June 2017 to July 2018, 169 Tibetan CHC patients were selected as the study objects(CHC group), and 170 Tibetan healthly examinees in Qinghai were selected as the control group.The levels of HCV-RNA in all cases were detected using PCR fluorescence probe, the PCR reverse hybridization was used to detect the HCV genotyping, and the levels of serum interferon-γ(INF-γ) and interleukin-4(IL-4) were measured by enzyme-linked immunosorbent assay(ELISA).The relationships between the genotype distribution, and disease severity, serum levels of INF-γ and IL-4;and between disease severity, and levels of INF-γ and IL-4 were analyzed.ResultsCompared with the control group, the positive rate of HCV-RNA and serum level of IL-4 increased, and the serum level of INF-γ decreased in CHC group(P < 0.01).The positive rates of HCV-RNA in severe chronic hepatitis group and acute hepatitis group were higher than that in mild and moderate chronic hepatitis group(P<0.05).The differences of the positive rates of HCV-RNA among different genotypes and different severity of CHC were statistically significant(P < 0.01).Compared with the HCV genotype 1a, the serum levels of INF-γ in patients with genotype 2a and 3b increased(P < 0.01).Compared with the HCV genotype 1b, the serum level of INF-γ increased(P < 0.01), and the serum level of IL-4 level decreased in genotype 2a(P < 0.01).The serum level of INF-γ in genotype 3b decreased compared with the genotype 2a(P<0.05).The differences of the serum levels of INF-γ and IL-4 among different severity of CHC were statistically significant(P < 0.01).There was no statistical significance in the levels of INF-γ between mild and moderate group, and between moderate and severe group(P>0.05).The serum level of INF-γ in the mild group was higher than that in severe group, it decreased gradually in moderate group, severe group and acute group, and the differences of which were statistically significant(P < 0.01).The serum level of IL-4 increased gradually in the mild group, moderate group, severe group and acute group, and the differences of which were statistically significant(P < 0.01).ConclusionsFour subtypes of HCV(1a, 1b, 2a and 3b) are detected in Tibetan patients with hepatitis C in Qinghai area, the proportions of 1b and 2a are high.The genotype 1b is closely related to the severity of the disease, the level of INF-γ decreases, and the level of IL-4 increases.

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  • 丙型肝炎(chronic hepatitis C，CHC)属传染性疾病，呈全球性，且受地域影响，表现的典型症状不尽相同，同一地区不同民族均可出现丙型肝炎病毒(hepatitis C virus，HCV)易感[1]。HCV RNA作为诊断CHC的“金标准”，在检测CHC中得到广泛应用[2]，但受不同基因型影响，诊断可能出现偏差。了解不同基因型与疾病严重程度的关系，对于诊断、分型治疗更有意义[3]。免疫活性T细胞在CHC病人感染中发挥重要作用，增强特异性相关T细胞比例可提高CHC治疗比例，但免疫活性细胞与HCV分型关系尚不明确。本研究通过检测青海地区藏族CHC病人HCV基因型，分析HCV基因型与疾病严重程度及免疫活性细胞表达的关系，以期为诊断CHC提供一定的临床依据。现作报道。

1.   资料与方法

    1.1.   一般资料

  • 选取2017年6月至2018年7月我院收治的169例青海地区藏族CHC病人(CHC组)为研究对象，其中男116例，女53例，年龄30~52岁，平均(41.16±6.15)岁；同期选取170名青海地区藏族健康体检者作为对照组，其中男120名，女50名，年龄31~53岁，平均(41.66±8.32)岁。2组性别、年龄一般资料均具有可比性。CHC诊断根据2015年中华医学会肝病学分会、中华医学会感染病学分会制定的《丙型肝炎防治指南》的诊断标准[4]。纳入标准：(1)符合CHC诊断标准；(2)藏族；(3)首次确诊；(4)无其他免疫缺陷类疾病。排除标准：(1)青海地区非藏族病人；(2)使用过药物治疗病人；(3)存在明显免疫缺陷病人；(4)伴有严重药物过敏者。本研究获病人及家属知情同意，本研究获本院伦理协会审核并通过。将CHC病人按照上述诊断标准分为慢性肝炎142例(轻度60例、中度46例、重度36例)，急性肝炎27例。

    • 1.2.   主要试剂与仪器

  • 人HCV-Ab酶联免疫吸附(ELISA)试剂盒(上海荣盛生物药业有限公司，货号：20170352)，PCR-荧光探针检测试剂盒(东北制药集团股份有限公司，货号：20153400239)；PCR-反向杂交试剂盒(中山达安基因股份有限公司，货号：20153402110)。人干扰素-γ(interferon-γ，INF-γ)、白细胞介素-4(interleukin-4，IL-4) ELISA试剂盒(美国abcam，货号分别为：ab108743、ab215089)。AU5800型全自动生化分析仪(美国Beckman)，DOF9648型全自动核酸提取仪(TIANFEN)，StepOneTM实时荧光定量(qRT-PCR)仪(美国应用生物系统公司)，HBHM-9000A型全自动核酸分子杂交仪(广东凯普生物科技股份有限公司)。

    • 1.3.   检测方法

  • 病人住院当日或体检者早晨空腹用促凝管采集静脉血约5 mL，对静脉血进行血清分离，血样3 000 r/min离心10 min，收集血清后分装，置于-20 ℃中待测。从-20 ℃冰箱中取出血清，冰上融化，采用ELISA试剂盒对样品初检；PCR-荧光探针检测(qRT-PCR法)检测HCV-RNA水平，检测下限为25 IU/mL，线性范围100~108 IU/mL；高于HCV-RNA检测下限的样品行PCR-反向杂交法对HCV病毒基因分型，可检测6种基因型(1a、1b、2a、3a、3b、6a)，本研究检测出4种基因型(1a、1b、2a、3b)。所有步骤均按试剂盒说明书进行。从-20 ℃冰箱中另取血清，严格按照INF-γ、IL-4 ELISA说明书操作进行，检测待检样品INF-γ、IL-4和标准品，450 nm波长处测定各孔的吸光度值(OD值)。以标准品浓度为横坐标、对应OD值为纵坐标绘制标准品线性回归曲线，按曲线方程计算各样本INF-γ、IL-4浓度。

    • 1.4.   统计学方法

  • 采用t(或t′)检验、方差分析、q检验和χ2检验。

2.   结果

    2.1.   2组血清中HCV-RNA比较

  • 与对照组(14.12%)比较，CHC组(73.40%)HCV-RNA阳性率升高，差异有统计学意义(χ2=154.94，P < 0.01)。

    • 2.2.   2组血清中INF-γ、IL-4比较

  • 与对照组比较，CHC组血清中INF-γ水平降低，IL-4水平升高，差异均有统计学意义(P < 0.01)(见表 1)。

    分组 n INF-γ/(IU/mL) IL-4/(IU/mL)
    对照组 170 4.21±0.36 23.62±1.52
    CHC组 169 3.16±0.25 24.56±1.69
    t — 31.21* 5.38
    P — < 0.01 < 0.01
    *示t′值

    • 2.3.   CHC不同严重程度病人HCV-RNA比较

  • CHC不同严重程度病人HCV-RNA阳性率比较差异有统计学意义(P < 0.01)，与慢性肝炎轻、中度组比较，慢性肝炎重度组、急性肝炎组HCV-RNA阳性率升高(P<0.05)(见表 2)。

    分组 n HCV-RNA(+) HCV-RNA(-)
    慢性肝炎轻度组 60 44(73.33) 16(26.67)
    慢性肝炎中度组 46 34(73.91) 12(26.08)
    慢性肝炎重度组 36 34(94.44) 2(5.56)*#
    急性肝炎组 27 26(96.30) 1(3.70)*#
    χ2 — 12.41
    P — < 0.01
    χ2分割检验：与慢性肝炎轻度组比较*P<0.05；与慢性肝炎中度组比较#P<0.05

    • 2.4.   基因型、亚型分布与CHC不同严重程度病人HCV-RNA阳性的关系

  • 不同基因型、亚型分布的病人在CHC不同严重程度分组中HCV-RNA阳性率差异有统计学意义(P < 0.01)(见表 3)。

    HCV基因型 n 慢性肝炎轻度组(n=44) 慢性肝炎中度组(n=34) 慢性肝炎重度组(n=34) 急性肝炎组(n=26)
    1a 4 2(50.00) 1(25.00) 0(0.00) 1(25.00)
    1b 81 16(19.75) 21(25.93) 21(25.93) 23(28.40)
    2a 50 23(46.00) 12(24.00) 13(26.00) 2(4.00)
    3b 3 3(100.00) 0(0.00) 0(0.00) 0(0.00)
    χ2 — 24.68
    P — < 0.01

    • 2.5.   基因型、亚型分布与INF-γ、IL-4水平的关系

  • 不同基因型病人INF-γ、IL-4水平差异有统计学意义(P < 0.01)。与HCV基因型1a比较，基因型2a、3b病人血清中INF-γ水平升高(P < 0.01)；与HCV基因型1b比较，基因型2a血清中INF-γ水平升高(P < 0.01)，IL-4水平降低(P < 0.01)；与HCV基因型2a比较，基因型3b血清中INF-γ水平降低(P < 0.01)(见表 4)。

    HCV基因型 n INF-γ/(IU/mL) IL-4/(IU/mL)
    1a 4 2.16±0.12 24.84±1.06
    1b 81 2.04±0.15 24.73±0.59
    2a 50 3.45±0.21**△△ 24.26±0.84△△
    3b 3 2.90±0.16**△△## 24.68±0.56
    F — 688.08 4.84
    P — < 0.01 < 0.01
    MS组内 — 0.030 0.496
    q检验：HCV与基因型1a比较**P < 0.01；HCV与基因型1b比较△△P < 0.01；HCV与基因型2a比较##P < 0.01

    • 2.6.   CHC不同严重程度HCV-RNA阳性与INF-γ、IL-4的关系

  • CHC不同严重程度INF-γ、IL-4水平差异有统计学意义(P < 0.01)。INF-γ水平，慢性肝炎轻度组和中度组、中度组和重度组，差异无统计学意义(P>0.05)，轻度组>重度组、中度组>重度组>急性组，差异均有统计学意义(P < 0.01)；IL-4水平，轻度组<中度组<重度组<急性组，差异均有统计学意义(P < 0.01)(见表 5)。

    分组 n INF-γ/(IU/mL) IL-4/(IU/mL)
    慢性肝炎轻度组 44 3.47±0.51 24.03±0.61
    慢性肝炎中度组 34 3.28±0.48 24.46±0.43**
    慢性肝炎重度组 34 3.08±0.43** 24.71±0.59**
    急性肝炎组 26 2.65±0.65**△△## 25.26±0.48**△△##
    F — 14.71 29.65
    P — < 0.01 < 0.01
    MS组内 — 0.265 0.294
    q检验：与慢性肝炎轻度组比较**P < 0.01；与慢性肝炎中度组比较△△P < 0.01；与慢性肝炎重度组比较##P < 0.01
3.   讨论

  • CHC为传染性疾病，全球影响严重，且HCV易感，促进HCV基因型G到A和C到U的诱导，对于治疗CHC意义重大[5]。CHC存在不同基因型对治疗造成一定困难，了解具体HCV基因型与疾病严重程度的关系可针对性治疗疾病。CHC病人病情亦与免疫相关，特别是细胞免疫发挥重要作用[6]。不同HCV基因型与免疫相关因子之间的关系对于疾病诊断是否存在关联是本研究重点。

    HCV有1~6基因型，50多种亚型，高度变异，受国家、地区甚至民族的影响，中国有1a、1b、2a、3a、3b、6a六种亚型[7]。其中1a是第一个被鉴定的HCV基因型；亚洲地区1b型较多；2a在北美、中国、日本等地均有分布；3b在中国分布较少[8]，本研究在青海地区发现1a、1b、2a、3b四种亚型，证明青海地区HCV出现多样化现象。可能受地域或样本量影响，本次未检测到3a、6a HCV基因型。在本研究中CHC病人中HCV阳性率达73.40%，其中HCV 1a型4(2.90%)例、HCV 1b型81(58.70%)例、HCV 2a型50(36.23%)例、HCV 3b型3(2.17%)例。HCV基因型比例可能受人口流动、不良习惯等多方面影响存在差异。不同疾病严重程度与HCV基因型关系密切，肝病中1a、1b型病毒载量明显高于2型、3型，1b型病人进展为肝硬化和肝癌的比例较高，2a型治疗后免疫应答概率较高[9]。不同基因型、亚型分布的病人在CHC不同严重程度分组中HCV-RNA阳性率差异有统计学意义。1a、1b型影响病毒载量；其中1a型导致病情恶化，疾病加重；1b型随着疾病严重比例逐渐升高，可能影响疾病进程。

    Th1/Th2比例失衡是导致CHC发病的主要原因之一，Th1、Th2各因子水平可反映机体免疫状态[10]。INF-γ作为Th1代表性细胞因子，可反映Th1在Th1/Th2中的比例状况。在卵巢癌等肿瘤中INF-γ水平降低可反映Th1状况[11]；在CHC晚期肝损伤中INF-γ比例降低，从而降低IL-17比例，促进疾病严重程度[12]。IL-4作为Th2代表性因子，可通过调节IL-4水平来调节Th2水平[13]。INF-γ/IL-4水平可反映Th1/Th2水平[14]。本研究发现，与对照组比较，CHC组血清中INF-γ水平降低，IL-4水平升高。不同HCV基因型比较发现，HCV 1b型血清中INF-γ水平最低，IL-4水平较高，低水平INF-γ、高水平IL-4可能促进HCV诱导成1b型从而影响疾病。同时发现随着疾病严重，血清中INF-γ水平降低，IL-4水平升高，INF-γ、IL-4水平与疾病严重程度有关，可能由HCV诱导成1b型引起。

    综上所述，青海地区藏族CHC病人发现HCV(1a、1b、2a、3b)四种亚型，其中1b亚型与疾病严重程度、Th1/Th2关系密切，推测Th1/Th2向Th2偏移使HCV诱导成1b型，从而加重疾病进程。但本研究并未进一步验证HCV 1b型与Th1/Th2的直接关系，且影响Th1、Th2因子众多，亦可能受别的因子影响，这是笔者接下来研究的重点。

Reference (14)

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