乙型肝炎病毒对胰岛素样生长因子结合蛋白7表达的影响及其临床意义

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宋惠, 刘兴晖, 祝成亮, 钟基大, 李毅. 乙型肝炎病毒对胰岛素样生长因子结合蛋白7表达的影响及其临床意义. 28(12): 1092-1094[SONG Hui, LIU Xinghui, ZHU Chengliang, ZHOANG Jida, LI Yi. The influence of hepatitis B virus on the expression of insulin-like growth factor-binding protein 7 and its clinical significance. 上海检验医学, 28(12): 1092-1094] 复制到剪切板

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乙型肝炎病毒对胰岛素样生长因子结合蛋白7表达的影响及其临床意义

宋惠¹, 刘兴晖², 祝成亮³, 钟基大⁴, 李毅¹

1. 湖北医药学院附属东风医院病理科,湖北十堰 442008

2. 上海市浦东新区公利医院检验科,上海 200135

3. 武汉大学人民医院检验科,湖北武汉 430060

4. 湖北医药学院附属东风医院传染科,湖北十堰 442008

宋惠,女,1976年生,主管技师,主要从事细胞病理学工作。

刘兴晖,联系电话:0719-8218427。

基金:国家自然科学基金资助项目(81101485); 中国博士后特别资助基金(201104485); 中国博士后科学基金资助项目(20100471144); 湖北省教育厅科学技术研究项目(D20122404);

摘要

目的

探讨乙型肝炎病毒(HBV)对胰岛素样生长因子结合蛋白7(IGFBP7)表达的影响及其临床意义。

方法

逆转录-聚合酶链反应(PCR)检测IGFBP7 mRNA的表达水平,酶联免疫吸附试验(ELISA)检测血清IGFBP7含量,分析IGFBP7在慢性乙型肝炎(CHB)、肝纤维化(LC)和肝细胞癌(HCC)患者间血清含量的差异。

结果

IGFBP7 mRNA在HepG2.2.15细胞中的表达水平较HepG2高;与健康对照组相比,HBV感染者IGFBP7血清学水平明显升高(P<0.05);IGFBP7在LC和HCC患者低于CHB患者(P<0.05)。

结论

HBV能够上调IGFBP7的表达,其血清水平与疾病进程呈负相关。

关键词: 乙型肝炎病毒; 胰岛素样生长因子结合蛋白7; 肝纤维化; 肝细胞癌

中图分类号:R446.62 文献标志码:A 文章编号:1673-8640(2013)12-1092-03

The influence of hepatitis B virus on the expression of insulin-like growth factor-binding protein 7 and its clinical significance

SONG Hui¹, LIU Xinghui², ZHU Chengliang³, ZHOANG Jida⁴, LI Yi¹

1.DepartmentofPathology,DongfengHospital,HubeiUniversityofMedicine,HubeiShiyan 442008,China

2.DepartmentofClinicalLaboratory,PudongNewAreaGongliHospital,Shanghai 200135,China

3.DepartmentofClinicalLaboratory,People'sHospitalofWuhanUniversity,HubeiWuhan 430060,China

4.DepartmentofInfection,DongfengHospital,HubeiUniversityofMedicine,HubeiShiyan 442008,China

Abstract

Objective

To investigate the influence of hepatitis B virus(HBV) on the expression of insulin-like growth factor-binding protein 7(IGFBP7) and its clinical significance.

Methods

The mRNA expression of IGFBP7 was measured by reverse transcription-polymerase chain reaction (PCR), and serum levels of IGFBP7 were measured by enzyme-linked immunosorbent assay(ELISA). The different expressions of IGFBP7 among patients with chronic hepatitis B(CHB), liver cirrhosis(LC) and hepatocellular carcinoma(HCC) were analyzed.

Results

The expression of IGFBP7 mRNA was higher in HepG2.2.15 cells than in HepG2 cells. Compared with healthy controls, serum IGFBP7 levels were much higher in HBV patients (P<0.05), and the serum levels of IGFBP7 were detected at lower levels in patients with LC and HCC than those with CHB(P<0.05).

Conclusions

HBV can upregulate the expression of IGFBP7, which is negatively correlated with the disease progression.

Keyword: Hepatitis B virus; Insulin-like growth factor-binding protein 7; Liver cirrhosis; Hepatocellular carcinoma

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引言

乙型肝炎病毒(hepatitis B, HBV)感染引起慢性乙型肝炎(chronic hepatitis B, CHB),并发展成肝纤维化(liver cirrhosis, LC),最终发展为肝细胞癌(hepatocellular carcinoma, HCC),严重危害人类健康。但HBV的致病机制还不清楚。前期工作中,我们采用基因芯片筛选了肝癌细胞系HepG2和HepG2.2.15的差异表达基因^{[ 1]},发现胰岛素样生长因子结合蛋白7(insulin-like growth factor-binding protein 7, IGFBP7)在HepG2.2.15细胞中表达升高。本研究主要探讨HBV对IGFBP7表达的影响,并分析IGFBP7血清学水平与疾病进程的关系,为揭示HBV的致病机制奠定基础。

材料和方法

一、研究对象

收集临床确诊肝炎患者268例,男146例,女122例,平均年龄(45.8±16.5)岁,其中CHB患者114例,LC患者81例 ,HCC患者73例,所有患者均排除心、脑、肾等重要器官疾病及其他嗜肝病毒的感染。对照组为94名健康体检者,男52名,女42名,平均年龄(43.7±15.3)岁。所有研究对象空腹采血分离血清。

二、材料

人肝癌细胞系HepG2和HepG2.2.15购自武汉大学典型培养物保藏中心,后者为稳定转染了HBV质粒的HepG2细胞;RNA提取试剂TRIzolR购自Invitrogen公司;M-MLV逆转录酶购自美国Promega公司;IGFBP7酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测试剂盒购自美国Santa Cruz公司;引物由上海英俊生物技术有限公司合成。

三、方法

1. HepG2和HepG2.2.15细胞的培养

肝癌细胞系HepG2和HepG2.2.15用含10%的胎牛血清、100 U/mL青霉素和100 mg/L链霉素的RPIM1640培养基,在37 ℃恒温箱中培养。

2. 逆转录-聚合酶链反应(polymerase chain reaction, PCR)检测

采用TRIzolR试剂分别提取HepG2和HepG2.2.15细胞的总RNA,反转录出相应的cDNA后,用 IGFBP7基因的检测引物5'-GCAAGAGGCGGAAGGGTAAA-3'(上游),5'-CTCAAGTACACCTGGGCACC-3'(下游) 进行PCR 扩增,扩增产物大小为268 bp,并设立β-actin作为内参照,引物序列:5'-CACTGGCATCGTGATGGACT-3'(上游),5'-AATGCCAGGGTACATGGTGG-3'(下游),扩增产物大小484 bp,产物经1%琼脂糖凝胶电泳检测。

3. IGFBP7的检测

患者和健康对照者血清IGFBP7检测采用ELISA。

四、统计学方法

所有资料的统计学分析均应用SPSS 13.0软件,数据以 ±s表示,2组间IGFBP7水平的比较采用 t检验,多组间比较采用方差分析和 q检验, P<0.05为差异具有统计学意义。

结果

一、HBV对IGFBP7 mRNA的表达影响

HepG2和HepG2.2.15细胞IGFBP7 mRNA的表达情况见图1。HepG2.2.15细胞中IGFBP7 mRNA表达升高,表明HBV能够在HepG2细胞上调IGFBP7 mRNA的表达。

	Figure Option View Download New Window
	图1 HepG2和HepG2.2.15细胞IGFBP7 mRNA检测

注:M为Marker;1和2分别为HepG2.2.15和HepG2中β-actin mRNA;3和4分别为HepG2.2.15和HepG2中IGFBP7 mRNA

二、HBV感染者血清IGFBP7水平

HBV感染者血清IGFBP7的水平[(45.8±18.4) ng/mL]明显高于健康对照组[(23.6±9.8)ng/mL],差异有统计学意义( P<0.05)。

三、IGFBP7血清学水平与疾病进程的相关性

CHB组IGFBP7水平为(58.7±17.5) ng/mL,LC组为(43.1±14.6) ng/mL,HCC组为(33.8±11.6) ng/mL。CHB患者血清IGFBP7水平明显高于其他2组( P<0.05),HCC患者IGFBP7含量最低。

讨论

IGFBP7是一种分泌型糖蛋白,是IGFBPs的家族成员之一,广泛表达于不同组织来源的肿瘤细胞,在人体多种组织器官中都可以检测到其表达,主要参与调节上皮细胞生长、刺激纤维母细胞生长、诱导细胞凋亡等^{[ 2, 3, 4]}。

CHB感染最终导致HCC的发生,但其机制不明。为探讨HBV致病和致癌的机制,前期工作我们对HepG2和HepG2.2.15细胞的差异表达基因进行了检测,发现补体C3和C4表达水平在HepG2.2.15细胞表达降低^{[ 1]},而IGFBP7在HepG2.2.15细胞表达升高,其细胞荧光强度cy3/cy5比值(R/G值)为17.31,为排除基因芯片的假阳性结果,我们采用逆转录-PCR进行了验证,结果与芯片测定结果一致,表明HBV感染HepG2后能够明显促进IGFBP7的转录。

IGFBP7是一种分泌型糖蛋白,可分泌到人体血液中,我们检测了HBV感染者和健康对照者IGFBP7的血清学含量,结果表明IGFBP7在HBV感染者血清中明显升高,与基因芯片和逆转录-PCR结果相符,表明HBV能够在体内和体外促进IGFBP7的合成和分泌。

本研究结果表明,CHB、LC和HCC患者血清IGFBP7水平差异有统计学意义。IGFBP7在CHB患者表达最高,其次是LC患者,在HCC患者表达最低。因此,IGFBP7可能作为HBV相关疾病鉴别的指标之一。

Chen等^{[ 5]}研究HCC患者肝组织标本IGFBP7的表达,发现IGFBP7的表达与HCC的分期和分级呈负相关,认为IGFBP7是HCC的肿瘤抑制因子,提出“以IGFBP7过度表达为靶点的策略可能成为治疗HCC的潜在新疗法。”本研究结果表明,HBV感染者血清IGFBP7水平随HBV相关疾病的进展而下降,HCC患者最低,支持Chen等^{[ 5]}的观点。

本研究中,观察到HBV感染上调IGFBP7 mRNA的表达,而HBV在 IGFBP7基因表达的各个环节(转录、mRNA成熟、翻译及翻译后修饰)中的调节机制尚待进一步探讨。

总之,本研究探讨了HBV对IGFBP7表达的调节作用,并分析了其血清水平与疾病进程的关系,为HBV相关疾病的病因探讨和病程分析提供了有益的资料。

The authors have declared that no competing interests exist.

参考文献

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2011

0.0

... 15的差异表达基因^[1],发现胰岛素样生长因子结合蛋白7(insulin-like growth factor-binding protein 7, IGFBP7)在HepG2 ...

... 15细胞表达降低^[1],而IGFBP7在HepG2 ...

2011

3.287

0.0

... CHB患者血清IGFBP7水平明显高于其他2组(P讨论IGFBP7是一种分泌型糖蛋白,是IGFBPs的家族成员之一,广泛表达于不同组织来源的肿瘤细胞,在人体多种组织器官中都可以检测到其表达,主要参与调节上皮细胞生长、刺激纤维母细胞生长、诱导细胞凋亡等^[2,3,4] ...

2011

3.392

0.0

2012

4.469

0.0

. 2012, 133(2):563-573 DOI:10.1007/s10549-011-1816-4

IGFBP7 reduces breast tumor growth by induction of senescence and apoptosis pathways

<li>1.Division of Molecular and Cellular Biology, Sunnybrook Research Institute, 2075 Bayview Avenue, S-238, Toronto, ON, M4N 3M5, Canada</li><li>4.Genomics Core Facility, Sunnybrook Research Institute, Toronto, ON, Canada</li><li>6.Institute of Protein Research, Pushchino, Moscow Region, 142290, Russia</li><li>2.Department of Anatomic Pathology, Sunnybrook Health Science Center, Toronto, ON, Canada</li><li>5.Department of General Surgery, Odette Cancer Center, Sunnybrook Health Science Center, Toronto, ON, Canada</li><li>3.Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada</li>

Abstract
Insulin-like growth factor binding protein 7 (IGFBP7) has been shown to be a tumor suppressor in a variety of cancers. We previously have shown that IGFBP7 expression is inversely correlated with disease progression and poor outcome in breast cancer. Overexpression of IGFBP7 in MDA-MB-468, a triple-negative breast cancer (TNBC) cell line, resulted in inhibition of growth and migration. Xenografted tumors bearing ectopic IGFBP7 expression were significantly growth-impaired compared to IGFBP7-negative controls, which suggested that IGFBP7 treatment could inhibit breast cancer cell growth. To confirm this notion, 14 human patient primary breast tumors were analyzed by qRTPCR for IGFBP7 expression. The TNBC tumors expressed the lowest levels of IGFBP7 expression, which also correlated with higher tumorigenicity in mice. Furthermore, when breast cancer cell lines were treated with IGFBP7, only the TNBC cell lines were growth inhibited. Treatment of NOD/SCID mice harboring xenografts of TNBC cells with IGFBP7 systemically every 3–4 days inhibited tumorigenesis, with associated anti-angiogenic effects, together with increased apoptosis. Upon examining the mechanism of IGFBP7-mediated growth inhibition in TNBC cells, we found that cells not only were arrested in G1 phase of the cell cycle but also underwent senescence as a result of treatment with IGFBP7. Interestingly, IGFBP7 treatment was also associated with strong activation of the stress-associated p38 MAPK pathway, together with upregulation of p53 and the cyclin-dependent protein kinase (CDK) inhibitor, p21^cip1. Prolonged treatment of cells with IGFBP7 resulted in increased cell death, marked by an increase in apoptotic cells and associated cleaved PARP. This is the first study showing that exogenous IGFBP7 inhibits TNBC cell growth both in vitro and in vivo. Taken together, these results suggest IGFBP7 treatment might have therapeutic potential for TNBC.

2011

2.914

0.0

... Chen等^[5]研究HCC患者肝组织标本IGFBP7的表达,发现IGFBP7的表达与HCC的分期和分级呈负相关,认为IGFBP7是HCC的肿瘤抑制因子,提出#cod#x0201c ...

... 本研究结果表明,HBV感染者血清IGFBP7水平随HBV相关疾病的进展而下降,HCC患者最低,支持Chen等^[5]的观点 ...