E-钙黏附素和β-连环素在胰腺上皮内瘤变和胰腺癌组织中的表达

摘要

目的: 探讨胰腺上皮内瘤变PanIN和胰腺癌组织中E-钙黏附素(E-Cad)和β-连环素(β-Cat)异常表达的意义.

方法: 回顾性研究长海医院2001-01/2003-12间外科切除和同期尸检的156例胰腺标本, 并构建了组织芯片, 其中含有129灶PanIN-1A , 104灶 PanIN-1B, 22灶PanIN-2, 11灶PanIN-3和121例导管腺癌和相应癌旁组织. 用EnVision免疫组化技术检测上述病变组织中E-Cad和β-Cat的表达变化, 并结合临床病理资料进行相关分析.

结果: 导管腺癌中E-Cad异常表达率明显高于PanINs和正常导管(64.5%, 32.3%, 0%), 且与胰腺癌的分化程度、淋巴结转移和神经浸润密切相关(P<0.05). PanINs和导管腺癌中E-Cad胞质表达较正常导管明显增加. β-Cat的异常表达与胰腺癌淋巴结转移和神经浸润有明显相关性(P<0.05). 高级别PanINs和导管腺癌中α-Cat胞质和胞核的表达率明显高于低级别PanINs和正常导管(P<0.05). PanINs和导管腺癌中E-Cad和β-Cat表达间呈正相关性(P<0.01, P<0.05).

结论: 胰腺癌和PanINs中E-Cad和β-Cat的异常改变提示他们不仅与胰腺癌的生物学行为和预后有关, 而且也参与了胰腺癌的发生.

关键词: N/A

E-cadherin and β-catenin expression in pancreatic intraepithelial neoplasia and pancreatic adenocarcinoma

Jian-Ming Zheng, Ming-Hua Zhu, Can-Rong Ni, Guan-Zhen Yu, Xiao-Hong Liu, Wan-He Lin, Zhi-Jing Gong

Jian-Ming Zheng, Ming-Hua Zhu, Can-Rong Ni, Guan-Zhen Yu, Xiao-Hong Liu, Wan-He Lin, Zhi-Jing Gong, Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China

Supported by: the Fund for New Projects in Changhai Hospital and Subject Constructive Program of Basic and Clinical Research on Pancreatic Diseases, No. 123258.

Correspondence to: Ming-Hua Zhu, Department of Pathology, Changhai Hospital, Second Military Medical University, 174 Changhai Road, Shanghai 200433, China. mhzhu@smmu.edu.cn

Received: July 23, 2004
Revised: September 1, 2004
Accepted: September 4, 2004
Published online: November 15, 2004

Abstract

AIM: To investigate the significance of abnormal E-cadherin (E-Cad) and β-catenin (β-Cat) expression in pancreatic intraepithelial neoplasia (PanIN) and pancreatic adenoc-arcinoma.

METHODS: One hundred and fifty-six cases of pancreatic samples, which were surgically removed and autopsied in Changhai Hospital from January 2001 to December 2003, were retrospectively studied. Tissue microarray blocks containing 129 PanIN-1A lesions, 104 PanIN-1B lesions, 22 PanIN-2 lesions, 11 PanIN-3 lesions, and 121 cases of pancreatic ductal adenocarcinoma and corresponding para-cancerous tissues were constructed. EnVision method of immunohistochemistry was used to detect the E-Cad and β-Cat expression in the above tissues. The correlation between the abnormal E-Cad, β-Cat expression and clinicopathological parameters was analyzed.

RESULTS: The rate of E-Cad abnormal expression was significantly higher in ductal adenocarcinomas than that in PanIN lesions and normal ducts (64.5% vs 32.3%, 64.5% vs 0%, P < 0.05), and the rate of E-Cad abnormal expression was in significant relation to differentiation, lymph node metastasis and perineural invasion of pancreatic adenocarcinoma (P < 0.05). There was remarkably increase of the E-Cad cytoplasmic expression in PanIN lesions and ductal adenocarcinomas compared with that in normal ducts (21.8%, 34.7% vs 1.5%; P < 0.05). The rate of β-Cat abnormal expression was found to relate to lymph node metastasis and perineural invasion of pancreatic adenocarcinoma (P < 0.05). The expression of β-Cat cytoplasm and/or nucleus was significant in high-grade PanIN lesions (27.3%) and ductal adenocarcinomas (29.8%) compared with those in low grade PanIN lesions and normal ducts (3.0%, 2.3%; P < 0.05). There was a positive relationship between the E-Cad and β-Cat expression in PanIN lesions and ductal adenocarcinomas (P < 0.01, P < 0.05).

CONCLUSION: There was aberration in the expression of the E-Cad and β-Cat in PanIN lesions and ductal adenocar-cinomas, suggesting that E-Cad and β-Cat not only relate to the biological behaviour and prognosis of pancreatic cancer, but also get involved in pancreatic carcinogenesis.

Key Words: N/A

0 引言

E-钙黏附素(epithelial-cadherin, E-Cad)是维持上皮细胞极性和细胞间黏附连接的主要分子, 许多恶性上皮肿瘤的侵袭转移与E-Cad表达及功能障碍有关[1-5]. β-连环素(β-catenin, β-Cat)是一种多功能蛋白, 参与细胞增生、分化的调节. β-Cat的异常改变与多种肿瘤的发生密切相关[6-8]. 胰腺癌组织中亦存在E-Cad和β-Cat的异常表达, 且与胰腺癌的生物学行为和预后有关[9-11]. 胰腺上皮内瘤变(pancreatic intraepithelial neoplasia, PanIN)是新近提出的用来描述胰腺小导管上皮细胞非典型增生至原位癌这一系列癌前病变连续过程的新概念[12]. PanIN, 特别是高级别PanIN, 被认为是胰腺癌的癌前病变[13]. 那么在由正常导管上皮→低级别PanIN→高级别PanIN→胰腺癌的过程中E-Cad和β-Cat的变化如何？ 目前尚未见相关报道, 我们用组织芯片和免疫组化技术比较研究了PanIN和胰腺癌组织中E-Cad和β-Cat的表达变化, 并联系临床病理资料和相关指标进行分析, 以探讨其意义.

1 材料和方法

1.1 材料

2001-01/2003-12我院病理科资料较完整的手术切除及同期尸检获得的胰腺标本250例, 复读全部病理切片, 根据胰腺上皮内瘤变最新概念和诊断标准[12], 检出PanIN病灶156例, 检出率62.4%, 包括导管腺癌121例、慢性胰腺炎23例, 正常胰腺12例(尸检9例, 胰腺外伤1例, 脾亢1例, 异位1例). 男93例, 女63例, 年龄30-80(平均58.6岁). 胰腺导管腺癌位于胰头部92例, 胰体尾27例, 全胰2例. 高分化21例, 中分化68例, 低分化32例.

1.2 方法

组织芯片构建参照Kononen et al[14]方法, 应用组织芯片构建仪(Tissue Arrayer, Beecher Instruments, Silver Spring, MD, USA)制备组织芯片. 流程:应用构建仪在预先制备的空白蜡块(45 mm×20 mm)中穿孔(直径2 mm, 深度2-3 mm), 然后在组织蜡块标记点处穿取组织(直径及深度同上), 准确放入空白蜡块的小孔内, 依次按序操作, 直至完成, 并作好记录. 然后对制备好的芯片蜡块进行加热处理, 使嵌入的组织条与蜡块密切融合. 选取典型病变, 构建了6张组织芯片(图1), 共计899个点, 包括不同级别的PanIN病灶、胰腺病变和病变周围正常组织. PanIN病灶共266灶, 其中PanIN-1A有129灶, PanIN-1B有104灶, PanIN-2有22灶, PanIN-3有11灶. 石蜡切片厚4 μm, 采用免疫组化EnVision二步法, 兔抗人E-Cad和β-Cat抗体购置Santa Cruz公司, 工作浓度均为1:50, 用PBS替代一抗作阴性对照, DAB显色. 具体操作流程按说明书进行. 以正常胰腺导管上皮作为阳性对照, E-Cad和β-Cat免疫组化染色根据细胞定位(胞膜、胞质或胞核)、染色强度和分布来决定. 染色强度: 0分: 阴性; 1分: 弱阳性; 2分: 强阳性. 染色分布: 0分: 阳性细胞数<10%; 1分: 阳性细胞数10 -50%; 2分: 阳性细胞数>50%; 由二者得分的和来决定染色结果: 对于胞膜或胞质来说, 0分: 阴性(-); 2分: 弱阳性(±); 3-4分: 强阳性(＋). 对于胞核来说, 0分: 阴性(-); 2-4分: 阳性(+). 将正常胰腺导管上皮细胞膜E-Cad和α-Cat的强阳性表达和个别胞质的弱阳性表达视为正常, 其余均为异常表达.

图1 胰腺病变组织芯片, 含144个位点, HE×4.

统计学处理 PanIN-1A和 PanIN-1B 为低级别胰腺上皮内瘤变; PanIN-2和PanIN-3为高级别胰腺上皮内瘤变. 统计软件为SPSS/PC, 采用χ²检验.

2 结果

2.1 E-Cad和β-Cat在PanINs的表达

E-Cad和β-Cat在正常胰腺腺泡、导管上皮和部分胰岛细胞膜上显示均质的强阳性染色, 呈棕黄色颗粒状, 个别细胞呈胞质弱阳性, 胰腺间质细胞不着色. PanINs和胰腺癌中E-Cad膜表达明显降低或缺失, 而细胞质表达则明显升高, 未见有胞核染色者. 高级别PanINs和胰腺癌中, 可见有β-Cat膜表达的降低或缺失, 而出现核和/或胞质的表达(图2).

图2 胰腺病变中E-Cad和β-Cat的表达(EnVision法×200). A: 正常胰腺腺泡和导管上皮E-Cad呈膜强阳性; B: 正常胰腺腺泡和导管上皮β-Cat膜强阳性; C: PanIN中E-Cad膜表达降低而胞质表达增强; D: 高级别PanIN中β-Cat膜表达缺失而部分细胞核表达阳性; E:胰腺癌中E-Cad膜表达降低, 而细胞质表达增强; F: 胰腺癌中β-Cat膜表达缺失, 而部分细胞核表达阳性.

2.2 E-Cad和β-Cat表达与胰腺癌临床病理学的关系

E-Cad和β-Cat异常表达与胰腺癌患者年龄、性别及肿瘤大小无关(P>0.05). 胰腺癌中E-Cad异常表达率明显高于正常胰腺导管和PanINs(P<0.05), 且与胰腺癌分化程度、淋巴结转移和神经浸润密切相关(P<0.05). 而β-连环素异常表达则与胰腺癌淋巴结转移和神经浸润明显相关(P<0.05, 表1). 我们还比较了导管腺癌、PanINs和正常导管上皮中E-Cad胞质的表达, 发现正常胰腺导管上皮仅有个别呈胞质弱阳性, 而在胰腺导管腺癌和PanINs中分别达到21.8%和34.7%, 明显高于正常导管(1.5%). 高级别PanINs和胰腺癌中b-Cat胞核和/或胞质表达率(27.3%, 29.8%)显著高于低级别PanINs和正常导管(3.0%, 2.3%).

表1 E-Cad和β-Cat异常表达与PanINs分级和胰腺癌临床病理学的关系.

临床病理学参数	n	E-Cad		β-Cat
		异常	正常	异常	正常
PanIN-1A	129	38	91	55	74
PanIN-1B	104	36	68	46	58
PanIN-2	22	8	14	14	8
PanIN-3	11	4	7	7	4
高分化癌	21	8	13	13	8
中分化癌	68	43	25a	47	21
低分化癌	32	27	5	24	8
男	72	47	25	50	22
女	49	31	18	34	15
年龄≤40	4	2	2	2	2
40-60	48	30	18	33	15
>60	69	46	23	49	20
肿瘤<3 cm	32	19	13	21	11
3-6 cm	62	40	22	43	19
>6 cm	27	19	8	20	7
淋巴结转移+	23	20	3a	21	2a
-	98	58	40	63	35
神经浸润+	55	41	14a	44	11a
-	66	37	29	40	26

^aP<0.05, 组内比较.

2.3 E-Cad和β-Cat在PanIN和胰腺癌中表达的相关性

266灶PanIN病变中同时E-Cad和β-Cat异常表达者72灶, 正常表达者126灶, 不一致者68灶. 二者呈非常显著的正相关性(P<0.01). 胰腺癌中, E-Cad和β-Cat均有异常表达者46例, 正常表达者28例, 不一致者47例, 经统计学分析二者亦呈显著的正相关性(P<0.05).

3 讨论

黏附素与连环素在上皮细胞间黏附和信号传导中起着十分重要的作用. E-Cad为钙依赖黏附蛋白家族成员之一, 相邻细胞E-Cad胞外区彼此结合形成拉链样结构介导同型细胞相互黏附, 胞质区则与β-Cat和β-Cat结合, 后者再与细胞骨架连接, 一起形成连接复合体, E-Cad功能的发挥离不开连环素的作用[15-17]. β-连环素的另一个重要作用是作为Wnt信号通路的关键分子, 介导信号从胞膜至胞质再到胞核的传递. 在正常细胞中, β-Cat的表达主要在细胞膜, 胞质中很少, 而一旦在胞质中发生累积, β-Cat转位入核增多, 与转录因子TCF(Tcell transcription factor)形成复合物, 促进下游靶基因, 如Cyclin D1, c-myc等表达增多, 从而引起细胞增生[18].

研究显示, 在人类多种肿瘤中均存在E-Cad和β-Cat的异常[19-22]. 胰腺癌组织E-Cad和α-Cat的表达研究亦见有报道[23-24]. Joo et al[25]用免疫组化法检测了30例胰腺癌组织中E-Cad和β-Cat的表达, 结果显示, 正常胰腺上皮细胞E-Cad和β-Cat呈一致性较强的膜表达, 而胰腺癌组织中60.0%病例E-Cad和56.7%病例β-Cat的膜表达减弱或缺失, 且与胰腺癌的分化差有关. 另外, E-Cad膜表达减弱或缺失与胰腺癌的临床分期和淋巴结转移相关. Qiao et al[26]对43例胰腺癌组织进行α-Cat免疫组化研究, 发现膜表达减少者为58.1%, 有65.1%的病例出现胞质染色, 二者都与Cyclin D1高表达明显相关. 我们应用高通量组织芯片技术, 分析了胰腺癌及其癌前病变-胰腺上皮内瘤变中E-Cad和β-Cat的表达, 结果显示在64.5%和69.4%的胰腺癌组织中存在E-Cad和β-Cat的异常表达, 且E-Cad异常表达与胰腺癌组织学分级、淋巴结转移和神经浸润密切相关, β-Cat异常表达与胰腺癌淋巴结转移和神经浸润密切相关. 胰腺癌组织中E-Cad和β-Cat的异常改变, 一方面导致细胞间黏附功能下降, 另一方面β-Cat在胞质内异常集聚, 并进入核中进而激活靶基因的表达, 促进细胞增生. 上述结果提示E-Cad和β-Cat的异常与胰腺癌的生物学行为和预后有关. 此外, 我们还发现在胰腺上皮内瘤变中, E-Cad和β-Cat胞膜表达均减弱或缺失, 而胞质表达较正常胰腺导管为强; 在高级别胰腺上皮内瘤变和胰腺癌中β-Cat胞核和/或胞质的表达率明显高于低级别胰腺上皮内瘤变和正常导管. 提示E-Cad和β-Cat的异常改变在胰腺癌的发生阶段就已出现了, 说明他们也参与了胰腺癌的形成. 分析β-Cat在胞质内积聚的机制可能包括APC基因突变或缺失, β-连环素基因突变和Wnt信号途径的活化. 但在胰腺癌中有人未检测出β-Cat基因的突变, 推测可能系β-Cat基因转录增加所致[27-28].

目前, 对胰腺上皮内瘤变中基因变化的规律已有了部分认识, 了解到胰腺导管癌中的某些基因改变在PanIN中也存在, 如K-ras等癌基因的活化, P16, P53和DPC4等抑癌基因的失活等[29-31]. Hruban et al通过检测胰腺正常导管、各级胰腺上皮内瘤变和胰腺癌的基因改变, 将胰腺癌的组织学进展与分子遗传学改变联系了起来, 并建立了胰腺癌发生、发展的分子模型[32], 该模型初步揭示了胰腺癌发生过程中部分基因的变化规律. 我们的研究显示出在胰腺上皮内瘤变中亦存在着E-Cad和β-Cat的异常改变, 这是对胰腺上皮内瘤变中基因变化规律的补充, 由于胰腺上皮内瘤变中分子改变极其复杂, 要全面认识他尚有大量工作要做.

备注

编辑: N/A

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