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. 2004-10-15; 12(10): 2349-2352
在线出版日期: 2004-10-15. doi: 10.11569/wcjd.v12.i10.2349
胰腺癌组织化学趋化因子表达与肿瘤相关巨噬细胞计数的相关性
杨竹林, 邓星辉, 杨乐平, 苗雄鹰, 刘栋才
杨竹林, 邓星辉, 杨乐平, 苗雄鹰, 刘栋才, 中南大学湘雅二医院肝胆疾病研究室 湖南省长沙市 410011
杨竹林, 男, 1962-08-08生, 湖南省涟源市人, 汉族, 1998年湖南医科大学博士, 教授, 博士生导师, 主要从事消化系统疾病的病理研究.
通讯作者: 杨竹林, 410011, 湖南省长沙市, 中南大学湘雅二医院肝胆疾病研究室.
收稿日期: 2004-06-08
修回日期: 2004-07-09
接受日期: 2004-08-30
在线出版日期: 2004-10-15

目的: 研究胰腺癌组织中IL-8, MCP-1和 MIP-1a表达特征及与TAM计数的相互关系及临床病理意义.

方法: 胰腺癌(n = 51)手术切除标本经40 g/L中性甲醛固定后常规制作石蜡包埋切片, IL-8, MCP-1和 MIP-1a表达及TAM染色均采用常规ABC免疫组化法.

结果: 胰腺癌组织IL-8, MCP-1和 MIP-1a表达阳性率分别为62.7%, 66.7%和66.7%, 其评分值分别为2.9±1.9, 2.6±1.8和2.4±1.7, TAM计数均值为18.0±6.0个/HP; 高分化腺癌IL-8评分及TAM计数明显低于低分化腺癌(P <0.05); 转移病例IL-8, MCP-1, MIP-1a表达阳性率及其评分及TAM计数均明显高于未转移病例(P <0.05或P <0.01); 化学趋化因子阳性表达病例TAM计数均明显高于阴性病例(P <0.01), 且其评分值与TAM计数均呈密切正相关(r IL-8 = 0.52, P <0.01; r MCP-1 = 0.50, P <0.01; r MIP-1a = 0.46, P <0.01), 同样IL-8与MCP-1(r = 0.54, P <0.01)和MIP-1a(r = 0.52, P <0.01)及MCP-1与MIP-1ar = 0.64, P <0.01)之间也均呈密切正相关; 化学趋化因子表达和TAM计数与胰腺癌其他临床病理特征均无明显关系.

结论: 化学趋化因子及TAM计数与胰腺癌进展、血管生成、转移发生及预后有密切关系, 阳性表达者或TAM计数高者可能进展快和预后差.

关键词: N/A
引文著录: 杨竹林, 邓星辉, 杨乐平, 苗雄鹰, 刘栋才. 胰腺癌组织化学趋化因子表达与肿瘤相关巨噬细胞计数的相关性. 世界华人消化杂志 2004; 12(10): 2349-2352
Relationship between expression of chemokines and tumor-associated macrophage counting in pancreatic cancer
Zhu-Lin Yang, Xing-Hui Deng, Le-Ping Yang, Xiong-Ying Miao, Dong-Cai Liu
Zhu-Lin Yang, Xing-Hui Deng, Le-Ping Yang, Xiong-Ying Miao, Dong-Cai Liu, Laboratory of Hepatobiliary Diseases, Xiangya Second Hospital of Central South University, Changsha 410011, Hunan Province, China
Correspondence to: Zhu-Lin Yang, Laboratory of Hepatobiliary Diseases, Xiangya Second Hospital of Central South University, Changsha 410011, Hunan Porvince, China.
Received: June 8, 2004
Revised: July 9, 2004
Accepted: August 30, 2004
Published online: October 15, 2004

AIM: To investigate the relationship among the expression of IL-8, MCP-1, MIP-1a and tumor-associated macrophage (TAM) counting in pancreatic cancer.

METHODS: Paraffin section of pancreatic cancer specimen was made using routine method. TAM was counted after staining. The expression of IL-8, MCP-1, MIP-1a was detected by immunohistochemical method

RESULTS: The positive rates of IL-8, MCP-1 and MIP-1a were 62.7%, 66.7% and 66.7%, and their scores were 2.9±1.9, 2.6±1.8 and 2.4±1.7, respectively. Mean counting of TAM was 18.0±6.0 in each pancreatic cancer specimen. The scores of IL-8 and TAM counting were significantly less in well-differentiated cancer than those in poorly-differentiated cancer (IL-8:2.7±1.7 vs 3.2±1.2, P <0.05; TAM:15.9±6.4 vs 21.2±5.2, P <0.05). The positive rates and levels of IL-8, MCP-1, MIP-1a expression as well as TAM counting were significantly higher in metastatic cancer than those in the cancer without metastasis (P <0.05 or P <0.01). TAM counting was significantly higher in IL-8, MCP-1 and MIP-1a positive cancer than that in the negative one. The levels of IL-8, MCP-1, MIP-1a expression positively related to TAM counting (rIL-8 = 0.52, P <0.01; rMCP-1 = 0.50, P <0.01; rMIP-1a = 0.46, P <0.01). At the same time, positive relationship existed between expression of IL-8 and MCP-1a (r = 0.52, P <0.01), IL-8 and MCP-1 (r = 0.54, P <0.01), MCP-1 and MCP-1a (r = 0.64, P <0.01).

CONCLUSION: The expression of IL-8, MCP-1, MIP-1a and TAM counting may relate to the progression, angiogenesis, metastasis and prognosis of pancreatic cancer. Positive expression of IL-8, MCP-1, MIP-1a and high TAM counting may indicate rapid progression and poor prognosis of pancreatic cancer.

Key Words: N/A
0 引言

化学趋化因子(chemokines)是一族具有趋化作用的细胞因子, 分子量较小, 对白细胞迁移有关键作用[1-2]. 研究发现一些趋化因子表达与恶性肿瘤进展[3-6]、血管生成[7-10]、转移发生[11-14]及预后[15-18]有密切关系[19-20]. 新近研究显示, 恶性肿瘤内浸润的巨噬细胞分化成肿瘤相关巨噬细胞(tumor-associated macrophage, TAM)数量与恶性肿瘤进展[21-24]、血管生成[25-28]及预后[29-31]也密切相关[32-33]. 胰腺癌组织趋化因子表达与TAM计数的关系研究, 国内外未见文献报道. 我们应用免疫组化方法研究胰腺癌组织中IL-8、单核细胞趋化蛋白-1(monocyte chemoattracant protein-1, MCP-1)、巨噬细胞炎性蛋白-1a(macrophage inflammatory protein-1 alpha, MIP-1a)表达及TAM计数, 并探讨其相互关系和临床病理意义.

1 材料和方法
1.1 材料

我院及湘雅医院胰腺癌手术切除标本51例, 男38例, 女13例, 年龄21-73(51±17岁); 均为胰腺导管上皮癌, 包括高分化腺癌20例, 中分化腺癌12例和低分化腺癌19例; 临床病理证实胰腺外转移(包括区域淋巴结、网膜、邻近组织器官等)35例(68.6%). 标本经40 g/L甲醛固定后常规制作石蜡包埋切片厚4 mm. HE染色复述病理组织学特征, 其他切片行免疫组化染色. 兔抗人IL-8, MCP-1和 MIP-1a多克隆抗体, 鼠抗人CD68 mAb, 生物素标记羊抗兔lgG, 生物素标记羊抗鼠IgG, ABC试剂以及DAB-Hcl显色试剂盒均购自武汉博士德公司.

1.2 方法

TAM 和IL-8, MCP-1, MIP-1a染色方法均为常规 ABC 免疫组化法, 胞质内出现明显棕黄色颗粒者为阳性细胞. 趋化因子表达评分标准[34]: 将癌细胞染色强度评分(无, 0分; 弱, 1分; 中度, 2分; 强度, 3分)和阳性细胞率评分(小于5%, 0分; 5-10%, 1分;10-20%, 2分; 20-50%, 3分; 大于50%, 4分)之和为该病例评分值, 将评分值小于或等于2分定为阴性病例, 大于2分定为阳性病例. TAM计数方法[21,25,27,29]: 随机观察10个癌组织高倍视野, 计数各视野内TAM数, 求其均值为该病例TAM数.以博士德公司提供的阳性切片作为四项指标染色的阳性对照, 以0.01 mol/L PBS液(pH7.4)替代-抗作为每次染色的阴性或替代对照.

统计学处理 采用SPSS10.0统计软件包进行t 检验, χ2检验或Fischer精确概率法, 检验水准a=0.05.

2 结果

IL-8, MCP-1, MIP-1免疫反应产物定位于胞质, TAM免疫反应产物定位于胞质和/或胞核(图1-4). 胰腺癌51例TAM均值为18.0±6.0个/HP, IL-8, MCP-1和MIP-1a表达阳性病例分别为32例(62.7%), 34例(66.7%)和34例(66.7%), 三者评分分别为2.9±1.9、2.6±1.8和2.4±1.7(表1), 高分化腺癌和未转移癌TAM计数明显低于低分化腺癌和转移癌, 均有显著或高度显著性差异(P <0.05或P <0.01); 高分化腺癌IL-8表达评分明显低于低分化腺癌(P <0.05), IL-8表达阳性率及MCP-1, MIP-1a表达阳性率及评分高分化腺癌低于低分化腺癌, 但均无显著性差异(P >0.05); 未转移病例IL-8, MCP-1, MIP-1a表达阳性率及其评分均明显低于转移病例, 均有显著或高度显著性差异(P <0.05或P <0.01, 表2), IL-8, MCP-1和MIP-1a表达阳性病例TAM值明显高于阴性病例, 均有高度显著性差异(P <0.01); 经Spearm相关分析, TAM计数与IL-8(r = 0.52, P <0.01), MCP-1(r = 0.50, P <0.01)和MIP-1a(r = 0.46, P <0.01), 评分值之间均存在密切正相关; IL-8与MCP-1 (r = 0.54, P <0.01), MIP-1a(r = 0.52, P <0.01)评分值之间均存密切正相关; MCP-1与MIP-1a评分值之间也存在密切正相关(r = 0.64, P <0.01).

表1 胰腺癌组织TAM和IL-8, MCP-1, MIP-1a表达与分化程度和转移状况的关系.
胰腺癌组织nTAMIL-8
MCP-1
MIP-1a
阳性率评分阳性率评分阳性率评分
高分化2015.9±6.445.0%2.7±1.755.0%2.3±1.860.0%2.4±1.8
中分化1216.7±6.066.7%2.6±1.566.7%2.3±1.558.3%2.1±1.6
低分化1921.2±5.2a78.9%3.2±1.2a78.9%3.1±1.878.9%2.7±1.6
无转移1613.2±2.725.0%1.4±1.437.5%1.4±1.737.5%1.5±1.8
有转移3520.2±5.8b80.0%b3.2±1.2b80.0%b3.1±1.5b80.0%b2.9±1.5b
aP <0.05 vs 高分化;
bP <0.01 vs无转移.
图1
图1 TAM计数, 18个/HP, 胰腺中分化腺癌, ABC免疫组化法, ×200.
表2 胰腺癌组织趋化因子表达与TAM计数的关系.
表达状况nTAM (mean±SD)
IL-8(+)3220.5±5.8
IL-8(-)1913.8±3.4b
MCP-1(+)3420.0±5.8
MCP-1(-)1713.9±4.0b
MIP-1a (+)3420.0±6.0
MIP-1a (-)1714.0±3.5b
bP <0.01 vs阳性.
图2
图2 IL-8阳性表达, 评分6分, 胰腺低分化腺癌, ABC免疫组化法, ×200.
图3
图3 MCP-1阳性表达, 评分4分, 胰腺高分化腺癌, ABC免疫组化法, ×200.
图4
图4 MIP-1a阳性表达, 评分5分, 胰腺中分化腺癌, ABC免疫组化法, ×200.
3 讨论

化学趋化因子IL-8, MCP-1和MIP-1a均为具有重要生理作用的化学趋化因子, 其表达水平与许多恶性肿瘤进展、血管生成、局部炎性细胞浸润、转移或侵袭能力及预后密切相关, 阳性表达者进展快[1-4]、血管生成密度高[5-8], 炎性细胞浸润量高[9-12]、易发生转移[13-16]、侵袭能力强[17-18]及预后差[19-20]. 肿瘤细胞分泌的趋化因子与相应受体或配体结合后能促进癌间质炎性细胞浸润、肿瘤血管生成、癌基质蛋白降解及影响癌细胞本身生物学行为(如增生活性增加、促血管生成因子合成增加等), 但其确切机制仍需进一步研究[17-20]. 我们发现高分化和未转移胰腺癌三者表达阳性率及其评分明显高于低分化和转移病例, 阳性表达者TAM计数明显高于阴性表达者, 且三者评分值与TAM计数均呈密切正相关及三者本身评分值之间也呈密切正相关. 提示3种趋化因子具有促进胰腺癌进展、转移发生及TAM浸润作用, 可能是反映胰腺癌进展、生物学行为及预后的重要生物学标记物, 且三者可能具有相互协同和共同促进作用.

巨噬细胞是单核巨噬细胞系统内某个时期的分化类型, 是肿瘤间质中炎性细胞浸润的一个关键成分, 单核巨噬细胞在癌间质通常分化为TAM, 与肿瘤血管生成关系密切[29-33]. 研究发现TAM可产生许多生长刺激因子和抑制因子、细胞因子及蛋白水解酶, 在肿瘤血管生成的级链反应中起重要作用, 除产生VEGF, bFGF, TGF-α, PDGF以及IL-8, MCP-1, MIP-1a外, 活化的TAM还可产生IL-1, TNF-α, 可提高VEGF和bFGF在血管内皮中表达水平, 从而促进肿瘤血管生成. 许多恶性肿瘤细胞自分泌多种化学趋化因子, 已证实IL-8, MCP-1, MIP-1a等趋化因子对TAM局部迁移、浸润及其活化起重要作用[31-33]. 最近, 一些文献报道TAM除与肿瘤血管生成有关外, 与恶性肿瘤进展, 转移及预后关系密切, TAM数高者进展快, 易发生转移及预后差, TAM可作为恶性肿瘤预后评估的客观指标[31-33]. 本组资料显示, 高分化和未转移癌TAM计数明显低于低分化和转移病例, IL-8, MCP-1和MIP-1a阳性表达病例TAM明显高于阴性病例, 且其三者评分值与TAM计数呈密切正相关. 提示TAM与胰腺癌分化和转移发生关系密切, 胰腺癌细胞合成和分泌IL-8, MCP-1和MIP-1a具有趋化巨噬细胞向肿瘤局部迁移、浸润和活化作用, 其结果与国外文献报道较一致.

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