中国公共卫生  2010, Vol. 26 Issue (7): 881-883   PDF    
引用本文
黄志刚, 段广才, 唐焕文, 胡利人, 杜进林, 潘海燕. 幽门螺杆菌cagA基因影响胃粘膜上皮细胞肿瘤相关蛋白表达[J]. 中国公共卫生, 2010, 26(7): 881-883.
HUANG Zhi-gang, DUAN Guang-cai, TANG Huan-wen,et al. Cytotoxin-associated gene A of Helicobacter pylori affects cancer-related protein expression in gastric epithelial Ges-1 cells[J]. Chinese Journal of Public Health, 2010, 26(7): 881-883.
幽门螺杆菌cagA基因影响胃粘膜上皮细胞肿瘤相关蛋白表达
黄志刚1, 段广才2, 唐焕文1, 胡利人1, 杜进林1, 潘海燕1     
1. 广东医学院公共卫生学院流行病学教研室, 广东东莞523808;
2. 郑州大学公共卫生学院流行病学教研室
收稿日期: 2009-10-09.
基金项目: 河南省创新人才基金资助项目(2000-84);广东医学院博士启动项目
作者简介: 黄志刚(1971- ),男,河北武安人,副教授,博士,主要从事慢性病分子流行病学研究。
通讯作者: 段广才
摘要: 目的 筛选和鉴定与幽门螺杆菌(Hp)细胞毒素相关基因A(cagA)有关的宿主细胞肿瘤相关蛋白。方法 用幽门螺杆菌野生株和cagA基因敲除株分别与胃粘膜上皮细胞Ges-1共培养10h(细菌与细胞的比例为100:1),提取2组细胞总蛋白,进行等电聚焦和十二烷基磺酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳,比较2组细胞蛋白质表达谱差异,选取差异蛋白点进行质谱分析鉴定。结果 经肽质量指纹图(PMFs)检索分析鉴定,与cagA基因敲除株比对,幽门螺杆菌野生株攻击后的胃粘膜上皮细胞Ges-1总蛋白中的磷酸丙糖异构酶、膜联蛋白A1和α-烯醇酶(2个相邻位点)4个差异蛋白点均呈高表达,质谱评分依次为:325,254,395,347;肽段匹配率分别为:95%,79%,77%,76%。结论 3种差异表达的蛋白均与幽门螺杆菌cagA基因有关,属于胃粘膜上皮细胞肿瘤相关蛋白。
关键词: 幽门螺杆菌(Hp)     细胞毒素相关基因A(cagA)     胃粘膜上皮细胞     蛋白质组    
Cytotoxin-associated gene A of Helicobacter pylori affects cancer-related protein expression in gastric epithelial Ges-1 cells
HUANG Zhi-gang, DUAN Guang-cai, TANG Huan-wen,et al    
Department of Epidemiology, School of Public Health, Guangdong Medical College, Dongguan 523808, China
Abstract: Objective To screen and identify the cancer-related proteins in host cells with relation to cagA gene of Helicobacter pylori(H pylori).Methods Gastric epithelium cell line Ges-1 cells were co-cultured with the wild-type strains Hp 27 and the isogenic cagA-deleted mutants Hp 27△cagA at a ratio of 100:1 for the bacteria and the cells in culture medium for 10 hr.Total cellular proteins of two groups were extracted with Sonication-Urea-CHAPS-DTT method and the protein concentration was measured with Bradford's method.The soluble proteins of two groups were separated firstly by isoelectric focusing(IEF)electrophoresis and then by SDS-PAGE to obtain 2-DE maps,which were digitalized and analysed to search the protein spots of differential expression.Spots of interest were cut out and subjected to in-gel digestion.Then the peptide mass finger-printer(PMF)was obtained with matrix assisted laser adsorption ionization time of flight mass spectrometry (MALDI-TOF-MS).Then the biology information was used to search protein database of these PMF and to identify the proteins,and analyze the biological significance and function of the proteins.Results Four protein spots of differential expression,which were high abundance in the cellular proteins of wild-type strain group and were down-regulated or absently expressed in the proteins of mutant group,were selected and identified.The identified proteins were triosephosphate isomerase, annexin A1 and a-enolase,which were reported to have close relationship with the pathophysiological processes of many kinds of cancers.Conclusion Three tumor marker proteins of triosephosphate isomerase,a-enolase and annexin A1 are associated with cagA gene of H pylori.The result suggests that the cagA gene is involved in the carcinogenesis of H pylori.
Key words: Helicobacter pylori     cytotoxin-associated gene A     gastric epithelium cell     proteomics    

幽门螺杆菌(Helicobacter pylori,Hp)感染是导致胃癌发生的重要危险因素,细胞毒素相关基因A (cytotoxin-associated gene A,cagA)是其主要毒力基因之一〔1, 2〕。Ohnishi等〔3〕在表达幽门螺杆菌CagA蛋白的转基因小鼠实验中获得了 cagA致癌的直接证据,但其致癌分子机制尚不明确。为了进一步探寻CagA致癌的分子机制,筛选和鉴定与cagA基因有关的宿主细胞效应蛋白,本研究采用比较蛋白质组学方法,对幽门螺杆菌野生株(cagA+)与cagA基因敲除株(cagA-)攻击正常胃粘膜上皮细胞Ges-1后细胞总蛋白表达谱的差异进行了比较。现将结果报告如下。

1 材料与方法 1.1 材料 1.1.1 菌株与细胞

(1)幽门螺杆菌野生株:Hp27,分离自郑州大学第一附属医院慢性萎缩性胃炎患者胃粘膜标本(cagA+);(2)幽门螺杆菌cagA基因敲除株:自行构建Hp27 △cagA (cagA-)〔4〕。(3)细胞:永生化胃粘膜上皮细胞Ges-1 (郑州大学公卫学院流行病学教研室王凯娟教授惠赠):来源于9月龄胎儿正常胃粘膜上皮细胞,用SV40病毒感染3周左右,分离出转化细胞克隆;细胞除基因组整合SV40基因后表现的转化特性外,基本保留正常细胞结构和功能,接种于裸鼠6个月无致癌性。

1.1.2 主要试剂与仪器

变性剂尿素(urea),表面活性剂 (CHAPS),载体两性电解质(ampholyte),还原剂二硫苏糖醇 (DTT),碘乙酰胺(Iodoacetamide),十二烷基磺酸钠 (SDS),丙烯酰胺(Arc),过硫酸胺(APS),矿物油,低熔点琼脂糖,ReadyStripTM IPG胶条(17cm)(美国BIO-RAD公司); PROTEAN IEF系统组件、PROTEAN II xi系统组件,GS-800校准型光密度扫描成像系统及PDQuest 7.1双向电泳图像分析软件(美国BIO-RAD公司)。

1.2 方法 1.2.1 菌株与细胞培养

(1)幽门螺杆菌培养:将野生株 (Hp27)和cagA基因敲除株(Hp27△cagA)分别接种于含 10%羊血的布氏平板培养基(万古霉素10 mg/L、多粘菌素B 2500 U/L、甲氧苄氨嘧啶(TMP)5 mg/L、两性霉素5 mg/L),37℃、微需氧条件下(10% CO2 ,5% O2 ,85% N2 )培养,3 d后收集细菌,分别用不含抗生素的1640培养液制成细菌悬液备用。(2)细胞培养:永生化胃粘膜上皮细胞Ges-1,在含有10%胎牛血清的1640培养液中常规培养,根据生长情况2~3 d传代1次,取处于对数生长期细胞用于实验。 (3)幽门螺杆菌与Ges-1细胞共培养:Ges-1细胞以每瓶5 × 105个细胞接种于50 mL细胞培养瓶中,37℃含5% CO2培养箱中培养过夜;弃去细胞培养液,分别加入幽门螺杆菌野生株菌悬液(野生株组)和敲除株菌悬液(敲除株组),置37℃微需氧环境中共培养(细菌与细胞比例均为100:1)10 h,胰蛋白酶消化细胞,收集、洗涤、离心收集细胞,冷磷酸盐缓冲液 (PBS)洗3遍,抽提细胞总蛋白。

1.2.2 共培养细胞总蛋白提取

每2 × 106个细胞加入预冷的细胞样品裂解液350 μL,吹打混匀,冰上静置30 min,经超声(200 W,10 s,间隔10 s,共20次)后于4℃ 20 000 r/min离心1 h,取上清,Bradford定量,-80℃保存。

1.2.3 双向电泳

(1)固相pH梯度等电聚焦(IEF,pH3~ 10):向17 cm聚焦盘中加入350 μL水化上样缓冲液(约含样品1 000 μg),使Ready StripTM IPG胶条覆盖到液面上,并覆盖矿物油。将聚焦盘置于PROTEAN IEF电泳仪中等电聚焦,使蛋白质按等电点不同进行分离。(2)十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE电泳):取出聚焦好的IPG胶条,经平衡缓冲液平衡后移至13% SDS-PAGE凝胶顶端,与胶面完全接触,选择恒功率电泳,起始用低功率(2.5 w/ gel/17 cm),然后加大功率(15w/凝胶/17 cm),待溴酚蓝指示剂到达凝胶底部边缘时停止电泳,取出凝胶。

1.2.4 图像扫描与分析

凝胶经考马斯亮蓝染色,GS-800光密度扫描仪扫描成像,然后经PDQuest 7.1软件分析得到2组凝胶上蛋白质点的标准化体积及差异,选择与确定具有统计学意义的差异蛋白质点(P < 0.05)进行比对分析;为保证二维电泳的重复性,每个样品的蛋白质电泳至少重复3次。具体操作按PDQuest 7.1版本的指导手册。

1.2.5 蛋白质分子鉴定

从凝胶上挖下差异蛋白点,经脱色、酶解、肽提取后,进行基质辅助电离解析飞行时间质谱 (MALDI-TOF-MS)分析,获得差异表达的蛋白质点肽质量指纹图谱(PMF),然后利用蛋白质数据库NCBInr网站(http:// prospector.ucsf.edu)提供的程序(MS-Fit)进行数据库检索,鉴定出差异蛋白点的命名与特征〔5〕。

1.3 统计分析

采用SPSS 15.0软件进行t检验,检验水准取0.05。

2 结 果 2.1 野生株与敲除株组细胞总蛋白分布比较(图 1)
注:A:野生株组; B:敲除株组。 图 1 野生株组与敲除株组Ges-1 细胞总蛋白考染图谱

2组细胞总蛋白分布模式相似,大部分蛋白质点在2组细胞总蛋白表达谱中可以很好地匹配。经GS-800图像采集和 PDQuest 7.1软件分析结果表明,与野生株组比较,敲除株组细胞总蛋白中有20个明显改变的蛋白质点,其中8个蛋白质点缺失表达,11个蛋白质点表达降低,1个蛋白质点表达上调。选取在野生株组细胞总蛋白中呈高表达而在敲除株组不表达或低表达的4个蛋白点分别记作G1~G4(图 1A所示),经剪切放大后,可见敲除株组的4个蛋白点缺失或呈极弱表达(图略)。

2.2 差异蛋白点质谱解析(表 1)
表 1 4 个差异表达的蛋白质位点PMFs 检索比对结果

4个差异表达的蛋白点经肽质量指纹图(PMF)检索分析结果显示,G1为磷酸丙糖异构酶(triosephosphate isomerase,TIM),G2为膜联蛋白A1 (annexin A1),G3和G4均为α-烯醇酶(Alpha-enolase)。后2个位点分子量相同,但等电点不同,可能与蛋白质翻译后修饰不同有关。

3 讨 论

本研究通过比较经遗传背景相同的Hp野生株和cagA基因敲除株攻击后,Ges-1细胞蛋白质表达谱的差异,初步筛选得到3种与cagA基因有关的肿瘤相关蛋白:磷酸丙糖异构酶(triosephosphate isomerase,TIM)、膜联蛋白A1(annexin A1)和α-烯醇酶(α-enolase)。其中,TIM与肺腺癌、肾细胞癌、结直肠癌等病理进展明显相关,并且可能还涉及肿瘤的转移过程〔6, 7, 8〕。本研究结果表明,在Hp野生株处理的Ges-1细胞总蛋白中TIM也呈高表达,而在cagA基因敲除株组缺失表达,提示cagA基因可能与TIM的表达变化有关。

Annexin A1是重要的炎症调控蛋白之一,在炎性代谢产物产生、中性粒细胞/单核细胞与内皮细胞黏附过程中起重要作用〔9〕。Wang等〔10〕研究发现,annexin A1在食管及食管胃连接处的腺癌组织呈高表达,并且与肿瘤的病理分期和远端转移有关。更多研究报道,annexin A1在肺腺癌、肝癌、胰腺癌及肾癌中表达增强,而在前列腺癌、B细胞淋巴瘤、头颈癌中表达下调,推测annexin A1可能通过表达异常、磷酸化状态及细胞定位的改变等方式参与了肿瘤的病理生理过程〔11, 12, 13〕。本研究发现,annexin A1蛋白在Hp野生株处理的Ges-1细胞总蛋白中呈高表达,而在cagA基因敲除株组呈低表达,提示 annexin A1的表达变化与cagA基因有关,annexin A1可能是参与幽门螺杆菌致癌过程的相关蛋白之一。

α-enolase是参与糖酵解的一种酶,在能量代谢过程中发挥作用。在乳腺癌、肝内胆管癌、小细胞肺癌的蛋白质组研究中,发现此酶在肿瘤细胞表达上调〔14, 15〕。Lim等〔16〕发现α- enolase可在Hp感染的胃癌细胞内高表达。本研究结果与 Lim JW等的结果一致,用Hp野生株攻击Ges-1细胞,可发现 α-enolase表达升高,而cagA基因敲除株组却未发现此现象,可见宿主细胞α-enolase表达升高与cagA基因有关。

本研究运用蛋白质组学技术,从胃粘膜上皮细胞Ges-1总蛋白表达谱中筛选出与幽门螺杆菌cagA基因有关的3种肿瘤相关蛋白,这些蛋白在宿主细胞的表达异常可能是幽门螺杆菌导致细胞恶性转化的机制之一,而CagA分子也许在启动细胞恶性转化过程中扮演了重要角色,本研究结果为揭示CagA参与幽门螺杆菌致癌的分子机制提供了实验证据。

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