HIV-1慢性感染者B淋巴细胞表型和功能损伤的研究

发布时间：2018-04-13 23:28

本文选题：HIV-1 + B细胞　；参考：《复旦大学》2013年硕士论文

【摘要】：背景：人类免疫缺陷病毒I型(human immunodeficiency virus type1, HIV-1)感染会造成严重的免疫功能损伤,除了CD4+T细胞的不断耗损和功能损伤,HIV-1感染也能导致严重的体液免疫应答缺损。有报道称HIV-1感染者外周血B细胞数目减少,B细胞的亚群比例也出现紊乱,其中活化、耗竭和终末分化的B细胞亚群以及未成熟/过渡型B细胞增加,而CD27+记忆B细胞减少。B细胞表面活化分子、耗竭分子和凋亡分子表达增高,而抗凋亡分子表达降低,出现多克隆活化和抗原反应性低等功能损伤现象。这些研究进展多采用不同地区、以及不同疾病进展阶段的感染者为研究对象,病毒的分子特征也存在差异性。尤其是ART后长期存活的感染者处于慢性感染的过程中,这部分人的B细胞亚群的表型和功能损伤的免疫学特征与机制、以及上述它们与疾病进展相关性等均有待深入探索,以建立有效的免疫干预策略,修复B细胞的表型异常与功能损伤。本研究选取中国1995-1996年因有偿献血而集中形成的HIV-1长期慢性感染人群为研究对象,该人群的人口学特征、遗传背景、以及病毒分子特征均相近,以期在上述研究方向上有所突破。方法：按照知情同意的原则招募研究对象,分为3组：HIV感染未进行抗病毒治疗组(HIV-viremic,简称VI), HIV感染接受ART组(HIV-aviremic,简称AV),与HIV-1阴性健康组(HIV-negative,简称HN)。采集HIV-1慢性感染者和HIV-1阴性健康人静脉血分离外周血单个核细胞(PBMC)。首先进行B细胞表面分子染色以界定B细胞亚群：未成熟B细胞(CD19+CD10+CD27)、初始B细胞(CD19+CD10-CD21+CD27-)、静息记忆B细胞(CD19+CD10-CD21+CD27+)、活化记忆B细胞(CD19+CD10-CD21-CD27+)、组织样记忆B细胞(CD19+CD10-CD21-CD27-)和浆母细胞(CD19+CD10-CD21-CD27+CD20-)。同时分析活化分子(CD38)、细胞增殖分子(Ki-67)、凋亡相关分子(CD95、Bcl-2和PD-1)以及共刺激分子(CD40和CD70)的表达。利用流式细胞仪检测上述标志性分子的表达以及ART对B细胞表型的修复作用。随后,利用免疫磁珠负选法分选B细胞,进行B细胞功能性研究：用酶联免疫斑点试验(ELISpot)检测抗原特异性(包括HIV-1膜蛋白gp120和流感膜蛋白HA特异性)的抗体分泌细胞表达水平；利用CBA(Cytometric Beads Assay)检测细胞培养上清中细胞因子的分泌水平。结果：所招募的HIV-1慢性感染者的外周血CD4+T细胞和B细胞数目都显著低于HN组(422±33个/μ1vs814±57个/μ1,p0.01；156±19个/μl vs216±33个/μ1,p=0.05),在VI二者显著正相关(r=0.376,p=0.037),经ART后CD4+T细胞和B细胞数目有所恢复。VI组与HN组相比,B细胞亚群变化显著,未成熟B细胞、初始B细胞、静息记忆B细胞和浆母细胞显著降低,而组织样记忆B细胞显著增加,并且AV组初始B细胞和组织样记忆B细胞比例都有所恢复,但是静息记忆B细胞比例却没有恢复。 HIV-1感染后,B细胞以及未成熟B细胞、初始B细胞、静息记忆B细胞和组织样记忆B细胞4个亚群上调表达活化分子CD38,和细胞增殖分子Ki-67的表达呈正相关(r=0.432,p0.01)。凋亡分子CD95在静息记忆B细胞、活化记忆B细胞和浆母细胞上的表达水平较高；而抗凋亡分子Bcl-2的表达在初始B细胞、组织样记忆B细胞和浆母细胞3个亚群上显著降低；同时静息记忆B细胞和浆母细胞上调表达PD-1。共刺激分子CD40在所有B细胞亚群上的表达强度降低,而初始B细胞、静息记忆B细胞、活化记忆B细胞和组织样记忆B细胞上CD70的表达水平显著降低。HIV-1感染引起B细胞及其亚群上活化分子、凋亡分子以及共刺激分子的异常表达经抗病毒治疗后获得部分恢复。 HIV-1慢性感染者B细胞功能损伤明显,Total IgG抗体分泌细胞(antibody-secreting cells, ASCs)由HN组的10600±2623ASCs/106B细胞显著减少至IV组的3818±931ASCs/106B细胞(p=0.05),且流感蛋白特异性ASCs也显著低于HN(22±7ASCs/106B细胞vs53±10ASCs/106B细胞,p=0.028),ART后改善不明显。HIV-1膜蛋白特异性ASCs在HIV-1感染后升高(163±58ASCs/106B细胞),抗病毒治疗后显著降低(30±8ASCs/106B细胞,p0.01)。同时,HIV-1感染者B细胞分泌炎症细胞因子IL-10、IL-6和IL-8,以及LT-α和趋化因子CCL-3的能力显著低于健康人对照,ART并不能恢复B细胞分泌细胞因子的能力。结论：HIV-1长期慢性感染造成外周血B细胞数目减少以及亚群比例的变化,并且B细胞亚群表现出过度活化,极易发生凋亡以及B-T细胞相互作用受损等多种损伤表型。HIV-1长期慢性感染造成B细胞功能损伤,表现在抗原特异性抗体分泌细胞减少和细胞因子分泌能力降低。ART仅能部分修复HIV-1慢性感染者B细胞表型和功能的损伤。
[Abstract]:BACKGROUND : Human immunodeficiency virus type 1 ( HIV - 1 ) infection can cause severe immune function damage . In addition to the increasing of CD4 + T cells and the impairment of function , HIV - 1 infection can lead to severe humoral immune response .

In this study , the population of HIV - 1 chronically infected with HIV - 1 in China from 1995 to 1996 was studied . The demographic characteristics , genetic background and viral molecular characteristics of the population were similar , with a view to making a breakthrough in the research direction .

Methods : The subjects were recruited according to the principle of informed consent , divided into 3 groups : HIV - viremic ( VI ) , HIV - aviremic , and HIV - 1 negative health group ( HN ) . Peripheral blood mononuclear cells ( PBMC ) were isolated from peripheral blood of HIV - 1 chronic infection and HIV - 1 negative healthy human venous blood .

First , B - cell surface molecule staining was performed to define B - cell subsets : immature B - cells ( CD10 + CD27 ) , initial B - cells ( CD19 + CD10 - CD21 + CD27 + ) , resting memory B cells ( CD19 + CD10 - CD21 + CD27 + ) , tissue - like memory B cells ( CD19 + CD10 - CD21 - CD27 + ) , tissue - like memory B cells ( CD19 + CD10 - CD21 - CD27 + ) , and costimulatory molecules ( CD10 - CD21 - CD27 + CD20 - ) .

Subsequently , B cells were sorted by negative selection with immunomagnetic beads to perform the functional study of B cells : antibody secretion cell expression levels of antigen specificity , including HIV - 1 membrane protein and influenza - membrane protein HA - specific ) , were detected by enzyme - linked immunodot assay ( ELISA ) .
The level of cytokine secretion in the culture supernatant was detected using the CBA ( Cytometric Assay Assay ) .

Results : The number of CD4 + T cells and B cells in peripheral blood of HIV - 1 patients were significantly lower than that in HN group ( 422 卤 33 / 渭1 vs 814 卤 57 / 渭1 , p0.01 ) .
There was significant positive correlation between group VI and HN group ( r = 0.376 , p = 0.037 ) , and the number of CD4 + T cells and B cells increased significantly after ART ( r = 0.376 , p = 0.037 ) .

After infection with HIV - 1 , B cells and immature B cells , initial B cells , resting memory B cells and tissue - like memory B cells up - regulated the expression of activated molecule CD38 and the expression of Ki - 67 in cell proliferation molecules ( r = 0.432 , p0.01 ) . The expression level of apoptotic molecules CD95 in resting memory B cells and activated memory B cells and plasma cells was higher .
The expression of anti - apoptotic molecules Bcl - 2 decreased significantly in the initial B cells , the tissue - like memory B cells , and the 3 subgroups of the plasma cells ;
At the same time , the expression level of CD70 in B cells and plasma cells decreased significantly , while the level of CD70 expression in B cells , resting memory B cells , activated memory B cells and tissue - like memory B cells decreased significantly . The abnormal expression of activated molecules , apoptotic molecules and co - stimulatory molecules on B cells and their sub - populations caused by HIV - 1 infection was partially recovered after antiviral treatment .

Anti - HIV - 1 membrane protein - specific ASCs were significantly lower than HN ( 22 卤 7ASCs / 106b cells vs 53 卤 10ASCs / 106b cells , p = 0.028 ) . The anti - HIV - 1 membrane protein - specific ASCs increased after HIV - 1 infection ( 163 卤 58ASCs / 106b cells ) and decreased significantly after antiviral therapy ( 30 卤 8ASCs / 106b cells , p0.01 ) . At the same time , the IL - 10 , IL - 6 and IL - 8 secretion of IL - 10 , IL - 6 and IL - 8 , as well as LT - 伪 and chemokine CCL - 3 , were significantly lower than those of healthy controls , and ART did not restore the ability of B cells to secrete cytokines .

Conclusion : Chronic infection of HIV - 1 caused the decrease of the number of B cells in peripheral blood and the change of the proportion of B - T cells , and the B - cell subsets showed a variety of damage phenotype , such as excessive activation , apoptosis and impaired B - T cell interaction .

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91

【共引文献】