T细胞受体BV家族特异性表达与造血干细胞移植术后巨细胞病毒感染

发布时间：2018-01-26 05:54

本文关键词： T细胞受体 CDR3 人巨细胞病毒异基因造血干细胞移植术受者抗原血症　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:异基因造血干细胞移植(hematopoietic stem cell transplantation,HSCT)是白血病、淋巴瘤和重型再生障碍性贫血等血液系统疾病的有效治疗方法。然而,移植术后长期的免疫抑制疗法导致了免疫重建严重迟缓,持续的细胞免疫功能低下,导致感染。在众多感染因素中,人巨细胞病毒(HumanCytomegalovirus,HCMV)是异基因造血干细胞移植术后感染的常见病原体,是导致受者术后感染和死亡的重要原因之一。为长期抑制HSCT术后HCMV激活,早期诊断抢先治疗与受者自身免疫重建的恢复两者必不可少。HCMV抗原的检测为早期诊断及抢先治疗提供了重要依据,病毒磷蛋白65(phosphoprotein65,pp65)抗原,是HCMV病毒在复制过程中出现的抗原表达,可直接反映体内病毒的复制状态,对HCMV感染和HCMV病有早期诊断的作用,其水平的高低能帮助临床预测发生HCMV感染的可能性。除此之外,HSCT受者HCMV高感染率增加与术后免疫重建延迟同样密切相关,而细胞毒T淋巴细胞(CTLs)通过T细胞受体(T-cellreceptor,TCR)识别HCMV抗原产生特异性免疫反应是机体长期抵抗HCMV感染最重要的保护性机制。TCR通过位于TCRβ链可变区域(TCRBV)的互补决定区(CDR3)与抗原肽结合,在HCMV感染状态下,TCR和HCMV特异性抗原通过CDR3介导相互作用,CDR3序列会出现特异性频率,反映特定T细胞克隆扩增程度,同时也从一个侧面反映了 HSCT受者术后免疫重建的状况。研究表明病毒(如HBV、HIV)感染下,TCRBV出现特定的CDR3序列,本研究分析HSCT受者术后的HCMV感染状态,结合TCRBV家族分子特征分布,了解HCMV激活状态下HSCT受者体内的免疫应答状态,分析HSCT受者术后TCR表达与HCMV激活感染的关系。研究与HCMV激活相关的特定TCRBV家族或有助于早期诊断,为将来可能作为免疫治疗靶点打下基础。方法和目的:选择了 2009年8月至2012年6月在浙江大学附属第一医院接受HSCT受者7例,术后3个月分别采集新鲜外周血3～5 mL,抗凝,用淋巴细胞分离液提取外周血单个核细胞(peripheral blood mononuclear cells,PBMC)用于TCR检测和抗原血症检测。术后第3个月开始每月1次直至第12个月,共10次,每次抽取新鲜外周血约10 mL,分别用于HCMV-pp65抗原和HCMV-IgM、HCMV-IgG抗体检测。HCMV-pp65抗原检测采用免疫组化法,HCMV-IgM、HCMV-IgG抗体采用ELISA法进行检测,TCR BV家族测序分析采用荧光定量PCR溶解曲线法,统计分析HSCT术后TCRBV序列与HCMV感染指标的关系。结果:1.移植后第3个月,7例受者中抗原血症检测阳性5例,阴性2例,在术后3～12月随访期间,7例受者均出现过不同程度的HCMV-pp65抗原血症,HCMV-IgM阳性组的pp65抗原阳性率明显高于HCMV-IgM阴性组,两组之间存在显著性差异(p0.05)。2.术后3个月～12个月,7例HSCT受者HCMV-IgG均为阳性,4例受者HCMV-IgM为阴性,3例受者HCMV-IgM检测为阳性;3例HCMV-IgM阳性受者之间的IgMS/CO值有统计学差异(p0.05)。3.7例受者移植术后TCR BV家族CDR3谱型PCR扩增结果显示TCR BV部分家族呈单克隆峰、寡克隆峰。单克隆增殖的TCR家族BVCDR3测序显示为BV9、BV11、BV17、BV20 等 BV 家族序列;TCR BV9 含"QVRGGTDTQ",TCR BV11含 "VATDEQ" 和 "LGDEQ",TCR BV17 含 "IGQGNTEA",TCR BV20 含"VGLAANEQ"等共有氨基酸序列。4.TCRBV家族在HCMV抗原血症阳性受者和阴性受者中的表达无显著性差异(p0.05);而TCR BV11家族的表达在HCMV-IgM阳性组与阴性组之间差异有统计学意义(p0.05)。结论:TCRBV11的表达可能与HSCT受者术后HCMV激活有关,与HSCT受者术后参与HCMV的清除及预后有关。
[Abstract]:Background: allogeneic hematopoietic stem cell transplantation (hematopoietic stem cell transplantation, HSCT) is an effective method for the treatment of leukemia, lymphoma and severe aplastic anemia and other hematologic diseases. However, after transplantation of long-term immunosuppressive therapy resulted in immune reconstruction severely slow, sustained low cellular immune function in lead to infection. Many infection factors, human cytomegalovirus (HumanCytomegalovirus, HCMV) is a common pathogen infection after allogeneic hematopoietic stem cell transplantation is the cause of death, and one important reason for the infection of the recipients. For the long-term inhibition of HSCT after HCMV activation, early diagnosis and treatment to the recipient's own immune recovery in two the essential of.HCMV antigen detection provides an important basis for early diagnosis and preemptive therapy, virus phosphoprotein 65 (phosphoprotein65, pp65) - the original, is the HCMV virus in the complex The expression of the antigen in the process, can directly reflect the in vivo replication of virus, HCMV infection and HCMV disease early diagnosis, the level can help predict clinical possibility of HCMV infection. In addition, HSCT HCMV high infection rate increase is also closely related with immune reconstitution after delay however, cytotoxic T lymphocyte (CTLs) through the T cell receptor (T-cellreceptor, TCR) identification of HCMV antigen specific immune response against HCMV infection is the body's long-term protective mechanism of.TCR most important through the TCR beta chain variable region (TCRBV) of the complementarity determining region (CDR3) combined with antigen peptide, in the state HCMV infection, TCR and HCMV specific antigen mediated interactions mediated by CDR3, CDR3 sequence will specific frequency, reflect the specific T cell clone, but also from one side to reflect the immune HSCT after operation The reconstruction of the situation. The research showed that the virus (HBV, HIV) infection, TCRBV specific CDR3 sequences, this study analyzed HSCT recipients HCMV infection status, combined with the TCRBV family molecular distribution, understanding of HCMV activated HSCT by immune response in vivo condition, analysis of HSCT recipients TCR the expression of the active infection with HCMV. Study on the activation of HCMV and related specific TCRBV family or contribute to the early diagnosis, for the future may serve as a target for immunotherapy basis. Methods and objective: selected from August 2009 to June 2012 in Zhejiang University First Affiliated Hospital of 7 cases of HSCT, 3 months after operation respectively. Fresh peripheral blood was collected from 3 to 5 mL, anticoagulation, separation of liquid extracted by lymphocytes in peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC) for TCR detection and antigenemia test. Third months after the operation started 1 times a month straight To twelfth months, a total of 10 times, each time from fresh peripheral blood was about 10 mL, HCMV-pp65 and HCMV-IgM were used to detect HCMV-IgG antigen, antibody detection of.HCMV-pp65 antigen by immunohistochemistry, HCMV-IgM, HCMV-IgG antibody was detected by ELISA method, TCR BV sequencing analysis using family fluorescence quantitative PCR dissolution curve method, statistical analysis the relationship between TCRBV sequence and HCMV index of infection after HSCT. Results: 1. third months after transplantation, 5 cases of 7 cases of recipients in the antigenemia test positive, 2 cases were negative, after 3~12 month follow-up period, 7 patients had HCMV-pp65 antigen in different degree, pp65 antigen positive rate HCMV-IgM the positive group was significantly higher than HCMV-IgM negative group, there were significant differences between the two groups (P0.05) 3 ~ 12 months after.2., 7 cases of HSCT recipients were positive for HCMV-IgG, HCMV-IgM of 4 cases were negative, 3 cases were HCMV-IgM positive; 3 cases of HCM Among V-IgM positive recipients IgMS/CO value had significant difference (P0.05).3.7 cases of recipients in TCR BV family CDR3 spectral type PCR amplification showed that TCR BV was part of a family of monoclonal peak, oligo TCR family BVCDR3 sequencing. G Longfeng monoclonal proliferation show for BV9, BV11, BV17, BV20 and other BV family sequence; TCR BV9, TCR BV11 with "QVRGGTDTQ" with "VATDEQ" and "LGDEQ", TCR BV17, TCR BV20 with "IGQGNTEA" with "VGLAANEQ" co expression of.4.TCRBV family in the amino acid sequence of HCMV antigen in positive and negative recipients had no significant difference (P0.05); the expression of TCR and BV11 the family between HCMV-IgM positive group and negative group had significant difference (P0.05). Conclusion: the expression of TCRBV11 and HSCT recipients after the activation of HCMV, the clearance and HSCT and the prognosis of recipients in HCMV.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7;R511

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