本文關(guān)鍵詞： T細(xì)胞受體 CDR3 人巨細(xì)胞病毒 異基因造血干細(xì)胞移植術(shù) 受者 抗原血癥　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:異基因造血干細(xì)胞移植(hematopoietic stem cell transplantation,HSCT)是白血病、淋巴瘤和重型再生障礙性貧血等血液系統(tǒng)疾病的有效治療方法。然而,移植術(shù)后長期的免疫抑制療法導(dǎo)致了免疫重建嚴(yán)重遲緩,持續(xù)的細(xì)胞免疫功能低下,導(dǎo)致感染。在眾多感染因素中,人巨細(xì)胞病毒(HumanCytomegalovirus,HCMV)是異基因造血干細(xì)胞移植術(shù)后感染的常見病原體,是導(dǎo)致受者術(shù)后感染和死亡的重要原因之一。為長期抑制HSCT術(shù)后HCMV激活,早期診斷搶先治療與受者自身免疫重建的恢復(fù)兩者必不可少。HCMV抗原的檢測為早期診斷及搶先治療提供了重要依據(jù),病毒磷蛋白65(phosphoprotein65,pp65)抗原,是HCMV病毒在復(fù)制過程中出現(xiàn)的抗原表達(dá),可直接反映體內(nèi)病毒的復(fù)制狀態(tài),對HCMV感染和HCMV病有早期診斷的作用,其水平的高低能幫助臨床預(yù)測發(fā)生HCMV感染的可能性。除此之外,HSCT受者HCMV高感染率增加與術(shù)后免疫重建延遲同樣密切相關(guān),而細(xì)胞毒T淋巴細(xì)胞(CTLs)通過T細(xì)胞受體(T-cellreceptor,TCR)識別HCMV抗原產(chǎn)生特異性免疫反應(yīng)是機(jī)體長期抵抗HCMV感染最重要的保護(hù)性機(jī)制。TCR通過位于TCRβ鏈可變區(qū)域(TCRBV)的互補(bǔ)決定區(qū)(CDR3)與抗原肽結(jié)合,在HCMV感染狀態(tài)下,TCR和HCMV特異性抗原通過CDR3介導(dǎo)相互作用,CDR3序列會出現(xiàn)特異性頻率,反映特定T細(xì)胞克隆擴(kuò)增程度,同時也從一個側(cè)面反映了 HSCT受者術(shù)后免疫重建的狀況。研究表明病毒(如HBV、HIV)感染下,TCRBV出現(xiàn)特定的CDR3序列,本研究分析HSCT受者術(shù)后的HCMV感染狀態(tài),結(jié)合TCRBV家族分子特征分布,了解HCMV激活狀態(tài)下HSCT受者體內(nèi)的免疫應(yīng)答狀態(tài),分析HSCT受者術(shù)后TCR表達(dá)與HCMV激活感染的關(guān)系。研究與HCMV激活相關(guān)的特定TCRBV家族或有助于早期診斷,為將來可能作為免疫治療靶點(diǎn)打下基礎(chǔ)。方法和目的:選擇了 2009年8月至2012年6月在浙江大學(xué)附屬第一醫(yī)院接受HSCT受者7例,術(shù)后3個月分別采集新鮮外周血3～5 mL,抗凝,用淋巴細(xì)胞分離液提取外周血單個核細(xì)胞(peripheral blood mononuclear cells,PBMC)用于TCR檢測和抗原血癥檢測。術(shù)后第3個月開始每月1次直至第12個月,共10次,每次抽取新鮮外周血約10 mL,分別用于HCMV-pp65抗原和HCMV-IgM、HCMV-IgG抗體檢測。HCMV-pp65抗原檢測采用免疫組化法,HCMV-IgM、HCMV-IgG抗體采用ELISA法進(jìn)行檢測,TCR BV家族測序分析采用熒光定量PCR溶解曲線法,統(tǒng)計分析HSCT術(shù)后TCRBV序列與HCMV感染指標(biāo)的關(guān)系。結(jié)果:1.移植后第3個月,7例受者中抗原血癥檢測陽性5例,陰性2例,在術(shù)后3～12月隨訪期間,7例受者均出現(xiàn)過不同程度的HCMV-pp65抗原血癥,HCMV-IgM陽性組的pp65抗原陽性率明顯高于HCMV-IgM陰性組,兩組之間存在顯著性差異(p0.05)。2.術(shù)后3個月～12個月,7例HSCT受者HCMV-IgG均為陽性,4例受者HCMV-IgM為陰性,3例受者HCMV-IgM檢測為陽性;3例HCMV-IgM陽性受者之間的IgMS/CO值有統(tǒng)計學(xué)差異(p0.05)。3.7例受者移植術(shù)后TCR BV家族CDR3譜型PCR擴(kuò)增結(jié)果顯示TCR BV部分家族呈單克隆峰、寡克隆峰。單克隆增殖的TCR家族BVCDR3測序顯示為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抗原血癥陽性受者和陰性受者中的表達(dá)無顯著性差異(p0.05);而TCR BV11家族的表達(dá)在HCMV-IgM陽性組與陰性組之間差異有統(tǒng)計學(xué)意義(p0.05)。結(jié)論:TCRBV11的表達(dá)可能與HSCT受者術(shù)后HCMV激活有關(guān),與HSCT受者術(shù)后參與HCMV的清除及預(yù)后有關(guān)。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R457.7;R511

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