人外周血单个核细胞<italic>miR-UL</italic>112 水平在HCMV感染中的诊断价值研究

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吴蓉, 孔倩倩, 吕志异, 许健, 倪振华, 相芬芬, 康向东. 人外周血单个核细胞miR-UL112 水平在HCMV感染中的诊断价值研究 . 检验医学2014, 29(6):631-634
WU Rong, KONG Qianqian, LÜ Zhiyi, XU Jian, NI Zhenhua, XIANG Fenfen, KANG Xiangdong. The diagnosis significance ofmiR-UL112 level in human peripheral blood mononuclear cells during HCMV infection . Labratory Medicine, 2014, 29(6):631-634 复制到剪切板

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人外周血单个核细胞miR-UL112 水平在HCMV感染中的诊断价值研究

吴蓉¹, 孔倩倩¹, 吕志异², 许健³, 倪振华³, 相芬芬¹, 康向东¹

1. 上海中医药大学附属普陀医院检验科,上海 200062

2. 重庆医科大学检验医学院,重庆 400016

3. 上海中医药大学附属普陀医院中心实验室,上海 200062

通讯作者:康向东,联系电话:021-62160801。

作者简介:吴蓉,女,1970年生,本科,副主任技师,主要从事微生物检验工作。

基金:上海市教委预算项目(2012JW70);

摘要

目的

探讨单个核细胞中人巨细胞病毒(HCMV)微小RNA(miR)-UL112-3p、miR-UL112-5p水平对HCMV潜伏和激发感染的诊断价值。

方法

选取非肿瘤患者92例[其中HCMV特异性CD8⁺ T淋巴细胞高水平组(简称CD8⁺细胞高水平组)57例、HCMV特异性CD8⁺ T淋巴细胞低水平组(简称CD8⁺细胞低水平组)35例]、肿瘤化疗患者30例,提取外周血单个核细胞,采用实时荧光定量-聚合酶链反应(PCR)检测HCMV DNA、miR-UL112-3p及miR-UL112-5p水平。采用受试者工作特征(ROC)曲线评价miR-UL112-3p和miR-UL112-5p水平对HCMV潜伏感染的诊断价值。

结果

肿瘤化疗组HCMV DNA、miR-UL112-3p及miR-UL112-5p水平明显高于非肿瘤组(P<0.05)。非肿瘤组中CD8⁺细胞高水平组HCMV DNA、miR-UL112-3p及miR-UL112-5p水平明显高于CD8⁺细胞低水平组(P均<0.01);以CD8⁺细胞低水平组miR-UL112-3p、miR-UL112-5p水平为标准,CD8⁺细胞高水平组miR-UL112-3p、miR-UL112-5p阳性率分别为59.65%、64.91%。分别以1.09 、1.52作为miR-UL112-3p和miR-UL112-5p诊断HCMV较高水平潜伏感染状态的Cut-off值,敏感性分别为59.65%、64.91%,特异性分别为97.1%、97.1%。以非肿瘤组miR-UL112-3p、miR-UL112-5p水平的+s(即5.24、6.63)作为诊断HCMV激发感染的标准,肿瘤化疗组miR-UL112-3p、miR-UL112-5p阳性率分别为50.00%、73.33%。

结论

miR-UL112-3p及miR-UL112-5p对HCMV潜伏和激发感染可能有一定的诊断价值。

关键词: 人巨细胞病毒; 微小RNA-UL112-3p; 微小RNA-UL112-5p; 潜伏感染; 激发感染

中图分类号:Q503 文献标志码:A 文章编号:1673-8640(2014)06-0631-04

The diagnosis significance ofmiR-UL112 level in human peripheral blood mononuclear cells during HCMV infection

WU Rong¹, KONG Qianqian¹, LÜ Zhiyi², XU Jian³, NI Zhenhua³, XIANG Fenfen¹, KANG Xiangdong¹

1. Department of Clinical Laboratory, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China

2. College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China

3. Central Laboratory, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China

Fund:

Abstract

Objective

To investigate the levels of micro RNA(miR)-UL112-3p and 5p in human peripheral blood mononuclear cells, and to evaluate the significance ofmiR-UL112 in the diagnosis of human cytomegalovirus (HCMV) atent and active infections.

Methods

Human peripheral blood mononuclear cells were isolated from 92 non-cancer patients (57 patients with high-level HCMV specific CD8⁺ T lymphocyte and 35 patients with low-level HCMV specific CD8⁺T lymphocyte) and 30 cancer patients receiving chemotherapy. HCMV DNA,miR-UL112-3p andmiR-UL112-5p were determined by real time fluorescence quantitation polymerase chain reaction (PCR). The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic significance of HCMV latent infections.

Results

In cancer group, the levels of HCMV DNA,miR-UL112-3p andmiR-UL112-5p were higher than those in non-cancer group (P<0.05). The levels of HCMV DNA,miR-UL112-3p andmiR-UL112-5p in patients with high-level CD8⁺ T lymphocyte were significantly higher than those in patients with low-level CD8⁺ T lymphocyte (P<0.01). As the levels ofmiR-UL112-3p andmiR-UL112-5p in patients with low-level CD8⁺ T lymphocyte for standards, the positive rates in patients with high-level CD8⁺ T lymphocyte were 59.65% and 64.91%. As 1.09 and 1.52 for the cut-off values ofmiR-UL112-3p andmiR-UL112-5p in HCMV high-level latent infection, the sensitivities were 59.65% and 64.91%, and the specificities were 97.1% and 97.1%. In the+s (5.24 and 6.63) standard ofmiR-UL112-3p andmiR-UL112-5p levels of non-cancer group, the positive rates of cancer group were 50.00% and 73.33%.

Conclusions

There might be diagnostic significance for HCMV latent and active infections by determining the levels ofmiR-UL112-3p and 5p.

Keyword: Human cytomegalovirus; Micro RNA-UL112-3p; MicroRNA-UL112-5p; Latent infection; Active infection

Show Figures

人巨细胞病毒(human cytomegalovirus,HCMV)在人群中普遍易感,成人对HCMV的感染率高达90%以上,通常无明显症状。HCMV潜伏在宿主细胞中,当机体免疫功能低下或缺陷时(如移植受者、艾滋病患者等)常呈激发感染,引起疾病,严重可致死亡^{[ 1]}。目前还没有明确的方法可区分潜伏、激发感染。Shen^{[ 2]}报道细胞感染HCMV病毒后微小RNA(micro RNA,miR) UL112水平明显升高,与病毒复制及免疫逃逸密切相关。提示HCMV miR-UL112对HCMV激发感染具有潜在诊断价值。我们提取非肿瘤以及肿瘤化疗患者外周血单个核细胞,采用实时荧光定量-聚合酶链反应(real time fluorescence quantitative polymerase chain reaction,RT-PCR)检测 miR-UL112-3 p、 miR-UL112-5 p及HCMV DNA水平,探讨 miR-UL112在诊断HCMV潜伏、激发感染状态中的价值。

材料和方法

一、材料

1. 标本来源选取 2012 年3月至2014 年3月门诊非肿瘤患者92例,年龄50~70岁。前期应用HCMV PP65合成多肽片段刺激患者外周血单个核细胞,采用酶联免疫斑点试验(ELISPOT)、细胞增殖实验等方法检测对HCMV特异性多肽反应的CD8⁺T淋巴细胞,根据CD8⁺T淋巴细胞水平的高、低将患者分为两组,其中HCMV特异性CD8⁺ T淋巴细胞高水平组(简称CD8⁺细胞高水平组)57例,HCMV特异性CD8⁺ T淋巴细胞低水平组(简称CD8⁺细胞低水平组)35例。另选取肿瘤化疗患者30例,年龄52~72岁。常规方法分离外周血单个核细胞,-80 ℃保存待测。

2. 仪器和试剂 HCMV DNA、 miR-UL112-3 p、 miR-UL112-5 p引物由北京天根生化公司设计合成,引物序列见表1;细胞基因组DNA提取(DP315)、细胞miRNA提取(DP501)、miRNA RT-PCR试剂盒(MP401)均购自北京天根生物有限公司;DNA RT-PCR(RR420)试剂盒购自TaKara生物有限公司。聚合酶链反应(polymerase chain reaction,PCR)仪购自美国应用生物系统公司。

表1 miRNA反转录与定量PCR引物、DNA定量PCR引物

二、方法

1. HCMV DNA提取每份标本取2.5×10⁵个外周血单个核细胞,按照细胞基因组DNA提取试剂盒说明书提取HCMV DNA,以HCMV DNA-F、HCMV DNA-R为引物,HCMV DNA为模板进行PCR扩增,检测HCMV载量。扩增条件:95 ℃ 30 s,95 ℃ 5 s,60 ℃ 30 s,95 ℃ 15 s,60 ℃ 60 s,95 ℃ 15 s,40个循环。以β-actin为内参进行相对定量分析。

2. miR-UL112水平测定每份标本取2.5×10⁵个外周血单个核细胞,按照 miRNA提取试剂盒说明书提取总miRNA,采用随机引物进行逆转录反应。以 miR-UL112-3 p-Forward、 miR-UL112-5 p-Forward为引物,逆转录产物为模板进行PCR扩增。反应条件:95 ℃ 30 s,95 ℃ 5 s,60 ℃ 34 s,40个循环。以U6为内参进行相对定量分析。

三、统计学方法

采用SPSS 18.0软件进行统计分析。计量数据成偏态分布,采用中位数( M)[四分位数( P₂₅ ~P₇₅)]表示,组间比较用单因素秩和检验。 P<0.05为差异有统计学意义。

结果

一、非肿瘤组及肿瘤化疗组HCMV DNA、 miR-UL112-3 p及 miR-UL112-5 p水平比较

肿瘤组HCMV DNA、 miR-UL112-3 p及 miR-UL112-5 p水平均明显高于非肿瘤组( P<0 .05、 P<0.01)。非肿瘤组中CD8⁺细胞高水平组HCMV DNA、 miR-UL112-3 p及 miR-UL112-5 p水平均明显高于CD8⁺细胞低水平组( P均<0.01)。见表2。

表2 肿瘤化疗组与非肿瘤组HCMV DNA、 miR-UL112-3 p及 miR-UL112-5 p水平比较[ M( P₂₅ ~P₇₅)]

二、 miR-UL112-3 p、 miR-UL112-5 p水平对HCMV感染诊断价值的评估

1. 对HCMV潜伏感染的诊断价值根据非肿瘤组 miR-UL112-3 p、 miR-UL112-5 p水平,绘制ROC曲线,见图1。分别以1.09 、1.52作为 miR-UL112-3 p和 miR-UL112-5 p诊断HCMV较高水平潜伏感染诊断的Cut-off值,敏感性分别为59.65%、64.91%,特异性分别为97.1%、97.1%,ROC曲线下面积分别为0.947、0.945。

	Figure Option View Download New Window
	图1 miR-UL112-3 p、 miR-UL112-5 p诊断HCMV潜伏感染的ROC曲线图

2. 对HCMV激发感染的诊断价值以非肿瘤组 miR-UL112-3 p、 miR-UL112-5 p水平的 +s(即5.24、6.63)作为诊断HCMV激发感染的标准,肿瘤组 miR-UL112-3 p、5 p阳性率分别为50.00%(15/30)、73.33%(22/30),非肿瘤组阳性率分别为6.52%(6/92)、5.43%(5/92)。其中40.00%(12/30)的患者 miR-UL112-3 p水平、63 .33 %(19 /30)的患者 miR-UL112-5 p水平升高10倍以上。

讨论

HCMV miR-UL112由Grey等^{[ 3]}首次采用生物信息方法与荧光素酶分析相结合鉴定出,与HCMV的激发感染密切相关^{[ 4]}。Zhang等^{[ 5]}利用HCMV感染细胞,对HCMV复制过程中的16种miRNA水平进行跟踪监测,结果显示随着HCMV的复制, miR-UL112表达水平不断升高,在48 h达到最高值(升高至原来的30倍),在72 h时伴随着细胞凋亡,病毒复制减弱, miR-UL112表达水平开始降低,但仍维持在一个较高水平,证实 miR-UL112与病毒的复制密切相关。Huang 等^{[ 6]}的研究结果也发现 miR-UL112在HCMV体外感染细胞24 h就可以检测到,随着HCMV的感染其水平有所增高。因此, miR-UL112可能作为HCMV潜伏、激发感染的诊断标志物。

在健康成人体内,当HCMV再激活和HCMV特异性CD8⁺细胞的抗病毒免疫控制处于一种平衡状态时呈潜伏感染。而肿瘤患者因为长期接受放、化疗,免疫功能受损,此平衡被打破,HCMV容易被激活而呈激发感染。本研究检测了30例肿瘤化疗患者、92例非肿瘤患外周血单个核细胞HCMV DNA、 miR-UL-112-3 p及 miR-UL112-5 p水平。结果表明肿瘤化疗组HCMV DNA、 miR-UL-112-3 p及 miR-UL112-5 p水平明显高于非肿瘤组( P<0 .05、 P<0 .01)。以非肿瘤组 miR-UL112-3 p、 miR-UL112-5 p水平的 +s作为诊断HCMV激发感染的标准,肿瘤组 miR-UL112-3 p、 miR-UL112-5 p阳性率分别为50 .00 %、73 .33 %;其中部分患者 miR-UL112大幅度升高(40 .00 %的患者 miR-UL112-3 p水平、63 .33 %的患者 miR-UL112-5 p水平升高10倍以上),与文献报道^{[ 7]}相一致。由此可见, miR-UL112-3 p、 miR-UL112-5 p水平对HCMV激发感染的诊断可能具有一定意义。

研究^{[ 8, 9]}表明HCMV高水平潜伏感染时,HCMV特异性CD8⁺细胞维持在较高水平,外周血单个核细胞中HCMV DNA水平也较高,这可能与正常人HCMV不断激活以及免疫逃逸相关。本研究按HCMV特异性CD8⁺ 细胞水平将非肿瘤患者分为两组,分别检测单个核细胞 miR-UL112水平,进一步研究 miR-UL112对于HCMV潜伏感染诊断价值。 miR-UL112-3 p对较高水平HCMV潜伏感染的阳性检出率(敏感性)为59.65%, miR-UL112-5 p为64.91%,ROC曲线下面积分别为0.947、0.945,具有良好的诊断效果。但其阳性检出率仍不够理想,这是由于CD8⁺细胞低水平组中有约8%的患者CD8⁺ 细胞假阴性,在一定程度上提高了Cut-off值。

综上所述,本研究采用RT-PCR分别检测了非肿瘤患者、肿瘤化疗患者外周血单个核细胞 miR-UL112-3 p、 miR-UL112-5 p水平,结果提示 miR-UL112-3 p、 miR-UL112-5 p对HCMV潜伏和激发感染的诊断可能具有一定的临床价值。

The authors have declared that no competing interests exist.

参考文献

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[1]	Cordier AG, Guitton S, Vauloup-Fellous C, et al. Awareness of cytomegalovirus infection among pregnant women in France[J]. J Clin Virol, 2012, 53(4): 332-337. [本文引用:1] [JCR: 3.287]
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2012

3.287

0.0

. 2012, 53(4):332-337 DOI:10.1016/j.jcv.2011.12.031

Awareness of cytomegalovirus infection among pregnant women in France

Abstract

Background

Cytomegalovirus (CMV) is the most frequent cause of congenital virus infection. Approximately 1% of newborns are infected by CMV at birth with severe consequences among 10% of them. Efficacy of hygienic counselling is nowadays established and should be spread.

Objective

To evaluate pregnant women's awareness of cytomegalovirus infection in France.

Study design

Pregnant women receiving prenatal care, at any moment of their pregnancy, in two different obstetrics clinics with different information policies, were asked to complete a written questionnaire about CMV infection.

Results

More than half (217/362, 60%) of the pregnant women had heard of congenital CMV infection, and most of them (72%) knew the hygiene measures to use to prevent infection. Nevertheless, most could not correctly identify the symptoms associated with congenital CMV disease. Awareness was associated with hospital's policy concerning CMV infection information, the mother's educational level, parity, and employment in health care. Indeed, when information is supposed to be given (hospital A), 74% (vs 34%) know congenital CMV infection and among them the knowledge is more precise.

Conclusions

This study tends to confirm that there is a large gap between knowledge of CMV and the burden of this disease. To bridge this gap, women should receive education about congenital CMV. Hospital-based prenatal education increases awareness and knowledge about CMV and CMV prevention.

... HCMV潜伏在宿主细胞中,当机体免疫功能低下或缺陷时(如移植受者、艾滋病患者等)常呈激发感染,引起疾病,严重可致死亡^[1] ...

2014

3.73

0.0

... Shen^[2]报道细胞感染HCMV病毒后微小RNA(micro RNA,miR)UL112水平明显升高,与病毒复制及免疫逃逸密切相关 ...

2005

5.076

0.0

... 讨论HCMV miR-UL112由Grey等^[3]首次采用生物信息方法与荧光素酶分析相结合鉴定出,与HCMV的激发感染密切相关^[4] ...

2007

8.136

0.0

... 讨论HCMV miR-UL112由Grey等^[3]首次采用生物信息方法与荧光素酶分析相结合鉴定出,与HCMV的激发感染密切相关^[4] ...

2011

9.737

0.0

... Zhang等^[5]利用HCMV感染细胞,对HCMV复制过程中的16种miRNA水平进行跟踪监测,结果显示随着HCMV的复制,miR-UL112表达水平不断升高,在48 h达到最高值(升高至原来的30倍),在72 h时伴随着细胞凋亡,病毒复制减弱,miR-UL112表达水平开始降低,但仍维持在一个较高水平,证实miR-UL112与病毒的复制密切相关 ...

2013

2.092

0.0

... Huang 等^[6]的研究结果也发现miR-UL112在HCMV体外感染细胞24 h就可以检测到,随着HCMV的感染其水平有所增高 ...

2012

3.287

0.0

... 33%的患者miR-UL112-5p水平升高10倍以上),与文献报道^[7]相一致 ...

2011

8.136

0.0

... 研究^[8,9]表明HCMV高水平潜伏感染时,HCMV特异性CD8⁺细胞维持在较高水平,外周血单个核细胞中HCMV DNA水平也较高,这可能与正常人HCMV不断激活以及免疫逃逸相关 ...

2011

4.084

0.0

. 2011, 33(4):607-614 DOI:10.1007/s11357-011-9205-9

Relationship between cytomegalovirus (CMV) IgG serology, detectable CMV DNA in peripheral monocytes, and CMV pp65(495-503)-specific CD8+ T cells in older adults

1.Division of Geriatric Medicine and Gerontology, Department of Medicine, Johns Hopkins University School of Medicine, 5505 Hopkins Bayview Circle, Baltimore, MD 21224 USA 2.Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 3.Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 USA 4.Clinical Research Branch, Longitudinal Studies Section, National Institute on Aging, Baltimore, MD, USA 5.Mailman School of Public Health, Columbia University, New York, NY, USA

In immunocompetent individuals, cytomegalovirus (CMV) is thought to persist in a latent state in monocytes and myeloid progenitor cells, establishing a lifelong infection. In CMV-seropositive older adults, aging has been associated with both expansion of CMV pp65_495–503-specific CD8⁺ T cell clones and shrinkage of the T cell repertoire that characterize T cell immunosenescence. In fact it has been suggested that chronic CMV infection is a driving force in age-related T cell immunosenescence. In older adults, chronic CMV infection is conventionally diagnosed by positive IgG serology which does not distinguish between past and persistent infections. To better define the relationship between chronic CMV infection and expansion of CMV pp65_495–503-specific CD8⁺ T cells, we directly assessed CMV viral DNA in monocyte-enriched peripheral blood mononuclear cells in 16 HLA-A2-positive elderly volunteers (mean age = 83 years). While all participants had positive CMV IgG serology by enzyme-linked immunosorbent assays, only nine (56%) had detectable CMV DNA by nested polymerase chain reaction. These nine individuals had significantly higher percentages of CMV pp65_495–503 tetramer-positive CD8⁺ T cells (median = 1.3%) than those without detectable CMV DNA (median = 0.1%; p < 0.001). Absolute CMV IgG antibody titers did not differ between these two groups (median = 54.6 vs 44.2 EU/ml, respectively, p = 0.4). CMV IgM titers were negative for all 16 participants, suggesting that recent primary CMV infection was unlikely. These results demonstrate a strong association between the presence of CMV DNA in peripheral monocytes and the expansion of CD8⁺ T cells specific for the CMV immunodominant epitope pp65_495–503. Although the sample size in this study is relatively small, these findings provide initial evidence suggesting the heterogeneity of CMV IgG-seropositive older adult population and CMV viral DNA detection in peripheral monocytes as an informative tool to better understand the relationship between chronic CMV infection and T cell immunosenescence.