本文選題：乙型肝炎病毒 + 細胞毒性T細胞��； 參考：《第三軍醫(yī)大學》2017年碩士論文

【摘要】：目的T細胞應答為主的細胞免疫在HBV感染的發(fā)生發(fā)展中發(fā)揮著重要并且復雜的作用[1-2]。一方面,它們通過殺傷HBV感染的肝細胞來清除病毒,促進感染消退,疾病痊愈,另一方面,它們可以通過旁臨細胞效應或者耗竭對正常的肝臟組織形成損害,從而造成慢性炎癥,進而發(fā)展成為肝硬化甚至肝癌[3-4]。T細胞的抗原受體庫數(shù)量可達1×1011到1×1014,并且具有非常豐富的多樣性,其在整體水平表現(xiàn)出來的狀態(tài)反應了機體對于多種抗原的潛在應答能力,同時也反映了機體曾經(jīng)的抗原暴露史和免疫調(diào)節(jié)的結果。而在慢性乙型肝炎的病程中,HBsAg常常作為病人是否痊愈的標志,其與乙肝病毒的cccDNA呈密切的聯(lián)系,而cccDNA又是介導乙肝復發(fā)的關鍵因素。因此,研究HBsAg轉陰相關的T細胞免疫庫譜意義深遠[5]。高通量測序技術的應用極大地提升了對于大樣本數(shù)據(jù)的獲取能力,通過對病人外周血T細胞中TCR mRNA進行測序,可以高效準確地了解T細胞庫譜的特征。本研究就通過對慢性乙型肝炎患者與健康對照外周血中T細胞受體β鏈(TCRβ,TRB)序列分析比較,探討如下問題:1.闡述HBV慢性感染時CD8+T細胞受體庫(TCR repertoire)的特征;2.尋找HBsAg血清學轉換相關的CD8+T細胞受體庫的特征,以期作為預測臨床療效以及評價治療效果的指標。方法1.選擇HBV感染病人及健康對照,收集其外周血,采用密度梯度離心法獲取淋巴細胞,液氮凍存。常規(guī)治療76周并隨訪檢測病毒學和生化指標,挑選HBV感染組61人及健康對照組40人進行對比研究。取出入組者的凍存的細胞,復蘇后提取RNA并將其逆轉錄為cDNA,采用多重PCR法對其TCRβ鏈CDR3區(qū)(TRB-CDR3)序列進行擴增及純化,采用高通量測序技術獲得序列信息后,將其輸入IMGT數(shù)據(jù)庫比對,通過組間對照,獲得HBV慢性感染病人外周血TRB-CDR3免疫組庫(immune repertoire)的特征,分析其多樣性及其他特征,并且找到HBV感染相關的T細胞克隆型。2.選擇HBV感染病人及健康對照,收集其外周血,采用密度梯度離心法獲取淋巴細胞,液氮凍存。常規(guī)治療76周并隨訪檢測病毒學和生化指標,挑選HBsAg未轉陰組27人和轉陰組34人進行對比研究。取出入組者的凍存的細胞,復蘇后提取RNA并將其逆轉錄為cDNA,采用多重PCR法對其TCRβ鏈CDR3區(qū)(TRB-CDR3)序列進行擴增及純化,采用高通量測序技術獲得序列信息后,將其輸入IMGT數(shù)據(jù)庫比對,通過組間對照,分析人外周血TRB-CDR3多樣性等特征與HBsAg轉陰之間的關系,探索TCR repertoire是否能夠作為HBsAg轉陰的預測標志物。同時,為了研究不同治療方式導致HBsAg轉陰時TCR repertoire的差異,以期探索免疫組庫是否能作為患者選擇治療方法時的參考標準,我們將HBsAg轉陰組進一步分成了自發(fā)轉陰組和IFN治療轉陰組,以研究包括未轉陰組在內(nèi)的三組病人的TCR repertoire情況。另外,為了初步探討TCR repertoire作為HBsAg轉陰預測標志物的機制,我們對兩組病人的T細胞進行了體外培養(yǎng),檢測抗原特異性T細胞的功能;并且,對外周血總T細胞進行了RNA測序,檢測其基因表達情況。結果1.HBV慢性感染患者組的reads數(shù)明顯高于健康對照;慢性感染組的TCR組庫多樣性明顯低于健康對照組;雖然CDR3的平均長度沒有差異,但是感染組的某些個體的CDR3長度呈現(xiàn)偏態(tài)分布;N端去除或者添加的堿基數(shù)沒有差異;V、J基因的取用頻率以及其組合沒有差異。此外,我們找到了若干HBV感染相關的TCR克隆型,這些克隆在很多HBV慢性感染的機體中共同存在,而不存在于健康個體中。2.HBsAg轉陰組相較于未轉陰組,其CDR3的長度,插入堿基數(shù)量,V、J基因之間堿基總長度(ndn size),V、J基因的取用分布以及其組合等均無差異,但是從CDR3的分布圖可以看出,HBsAg轉陰組呈現(xiàn)正態(tài)分布,而未轉陰組呈偏態(tài)分布。HBsAg轉陰組的免疫組庫多樣性明顯高于未轉陰組。通過比較發(fā)現(xiàn),自發(fā)轉陰組和IFN誘導的轉陰組之間各項指標均無差異�？寺⌒途垲惙治鲆诧@示,這兩組的克隆型基本類似,都明顯區(qū)別于未轉陰組和健康對照組。對各轉陰組以及未轉陰組的抗原特異性T細胞的功能分析沒有發(fā)現(xiàn)統(tǒng)計學差異。但是對于總的T細胞進行轉錄組分析,發(fā)現(xiàn)HBsAg轉陰組的T細胞活化以及效應相關基因的表達高于未轉陰組。結論1.相較于健康對照,HBV慢性感染患者體內(nèi)的T細胞長期受到抗原刺激,因此處于增殖狀態(tài),導致其CDR3長度呈現(xiàn)偏態(tài)分布,免疫組庫的多樣性也發(fā)生明顯降低。經(jīng)過長期的抗原刺激,其體內(nèi)的確存在有HBV感染相關的克隆型。有些克隆型在HBV感染病人中廣泛存在,其在病人群體中的分布率甚至高達59%。2.HBsAg轉陰組的多樣性明顯高于未轉陰組,并且其CDR3長度分布幾乎全部處于正態(tài)分布,這提示其增殖水平低于未轉陰組,可能是由于感染初期該組患者發(fā)生了快速有力的免疫反應,及時清除了HBV,使得整體的炎癥水平明顯降低所致。通過比較各種repertoire指標和克隆型聚類分析,未發(fā)現(xiàn)IFN誘導轉陰組和自發(fā)轉陰組有差異,因此,我們認為在免疫組庫水平上不能分辨二者的差異。就目前的檢測手段及指標來看,免疫組庫檢測尚不能作為患者選擇治療方式的參考指標。對抗原特異性T細胞的功能檢測結果可知,各治療方式致HBsAg轉陰組及未轉陰組患者之間的抗原特異性T細胞的功能無明顯差異,這提示可能有其他機制參與轉陰過程�？俆細胞的RNA sequencing檢測提示,就T細胞總體來看,轉陰組患者體內(nèi)的T細胞功能應該更加活躍。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62

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相關期刊論文 前1條

1 王晨輝;劉明;姜瓊;唐小琴;董惠;尚小云;毛青;吳玉章;;IFN-α治療后發(fā)生HBsAg轉陰的慢性乙型肝炎患者外周血免疫庫譜特征分析[J];免疫學雜志;2017年04期

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本文編號：2094672