甲基化干预对小鼠大肠癌生长及p16／CDKN2基因表达的影响  

Effects of 5-Aza-2＇-deoxycytidine on the carcinogenesis of colorectal cancer in mouse and the in vivo expression of p16/CDKN2 mRNA

作　　者：方晓明[1] 姜朝晖[1] 姚宁[1] 丁小文[2] 彭佳萍[3] 郑树[3] 

机构地区：[1]解放军第一一七医院普通外科,杭州310013 [2]浙江省肿瘤医院乳腺外科 [3]浙江大学医学院肿瘤研究所

出　　处：《中华医学杂志》2011年第33期2362-2365,共4页National Medical Journal of China

基　　金：国家“973”重点基础研究发展计划（G1998051200）;浙江省科技计划项目（011110541）

摘　　要：目的探讨甲基转移酶抑制剂5-氮-2’脱氧胞苷（5-Aza-CdR）对小鼠大肠癌发生发展的影响及其与细胞周期依赖性激酶抑制因子p16／CDKN：mRNA表达的关系。方法4JD只雄性KM小鼠按随机数字表法随机分为2组：1，2-二甲肼（DMH）诱癌组和甲基化干预组，每组20只。小鼠皮下注射DMH25mg／kg，每周1次，连续22周，以诱发小鼠大肠癌，甲基化干预组于12周开始皮下注射5-Aza-CdR。另选同来源雄性KM小鼠作为未诱癌对照组（阴性对照组）10只，于皮下按25mg／kg注射生理盐水，每周1次，连续22周。“席卷法”收集肿瘤组织，HE染色观察肿瘤发生的数目、类型及浸润程度；免疫组织化学染色观察细胞增殖核抗原（PCNA）蛋白表达情况；原位杂交技术检测肠癌组织抑癌基因p16／CDKN，mRNA的表达。结果（1）DMH诱癌组肿瘤数达（7．6±3．1）个／只，而甲基化干预组诱癌数为（3．4±1．8）个／只，且以重度异性增生为主，浸润癌的发生率明显低于DMH诱癌组（P〈0．05〉。（2）DMH诱癌组19只小鼠中PCNA阳性表达16只，明显多于甲基化干预组（19只中11只阳性表达）及未诱癌对照组（0只，均P〈0．05）。（3）DMH诱癌组有明显p16／CDKN：mRNA杂交信号（蓝紫色），主要位于胞质，但甲基化干预组染色显然较DMH诱癌组强，尤其以瘤灶部位明显。结论早期5-Aza—CdR的干预能显著抑制小鼠大肠癌的发生发展，其抑制肿瘤机制可能是通过激活抑制因子p16／CDKN2的表达。Objective To explore the effects and relationship of specific demethylation agent 5-Aza- 2＇-deoxycytidine （5-Aza-CdR） on colorectal cancer （ CRC ） induced by 1,2-dimethylhydrazine （ DMH ） in mouse and the in vivo expression of cyclin-dependent kinases inhibitor pl6/CDKN2 mRNA. Methods A total of 40 male KM mice were randomized into 2 groups and CRC was induced by a 22-week injection of DMH. One group was interfered by specific DNA methyltransferase inhibitor 5-Aza-CdlL Another 10 the same source male KM mice were induced by a 22-week injection of saline as none induced cancer control group （negative control group ）. All mice were sacrificed to examine for eolorectal neoplasm. Immunohistoehemieal staining was used to assess the expression of proliferating cell nuclear antigen （PCNA）. The expression of pl6/CDKN2 mRNA was detected by in situ hyhridizatlon. Results The average numbers of neoplasm was higher in the DMH group （7.6 ±3.1 ） than that of the group DMH ＋ 5-Aza-CdR （3. 4 ± 1.8, P 〈0. 05 ）. Immtmohistochemical staining showed there was a significant elevation of PCNA in the group DMH（16/19） as compared with that in the group DMH ＋ 5-Aza-CdR （ 11/19, P 〈 0. 05 ）. In situ hybridization revealed that the level of tumor suppressor gene pl6/CDKN2 mRNA was significantly lower in the group DMH than that in the group DMH ＋ 5-Aza-CdR. Conclusion The specific demethylation agent 5-Aza-2＇-deoxycytidine may inhibit the carcinogenesis of CRC. Its mechanism may be related with a high expression of pl6/CDKN2 mRNA.

关 键 词：甲基化 肠肿瘤 原位杂交 p16/CDKN2 

分 类 号：R735.34[医药卫生—肿瘤]