肝癌细胞BEL7402中神经元特异性烯醇化酶的表达

摘要

目的: 分别从转录水平、蛋白水平及细胞水平检测人神经元特异性烯醇化酶(NSE)在肝癌细胞BEL7402中的表达情况.

方法: 利用NSE特异引物, 通过逆转录聚合酶链式反应(RT-PCR)从人肝癌细胞BEL7402中扩增人NSE基因的转录产物, 采用免疫印迹(Western blot)、免疫细胞化学(ICC)染色技术检测NSE在肝癌细胞中的表达.

结果: 通过RT-PCR方法可以从BEL7402中扩增1 305 bp的NSE产物; Western blot方法证实BEL7402细胞可以表达Mr50 000的NSE蛋白; 免疫细胞化学染色显示BEL7402与抗NSE单抗呈阳性反应, 这说明从转录水平、蛋白水平及细胞水平均检测到了NSE在肝癌细胞BEL7402中的表达.

结论: NSE可在肝癌细胞BEL7402转录和表达.

关键词: 神经元特异性烯醇化酶; 肝癌细胞; BEL7402细胞

Expression of human neuron-specific enolase gene in human hepatocellular cancer cells BEL7402

Ai-Ping Zhu, Qing-Yun Zhang, Ya-Ming Wang, Jian-Jun Xu, Li Sun

Ai-Ping Zhu, Department of Medical Laboratory, the Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China

Qing-Yun Zhang, Department of Medical Laboratory, Beijing Cancer Hospital, Peking University, Beijing 100036, China

Qing-Yun Zhang, Department of Immunology, Beijng Institute for Cancer Research, Beijing 100036, China

Qing-Yun Zhang, Ya-Ming Wang, Jian-Jun Xu, Li Sun, the School of Oncology, Peking University, Beijing 100036, China

Correspondence to: Dr. Qing-Yun Zhang, Department of Immunology, Beijng Institute for Cancer Research, Beijing 100036, China. qingyzhang@btamail.net.cn

Received: July 28, 2005
Revised: July 31, 2005
Accepted: August 3, 2005
Published online: September 28, 2005

Abstract

AIM: To investigate the expression of neuron-specific enolase(NSE) in hepatocellular cancer cells BEL7402 at the levels of transcription, protein and cells.

METHODS: The fragment of NSE gene was amplified from human hepatocellular cancer cell strain BEL7402 by reverse transcription polymerase chain reaction (RT-PCR) with NSE specific primers. The NSE expression was detected in the BEL7402 cells by Western blot and immunocytochemical (ICC) staining.

RESULTS: NSE product with a length of 1 305 bp was obtained by by RT-PCR. Western blot confirmed that Mr 50 000 NSE protein was expressed in BEL7402 cells. Positive reaction of BEL7402 with anti-NSE monoclonal antibody was observed by immunocytochemistry. The expression of NSE existed in BEL7402 cells at the levels of transcription, protein and cells.

CONCLUSION: The NSE can be expressed and transcribed in hepatocellular carcinoma cells BEL7402.

Key Words: Neuron-specific enolase; Hepatocellular carcinoma; BEL7402 cells

0 引言

神经元特异性烯醇化酶(neuron-specific enolase, NSE)广泛分布于人类及各种动物的成熟神经元、周围神经的神经内分泌细胞和一些感觉细胞中[1-4]. 它在正常人脑组织中含量最高, 起源于神经内分泌细胞的肿瘤组织也有异常表达. 常见于神经母细胞瘤、小细胞肺癌、胰腺癌、前列腺癌[5]、嗜铬细胞瘤等. 目前在临床上, NSE可作为神经元损伤的标志酶[6-11]; 也是小细胞肺癌首选的肿瘤标志物[12-14], 可用于对SCLC的鉴别诊断, 放化疗效果、预后、恶变情况等的监测[15-20]. 我们应用PCR, Western-blot和免疫细胞化学方法在不同的水平上, 发现NSE可在肝癌细胞中表达, 报告如下.

1 材料和方法

1.1 材料

人肝癌细胞BEL 7402及肺癌细胞A549由北京市肿瘤防治研究所免疫208研究室保存; 鼠抗人NSE mAb由北京市肿瘤防治研究所免疫研究室208室研制[21]; 羊抗鼠-HRP二抗由北京市肿瘤防治研究所生化202研究室惠赠; 胎牛血清为天津市川页生化制品有限公司产品; RPMI 1640培养基、DMEM为GIBCO公司产品; Trizol为Sigma公司产品; RNA酶抑制剂、M-MLV逆转录酶、5×M-MLV Buffer、dNTPs、Taq DNA Polymerase为Promega公司产品; EnVision 试剂盒购自Dako公司; 硝酸纤维素膜购自Amersham 公司; DAB、丙烯酰胺、甲叉双丙烯酰胺为北京鼎国生物技术公司产品; 甘氨酸、甲醇、十二烷基磺酸钠、三羟甲基氨基甲烷(Tris碱)等为北京化学试剂公司产品.

1.2 方法

肝癌细胞BEL7402和人肺癌细胞A549在含120 mL/L胎牛血清的RPMI 1640培养基, 5 mL/L CO₂, 37 ℃温箱中培养. Trizol法提取细胞总RNA(按说明书操作). 逆转录合成cDNA, 逆转录(RT)反应体系为: 细胞总RNA 5 mL、Oligo dT(0.5 g/L)1 mL、DEPC H₂O 6 mL, 在70 ℃孵育10 min. 再加RNAsin(50 MU/L) 1 mL、5×M-MLV Buffer 4 mL, dNTP(10 mmol/L)2 mL, M-MLV(200 MU/L)1 mL, 反应总体积20 mL, 37 ℃ 1 h后, 立刻放入冰中, 即为逆转录后的NSE-cDNA. 随后进行PCR反应扩增NSE基因片断. 上、下游引物分别为5'-GAATTCATGTCCATAGAGAAGATCTG-3'和5'-TCTAGAAGAGGAATCACAGCACACTG-3', 及通用β-actin引物(均由上海申友生物技术公司合成并测序). PCR条件为: 94 ℃ 5 min; 94 ℃ 30 s, 64 ℃ 30 s, 72 ℃ 30 s, 扩增30个循环; 72 ℃延长反应10 min.10 g/L琼脂糖凝胶电泳鉴定.

1.2.1 Western blot检测肿瘤细胞中NSE的表达: 将BEL 7402, A549细胞用0.2 g/L EDTA消化后, 细胞计数, 1 000 r/min离心5 min. 加入PBS洗1次, 取2×106细胞加入PBS 100 mL、PMSF 2 mL, 液氮冷冻, 37 ℃溶化反复3次, 混匀后与2×SDS凝胶加样缓冲液等量混合, 煮沸5 min, 100 g/L SDS-PAGE电泳. 120 mA, 10 ℃条件下电转移硝酸纤维素膜, 105 min. 取出NC膜, 用1´丽春红S染色液染色2-3 min, 用蒸馏水洗净染料. 50 g/L脱脂奶粉37 ℃ 2 h封闭NC膜, TBST洗3次. 加入NSE mAb作为一抗, 4 ℃过夜, 0.5 g/L TBST洗3次. 加入羊抗鼠-HRP(1:1000), 室温孵育1 h. 0.5 g/L TBST 洗3次, 再用TBS洗1次. 加入10 mL DAB染液染色至有条带出现, PBS终止显色. 晾干, 室温保存.

1.2.2 细胞化学方法检测肿瘤细胞中NSE的表达: 0.2 g/L的EDTA消化细胞, 加培养液调整细胞浓度为1-1.5×10⁹ 个/L, 滴于干净的玻片上, 37 ℃, 50 mL/L CO₂培养箱中孵育4-6 h. 冷丙酮/甲醇(1:1)固定5-7 min, 自来水冲洗3遍, 晾干后-20 ℃保存. 从-20 ℃取出固定好的片子, 在30 mL/L H₂O₂/PBS中浸泡10 min以去除内源性的过氧化物酶, PBS浸洗3次, 100 mL/L 马血/PBS 37 ℃封闭60 min, 加入抗NSE mAb 30 mL 37 ℃ 1 h, PBS浸洗3次, 加EnVision工作液30-50 mL, 室温放置60 min, PBS浸洗3次; H₂O₂-DAB显色, 苏木精复染13 h, 10 mL/L 盐酸/酒精分色30 s. 将复染后的细胞片脱水后用树脂胶封片, 镜下观察.

2 结果

2.1 NSE- cDNA PCR结果

以逆转录的NSE cDNA为模板进行PCR扩增, 扩增产物经10 g/L琼脂糖凝胶电泳分析, 可见在1375 bp附近出现一特异条带, 与NSE片断的1 305 bp分子大小相符, 300-400 bp之间有一条带与β-actin看家基因扩增片段大小相符(图1).

图1 NSE片段的PCR扩增. M1: DNA 分子质量标准: Lambda DNA/ EcoRI+HindⅢ; M2: 100 bp DNA 分子质量标准. 1: RT-PCR扩增A549细胞中NSE 产物; 2: RT-PCR扩增BEL 7402细胞中NSE 产物.

2.2 Western blot检测肿瘤细胞中NSE的表达

MS2-NSE融合蛋白在约Mr57 000处有一特异性条带(图2A第1道), 肝癌BEL7402及肺癌A549细胞蛋白在约Mr50 000之间有一条较强的杂交带(图2A第3, 4道), 其分子质量大小与NSE的分子质量相符(图2). 虽然在聚丙烯酰胺凝胶电泳可以见到无关蛋白MS2-GRP呈Mr18 000的蛋白带(图2B第2道), 但在Western blot反应中因为与NSE mAb不相关而不能呈现特异反应带(图2A第2道).

图2 抗NSE 单抗Western blot蛋白印迹. A: 和聚丙烯酰胺凝电泳. B: 结果. M: 蛋白分子质量标准; 1: 融合蛋白 MS2-NSE; 2: 融合蛋白MS2-GRP; 3: A549细胞裂解总蛋白; 4: BEL7402细胞裂解总蛋白.

2.3 免疫细胞化学方法检测肿瘤细胞中NSE的表达

NSE在以上两种肿瘤细胞中经细胞免疫化学染色均呈棕黄色阳性反应, 着色部位主要位于胞质(图3A, B).而在使用正常鼠血清作一抗对人肝癌细胞BEL7402染色的阴性对照中则呈阴性反应 (图3C).

图3 肝癌BEL7402和肺癌A549细胞中NSE的表达(EnVision×400). A: 肺癌细胞A549; B: 肝癌细胞BEL7402; C: 肝癌细胞BEL7402 (阴性对照).

3 讨论

从人肝癌细胞BEL7402及A549肺癌细胞中提取总RNA, RT-PCR得到NSE cDNA. 用特异性引物、Taq DNA聚合酶扩增了NSE基因片断, 长度为1 305 bp. 从人BEL7402肝癌细胞及A549肺癌细胞系中都能扩增出NSE和β-actin cDNA, 表明这两种细胞都有NSE的转录. BEL7402细胞和A549细胞表达的蛋白在Mr50 000处有一特异性条带, MS2-NSE融合蛋白在57 000处有一特异性条带, GRP融合蛋白则没有任何条带, 这与预测结果完全一致. 我们克隆的NSE编码序列可编码434个氨基酸, 分子量约为Mr50 000. MS2蛋白Mr9 000, MS2-NSE融合蛋白为Mr57 000. A549细胞和BEL7402细胞都能特异性表达NSE蛋白. NSE在人肝癌细胞BEL7402及肺癌细胞A549中均呈阳性反应, 着色部位主要位于胞质. 这与PCR, Western blot检测结果完全一致. 说明从转录水平、蛋白水平及细胞水平都能检测到NSE的表达. 烯醇酶是参与糖酵解的关键酶, 由α、β、γ三种亚基以二聚体的形式组成αα、ββ、γγ、αγ、αβ五种同工酶. 其中γγ型特异性存在于神经元和神经内分泌细胞中, 称神经元特异性烯醇化酶(NSE), 约占脑内可溶性蛋白的1.5%, 最初NSE用于神经母细胞瘤及小细胞肺癌(神经内分泌瘤)的诊断, 但近年来一些研究证实在急性脑血管病、癫痫、急性脑外伤、新生儿缺氧性脑病、心源性缺氧性脑损伤等多种中枢神经系统疾病中血清NSE均有明显改变[22-29], 并提出NSE可作为诊断中枢神经系统疾病及脑损伤的一个有价值的指标. 如肝硬化时NSE的变化可特异反映毒性物质对中枢的影响情况, 可能作为亚临床期肝性脑病的临床判断指标[30]. 但未见到NSE在肝癌细胞中表达的报道, 且肝癌并不是起源于神经内分泌细胞的肿瘤. NSE在肝癌中表达可能(1)由于NSE为涉及能量代谢的酶, 肿瘤组织糖酵解加强, 细胞增殖周期加快, 细胞内的NSE释放进入血液增多, 导致此酶在血清内含量增高; (2)NSE存在于细胞质内, 在细胞被破坏时释放出来进入血液循环. 因此NSE在肝癌细胞中表达的机制及在肝癌中的表达和意义还有待进一步研究, 以便为肝癌的发生预测和早期诊断寻求新的指标, 为肝癌的预防和早期治疗开辟新的途径. 此外, 本研究是一种定性实验, 若能从转录水平、蛋白水平及细胞水平定量分析肝癌细胞中NSE的表达水平将会有助于研究NSE在肝癌的临床诊断及预后判断中的意义.

备注

电编:李琪 编辑:潘伯荣 审读:张海宁

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