CHB患者外周血TCR CDR3谱型特征及与干扰素近期疗效的关系

发布时间：2018-04-05 07:46

  本文选题：基因型　切入点：慢性乙型肝炎　出处：《新乡医学院》2014年硕士论文

【摘要】：背景 慢性乙型肝炎(chronic hepatitis B, CHB)的发病机制目前普遍认为与免疫损伤有关。干扰素用于CHB治疗时,具有抗病毒和免疫调节的双重作用,病人的感染状态和基础免疫状况可能决定其治疗的疗效和稳定性,但其机制尚不清楚。T细胞受体(T cell receptor, TCR)互补决定区3(complementarity determining region3,CDR3)谱型分析技术是一项很好的反映机体细胞免疫功能的新方法。本研究采用该技术分析CHB患者基线状态的细胞免疫功能情况,并探讨其与干扰素治疗早期病毒学应答的关系。 目的 通过对入组患者的血清HBV基因型进行检测,初步了解本地区HBV基因型的分布情况；利用实时荧光定量PCR溶解曲线分析技术了解不同基因型CHB患者基线状态的T细胞克隆性增生情况,探讨基线状态的细胞免疫功能与干扰素抗病毒治疗近期疗效的关系,以了解基础免疫状况对干扰素抗病毒近期疗效的评估作用,指导临床治疗。 方法 (1)选取2013年4月至2013年10月在新乡医学院第一附属医院感染内科住院接受干扰素-α治疗的CHB患者23例,治疗前留取外周静脉血5m1,肝素钠抗凝,采用密度梯度离心法分离血清和外周血单个核细胞(peripheral blood mononuclearcell,PBMC)；(2)采用S基因测序法检测HBV基因型：根据文献设计并合成所用引物,提取血清中的HBV-DNA用于PCR扩增模板,扩增产物进行琼脂糖凝胶电泳和S基因区测序,测序结果用DNASTAR处理,并与GenBank/EMBL/DDBJ数据库中的A-G7种HBV基因型参考序列进行比较,进化树分析用MEGA5.1软件,确定出基因型；(3)提取PBMC总RNA,逆转录为cDNA,设计并合成TCRβ链24个可变区基因(BV)家族上游引物,并在p链恒定基因区(BC)设计一条共同的无荧光标记的下游引物。采用荧光定量PCR扩增TCR各BV家族CDR3区基因,溶解曲线分析各样本PCR产物形成的24个TCR BV家族溶解曲线谱型图,溶解后的产物重新短暂扩增后,扩增产物进行1.5%的琼脂糖凝胶电泳分析,了解各BV家族的表达情况；(4)同时观察基线及治疗3个月HBV-DNA、肝功能水平变化;(5)统计学分析应用PSS16.0软件进行,计量资料以均数±标准差(x±s)或中位数/四分位数间距(M/QR)表示,对数据进行独立样本t检验、配对样本比较Wilcoxon符号秩检验、两独立样本比较的Wilcoxon符号秩检验,P0.05为差异有统计学意义。 结果 (1)入组的23例CHB患者中,C2基因型21例(91.3%),B2基因型1例(4.34%),D基因型1例(4.34%)。 (2)23例CHB患者基线时外周血TCR CDR3各BV家族谱系在溶解曲线谱型图上均表现不同程度的谱型偏移,出现单峰、寡峰及偏峰图,多数家族表现为多峰图,小部分家族由于表达频率极低或缺失表现为无峰图。对照的GAPDH表现为明显的单峰图形,阴性对照无熔解曲线峰。扩增产物在1.5%的琼脂糖凝胶上电泳,结果显示多数BV家族均有表达,在预测位置处显示一条模糊的条带,部分家族表达增强或缺失,显示一条清晰条带或条带缺失。GAPDH对照呈现单条带,阴性对照无条带。 (3)23例CHB患者干扰素-a治疗3个月与基线比较,血清谷丙转氨酶(alanine aminotransferase, ALT)、谷草转氨酶(glutamic-oxaloacetic transaminase, AST/GOT)水平及HBV-DNA载量均明显下降(P0.01)；根据谱型分析结果,将21例C基因型CHB患者分为TCR CDR3高谱型偏移率组(谱型偏移率≥35%)和低谱型偏移率组(谱型偏移率35%)。TCR CDR3高谱型偏移率组与低谱型偏移率组比较,干扰素治疗过程中HBV-DNA载量下降幅度更明显(P0.05), ALT、AST下降水平间差异均无显著性(P0.05)。 结论 本地区HBV基因型以C2基因型为主,极少数为B型或D型；不同基因型的CHB患者基线状态外周血T淋巴细胞存在不同程度的克隆性增生；C基因型CHB患者中基线外周血T细胞表现为高克隆性增生的感染者在干扰素抗病毒治疗过程中HBV-DNA载量下降幅度更明显,此可能与干扰素治疗较好的近期疗效相关。
[Abstract]:Background

The pathogenesis of chronic hepatitis B and chronic hepatitis B is generally considered to be related to immune injury . When the interferon is used in the treatment of chronic hepatitis B , it has the double effect of anti - virus and immunoregulation . The infection state and the basal immune status of the patient may determine the therapeutic effect and stability of the patients . The technique is used to analyze the cellular immune function of the patients with chronic hepatitis B . The relationship between the immune function and the early virological response is discussed .

Purpose

The distribution of HBV genotypes in the region was preliminarily determined by the detection of HBV genotypes in patients with enrolled patients .
Using real - time fluorescence quantitative PCR dissolution curve analysis technique to understand the T cell clonal expansion of different genotypes and patients with different genotypes , the relationship between the cellular immune function of baseline status and the short - term efficacy of interferon antiviral therapy was discussed to understand the effect of basal immune status on the short - term efficacy of interferon antiviral therapy , and to guide clinical treatment .

method

( 1 ) From April 2013 to October 2013 , 23 patients who received interferon - 伪 were treated with interferon - 伪 in the First Affiliated Hospital of Xinxiang Medical College from April 2013 to October 2013 . Peripheral blood mononuclear cells ( PBMC ) were isolated from peripheral blood mononuclear cells ( PBMC ) by density gradient centrifugation .
( 2 ) detecting HBV genotypes by using S gene sequencing method : designing and synthesizing the primer according to the document , extracting HBV - DNA in the serum for PCR amplification template , carrying out agarose gel electrophoresis and S gene region sequencing , sequencing the amplified products by DNASTAR , and comparing with the reference sequence of the A - G7 type HBV genotype in the GenBank / EMBL / DDBJ database , and determining the genotype by using the MEGA5.1 software ;
( 3 ) The total RNA of PBMC was extracted , and the upstream primer of TCR 尾 chain 24 variable region genes ( BV ) was designed and synthesized .
( 4 ) The baseline and 3 months of HBV - DNA and liver function changes were observed at the same time ; ( 5 ) Statistical analysis was performed using PSS16 . 0 software . The measurement data was expressed by mean 卤 standard deviation ( x 卤 s ) or median / quartile range ( M / QR ) . The data was subjected to independent sample t test . The paired samples were compared with Wilcoxon signed rank test , and the Wilcoxon signed rank test between two independent samples was statistically significant .

Results

( 1 ) Of the 23 patients , the C2 genotype was 21 ( 91.3 % ) , B2 genotype was 1 ( 4.34 % ) , genotype D was 1 ( 4.34 % ) .

( 2 ) Twenty - three patients with hepatitis B showed a different degree of spectral shift on the spectrum of dissolution profiles . There were single peaks , few peaks and partial peaks . Most of the families showed multiple peaks . The results showed that most of the families of BV had a single peak pattern , and the negative control showed no melting curve . The results showed that most BV families were expressed and a clear band or band deletion was displayed at the predicted position .

( 3 ) The levels of alanine aminotransferase ( ALT ) , aspartate - oxaloacetic transaminase ( AST / GOT ) and HBV - DNA were significantly decreased ( P0.01 ) .
According to the results of the spectral analysis , 21 patients with genotype C were divided into three groups with high spectral type ( 鈮，

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