修回日期: 2002-09-20
接受日期: 2002-10-03
在线出版日期: 2003-05-15
CagA＋H.pylori感染与胃癌的相关性在我国报道不一, 且致癌机制不祥. 本研究应用ELISA法检测H.pylori相关胃癌和慢性胃病血清中抗CagA抗体和免疫组化方法检测全部组织标本中Bcl-2、p53蛋白的表达, 研究H.pylori CagA 阳性株感染与胃癌、慢性胃病的相关性及与胃黏膜组织中Bcl-2、p53蛋白表达的相互关系, 探讨H.pylori的致癌机制．
79例患者(胃癌50例、萎缩性胃炎17例、胃溃疡伴不典型增生5例、浅表性胃炎7例)胃镜诊断明确后取胃窦或病变部位活检, 用于H.pylori的诊断、病理诊断及免疫组化检测Bcl-2、p53蛋白的表达． H.pylori的诊断采用快速尿素酶试验和ELISA法血清H.pylori抗体或病理Warthin-Starry银染色, 两项阳性诊断为H.pylori阳性． 用ELISA法检测患者血清CagA- H.pylori抗体, 用免疫组化方法检测全部组织标本中Bcl-2、p53蛋白的表达．
胃癌组CagA＋H.pylori感染阳性率86%(43/50)高于慢性胃病组45%(13/29)(P<0.01), 有显著性差异. Bcl-2蛋白表达: 胃癌组高于慢性胃病组(62% vs 41%; P>0.05), 胃癌CagA＋组高于CagA－组(65% vs 43%; P>0.05), 慢性胃病CagA＋组显著高于CagA－组(77% vs 12%; P<0.01)．p53蛋白表达: 胃癌组显著高于慢性胃病组(54% vs 28%; P<0.05), 胃癌CagA＋组显著高于CagA－组(60% vs 14%; P<0.05), 慢性胃病CagA＋组显著高于CagA－组(46% vs 12%; P<0.05)．
胃癌患者CagA＋H.pylori感染明显高于慢性胃病者, CagA＋H.pylori感染与胃癌发生明显相关, 而H.pylori的CagA可使癌前疾病组织中出现Bcl-2蛋白表达, 且随着Bcl-2蛋白表达增加疾病的严重程度也增加, 即在细胞恶性转化的早期阶段就发挥作用, 并逐渐增强, 导致胃上皮细胞增生增加, 凋亡减少． 增生的过程中继而出现p53基因的突变, 即突变型p53蛋白表达增强, 诱导凋亡作用减弱．
引文著录: 杜雅菊, 赵晶, 赵瑞波, 李宝杰. 幽门螺杆菌CagA蛋白与胃癌组织中Bcl-2、p53蛋白表达的关系. 世界华人消化杂志 2003; 11(5): 554-557
Revised: September 20, 2002
Accepted: October 3, 2002
Published online: May 15, 2003
The relationship between cytotoxin-associated protein (CagA)+Helicobacter pylori (H.pylori) infection and gastric cancer in China was reported inconsistently. Using enzyme-linked immunosorbent assay (ELISA) to detect serum anti-CagA antibody and ABC immunohistochemical staining to detect the expression of Bcl-2 and p53 proteins, this study is to explore the relationship between CagA H.pylori infection and expressions of p53 and Bcl-2 proteins in gastric cancer and chronic gastric diseases.
Seventy-nine patients (50 with gastric cancer, 17 with chronic atrophic gastritis, 5 with gastric ulcer and 7 with chronic superficial gastritis) were diagnosed by endoscopy and biopsied under endoscopy for detection of H.pylori infection, and confirmed by pathological examination. Expression of Bcl-2 and p53 proteins was detected by immunohistoche-mistry. Rapid urease test and serum H.pylori antibodies with ELISA or Warthin-Starry silver stains were used for H.pylori diagnosis. H.pylori was defined as positive when 2 or 3 of these tests were positive. ELISA was used for the detection of serum anti-CagA antibody.
Anti-CagA antibody was present in 43/50(86%) gastric cancer and in 13/29 (41%) chronic gastric disease, CagA+H.pylori rate in gastric cancer was higher than those in chronic gastric diseases (P<0.01). The positive rate of Bcl-2 expression in gastric cancer was higher than that in chronic gastric diseases (62% vs 41%, P>0.05). In CagA+ gastric cancer it was higher than that in CagA- one (65% vs 43%, P>0.05), and in CagA+ chronic gastric diseases it was higher than that in CagA- one (77% vs 12%, P<0.01). The positive rate of mutant p53 expression in gastric cancer was higher than that in chronic gastric diseases (54% vs 28%, P<0.05), in CagA+ gastric cancer it was significantly higher than that in CagA- one (60% vs 14%, P<0.05), and in CagA+ chronic gastric diseases it was significantly higher than that in CagA- ones (46% vs 12%, P<0.05).
Expression of anti-CagA antibody is present in a significantly higher percentage of gastric cancer subjects than that in chronic gastric diseases. It is suggested that CagA+H.pylori infection may increase the risk of gastric cancer. The CagA factor may be involved in the development of gastric cancer through influencing the expression of Bcl-2 gene and mutant p53 gene.
- Citation: Du YJ, Zhao J, Zhao RB, Li BJ. Relationship between expression of Bcl-2 and p53 protein and CagA+Helicobacter pylori in gastric cancer. Shijie Huaren Xiaohua Zazhi 2003; 11(5): 554-557
- URL: https://www.wjgnet.com/1009-3079/full/v11/i5/554.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v11.i5.554
大量临床研究已证明幽门螺杆菌(H.pylori)感染与慢性胃炎、消化性溃疡、胃癌以及胃MALT淋巴瘤等胃部疾病的发生有关[1-12], 尤其在胃癌发病中的作用越来越受到人们的重视[13-19]．细胞毒素相关蛋白A(cytotoxin-associated protein, CagA)是由H.pylori相应的CagA基因编码产生的一种外膜蛋白, 是H.pylori的重要致病因子[20-25]． 在感染H.pylori CagA＋株患者的血清中可监测到抗CagA抗体[26,27]. 胃癌的形成是一个多因素、多阶段、长期相互作用的正常细胞恶性转化的过程． 细胞增生和凋亡的失衡与肿瘤的发生发展有密切关系． H.pylori亦可能通过其毒力因子使癌基因活化或肿瘤抑制基因失活导致胃黏膜上皮细胞增生和凋亡之间失衡, 最终导致胃癌的发生．本文将通过检测H.pylori阳性胃癌与慢性胃病患者血清中CagA-H.pylori抗体和组织细胞中癌基因Bcl-2和抑癌基因p53蛋白表达, 进一步探讨H.pylori CagA与胃癌的相关性及致胃癌的可能分子机制．
2001-02/10我院就诊胃镜下诊断为胃癌、萎缩性胃炎、胃溃疡、浅表性胃炎, 距幽门5 cm以内钳取胃黏膜组织作快速尿素酶试纸测定, 阳性者为研究对象, 并钳取病变组织2块保存于40 g/L甲醛溶液中．同时抽取患者静脉血2 ml静置4 h, 离心后取血清置于-40 ℃保存．ELISA法血清H.pylori抗体或病理Warthin-Starry银染色, 两项阳性为H.pylori感染, 入选本实验胃癌组50例: 男40例, 女10例; 慢性胃病组29例: 其中萎缩性胃炎17例, 胃溃疡5例, 浅表性胃炎7例, 男18例, 女11例．
CagA蛋白的检测试剂盒购自上海晶莹生物技术有限公司．其抗体采用基因工程表达CagA抗原并经纯化而成．首先将待测血清及试剂盒平衡至室温, 血清样品作1:21倍稀释后, 将阳性对照液、阴性对照液及血清稀释标本各100 μL依次加入包被小孔中, A1孔作空白对照. 37 ℃保温30 min．洗涤后各孔加入酶标应用液100 μL, 37 ℃保温30 min．洗涤后加入底物A液、底物B液各50 μL, 37 ℃保温15 min．各孔加入终止液50 μL, 在450nm处读取A值．免疫组化方法测定Bcl-2和p53蛋白的表达. 将标本用石蜡包埋后切成4 μm的切片, 其中一张切片进行常规HE染色作组织学诊断, 另外的切片保存作免疫组化ABC法染色检测, Bcl-2、p53单克隆抗体被用作第一抗体.用缓冲盐液做阴性对照. 每个组织切片于高倍镜下记数1000个细胞以评价阳性细胞的百分数, <5%为阴性, >5%为阳性．
统计学处理 感染率和基因蛋白表达率用百分数表示, 比较采用x2检验, P<0.05有显著性差异.
胃癌组CagA＋株感染阳性率86%(43/50)高于慢性胃病组45%(13/29), 有显著性差异(P<0.01)． CagA＋H.pylori感染与胃癌明显相关．
Bcl-2蛋白表达: 胃癌组高于慢性胃病组(62% vs 41%; P>0.05), 胃癌CagA＋组高于CagA－组(65% vs 43%; P>0.05), 慢性胃病CagA＋组显著高于CagA－组(77% vs 12%; P<0.01)．p53蛋白表达: 胃癌组显著高于慢性胃病组(54% vs 28%; P<0.05), 胃癌CagA＋组显著高于CagA－组(60% vs 14%; P<0.05), 慢性胃病CagA＋组显著高于CagA－组(46% vs 12%; P<0.05)．
H.pylori感染的结局不同是由其基因多态性决定的, 只有60%的H.pylori有CagA基因． CagA进入细胞后, 被细胞内特定的激酶磷酸化, 从而引起细胞骨架的重排以及细胞内信号传导系统的变化, 最终导致更严重的炎症及免疫反应, 而长期的慢性炎症易导致胃黏膜萎缩, 并发展成为慢性萎缩性胃炎, 甚至胃癌[28]． Blaser et al[20]比较了H.pylori感染的103例胃癌患者和103例非胃癌患者, 在长达23 a时间内经血清学检查发现CagA抗体阳性者胃癌发生率比CagA阴性者危险性增加2倍, 尤其对发展为肠型胃癌的患者更显著． Parsonnet et al [24]研究发现H.pylori感染的胃癌患者CagA抗体阳性为77.8％, 感染CagA＋H.pylori的患者发生胃癌的危险性是对照组的5.8倍, 认为CagA＋H.pylori与胃癌明显相关．我国学者[25]在胃癌高发区福州对CagA蛋白与胃腺癌关系的研究也证实了上述观点, 认为CagA＋H.pylori的感染在胃癌发生的起始阶段起了"启动子"的作用． Crabtree et al[29]研究了55例H.pylori感染的胃癌患者, 其中50例(91%)血清抗CagA阳性, 明显高于H.pylori感染的非溃疡性消化不良患者(72%), 二者有显著的统计学差异. 本结果显示, 胃癌组中CagA＋H.pylori的感染率明显高于慢性胃病组(86% vs 45%), 萎缩性胃炎和胃溃疡CagA＋H.pylori的感染率(12/22, 54.6%)明显高于浅表性胃炎者(1/7, 14.3%), 认为CagA＋H.pylori的感染在胃癌发生的起始阶段起作用, 与上述研究结果相似．
胃癌的发生与细胞增生和凋亡的不平衡有关, 如果细胞凋亡活跃, 而增生减少, 可导致胃黏膜萎缩, 形成萎缩性胃炎; 如果细胞凋亡减少, 而增生活跃, 增加了自发性DNA复制错误的产生机率, 同时增加了内外源性致癌因子致DNA突变的机会, 导致肿瘤的发生. H.pylori的感染, 特别是CagA＋H.pylori感染的慢性浅表性胃炎其胃上皮细胞凋亡与增生均明显增加, 二者呈正相关, CagA＋H.pylori感染的萎缩性胃炎、不典型增生、胃癌, 细胞凋亡逐渐减少, 在胃癌时低于正常胃黏膜, 但细胞增生指数在萎缩性胃炎、不典型增生时减少, 在胃癌时明显增加[22-25].
Bcl-2基因是发现的第一个抑制细胞凋亡的基因, 是通过抵抗细胞凋亡、延长细胞的寿命、促进细胞生存, 从而使肿瘤发生发展． Bcl-2表达可增加细胞对多种促凋亡因素的抵抗性, 在细胞的程序化死亡中起着非常重要的作用． 野生型p53蛋白正常情况下是一种不稳定的调节蛋白, 其半衰期仅有15-30 min. 因此在正常细胞中几乎测不到, 故对细胞的生长无抑制作用. 当细胞中的DNA受到损伤后, p53蛋白出现积聚现象, 并诱导细胞凋亡. p53基因突变型蛋白表达增加, 但失去了诱导凋亡的作用, 细胞聚集增加, 最终导致癌变形成．野生型p53基因缺陷细胞中Bcl-2的表达水平升高[27-30]．
王思平et al[31]的实验中显示胃癌CagA＋株感染病例p53、bcl-2的阳性率(前者为57.4%, 后者为61.8%)均显著高于CagA-患者(0%)． 所以推测其可能机制为H.pylori及其CagA蛋白可通过促进Bax、bcl-2、 p16、p21的表达, 从而影响胃黏膜的增生和凋亡, 增加了DNA损伤的可能性, DNA的损伤又可引起p53的高表达又进一步促进了Bax、p16、p21的表达. 随着DNA的损伤、基因突变的累积、p53亦可能随之突变, 引起细胞增生与凋亡的失衡, 进而发生胃癌． 本研究结果提示, Bcl-2 和p53蛋白表达在CagA＋H.pylori感染者均高于CagA－H.pylori感染者(胃癌组和慢性胃病组), 说明CagA＋H.pylori可能导致Bcl-2蛋白表达增强和p53基因突变, 细胞凋亡抑制, 使慢性炎症导致的细胞增生无法控制, 促进胃癌的发生. 7例浅表性胃炎CagA＋H.pylori感染仅1例, 且组织中Bcl-2和p53的表达均阴性． 17例萎缩性胃炎和5例胃溃疡CagA＋H.pylori感染12例, Bcl-2的表达率最高, 为83%, 且Bcl-2在CagA＋组中表达率显著高于CagA-组, 说明Bcl-2蛋白在癌前疾病阶段已开始发挥作用, 且与CagA相关, 与其他研究结果相似[32-34]. 这提示我们, CagA＋H.pylori作为高毒株能够引起胃黏膜严重而持续的炎症, 导致胃上皮细胞Bcl-2的表达增强, 抑制细胞凋亡, 继而反应性增生增加[34]. 增生和凋亡的失衡增加了DNA损伤的可能性[35], 导致p53 基因突变[32-34]． 但确切机制有待于进一步的研究．
因此, 我们可以认为CagA＋H.pylori是H.pylori的高毒株, H.pylori的CagA是与胃癌形成密切相关的原因之一, 可能通过Bcl-2和p53蛋白表达增强使细胞增生与凋亡失衡, 导致细胞恶变．
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