肝硬化合并肝癌及单纯肝癌患者免疫功能检测结果分析

引用本文

张一超, 夏骏, 李雄. 肝硬化合并肝癌及单纯肝癌患者免疫功能检测结果分析. 29(11): 1128-1131
ZHANG Yichao, XIA Jun, LI Xiong. The detection result analysis on the immune functions in cirrhosis patients with liver cancer and patients with liver cancer alone. Labratory Medicine, 29(11): 1128-1131 复制到剪切板

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肝硬化合并肝癌及单纯肝癌患者免疫功能检测结果分析

张一超¹, 夏骏², 李雄³

1.临安市人民医院检验科,浙江临安 311300

2.浙江省人民医院检验科,浙江杭州 310014

3杭州艾迪康医学检验中心实验室,浙江杭州 310023

作者简介:张一超,男, 1983生,学士,主管技师,主要从事临床免疫学检验工作。

摘要

目的

探讨肝硬化合并肝癌患者及单纯肝癌患者细胞免疫及体液免疫功能的变化情况。

方法

选取肝硬化合并肝癌及单纯肝癌患者各40例,同期无肝病诊断史的健康人40名为对照。分别抽取对照者及肝癌患者外周血,以流式细胞术检测细胞免疫水平变化:T淋巴细胞亚群(CD3、CD4、CD8、CD4/CD8)的值;免疫透射比浊法检测体液免疫 (IgA、IgG、IgM)的浓度。

结果

与对照组相比,两组肝癌患者CD3、CD4、CD4/CD8比值明显降低(P均<0.05),而肝硬化合并肝癌CD3、CD4 、CD4/CD8比值与单纯肝癌患者相比明显下降(P均<0.05)。与对照组相比,两组肝癌患者IgA、IgG、IgM明显上升,而肝硬化合并肝癌较单纯肝癌患者lgA、lgG、lgM值上升更为明显(P均<0.05)。

结论

肝硬化合并肝癌患者免疫功能下降较单纯肝癌患者下降更为明显。因此,改善肝癌患者机体免疫功能,重建机体防御系统是很重要的治疗措施,同时监测机体免疫功能的改变,对估计预后、指导临床治疗有积极作用。

关键词: 肝癌; 肝硬化; 细胞免疫; 体液免疫

中图分类号:R446.11 文献标志码:A 文章编号:1673-8640(2014)11-1128-04

The detection result analysis on the immune functions in cirrhosis patients with liver cancer and patients with liver cancer alone

ZHANG Yichao¹, XIA Jun², LI Xiong³

1. Department of Clinical Laboratory, Lin'an People's Hospital, Zhejiang Lin'an 311300, China

2. Department of Clinical Laboratory, Zhejiang People's Hospital, Zhejiang Hangzhou 310014, China

3. Central Laboratory, Hangzhou ADICON Clinical Laboratories, Zhejiang Hangzhou 310023, China

Abstract

Objective

To investigate the changes of cellular and humoral immune functions in cirrhosis patients with liver cancer and patients with liver cancer alone.

Methods

A total of 40 cirrhosis patients with liver cancer and 40 patients with liver cancer alone were enrolled. At the same period, 40 healthy subjects with no liver disease history were enrolled as controls. Their peripheral blood samples were extracted, and their cellular immune level changes were determined by flow cytometry, including T lymphocyte subset (CD3, CD4, CD8 and CD4 / CD8) values. The concentrations of humoral immunity (IgA, IgG and IgM) were determined by immunoturbidimetry.

Results

The CD3, CD4 and CD4/CD8 significantly decreased in liver cancer patients than in controls (P<0.05), and the CD3, CD4 and CD4/CD8 in cirrhosis patients with liver cancer decreased obviously compared with those in patients with liver cancer alone (P<0.05). Compared with those in controls, the IgA, IgG and IgM increased obviously in liver cancer patients, and the IgA, lgG and lgM increased obviously in cirrhosis patients with liver cancer compared with those in patients with liver cancer alone (P<0.05).

Conclusions

The immune functions of cirrhosis patients with liver cancer decrease more significantly than those of patients with liver cancer alone. Therefore, improving the immune functions in patients with liver cancer and rebuilding the defense system are so important, and monitoring the changes of immune functions has a positive role for estimating prognosis and guiding clinical treatment.

Keyword: Liver cancer; Cirrhosis; Cellular immunity; Humoral immunity

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引言

肝癌是一种最常见的癌症,是世界癌症相关死亡的第三大原因^{[ 1]}。肝癌治疗的标准方法包括手术切除、化学治疗、放射治疗、超声消融或肝移植。然而,经过这些治疗,高复发率仍然是肝癌患者面临的一个大问题^{[ 2]}。鉴于这些事实,了解肝癌的发病机制的必要性变得迫切。研究表明肝癌的发病机制是伴随着免疫系统的紊乱,导致由免疫应答的肿瘤逃逸,而在许多情况下,肝癌患者具有慢性肝炎或肝硬化的背景发展,肝硬化合并肝癌患者及单纯肝癌患者的免疫功能是否具有各自特点,这对于目前越来越受重视的个体化免疫治疗具有重要参考意义。我们通过肝硬化合并肝癌患者及单纯肝癌患者免疫功能的检测,以期对肝硬化合并肝癌患者及单纯肝癌患者免疫功能的变化情况来估计预后、指导临床治疗产生积极作用。

材料和方法

一、研究对象

2013年6月~2014年5月在临安市人民医院肝病科就诊,经病理学和影像学确诊肝硬化合并肝癌患者31例,男19例,女12例,年龄4062岁,平均为42.2±3.9岁;单纯肝癌患者31例,男18例,女13例,年龄3860岁,平均为41.2±4.8岁。肝癌患者均严格符合2001年中国抗癌协会制定的《原发性肝癌的临床诊断标准》,肝硬化合并肝癌患者均符合2000年中华医学会肝病学分会、传染病与寄生虫病学分会联合制定的《全国病毒性肝炎防治方案》中病毒性肝炎诊断标准,且所有肝癌患者排除合并自身免疫性、遗传代谢性肝病及酒精脂肪肝等病因,无合并冠心病、肾脏病、风湿病、糖尿病等疾病。另选取同期在临安市人民医院体检的肝功能和病毒性肝炎标志物正常的同年龄段人群31名为对照组,年龄3961岁,平均为42.1±3.2岁,观察组与对照组之间年龄及性别构成差异均无统计学意义。

二、主要试剂与仪器

T淋巴细胞流式抗体CD3-异硫氰酸荧光素(FITC)、CD4-藻红蛋白荧光素(PE)、CD8-别藻蓝蛋白荧光素(APC)检测:BD FACSVerse^TM SYSTEM流式细胞仪及配套试剂,美国BD Biosciences公司;IgA、IgG、IgM免疫球蛋白检测:Roche E601全自动化学发光免疫分析仪及配套试剂盒,德国罗氏诊断有限公司;人淋巴细胞分离液:天津市灏洋生物制品科技有限责任公司。

三、方法

1. T淋巴细胞亚群检测

采集各观察组及对照组人群清晨空腹外周抗凝血2 mL,用人淋巴细胞分离液提取外周血单核细胞(PBMC,1×10⁶/mL),按T淋巴细胞流式抗体CD3-FITC、CD4-PE和CD8-APC检测试剂盒说明书操作。

2. 血清标本采集

空腹抽取各观察组及对照组人群肘静脉血3 mL,于无菌洁净试管[含0.2%乙二胺四乙酸(EDTA)抗凝管]中,4 ℃静置2 h,400× g离心10 min,取上清液,标记分装,-80℃低温冰箱保存待用。

3.免疫球蛋白IgA、IgG、IgM检测

严格按Roche E601全自动化学发光免疫分析系统说明书步骤进行操作。

四、统计学方法

全部数据采用SPSS 17.0和GraphPad Prism 5软件进行统计分析,所有数据用 ±s表示。数据比较先进行正态性和方差齐性检验。若两者均符,采用单因素方差分析和LSD- t检验进行多样本均数间的两两比较。若有1项不符,则采用Kruskal-Wallis H检验和Dunn's 多重检验对各组数据进行统计分析。以α=0.05作为检验水准, P<0.05为差异有统计学意义。

结果

一、肝硬化合并肝癌患者及单纯肝癌患者T淋巴细胞亚群的变化

两组肝癌患者与健康对照组T淋巴细胞亚群的检测结果见表1。

表1 CD3、CD4、CD4/CD8检测结果

二、肝硬化合并肝癌患者及单纯肝癌患者IgA、IgG、IgM的变化

观察组和对照组IgA、IgG、IgM的检测结果比较,见图1、2、3。

	Figure Option View Download New Window
	图1 各组患者IgA变化

	Figure Option View Download New Window
	图2 各组患者IgG变化

	Figure Option View Download New Window
	图3 各组患者IgM变化

讨论

肝癌是最常见的肿瘤之一,世界各地的报道表明其发病率和死亡率越来越高。肝癌通常来自肝炎纤维化或肝硬化,具有强烈的免疫细胞浸润^{[ 3]}。因此,免疫状态可能会在很大程度上影响肝癌的生物学行为。免疫状况可能在不同的肿瘤微环境而有所不同,而且对疾病进展的明显效果^{[ 4]}。然而,肝硬化合并肝癌患者及单纯肝癌患者细胞免疫及体液免疫功能的变化情况对肝癌的发生发展影响尚少见报道。

研究发现人体淋巴细胞中,CD3⁺、CD4⁺、CD8⁺、CD19⁺和CD56⁺ T细胞亚群被认为在抗肿瘤免疫中是非常重要的,特别是CD3⁺和CD4⁺细胞的表达水平与肝癌患者的总生存期正相关^{[ 5]}。而本研究发现肝癌患者的细胞免疫功能严重受损,单纯肝癌患者及肝硬化合并肝癌患者与健康对照组相比,CD3、CD4、CD4/CD8值明显降低,CD4⁺T淋巴细胞降低尤为明显, CD8细胞明显升高,肝硬化合并肝癌患者纯肝癌患者CD3、CD4、CD4/CD8值明显降低,CD4⁺T淋巴细胞降低尤为明显, CD8细胞明显升高,CD4⁺T淋巴细胞在免疫系统中起中心调节作用, CD4⁺T淋巴细胞表达减少,其产生的细胞因子亦减少, 从而削弱了宿主对肿瘤生长的监视、杀伤和清除功能^{[ 6]},因此对于肝癌患者特别是肝硬化合并肝癌患者在给予相应治疗的同时,进行相应的免疫功能的干预将有助于提高化疗的效果。

免疫标记预测患者预后或对治疗的反应可促进个性化治疗的潜在的癌症治疗^{[ 7]}。在本研究中,我们不仅评估了在肝癌患者外周血中的免疫细胞的多个子集,而且还确定与肝癌患者体液免疫相关的几个免疫球蛋白的表达情况。实验结果表明肝硬化合并肝癌较单纯肝癌组以及两组肝癌患者与对照组相比,IgA、IgG、IgM值均明显上升( P均为<0.05)。有研究指出肝癌患者受到自身肝癌特异性抗原的刺激导致其体液免疫水平的显著升高^{[ 8, 9]}。本研究结果表明,肝癌患者合并肝硬化后,随着肿瘤的发展,免疫球蛋白IgA、IgG、IgM为代表的体液免疫水平进一步升高。

在针对肝癌患者特别是肝硬化合并肝癌患者进行治疗时通过监测其免疫功能状态,并加以干预将会对肝癌患者估计预后、指导临床化疗产生积极作用。机体免疫功能的重建可能为肝癌特别是肝硬化合并肝癌患者提供了一个有前途的辅助免疫治疗策略。此外,在外周血筛查免疫状态是一种无创,简单的指标^{[ 10]},值得广泛用于肝癌患者的辅助诊断。由于此次入选样本量较小,随访时间不长等因素,故有待于增加样本量,进一步研究肝癌患者以及肝硬化合并肝癌患者免疫功能的改变,为指导临床治疗提供依据。

The authors have declared that no competing interests exist.

参考文献

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2014

0.0

... 引言肝癌是一种最常见的癌症,是世界癌症相关死亡的第三大原因^[1] ...

2014

0.0

... 然而,经过这些治疗,高复发率仍然是肝癌患者面临的一个大问题^[2] ...

2012

0.0

... 肝癌通常来自肝炎纤维化或肝硬化,具有强烈的免疫细胞浸润^[3] ...

2014

3.73

0.0

PLoS ONE. 2014, 9(5):e96080-null

Prevalence and Clinical Relevance of T-Helper Cells, Th17 and Th1, in Hepatitis B Virus-Related Hepatocellular Carcinoma

Gang Xiao 1 ,Xiao-Long Liu 1 ,Liang-Chun Yin 1 ,Ning-Lei Li 1 ,Li-Xin Liu 1 ,Lu-Zhe Han 1 ,Jian Yan 1 ,Li-Juan Ling 1 ,Yi-Nan Deng 2,3

1 Department of General Surgery, The Third Affiliated Hospital, Southern Medical University, Guangzhou, China 2 Guangdong Provincial Key Laboratory of Liver Disease Research, Guangzhou, China 3 Department of Hepatic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

Background and Aims

An immune imbalance in the cytokine profile exerts a profound influence on the progression of hepatitis B virus (HBV) infections and hepatocellular carcinoma (HCC). The present study evaluated the immune status of T helper (Th) 17 and Th1 cells in patients with HBV-related and non-HBV-related HCC.

Methods

We randomly enrolled 150 patients with HCC. Blood samples and tissue samples were obtained. The distributions and phenotypic features of Th17 and Th1 cells were determined by flow cytometry and/or immunohistochemistry.

Results

Compared to corresponding non-tumor regions, the levels of Th17 and Th1 cells were significantly increased in tumors of patients with HCC (P<0.001). The intratumoral densities of IL-17-producing cells and IFN-γ-producing cells were associated with overall survival (OS, P = 0.001) and disease-free survival (DFS, P = 0.001) of patients with HCC. The ratio of Th17 to Th1 in HBV-related HCC was higher than in non-HBV-related HCC. A multivariate Cox analysis revealed that the Th17 to Th1 ratio was an independent prognostic factor for OS (HR = 2.651, P = 0.007) and DFS (HR = 2.456, P = 0.002).

Conclusions

HBV infections can lead to an imbalance in immune status in patients with HCC. An elevated Th17 to Th1 ratio may promote tumor progression. The Th17 to Th1 ratio could serve as a potential prognostic marker for scoring the severity of HCC.

... 免疫状况可能在不同的肿瘤微环境而有所不同,而且对疾病进展的明显效果^[4] ...

2014

0.0

. 2014, 60(4):1231-1240

Retinoblastoma protein potentiates the innate immune response in hepatocytes: Significance for hepatocellular carcinoma

Jack Hutcheson 1 ,Ryan J. Bourgo 2,3 ,Uthra Balaji 1 ,Adam Ertel 2 ,Agnieszka K. Witkiewicz 1,4 andErik S. Knudsen 1,4,*

1Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 2Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 3Ben May Department for Cancer Research, University of Chicago, Chicago, IL 4Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX *Address reprint requests to: E.S. Knudsen, Ph.D., University of Texas Southwestern Medical Center, Department of Pathology, 5323 Harry Hines Blvd., Dallas, TX 75390-9072. E-mail: <a href="mailto:erik.knudsen@utsouthwestern.edu" title="Link to email address" shape="rect">erik.knudsen@utsouthwestern.edu</a>; fax: 214-648-4067.

Cancers mediated by viral etiology must exhibit deregulated cellular proliferation and evade immune recognition. The role of the retinoblastoma tumor suppressor (RB) pathway, which is lost at relatively high frequency in hepatocellular carcinoma (HCC), has recently been expanded to include the regulation of innate immune responsiveness. In this study we investigated the coordinate impact of RB-loss on cell cycle control and immune function in the liver. We found that RB depletion in hepatoma cells resulted in a compromised immunological response to multiple stimuli and reduced the potential of these cells to recruit myeloid cells. Viral-mediated liver-specific RB deletion in vivo led to the induction of genes associated with proliferation and cell cycle entry as well as the significant attenuation of genes associated with immune function, as evidenced by decreases in cytokine and chemokine expression, leukocyte recruitment, and hepatic inflammation. To determine if these changes in gene expression were instructive in human disease, we compared our liver-specific RB-loss gene signature to existing profiles of HCC and found that this signature was associated with disease progression and confers a worse prognosis. Conclusion: Our data confirm that RB participates in the regulation of innate immunity in liver parenchymal cells both in vitro and in vivo and to our knowledge describes the first gene signature associated with HCC that includes both immunoregulatory and proliferative genes and that can also be attributed to the alteration of a single gene in vitro. (Hepatology 2014;60:1231–1240)

... 研究发现人体淋巴细胞中,CD3⁺、CD4⁺、CD8⁺、CD19⁺和CD56⁺ T细胞亚群被认为在抗肿瘤免疫中是非常重要的,特别是CD3⁺和CD4⁺细胞的表达水平与肝癌患者的总生存期正相关^[5] ...

2014

0.0

... 而本研究发现肝癌患者的细胞免疫功能严重受损,单纯肝癌患者及肝硬化合并肝癌患者与健康对照组相比,CD3、CD4、CD4/CD8值明显降低,CD4⁺T淋巴细胞降低尤为明显, CD8细胞明显升高,肝硬化合并肝癌患者纯肝癌患者CD3、CD4、CD4/CD8值明显降低,CD4⁺T淋巴细胞降低尤为明显, CD8细胞明显升高,CD4⁺T淋巴细胞在免疫系统中起中心调节作用, CD4⁺T淋巴细胞表达减少,其产生的细胞因子亦减少, 从而削弱了宿主对肿瘤生长的监视、杀伤和清除功能^[6],因此对于肝癌患者特别是肝硬化合并肝癌患者在给予相应治疗的同时,进行相应的免疫功能的干预将有助于提高化疗的效果 ...

2013

0.922

0.0

... 免疫标记预测患者预后或对治疗的反应可促进个性化治疗的潜在的癌症治疗^[7] ...

2013

0.0

2011

0.0

2011

2.337

0.0

. 2011, 141(1):null-null

Effective inhibition of a Strongylocentrotus nudus eggs polysaccharide against hepatocellular carcinoma is mediated via immunoregulation in vivo

... 此外,在外周血筛查免疫状态是一种无创,简单的指标^[10],值得广泛用于肝癌患者的辅助诊断 ...