100023 北京市2345信箱 世界华人消化杂志  2001年9月15日;9(9):988-991
Email: wcjd@public.bta.net.cn 世界华人消化杂志  ISSN 1009-3079  CN  14-1260/R
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⊙研究原著⊙

HSP60和CD44V6在胃腺癌中的表达及其意义

陈志芬1 邓长生1 夏冰1 朱尤庆1 曾俊2 龚玲玲2

武汉大学中南医院 1消化科 2病理科 湖北省武汉市 430071
陈志芬，女，1968-09-25生，湖北省武汉市人，汉族. 1990年湖北医科大学 本科毕业，2000年获武汉大学医学硕士学位，讲师，主治医师.
国家教育部留学回国人员基金资助, No.GW9802
项目负责人  陈志芬, 430071, 湖北省武汉市武昌区东胡路, 武汉大学中南医院消化内科. Zhcxie@263.net
Tel: 027-87317915
收稿日期  2001-02-28    接受日期    2001-05-16


Expression of heat shock protein 60, CD44 splice variant V6 in human gastric cancer

Zhi-Fen Chen1, Chang-Sheng Deng1, Bing Xia1, You-Qing Zhu1, Jun Zeng2 and Ling-Ling Gong2
1Department of Gastroenterology, and 2Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, China
Supported by the fund of National Ministry of Education for the studied -abroad returnees, No.GW9802
Correspondence to  Zhi-Fen Chen, Department of Gastroenterology , Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, China. Zhcxie@263.net
Received  2001-02-28  Accepted  2001-05-16


Abstract
AIM  To determine the expression of HSP60 and CD44V6  in human gastric cancer tissues.

METHODS  Immunohistochemical staining was used in clinical specimens of 50 cases of human gastric cancer and 60 cases of noncancer chronic gastric mucosal lesion with monoclonal antibody against human HSP60, CD44V6.

RESULTS The positive rate of HSP60 in chronic superficial gastr itis, intestinal metaplasia, dysplasia and gastric cancer was 30%, 65%, 70% and 76%, respectively. The positive rate of CD44V6 in chronic superficial ga stritis, intestinal metaplasia, dysplasia and gastric cancer was 5%, 25%, 30% and 78%. The positive staining rates of HSP60 and CD44V6  in intestinal metaplasia, dysplasia and gastric cancer were higher than that in chronicsuperficial gastritis (P＜0.05). The overexpression rate of HSP60 in chronic superficial gastritis, intestinal metaplasia, dysplasia and gastric cancer was 10%, 25%, 35% and 58%. HSP60 was obviously over-expressed in gastric cancer as compared to that in intestinal and dysplasia (P＜0.05) . The positive rate of HSP60 in high, moderate and low differentiation of gastric cancer was 64.7%, 77.7% and 75.0%. The expressions of HSP60 in variant differentiation of gastric cancers were not different (P ＞0.05), but the positive rate of CD44V6 in high, moderate and low differentiation of gastric cancer was 64.7%, 66.2% and 91.7%, the expression  of CD44V6 in low differentiation of gastric cancer was more obvious than that in moderate and high differentiation of gastric cancer.

CONCLUSION  HSP60 and CD44V6 are expressed in human gastric cancer, which might contribute to the occurrence and development of the cancer. Determination of CD44V6 by immunohistochemistry might be useful in assessing tumor differentiation.

Subject headings stomach neoplasms/pathology; adenocarcin oma/pathology; chaperonin 60/biosynthesis; antigens, CD44/biosynthesis; glycoproteins/biosynthesis

Chen ZF, Deng CS, Xia B, Zhu YQ, Zeng J, Gong LL. Expression of heat shock protein 60, CD44 splice variant V6 in human gastric cancer. Shijie Huaren Xiaohua Zazhi, 2001;9(9):988-991


摘要
目的  研究热休克蛋白60（HSP60）、CD44V6在人胃癌组织 中的表达.

方法 对60例非癌胃粘膜病变及50例胃腺癌组织手术及内镜活检石蜡标本，应用抗人HSP60, CD44V6单克隆抗体，以免疫组织化Ъ觳夥椒觳釮SP60, CD44V6的表达.

结果 HSP60在慢性浅表性胃炎、肠上皮化生、不典型增生、胃腺癌的阳 性率为30%, 65%, 70%, 76%，过表达率为10%, 25%, 35%, 58%. CD44V6在慢性浅表性胃炎、肠上皮化生、不典型增生、胃腺癌的阳性率为5%, 25%, 30%, 78%, HSP60, CD44V6在肠上皮化生、不典型增生及胃腺癌中的阳性率和过表达率均明显高于慢性浅表性胃炎（P＜0.05）；HSP60在胃腺癌组织的过表达率高于肠上皮化生和不典型增生(P＜0.05). HSP60在高、中、低分化胃腺癌中的阳性表达率为64.7%,  77.7%, 75.0%. HSP60在不同分化程度胃腺癌中表达无明显差异(P＞0.05)，而CD44V6在高、中、低分化胃腺癌中的阳性表达率为64.7%, 66.2%, 91.7%, CD44V6在低分化胃腺癌的表达明显高于高、中度分化胃腺癌(P＜0.05).

结论 HSP60, CD44V6在人胃癌及癌前情况或病变中过度表达， 可能与胃腺癌的发生、发展有关，还可能有助于预测胃腺癌的分化程度.

主题词  胃肿瘤/病理学；腺癌/病理学；监控蛋白60/生物合成；抗原，CD44/生物合成；糖蛋白类/生物合成

陈志芬, 邓长生, 夏冰, 朱尤庆, 曾俊, 龚玲玲. HSP60和CD44V6在胃腺癌中的表达及其意义. 世界华人消化杂志, 2001;9(9):988-991


0  引言
HSP-S广泛存在于原核及真核细胞中，是一组与细胞耐受应激损伤关系密切的蛋白质［ 1-3]. CD44家族属粘附分子［4,5]. 近年，有学者报道HSP60, CD44V6与人体肿瘤关系密切［6-25]. 本文采用免疫组织化学方法检测胃癌组织及 非癌性胃粘膜组织中HSP60, CD44V6的表达, 以探讨其在胃腺癌发生、发展及分化程度的意义.

1  材料和方法
1.1 材料 收集1999-01-01/1999-12-30期间50例胃腺癌和60例非癌慢性胃粘膜病变病例的手术 及内镜活检的胃粘膜组织，标本来源于武汉大学中南医院及湖北省肿瘤医院病理科，所有标本均有病理依据. 其中，60例非癌性慢性胃粘膜炎性病变中，慢性浅表性胃炎20例， 肠上皮化生20例，不典型增生20例，男42例，女18例，年龄18岁～70岁，平均年龄49.5岁 ；50例胃腺癌中，按WHO分类标准，高分化癌17例，中等分化癌9例，低分化癌24例，男31例，女19例，年龄30岁～75岁, 平均年龄53.2岁.
1.2 方法 ① 切片制作：标本经10% Formalin液固定，

常规石蜡包埋，连续切片(切片厚度3μm). HE染色切片按常规程序制作，备作胃粘膜的病理检查. 用于免疫组织化学的切片，按免疫组化方法处理，并涂上多聚赖氨酸溶液，晾干后再贴片备用. ② 试剂： 鼠抗人HSP60单克隆抗体、鼠抗人CD44V6单克隆抗 体及免疫组化试剂盒、DAB显色剂，均购自福州迈新生物技术开发公司；多聚赖氨酸购自武 汉博士德生物工程有限公司. ③ 步骤： 切片常规脱蜡水化，经3% H2O2室温下浸泡15min，以灭活内源性酶，微波抗原修复20min，取出冷却，滴加过氧化酶阻断剂（3% H2O2甲醇）1滴，湿盒中放置10min, 再滴加正常羊血清湿盒中放置15min后甩干, 切片 分别滴加一抗(鼠抗人HSP60或CD44V6)，4℃下放置一夜，继之进入生物素标记的第二抗体（羊抗鼠IgG）孵育15min，滴加链霉素抗生物素蛋白-过氧化酶溶液，室温湿盒中 放置15min，DAB显色5min～10min，苏木素复染后，脱水透明封片，光镜下观察.  以上步骤 中，阴性对照以PBS代替一抗，阳性对照为已知的HSP60阳性、CD44V6阳性的人宫颈癌组织片. ④ 结果与判断： HSP60阳性结果判断，细胞浆出现棕黄色为阳性细胞，计数 20个高倍视野的阳性细胞，根据阳性细胞百分率分为阴性(-)：阳性细胞＜5%；弱阳性（+） ：阳性细胞数6%～30%；中等强度阳性（++）：阳性细胞数31%～60%；强阳性（+++）：阳性细胞数＞60%. 以中等强度阳性和强阳性为过表达病例［26]. CD44V6阳性结果判断： 细胞膜有明显棕黄色为阳性 细胞，按阳性细胞数＜5%，6%～25%，26%～50%和＞50%分为-, +, ++, +++四级［27]. 统计学处理 各样本间总体比较采用秩和检验，率间比较采用χ2检验. 相关分析应用Statistica 5.0软件分析.

2  结果
2.1   HSP60阳性细胞浆中出现棕黄色染色(见图1)，少数细胞出现胞膜着色，未见胞核着色，HSP60在胃腺癌及胃粘膜其他病变中染色情况见表1.

表1  HSP60在胃粘膜病变组织中的表达

病变类型 n 染色强度 阳性率(%) 过表达率(%)
- + ++ +++
慢性浅表性胃炎 20 14 4 2 0 30 10
肠上皮化生 20 7 8 3 2 65a 25c
不典型增生 20 6 7 4 3 70a 35c
胃腺癌 50 12 9 13 16 76a 58

aP＜0.05, vs 慢性浅表性胃炎；cP＜0.05, vs 胃腺癌.

HSP60在慢性浅表性胃炎中的表达多为弱阳性, 阳性为30%, 主要集中于腺颈部. 肠上皮化生与不典型增生相比HSP60表达阳性率差异不显著(P＞0.05)，胃腺癌与 肠上皮化生及不典型增生相比，阳性率差异不显著，但HSP60在胃腺癌中的过表达率与其在 肠上皮化生及不典型增生中的过表达率之间差异显著(P＜0.05).

2.2  CD44V6阳性表现 为细胞膜有明显棕黄色(见图2)，未见胞核、胞质着色，CD44V6在胃腺癌及胃粘膜其他病变中的染色情况见表2.

图1 胃腺癌HSP60胞质阳性表达. S-P法×200

图2 胃腺癌CD44V6胞膜阳性表达. S-P法×200

表2 CD44V6在胃粘膜病变组织中的表达

病变类型 n 染色强度 阳性率(%)
- + ++ +++
慢性浅表性胃炎 20 19 1 0 0 5
肠上皮化生 20 15 5 0 0 25ac
不典型增生 20 14 6 0 0 30ac
胃腺癌 50 11 11 14 14 78a

aP＜0.05, vs  慢性浅表性胃炎；cP＜0.05, vs  胃腺癌.

2.3  HSP60, CD44V6在不同分化程度胃腺癌中的表达 见表3.

表3 HSP60、CD44V6在不同分化程度胃腺癌中的表达

分化程度 n HSP60 (%) 总阳性率  CD44V6 (%) 总阳性率 
- + ++ +++ - + ++ +++
高分化 17 6 3 4 4 64.7a 6 4 4 3 64.7c
中等分化 9 2 2 2 3 77.7a 3 2 2 2 66.2c
低分化 24 6 4 7 7 75.0 2 5 8 9 91.7

aP＞0.05, cP＜0.05, vs 低分化胃腺癌.

2.4 将HSP60, CD44V6在不同胃粘膜病变组织中的表达阳性率作相关分析，其相关系数r=0.77598, P＜0.05(图3).

图3 HSP60与CD44V6表达阳性率的相关关系.

3  讨论
HSP又称应激蛋白，在正常细胞中表达很低，在高热、乙醇中毒、病毒感染、氨基酸类似物 储积、DNA损伤、缺氧、重金属离子过多、细胞内出现变性自身蛋白等应激因素影响下，细 胞内HSP-S表达增强，维持细胞内环境的稳定，使细胞耐受应激损伤，这是细胞的一种自身 保护［28-30]. HSP-S按其分子量大小分为若干家族，在众多的HSP-S中，以HSP60, HSP70在大多数生物细胞中含量最多［31]. 有报道HSP60在一些应激损伤 组织、炎症组织及肿瘤组织中表达增强［32]. 本研究显示，HSP60在慢性浅表性胃 炎、肠上皮化生、不典型增生及胃腺癌中的阳性表达率分别为30%, 65%, 70%, 76%；过表达 率分别为10%, 25%, 35%, 58%. HSP60在胃腺癌组织中过度表达. 正常胃粘膜是更新十分活 跃的组织（人类4～6d·次-1)，胃粘膜上皮及腺上皮细胞的更新修复是由胃腺颈 部增殖细胞（即多分化潜能干细胞）进行的［33-35]. 这种细胞及胃粘膜上皮尚未分化， 细胞最易受各种刺激因素的作用而损伤致癌. 胃粘膜在持续有害因素作用下，粘膜屏障被不断损伤而削弱. 粘膜病变由浅表性胃炎向萎缩性胃炎发展，同时刺激腺颈部增殖修复和向肠上皮分化，在此过程中,基因表达的不规则可使细胞出现异型性，进而癌变［36］ . 本研究中显示，在慢性浅表性胃炎病例中，HSP60阳性染色主要集中于腺颈部，反映HSP60参与胃粘膜上皮的更新及粘膜抗损伤修复. 而肠上皮化生和不典型增生是胃癌粘膜演变的前驱病变，属于慢性增殖性病变，本研究显示HSP60在肠上皮化生及不典型增生组织中 表达增强（分别为65%, 70%），并持续到胃癌阶段（76%）. 提示了肠上皮化生和不典型增 生的增殖属性. 胃癌是慢性胃粘膜病变演变的最后阶段，与其他肿瘤一样，胃癌具有无限增殖的特点. 在胃癌细胞中存在多种基因变异，包括原癌基因的突变、扩增、激活和抑癌基因的突变、缺失、失活等，存在DNA损伤等内环境紊乱，这些均可能导致HSP60在胃癌中过度表达［37,38]. 本研究中，HSP60在胃癌中的过表达率为58%，明显高于其在肠上皮化生及不典型增生中的过表达率. 有研究表明，HSP60的过度表达除了可能参与 胃癌增殖外，还可能对胃癌细胞有保护作用［39]. 过度表达的HSP可使细胞获得对 TNF及单核细胞杀伤的耐受，有助于肿瘤免疫逃逸［40]. 而胃癌中HSP60的过表达 是否有助于免疫逃逸尚待进一步研究. CD44家族是一类具有高度异质性的单链膜表面糖蛋白，参与细胞与细胞，细胞与间质之间的相互作用，被认为与细胞的运动、肿瘤生长和侵袭有关. 结肠癌、乳腺癌CD44 V6表达与肿瘤进展、淋巴转移以及手术预后有关［7,41-43]. 人类胃癌CD4 4V6表达阳性率为64%～77%, 且表明CD44V6阳性表达与胃癌的分化程度有［24,44-46］. 本研究显示胃腺癌CD44V6的阳性率为78%，与文献报道相近；还显示在不同分化程度胃腺癌中，低分化胃癌的CD44V6表达阳性率明显高于高、 中度分化胃癌，由此推测CD44V6表达率愈高，提示其恶性程度可能愈高. 然而，CD44V6表达在胃癌发生过程中是否属早期事件，目前尚未明确［13,47］， 本实验中，不典型增生的病例的CD44V6的阳性率是30%, 但明显低于胃腺 癌的78%，提示CD44V6的表达属于胃粘膜癌变早期.另外，HSP60, CD44V6在慢性浅表性胃炎、肠上皮化生、不典型增生、胃癌中的表 达逐级升高，且差异显著，表明在胃癌发生过程中，HSP60, CD44V6的表达上调. 值得注意的是，CD44V6强表达(染色强度≥+++)，仅发生 于胃癌组织，而非癌性胃粘膜病变中未发现，推断如果胃粘膜上皮出现CD44V6强阳性表达，应考虑是否有癌变. HSP60, CD44V6是两类不同的蛋白，但两者在浅表性胃炎、肠上皮化生、不典型增生、胃癌的表达逐级上升，通过两者阳性表达率的相关分析两者有相关性，且呈直线相关. 虽然两者不是互依的，但从这点上是否可以推断：在临床工作中，若同时检测两者均增高，对胃癌的临床诊断意义更大.
    总之，本研究应用免疫组织化学方法提示HSP60, CD44V6在人胃癌和癌前情况及病变中表达率增高，可能与胃癌的发生、发展有关，检测胃癌组织中HSP60, CD44V 6的表达，有助于胃腺癌分化程度的判定.

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