基础研究 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-04-15; 12(4): 897-901
在线出版日期: 2004-04-15. doi: 10.11569/wcjd.v12.i4.897
大肠癌组织HLA-I类抗原及相关分子的表达意义
申龙树, 黄培林, 张建琼
申龙树, 黄培林, 东南大学基础医学院病理学系 江苏省南京市 210009
张建琼, 东南大学遗传研究中心, HLA表达与肿瘤国际参考实验室 江苏省南京市 210009
申龙树, 男, 1970-02-03出生, 江苏省南京市人, 汉族, 东南大学医学院病理学硕士研究生, 研究方向为肿瘤免疫学.
通讯作者: 黄培林, 210009, 江苏省南京市丁家桥87号, 东南大学基础医学院病理学系. hpl@seu.edu.cn
电话: 025-83272450 传真: 025-83272392
收稿日期: 2003-09-08
修回日期: 2003-10-01
接受日期: 2003-10-27
在线出版日期: 2004-04-15

目的: 探讨大肠癌组织HLA-I类分子及相关分子表达及其意义.

方法: 用免疫组化的方法, 以切端黏膜作为正常对照, 检测大肠癌组织、癌旁组织和正常黏膜组织中HLA-I类分子及相关分子的表达, 并结合肿瘤的临床病理资料综合分析.

结果: 受检的大肠癌组织中重链HLA-A位点、B/C位点及轻链beta2微球蛋白(2M)较其相应正常黏膜表达明显下调(P = 0.0 001); 肠癌中低分子量多肽(LMP2)(P = 0.0 001), 钙联蛋白(calnexin)(P = 0.004)较正常黏膜表达明显下调; 肠癌中HLA-I类分子表达与肿瘤分化无关; 随着肿瘤的Dukes分期, 癌组织中HLA-I类分子表达渐次降低, 但各位点表达情况并不相同, HLA-B/C表达水平与肿瘤分期相关, 其在Dukes A期表达水平高于D期(P = 0.0262).

结论: 肠癌组织及癌旁黏膜中HLA-I类分子、低分子量多肽及钙联蛋白较正常黏膜表达明显下调; 肠癌组织中HLA-B/C位点随着肿瘤Dukes分期演进其表达下调.

关键词: N/A
引文著录: 申龙树, 黄培林, 张建琼. 大肠癌组织HLA-I类抗原及相关分子的表达意义. 世界华人消化杂志 2004; 12(4): 897-901
Expression of HLA molecule in colorectal cancer tissue
Long-Shu Shen, Pei-Lin Huang, Jian-Qiong Zhang
Long-Shu Shen, Pei-Lin Huang, Department of Pathology, Basic Medical College, Southeast University, Nanjing 210009, Jiangsu Province, China
Jian-Qiong Zhang, Department of Genetics, Southeast University Medical School, International "HLA Expression in Cancer" Chinese Research Center, Nanjing 210009, Jiangsu Province, China
Correspondence to: Pei-Lin Huang, Department of Pathology, Basic Medical College, Southeast University Medical School, 87 Dingjiaqiaolu, Nanjing 210009, Jiangsu Province, China. hpl@seu.edu.cn
Received: September 8, 2003
Revised: October 1, 2003
Accepted: October 27, 2003
Published online: April 15, 2004

AIM: To study the relationship between the expression of HLA class I and clinicopathological significance in colorectal cancers.

METHODS: Expression of HLA class I and associated proteins was studied by immunohistochemistry in colorectal cancer, histologically normal mucosa adjacent to cancer (<3 cm), and histologically normal mucosa distant from cancer. Several monoclonal antibodies (mAbs) were used in this study: HC10 and HCA2 reacted with the nonmorphic determinant of heavy chain of HLA class I antigen; L368 reacted with 2 microglobulin; SY1 reacted with LMP2 antigen, and TO-5 reacted with calnexin.

RESULTS: In colorectal cancer tissues, the expression of HLA -A,B/C, 2M, LMP2 and calnexin were reduced compared to that of both in histologically normal mucosa adjacent to cancer and histologically normal mucosa distant from cancer (P = 0.001).The expression of calnexin in cancer was also reduced compared to that of both in histologically normal mucosa adjacent to cancer and histologically normal mucosa distant from cancer (P = 0.004). The expression of HLA-B/C antigen in cancer was associated with diverse groups of pathological stage. With increase in Dukes staging of the cancer, the expression of HLA-B/C downregulated, in which that in Dukes A was higher than that in Dukes D (P = 0.0262).

CONCLUSION: The expression of HLA class I and associated protein is downregulated in the colorectal cancer tissue. It may be one important mechanism by which colorectal cancer cell escapes immune surveillance.

Key Words: N/A
0 引言

人类白细胞抗原(human leukocyte antigen, HLA)在肿瘤中表达下调或丢失使肿瘤抗原不能与HLA-I类抗原结合而递呈, 细胞毒性淋巴细胞(cytotoxic T lymphocyte, CTL)不能识别肿瘤抗原, 导致肿瘤细胞逃避CTL杀伤作用而存活, 这是肿瘤逃避机体免疫监视的重要途径, 成为肿瘤免疫研究热点[1-4]. 但各种肿瘤HLA-I抗原表达水平各家报道不一, 我们收集65例肠癌及其临床病理资料, 应用免疫组化的方法研究肠癌组织HLA-I类抗原及相关分子的表达并探讨其临床病理学意义.

1 材料和方法
1.1 材料

收集东南大学附属中大医院2000/2001年65例肠癌手术切除标本及其临床病理资料, 取肠癌组织及紧靠肠癌的癌旁组织(靠近肠癌组织3 cm范围内), 常规石蜡包埋, 切片, 经病理医师明确诊断. 同时以切端黏膜作正常对照. 其中直肠癌30例, 结肠癌35例, 男37例, 女28例. Dukes A期9例、B期28例, C期20例, D期8例; 高分化21例, 中分化25例, 低分化19例. HC10(抗HLA-A位点重链抗原决定基), HCA2(抗HLA-B／C抗原决定基), L368(抗2M抗原决定基), SY-1(抗LMP2抗原决定基), TO-5(抗Calnexin抗原决定基)单克隆抗体, 阴性对照: 无关抗体MK2-23. 以上抗体均由Soldano Ferrone教授 (Department of Immunology, Rosewell Park Cancer Institute, USA)提供.

1.2 方法

生物素亲合素复合物酶标法(ABC法)免疫组化方法参照Ferrone's Lab的方法, DAB显色, 试剂盒均为美国Vector公司产品. 取石蜡标本, 切成4 m薄片, 脱蜡至水, 在微波炉中用柠檬酸钠抗原修复液中100 ℃抗原修复15 min, PBS冲洗后用双氧水阻断15 min, 冲洗后用Casin室温下阻断40 min, 分别加入一抗HC10 (1: 10), HCA2 (1: 40), L368 (1: 40), SY-1(1: 180), TO-5(1: 40), MK2-23(1: 100). 4 ℃冰箱过夜, PBS冲洗后, 加入生物素化二抗(1: 200), 37 ℃ 60 min, PBS冲洗后, 加入生物素亲合素复合物(1: 20) 37 ℃ 40 min, 冲洗后浸入酶底物溶液中(0.5 g/L DAB 100 mL, 30 mL/L双氧水10 L), 室温下反应5 min. 蒸馏水洗片终止反应, 苏木精染核, 封片后观察[5-6]. 染色结果分别由两位病理医师按以下标准判断, 每张切片观察全部视野. 以组织中淋巴细胞作为阳性参照对象, 判断该片中淋巴细胞是否着色, 以反映染色成功与否; 染色呈棕黄色且阳性面积大于或等于75%记为3分; 染色呈黄色且阳性面积大于或等于75%或染色呈棕黄色且阳性面积在25%-75%之间记为2分; 染色呈淡黄色且阳性面积大于或等于75%或染色呈黄色且阳性面积在25%与75%之间记为1分; 阳性染色面积小于或等于25%分记为0分[4,6-7]. 以正常黏膜作为对照, 结合临床病理资料进行分期、分级[8-9], 统计每组各抗原的表达水平, 并进行相关性分析.

统计学处理 用SAS软件(SAS institute Inc USA 6.12版)对资料用配对t检验和χ2检验进行统计分析.

2 结果

所有组织切片中淋巴细胞都表达HLA-A, HLA-B/C, 2M, LMP2 和calnexin, 且明显高于正常黏膜细胞(P = 0.0 001), 成为本实验良好的内参照. HLA-A, B/C重链抗原在正常黏膜中多表达于细胞膜上, 呈黄色或棕黄色; 正常黏膜的HLA-A、HLA-B/C的表达水平高于肠癌组织(P = 0.0 001, 图1). 癌旁组织的HLA-A, HLA-B/C的表达水平介于肠癌组织和正常黏膜之间, 癌旁组织与肠癌组织的表达水平差异性无显著性. 在肠癌细胞表面HLA-A, HLA-B/C具有异质性即在同一肠癌组织中部分区域呈阳性, 部分区域呈阴性, 呈现异质性表达(图2-5).

图1
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图1 肠癌组织HLA-I类分子及相关分子的表达.
图2
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图2 HLA-B/C分子在正常黏膜中表达×400.
图3
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图3 HLA-B/C分子在肠癌中表达×200.
图4
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图4 HLA-B/C分子在肠癌中异质性表达×200.
图5
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图5 HLA-B/C分子在肠癌中不表达×200.

肿瘤组织HLA-A, HLA-B/C抗原表达水平与肿瘤分化程度无明显相关性; HLA-A表达随着Dukes分期依次降低, 但无明显差异; HLA-B/C位点抗原表达随着Dukes分期明显下调, A期与D期差异显著(P = 0.0 262); 而且肠癌中HLA-A, B/C抗原异质性表达现象只发生在肠癌浸润周围组织及淋巴结或出现远处转移时(Dukes B, C期, 表1). 2微球蛋白(2M)与HLA-I类抗原重链通过非共价键结合, 表达在细胞质及细胞膜表面, 呈黄色或淡黄色. 2M在肠癌组织中表达水平较正常黏膜低(P = 0.0 001); 癌旁黏膜表达强于肠癌组织而弱于正常黏膜, 与肠癌组织差异无显著性; 肠癌组织2M表达水平与肿瘤分期、分级、转移无明显相关性(图1, 表1). 低分子量蛋白2(LMP2)表达在细胞质内, 钙联蛋白(calnexin)呈膜浆型表达, 呈黄色或淡黄色(图6, 7), 在肠癌组织中呈现异质性表达, 表达水平较正常黏膜低(P = 0.004); 癌旁黏膜的LMP2和calnexin表达水平低于正常黏膜, 高于肠癌组织(P<0.01). LMP2和calnexin的表达水平与肠癌分级、分期无明显相关性(图1、表1).

表1 HLA-I类分子及相关分子表达与肠癌病理的关系(mean±SD).
HLA-AHLA-BC2MLMP2CALNEXIN
分化 高0.90±1.001.46±1.031.06±1.121.23±1.001.32±0.79
中1.20±0.981.25±1.261.13±0.961.60±1.031.60±1.03
低2.33±1.151.68±0.901.45±0.931.59±0.911.59±0.91
分期 A1.00±0.812.50±0.81b1.75±0.501.75±0.951.50±1.00
B1.42±1.101.76±0.861.23±1.051.75±0.881.60±0.96
C0.75±1.051.53±1.180.83±1.271.07±0.931.29±1.09
D1.00±1.000.90±0.740.75±1.500.90±1.001.12±1.43
bP<0.01 vs D期.
图6
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图6 Calnexin 分子在肠癌中表达×200.
图7
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图7 LMP2分子在肠癌中表达×200.
3 讨论

肿瘤细胞表面HLA-I类抗原表达下调是肿瘤逃避免疫监视的重要途径[10-11]. 本结果显示肠癌组织中HLA-A, B/C重链, 2M表达水平较正常黏膜明显下调(P<0.01), 在肿瘤的Dukes分期中, HLA-I抗原中各位点情况并不相同, HLA-B/C位点表达水平在Dukes A期与D期具有差异显著性. 抗原异质性表达现象只有出现在肠癌发生浸润及转移时. 肠癌细胞表面HLA-I类抗原表达降低使肿瘤可以逃避CTL的杀伤作用, 有利于癌细胞的生存[12-13]. 肠癌组织存在多个亚克隆, 在肠癌Dukes分期中, HLA-I类抗原高表达的亚克隆被CTL杀伤, HLA-I类抗原低表达的亚克隆得以演化成为优势克隆[10-11,14-15]. 肠癌细胞随着分期的变化, 该抗原表达逐渐降低, 并且在浸润至周围组织和/或发生淋巴结转移的肠癌细胞中HLA-I类抗原亦呈现低表达; 而在正常黏膜、肠癌早期未见该类抗原异质性表达现象.

肠癌细胞表面HLA-I类抗原表达降低逃避免疫监视的机制涉及许多方面因素, 其中2M表达下调或发生突变是影响HLA-I类抗原表达的重要因素[16-17]. 2M与HLA-I类抗原重链通过非共价键结合, 可使重链稳定地表达在细胞膜上, 发挥递呈抗原的作用. 若2M发生突变或缺失, 可致HLA-I类抗原重链不能有效表达在细胞膜上[18-19]. 在本研究中肠癌细胞2M的表达明显下调. 2M表达变化与HLA-A、B/C位点在肠癌中的改变基本保持一致, 随着2M表达下调, HLA-I类抗原重链表达亦下调, 提示2M参与影响HLA-I类抗原表达. LMP2主要作用是将抗原蛋白加工成适合与HLA-I类抗原结合的多肽, 它对HLA-I类抗原在细胞膜上表达的密度和稳定性起重要作用[20-22], LMP2缺陷或表达降低可导致HLA-I类抗原不能负载抗原肽, 使HLA-I类抗原表达下调[23]. 在宫颈癌, 卵巢癌, 恶性黑色素瘤, 肺小细胞癌, 肾癌中发现HLA-I类抗原低表达伴随抗原加工递呈系统分子低表达或缺陷[24-26]. 在本实验中, 比较正常黏膜与癌组织中LMP2分子表达, 发现肠癌中这种抗原表达低于正常黏膜(P<0.01). 该抗原与肠癌分级、分期无相关性, 但随着肿瘤的Dukes分期这种分子表达下降. 本实验结果显示同时存在LMP2、HLA-I类抗原下调, 提示LMP2下调是导致HLA-I类抗原下调的重要因素之一. 钙联蛋白(calnexin)是一种分子伴侣, 它帮助HLA-I类抗原正确折叠, 形成有功能的二聚体[27-28]. 低表达或缺乏calnexin可使HLA-I不能形成二聚体, 导致HLA-I类抗原表达下调[29-32]. 在本研究中, 比较切端黏膜与癌组织中calnexin分子表达, 显示肠癌中calnexin表达明显低于正常黏膜, calnexin分子与肠癌分级无相关性, 但随着Dukes分期, 这种分子表达逐步下调. 肠癌中这种分子表达与HLA-I类抗原改变不一致. 结果提示calnexin是一种影响HLA-I类抗原表达重要因素, 他影响HLA-I类抗原的表达与2M影响HLA-I类抗原表达下调的机制不同.

在癌旁组织中HLA-A, B/C, 2M, LMP2表达介于正常组织与癌组织之间, HLA-A, HLA-B/C, 2M在癌旁与肠癌组织表达水平无显著差异, LMP, calnexin在癌旁与肠癌组织表达差异性显著(P<0.01). 癌旁组织和肠癌组织均出现HLA-A, HLA-B/C, 2M表达下调, 提示肠癌组织通过某种细胞间的影响因子, 降低HLA-I类抗原表达, 这种作用对正常黏膜或癌细胞表面HLA-I类抗原的表达影响较为显著[33]. 癌旁组织LMP2, calnexin表达下调, 但结果与HLA-I类抗原的表达有差异, 提示影响HLA-I类抗原与LMP2, calnexin表达的机制不完全一样. 本结果为肠癌的免疫治疗提供重要的理论依据.

编辑: N/A

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