血管生成与胃癌发生之间关系的实验研究

摘要

目的: 研究血管生成与胃癌发生之间的关系.

方法: 利用化学致癌剂N-甲基-N'-硝基-亚硝基胍(MNNG)诱发建立大鼠胃癌前病变及胃癌模型, 观察胃癌发生过程中不同阶段血管生成情况, 并采用抗CD34抗体的免疫组化方法显示不同癌前病变期微血管数量(MVC)变化.

结果: 胃黏膜腺体处于增生期, 血管生成很少, 至异型增生阶段, 尤其是中、重度异型增生阶段或黏膜内癌时, 血管生成明显增多并呈明显的异质性分布, 至浸润性癌期, 浸润前缘周围血管异常丰富, 肿瘤组织间质也有血管生成. 与增生期和轻度异型增生相比, 中、重度异型增生的微血管数(MVC)明显增高(26.3±9.6 vs 17.1±5.6, 32.5±11.7 vs 17.1±5.6)(P分别<0.05, 0.01), 但中度与重度异型增生的微血管数差别不明显.

结论: 血管生成在胃癌发生早期即已启动, 随着病变损害向进展期发展而愈加明显, 与胃癌的发生发展关系密切.

关键词: N/A

Relationship between gastric carcinogenesis and angiogenesis

Hou-Quan Tao, Shou-Chun Zou, Rui-Nian Wang, Yan-Zhen Lin

Hou-Quan Tao, Shou-Chun Zou, Department of Surgery, Zhejiang Provincial People's Hospital, Hangzhou 310014, Zhejiang Province, China

Rui-Nian Wang, Department of Pathology, Shanghai Second Medical University, Shanghai 200025, China

Yan-Zhen Lin, Department of Surgery, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China

Correspondence to: Dr. Hou-Quan Tao, Department of Surgery, Zhejiang Provincial People's Hospital, Hangzhou 310014, Zhejiang Province, China. houquantao@yahoo.com

Received: March 29, 2002
Revised: May 25, 2002
Accepted: June 14, 2002
Published online: January 15, 2003

Abstract

AIM: To study the relationship between gastric carcinogenesis and angiogenesis.

METHODS: Experimental model of gastric precancerous lesions and carcinomas was established in rats by chemical carcinogen, N-methyl-N?nitro-soguanidine(MNNG). The specimens were collected in groups at different time points from pre-malignant precursor to gastric carcinoma, the sections were stained by HE and angiogenesis was observed. Tissue sections were also immunohistochemically stained for CD34 antigen, a marker for endothelial cells, and the microvessels were counted.

RESULTS: Only a few microvessels were found in the hyperplastic stage of gastric mucosal glands, but angiogenesis was increased markedly and heterogeneously distributed in the dysplastic stage, especially in specimens with moderate and severe degree of dysplasia as well as in the intra-mucosal carcinoma. Profuse vascularity in the frontier of tumor invasion and tumor stroma was found in the infiltrating carcinoma. Comparison of microvessel counts in low grade lesions(hyperplasia and mild dysplasia) with microvessel counts in moderate(26.3±9.6 vs 17.1±5.6 P < 0.05) and severe dysplasia lesions(32.5±11.7 vs 17.1±5.6 P < 0.01) showed a statistically significant increase in the more advanced lesions, but there was no significant difference in vessel count between moderate and severe degree of dysplasia.

CONCLUSION: Angiogenesis is switched on at the early stage of gastric carcinogenesis, and becomes more pronounced with the progress toward more advanced stage. There is a close relationship between gastric carcinogenesis and angiogenesis.

Key Words: N/A

0 引言

肿瘤生长和转移依赖于新生血管生成的概念早在30 a前就已提出[1-9], 由肿瘤产生的多种血管生成因子在近十几年也相继得以确认[10-15]. 在一些实体性肿瘤, 新生血管的数目与临床预后有一定的关系, 表明了肿瘤克隆的侵袭性与血管生成之间的相关性[16-20]. 抗血管生成治疗能明显阻止肿瘤生长和转移的实验性研究进一步提示, 肿瘤的生长需要新生血管生成[21-32]. Folkman et al发现在肿瘤发生的早期血管生成即已启动, 血管生成的启动是肿瘤表型的一个独特和必需的成分, 并自始至终陪伴着肿瘤的生长和发展. 由于人体恶性肿瘤发生的早期材料难以获得, 故研究较少, 仅在人乳腺导管内癌和子宫颈癌的活检组织中证实了转基因模型中所见到的现象. 为探索血管生成与胃癌发生的关系, 本研究采用化学致癌剂诱发建立大鼠胃癌模型, 观察胃癌前病变及胃癌形成过程中不同阶段的血管生成情况, 以期阐明血管生成在胃癌发生中的启动及演进.

1 材料和方法

1.1 材料

♂wistar大鼠50只, 10周龄, 体质量90±5 g, 分笼饲养, 饲以市售饲料, 饮用水按100 mg/L加入N-甲基-N'-硝基-N-亚硝基胍(MNNG), 隔日换新鲜配制液. 根据文献及以往胃癌实验模型制备的经验, 饲以MNNG 6-8 mo后, 大鼠可发生腺胃癌前和癌病变, 故自24-32 wk期间, 分批将大鼠处死, 肉眼观察胃肠道病变, 全胃壁取材, 40 g/L甲醛固定, 石蜡包埋切片, 一份作HE染色以观察胃组织病变及血管生成改变, 另一份用于免疫组织化学染色进行微血管计数.

1.2 方法

采用抗CD34单克隆抗体(1:50, Oncogen Inc.), 进行LSAB法免疫组织化学染色(LSAB试剂盒购自Dako Inc.), 方法简述如下: 切片经二甲苯脱蜡, 各级酒精水化, 3 ml/L H₂O₂/甲醇孵育30 min, 磷酸盐缓冲溶液(PBS)洗涤3次, 0.5 g/L蛋白酶K消化20 min, PBS洗涤, 1:400抗CD34抗体孵育2 h, PBS洗涤, 1:400稀释的生物素标记的第二抗体孵育1 h, PBS洗涤, 1:400稀释的过氧化物酶-链酶卵白素孵育1 h, PBS洗涤, DAB显色, 苏木素套染.20×10倍显微镜下, 随机分别选择5个病变区和邻近的正常胃黏膜组织进行微血管计数(MVC), 结果以5个区域的均数表示.

统计学处理 血管数(MVC)比较采用t检验.

2 结果

2.1 胃癌发生不同病变期血管生成改变

根据大鼠腺胃的病理变化, 将病变分成4个阶段: (1)充血反应期: 黏膜浅层有不同程度的充血反应, 但腺体无明显增生; (2)增生期: 胃小凹上皮及腺颈部增生, 固有层和黏膜下间质可见少量血管增生(图1);(3)异型增生期: 腺体形状不规则, 排列紊乱, 且分布稀疏不匀, 细胞核有不同程度的增大和深染, 呈多形性, 随异型性程度的加重, 血管增生也随病变的发展而增多, 突出的表现是血管在黏膜肌下或突破黏膜肌, 向异型增生腺体的某一区域生长, 但其分布具有明显异质性(图2);(4)浸润性癌时, 腺体极不规则, 有的已突破黏膜肌向黏膜下层呈浸润性生长, 细胞异型性明显, 核深染, 核分裂像多见, 浸润前缘血管密集, 腺体之间的间质中也有较多血管, 随着病变发展, 出现纤维增生, 血管腔被挤压变小(图3).

图1 大鼠腺胃腺颈部增生, 黏膜肌下微血管增生并突入黏膜肌(HE×200).

图2 大鼠腺胃异型增生, 固有层及黏膜下层微血管增生, 并突破黏膜肌向异型增生区生长 HE×100.

图3 大鼠腺胃浸润性腺癌, 癌腺体突破黏膜肌, 向黏膜下浸润, 浸润周围见大量血管增生HE×100.

2.2 大鼠不同癌前病变阶段血管数(MVC)的比较

在50例大鼠中, 6例呈增生性改变, 轻、中度异型增生分别为5例和6例, 9例呈重度异型增生, 其余均呈典型的胃癌组织学改变. 在抗CD34抗体的LSAB染色时, 与邻近正常上皮相比, 异型增生改变的胃黏膜下方有广泛网状结构的血管生成, 这种血管形成的区域在正常组织和异型增生之间有明显的分界线. 增生期和轻度异型增生期的MVC与邻近正常组织MVC间无明显差别(17.1±5.6 vs 15.6±5.2)(P>0.05), 而中、重度异型增生的MVC与邻近正常组织MVC之间有明显差别(26.3±9.6 vs 16.4±5.1, 32.5±11.7 vs 19.2±7.7)(P均<0.05), 至浸润性癌时, MVC为39.7±10.3, 高于邻近正常组织一倍以上(P<0.0 001)(表1). 同样由表1可知, 新生血管生成的程度随病变趋于严重而增加, 早期病变(增生期和轻度异型增生)与中、重度异型增生的MVC之间差别显著(P分别<0.05, 0.01), 而中度与重度异型增生的MVC之间无明显差别. 这些结果提示, 异型增生的血管形成随着病变损害向进展期发展而愈加明显.

表1 大鼠不同癌前病变阶段及浸润性癌期血管数变化(mean±SD).

病期	n	MVC		P
		正常组织区	病变区
增生期+轻度异型增生	10	15.6±5.2	17.1±5.6	>0.05
中度异型增生	6	16.4±5.1	26.3±9.6	>0.05
重度异型增生	9	19.2±7.7	32.5±11.7	>0.05
浸润性癌	25	18.4±8.2	39.7±10.3	<0.0 001

3 讨论

Saito et al用MNNG诱发建立大鼠模型, 对胃癌发生过程胃黏膜病变的演变进行动态观察后认为, 大鼠胃癌的发生分为3个阶段:(1)黏膜的糜烂与增生期(20 wk内).(2)腺瘤样增生期(20-30 wk, 表现为腺体过度增生.(3)腺癌发生期(30 wk后), 在腺体异型增生的基础上出现腺癌并向黏膜下层、肌层和浆膜层浸润. 本研究大鼠胃黏膜病变发生过程与Saito的报告相符. 在此模型上, 我们观察了不同病变期血管生成的改变, 以期阐明血管生成与胃癌发生之间的内在关系. 结果发现, 胃黏膜腺体处于增生期时, 血管生成很少, 当发展至异型增生阶段, 尤其是中、重度异型增生或黏膜内癌时, 血管生成明显增多, 至浸润性癌时, 浸润前缘周围血管异常丰富, 肿瘤间质也有血管生成. 这与Arbeit [33]将RIP-Tag转基因小鼠胰岛b-细胞分离, 放于含毛细血管内皮细胞的培养液中观察所见的现象相似, 在胰岛b-细胞高度增生和癌之间有一独特的阶段-血管化的胰岛, 即毛细血管内皮细胞呈放射状排列, 向胰岛迁移, 内皮细胞芽萌出和毛细血管腔形成. 同样在用转基因方法制成的小鼠鳞状细胞癌模型, 增生期的真皮下有少量血管生成, 异型增生期血管生成明显增多, 至侵袭性鳞状细胞癌时, 癌细胞突破基底膜向周围浸润, 并高度血管化. 在人类肿瘤如人乳腺导管内癌和子宫颈癌活检组织中也证实了上述的现象, 说明血管生成在肿瘤发生早期就已启动, 并可影响实体肿瘤发展速度的现象是肿瘤发生中的一个普遍规律. 我们在人胃黏膜活检组织中也发现异型增生的胃黏膜腺体周围有血管生成现象(图4), 提示人类胃癌的发生与血管生成之间也有同样的关系.

图4 人胃黏膜活检组织, 在肠化及轻度异型增生灶周围有血管增生及充血HE×100.

当大鼠胃黏膜腺体出现重度异型增生或黏膜内癌时, 血管生成不但明显增多而且呈明显的异质性分布, 即血管生成主要位于异型增生最明显区域的黏膜肌上下, 并可见新生血管正在穿透黏膜肌, 而附近相同或较轻病变区很少或无血管生成. 人胃黏膜中也可见异型增生腺体由血管围绕(图4). 这些结果提示, 在某些情况下, 血管生成先于浸润性肿瘤的生成. 这种在进行性病变时最明显改变是血管生成的事实提示:(1)营养血管的增加反映了病变区代谢活动的增强.(2)新生血管生成是浸润性克隆继发生长的一个重要先决条件. 在鼠纤维肉瘤形成的转基因鼠模型上发现, 癌前病变和癌能产生血管生成因子如碱性成纤维细胞因子(bFGF)等, 这些病变仅在发展至肿瘤形成性阶段时才分泌bFGF, 促进血管生成, 同时由于血管内皮细胞又可分泌生长因子(如bFGF)促进异型增生和癌的生长, 二者相互影响, 促进肿瘤发展[33], 因此有血管生成的病变区将成为肿瘤进展的中心, 此处黏膜肌常被新生血管突破, 肿瘤常沿血管浸润, 成为肿瘤组织向黏膜下浸润的突破口. 作者认为, 血管生成在胃癌发生的早期就已启动, 并促进异型增生向癌的转变.

异型增生是胃癌发生和发展的有价值模型, 在这一模型上可揭示血管生成与肿瘤发生之间的关系. 血管形成对于异型增生性损害的发生、维持和进展是必需的, 因此, 抗血管生成剂对于异型增生的治疗会有重要意义, 这为治愈胃癌前病变、阻止胃癌发生提供了一个新的方向.

备注

编辑: N/A

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