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浙江大学学报（医学版）
浙江大学学报（医学版）  2014, Vol. 43 Issue (4): 427-433    DOI: 10.3785/j.issn.1008-9292.2014.07.005
专题报道     
两种ErbB2/Neu阳性－PTEN缺失乳腺癌基因工程小鼠模型的建立及其生物学特征比较
王庆飞1，丁慧2，刘宝瑞2，张葵1
Generation and comparison of two genetically engineered mouse models of ErbB2/Neu positive-PTEN deficient breast cancer
WANG Qing-fei1，DING Hui2， LIU Bao-rui2，ZHANG Kui1
1. Department of Clinical Laboratory, Nanjing Drum Tower Hospital，School of Medicine，Nanjing University，Nanjing 210008，China 2. Department of Clinical Oncology， Nanjing Drum Tower Hospital，School of Medicine，Nanjing University，Nanjing 210008，China）
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摘要: 目的： 建立两种ErbB2/Neu阳性-PTEN缺失的乳腺癌基因工程小鼠模型并比较其生物学特征。 方法：利用先前建立的由鼠乳腺肿瘤病毒（MMTV） 启动子同时驱动活化型ErbB2/Neu基因和重组酶Cre表达的FVB/N-MMTV-NIC小鼠与Flox-PTEN小鼠交配；或将由内源性启动子驱动活化型ErbB2/Neu基因表达的FVB/N-ErbB2KI、FVB/N-MMTV-Cre及Flox-PTEN三种基因工程小鼠交配; PCR分别扩增Neu、Cre和PTEN基因，对其子代小鼠进行基因型鉴定；免疫组织化学法检测乳腺组织ErbB2/Neu和PTEN蛋白表达。对两种模型的成瘤时间、肿瘤数目、肺转移等生物学特征进行比较，观察肿瘤组织病理学形态，免疫组织化学法检测肿瘤细胞Ki-67表达，TUNEL法检测肿瘤细胞凋亡水平。 结果：经基因型鉴定和组织蛋白表达分析，获得了两种ErbB2/Neu阳性－PTEN纯合型缺失的乳腺癌基因工程小鼠模型：一是由MMTV外源性启动子同时驱动活化型ErbB2/Neu和Cre表达，抑癌基因PTEN条件性敲除的NIC/PTEN-/-模型；二是由MMTV-Cre使内源性启动子驱动ErbB2/Neu基因表达，PTEN条件性敲除的ErbB2KI/PTEN-/-模型。NIC/PTEN-/-模型的平均成瘤时间低于ErbB2KI/PTEN-/-模型（30 d与368 d,P<0.01）；肿瘤数目、肺转移率均高于ErbB2KI/PTEN-/-模型（分别为10个与1～2个，75.0%与37.5%，均P<0.01）；两种模型肿瘤呈现不同的组织病理形态，肿瘤细胞凋亡水平相当（P>0.05）；NIC/PTEN-/-模型Ki-67阳性细胞百分率高于ErbB2KI/PTEN-/-模型（86.9%±2.8%与37.4%±7.2%，P<0.01）。 结论：两种不同表型的ErbB2/Neu阳性－PTEN缺失的乳腺癌基因工程小鼠为探索ErbB2/HER2阳性乳腺癌的发生、发展规律及更有效的防治方案提供了理想的动物模型。
Abstract：Objective： To generate two genetically engineered mouse models of ErbB2/Neu positive-PTEN deficient breast cancer and to compare their biological properties. Methods： The genetically engineered mice previously developed with mouse mammary tumor virus （MMTV） promoter driven expression of activated ErbB2/Neu and recombinant Cre （FVB/N-MMTV-NIC）were interbred with Flox-PTEN mice; and FVB/N-ErbB2KI mice， harboring endogenous promoter driven activated ErbB2/Neu expression， FVB/N-MMTV-Cre mice and the flox-PTEN mice were interbred. Neu， Cre and PTEN genes were amplified by PCR for genotyping of the offsprings. ErbB2/Neu and PTEN expression in mammary tumors were detected by immunohistochemistry. Tumor formation time， tumor number， histopathology and lung metastasis were compared between two models， Ki-67 expression was detected by immunohistochemistry， and TUNEL staining of tumor tissues was performed. Results： Two genetically engineered mouse models of ErbB2/Neu positive-PTEN homozygous deficient breast cancer were generated. The models were confirmed by genotyping and immunohistochemistry. One model with exogenous MMTV promoter driven expression of activated ErbB2/Neu and Cre coupling PTEN disruption was designated as NIC/PTEN-/- mice， and the other with MMTV-Cre induced endogenous promoter driven expression of activated ErbB2/Neu with PTEN disruption was designated as ErbB2KI/PTEN-/- mice. The tumor formation time in NIC/PTEN-/- mice was significantly shorter than that of ErbB2KI/PTEN-/- mice （30 vs 368 d，P<0.01）; the number of tumor and incidence of lung metastasis was also significantly higher in NIC/PTEN-/- mice （10 vs 1-2 and 75.0% vs 37.5%， respectively，Ps<0.01）. The Two models displayed distinct histopathological morphology. NIC/PTEN-/- tumor showed more Ki-67 positive cells than ErbB2KI/PTEN-/- tumor did （86.9%±2.8% vs 37.4%±7.2%，P<0.01）， while the amount of cell apoptosis in tumors was not significantly different between two models. Conclusion： Two genetically engineered mouse models of ErbB2/Neu positive-PTEN homozygous deficient breast cancer with different phenotypes have been successfully generated， which may provide useful resource for further investigation of the initiation and progression of HER2/ErbB2 breast cancer， as well as for the development of novel prevention and treatment regimens of this malignance．
Key words： Breast neoplasms丨Genes, erbB-2丨Phosphoric monoester hydrolases/biosynthesis丨Immunohistochemistry    Chromosome deletion丨Tumor suppressor proteins/genetics丨Disease models, animal丨Genetic engineering
收稿日期: 2014-03-21
基金资助:国家自然科学基金（81302241）；南京市医学科技发展专项资金（YKK13066）．
作者简介: 王庆飞（1983-），男，博士，助理研究员，主要从事肿瘤免疫学的临床和基础研究工作；E-mail: wangqingfei1020@hotmail.com
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引用本文:

王庆飞，等. 两种ErbB2/Neu阳性－PTEN缺失乳腺癌基因工程小鼠模型的建立及其生物学特征比较[J]. 浙江大学学报（医学版）, 2014, 43(4): 427-433.
WANG Qing-fei，et al. Generation and comparison of two genetically engineered mouse models of ErbB2/Neu positive-PTEN deficient breast cancer. Journal of ZheJiang University(Medical Science), 2014, 43(4): 427-433.

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http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2014.07.005      或      http://www.zjujournals.com/xueshu/med/CN/Y2014/V43/I4/427

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