免疫库谱作为慢性乙型肝炎疗效预测生物标志物的研究

发布时间：2018-07-03 18:56

本文选题：乙型肝炎病毒 + 细胞毒性T细胞　；参考：《第三军医大学》2017年硕士论文

【摘要】：目的T细胞应答为主的细胞免疫在HBV感染的发生发展中发挥着重要并且复杂的作用[1-2]。一方面,它们通过杀伤HBV感染的肝细胞来清除病毒,促进感染消退,疾病痊愈,另一方面,它们可以通过旁临细胞效应或者耗竭对正常的肝脏组织形成损害,从而造成慢性炎症,进而发展成为肝硬化甚至肝癌[3-4]。T细胞的抗原受体库数量可达1×1011到1×1014,并且具有非常丰富的多样性,其在整体水平表现出来的状态反应了机体对于多种抗原的潜在应答能力,同时也反映了机体曾经的抗原暴露史和免疫调节的结果。而在慢性乙型肝炎的病程中,HBsAg常常作为病人是否痊愈的标志,其与乙肝病毒的cccDNA呈密切的联系,而cccDNA又是介导乙肝复发的关键因素。因此,研究HBsAg转阴相关的T细胞免疫库谱意义深远[5]。高通量测序技术的应用极大地提升了对于大样本数据的获取能力,通过对病人外周血T细胞中TCR mRNA进行测序,可以高效准确地了解T细胞库谱的特征。本研究就通过对慢性乙型肝炎患者与健康对照外周血中T细胞受体β链(TCRβ,TRB)序列分析比较,探讨如下问题:1.阐述HBV慢性感染时CD8+T细胞受体库(TCR repertoire)的特征;2.寻找HBsAg血清学转换相关的CD8+T细胞受体库的特征,以期作为预测临床疗效以及评价治疗效果的指标。方法1.选择HBV感染病人及健康对照,收集其外周血,采用密度梯度离心法获取淋巴细胞,液氮冻存。常规治疗76周并随访检测病毒学和生化指标,挑选HBV感染组61人及健康对照组40人进行对比研究。取出入组者的冻存的细胞,复苏后提取RNA并将其逆转录为cDNA,采用多重PCR法对其TCRβ链CDR3区(TRB-CDR3)序列进行扩增及纯化,采用高通量测序技术获得序列信息后,将其输入IMGT数据库比对,通过组间对照,获得HBV慢性感染病人外周血TRB-CDR3免疫组库(immune repertoire)的特征,分析其多样性及其他特征,并且找到HBV感染相关的T细胞克隆型。2.选择HBV感染病人及健康对照,收集其外周血,采用密度梯度离心法获取淋巴细胞,液氮冻存。常规治疗76周并随访检测病毒学和生化指标,挑选HBsAg未转阴组27人和转阴组34人进行对比研究。取出入组者的冻存的细胞,复苏后提取RNA并将其逆转录为cDNA,采用多重PCR法对其TCRβ链CDR3区(TRB-CDR3)序列进行扩增及纯化,采用高通量测序技术获得序列信息后,将其输入IMGT数据库比对,通过组间对照,分析人外周血TRB-CDR3多样性等特征与HBsAg转阴之间的关系,探索TCR repertoire是否能够作为HBsAg转阴的预测标志物。同时,为了研究不同治疗方式导致HBsAg转阴时TCR repertoire的差异,以期探索免疫组库是否能作为患者选择治疗方法时的参考标准,我们将HBsAg转阴组进一步分成了自发转阴组和IFN治疗转阴组,以研究包括未转阴组在内的三组病人的TCR repertoire情况。另外,为了初步探讨TCR repertoire作为HBsAg转阴预测标志物的机制,我们对两组病人的T细胞进行了体外培养,检测抗原特异性T细胞的功能;并且,对外周血总T细胞进行了RNA测序,检测其基因表达情况。结果1.HBV慢性感染患者组的reads数明显高于健康对照;慢性感染组的TCR组库多样性明显低于健康对照组;虽然CDR3的平均长度没有差异,但是感染组的某些个体的CDR3长度呈现偏态分布;N端去除或者添加的碱基数没有差异;V、J基因的取用频率以及其组合没有差异。此外,我们找到了若干HBV感染相关的TCR克隆型,这些克隆在很多HBV慢性感染的机体中共同存在,而不存在于健康个体中。2.HBsAg转阴组相较于未转阴组,其CDR3的长度,插入碱基数量,V、J基因之间碱基总长度(ndn size),V、J基因的取用分布以及其组合等均无差异,但是从CDR3的分布图可以看出,HBsAg转阴组呈现正态分布,而未转阴组呈偏态分布。HBsAg转阴组的免疫组库多样性明显高于未转阴组。通过比较发现,自发转阴组和IFN诱导的转阴组之间各项指标均无差异。克隆型聚类分析也显示,这两组的克隆型基本类似,都明显区别于未转阴组和健康对照组。对各转阴组以及未转阴组的抗原特异性T细胞的功能分析没有发现统计学差异。但是对于总的T细胞进行转录组分析,发现HBsAg转阴组的T细胞活化以及效应相关基因的表达高于未转阴组。结论1.相较于健康对照,HBV慢性感染患者体内的T细胞长期受到抗原刺激,因此处于增殖状态,导致其CDR3长度呈现偏态分布,免疫组库的多样性也发生明显降低。经过长期的抗原刺激,其体内的确存在有HBV感染相关的克隆型。有些克隆型在HBV感染病人中广泛存在,其在病人群体中的分布率甚至高达59%。2.HBsAg转阴组的多样性明显高于未转阴组,并且其CDR3长度分布几乎全部处于正态分布,这提示其增殖水平低于未转阴组,可能是由于感染初期该组患者发生了快速有力的免疫反应,及时清除了HBV,使得整体的炎症水平明显降低所致。通过比较各种repertoire指标和克隆型聚类分析,未发现IFN诱导转阴组和自发转阴组有差异,因此,我们认为在免疫组库水平上不能分辨二者的差异。就目前的检测手段及指标来看,免疫组库检测尚不能作为患者选择治疗方式的参考指标。对抗原特异性T细胞的功能检测结果可知,各治疗方式致HBsAg转阴组及未转阴组患者之间的抗原特异性T细胞的功能无明显差异,这提示可能有其他机制参与转阴过程。总T细胞的RNA sequencing检测提示,就T细胞总体来看,转阴组患者体内的T细胞功能应该更加活跃。
[Abstract]:Objective T cell response based cellular immunity plays an important and complex role in the development and development of HBV infection. On the one hand, they can clear the virus by killing the hepatocytes infected by HBV, promote the infection and cure the disease. On the other hand, they can form the normal liver through the paracellular effect or exhaustion. The number of antigen receptor libraries in the [3-4].T cells of liver cirrhosis and even liver cancer can reach 1 * 1011 to 1 * 1014, and has a very rich diversity, which reflects the body's potential response to a variety of antigens, and also reflects the body's once. In the course of chronic hepatitis B, in the course of chronic hepatitis B, HBsAg is often used as a symbol of the recovery of the patients, which is closely related to the cccDNA of hepatitis B virus, and cccDNA is the key factor for the recurrence of HBV. Therefore, the study of the T cell immune Library related to the HBsAg conversion related to a far-reaching [5]. high throughput test The application of sequence technology greatly improves the acquisition ability of large sample data. By sequencing the TCR mRNA in the peripheral blood T cells of the patient, the characteristics of the T cell pool spectrum can be effectively and accurately understood. This study compares the sequence analysis of the T cell receptor beta chain (TCR beta, TRB) sequence of the chronic hepatitis B patients and the healthy control peripheral blood of the peripheral blood. The following questions are discussed: 1. the characteristics of CD8+T cell receptor Library (TCR repertoire) in HBV chronic infection are described; 2. to find the characteristics of CD8+T cell receptor library related to HBsAg serological conversion, in order to predict the clinical efficacy and evaluate the therapeutic effect. Method 1. select HBV infected patients and healthy controls, collect peripheral blood and adopt density. Lymphocyte and liquid nitrogen cryopreservation were obtained by gradient centrifugation. 76 weeks of routine treatment and follow-up examination of Virology and biochemical indexes, 61 people in HBV infection group and 40 people in healthy control group were selected and 40 people were compared. The frozen cells in and out of the group were taken and RNA was extracted after resuscitation and then turned to cDNA, and the TCR beta chain CDR3 region (TRB-CDR3) sequence was determined by multiple PCR method. The column was amplified and purified. After the sequence information was obtained by high throughput sequencing technology, the column was compared with the IMGT database. The characteristics of the peripheral blood TRB-CDR3 immune group Library (immune repertoire) of the patients with HBV chronic infection were obtained by comparison between the groups. The diversity and other characteristics were analyzed, and the selection of the T cell cloned.2. selection related to HBV infection was found. HBV infected patients and health control, collect the peripheral blood, use density gradient centrifugation to obtain lymphocytes and liquid nitrogen cryopreservation. Routine treatment for 76 weeks and follow up the virology and biochemical indexes, select 27 people in the group of non negative Yin group and 34 people in Yin group, take the frozen cells from the group, extract RNA after resuscitation and reverse it. CDNA, using multiple PCR method to amplify and purify its TCR beta chain CDR3 region (TRB-CDR3) sequence, and use high throughput sequencing technology to obtain sequence information, to compare it into IMGT database, and to analyze the relationship between the characteristics of human peripheral blood TRB-CDR3 diversity and HBsAg conversion by comparison between groups, and explore whether TCR repertoire can be made. At the same time, in order to study the difference of TCR repertoire at the time of different treatments that lead to the change of the TCR repertoire, and to explore whether the immune group can be used as a reference standard for the choice of treatment for patients, we have further divided the HBsAg group into the spontaneous negative group and the IFN treatment group to study including the non conversion of Yin group. In the group of three patients, the TCR repertoire situation. In addition, in order to preliminarily explore the mechanism of TCR repertoire as a predictor of HBsAg conversion, we have cultured the T cells of the two groups of patients in vitro to detect the function of the antigen specific T cells. Moreover, the total T cells of the peripheral blood were sequenced and the gene expression was detected. The reads number of 1.HBV patients with chronic infection was significantly higher than that in the healthy control group. The diversity of TCR group in the chronic infection group was significantly lower than that in the healthy control group. Although the average length of the CDR3 was not different, the CDR3 length of some individuals in the infected group showed a partial distribution; the N terminal removal or addition of alkali base was not different; the frequency of V, J gene was used frequency. In addition, we found a number of TCR clones associated with HBV infection. These clones are common in many chronic HBV infected organisms, but not in the healthy individuals of the.2.HBsAg negative group than in the non negative group. The length of the CDR3, the number of base inserts, the total base length of the V, the J gene (NDN size), V, is not found in the healthy individuals. The distribution of J gene and its combination were not different, but the distribution map of the CDR3 showed that the HBsAg turned negative group showed a normal distribution, while the diversity of the immune group of the.HBsAg negative group in the unturned group was significantly higher than that in the unturned group. Cloned cluster analysis also showed that the clones of the two groups were basically similar to those in the non negative group and the healthy control group. There was no statistical difference in the functional analysis of the antigen specific T cells in the negative group and the non negative group. However, the total T cells were analyzed by the transcriptional analysis, and the T fine of the HBsAg negative group was found. The expression of cell activation and effect related genes was higher than that in the unturned group. Conclusion 1. of the T cells in the patients with HBV chronic infection were stimulated by the antigen for a long time compared to the healthy control. Therefore, the proliferation state resulted in the partial distribution of the CDR3 length and the decrease of the diversity of the immune group. After a long time of antigen stimulation, the body was in the body. There do exist clones associated with HBV infection. Some clones exist widely in patients with HBV infection, and their distribution in the group of patients is even higher than that in the group of 59%.2.HBsAg negative, and the distribution of the length of CDR3 is almost all in normal distribution, which suggests that the proliferation level is lower than that in the non negative group. It was due to the rapid and powerful immune response of the group in the early stage of infection, the HBV was cleared in time, and the overall inflammatory level was significantly reduced. By comparing various repertoire indicators and cloned cluster analysis, there was no difference between the IFN induced shift group and the spontaneous negative group. Therefore, we think that the level of the immune group is not on the level of the immune group. To distinguish the differences between the two, the detection of the immune group can not be used as a reference index for the treatment of the patients. The function test results against the original specific T cells showed that there was no significant difference in the function of the antigen specific T cells between the HBsAg conversion group and the non negative group. This suggests that there may be other mechanisms involved in the turning of the shade. The RNA sequencing detection of total T cells suggests that, as a whole, the function of T cells in the group of negative groups should be more active in T cells.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

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