Ptenfl/fl;Trp53fl/fl;Pbsn-iCre+基因编辑小鼠自发形成前列腺癌与乳腺癌的模型构建研究

doi:10.19401/j.cnki.1007-3639.2025.08.005

摘要/Abstract

摘要：

背景与目的：前列腺癌与乳腺癌作为高发恶性肿瘤，其发生、发展与抑癌基因10号染色体上缺失的磷酸酶与张力蛋白同源物基因（phosphatase and tensin homolog deleted on chremosome ten，Pten）及转化相关蛋白53基因（transformation related protein 53 gene，Trp53）的功能缺失密切相关，二者同时缺失可加速肿瘤恶性进展并诱导治疗抵抗。基于Cre-loxP系统的基因编辑小鼠自发性肿瘤模型是研究癌症机制的关键工具。研究表明，前列腺特异性启动子前泌蛋白（probasin, Pbsn）的基因驱动的iCre重组酶基因（Pbsn-iCre）可诱导雄性小鼠自发性前列腺癌，但其在雌性乳腺癌中的作用及跨性别表达特征尚未阐明。本研究构建 Pten^fl/fl;Trp53^fl/fl;Pbsn-iCre⁺ 转基因小鼠模型，旨在探究其在前列腺癌与乳腺癌中的自发性肿瘤表型，并验证Pbsn在乳腺组织中的表达特征。方法： 利用Cre-loxP系统，通过杂交及连续回交筛选获得基因型稳定的Pten^fl/fl;Trp53^fl/fl;Pbsn-iCre⁺子代小鼠（伦理审查批号：FUSCC-IACUC-2025115）。通过聚合酶链式反应及琼脂糖凝胶电泳验证Pten、Trp53及Pbsn-iCre基因型。通过H-E染色评估肿瘤组织病理学特征。免疫组织化学分析前列腺及乳腺肿瘤组织中Pten、p53蛋白表达水平，并检测Pbsn在乳腺、前列腺、卵巢、心脏、肝脏及肾脏中的分布。结果： Pten^fl/fl;Trp53^fl/fl;Pbsn-iCre⁺雄鼠与雌鼠分别自发前列腺癌或乳腺癌。前列腺癌病理学特征表现为肿瘤呈侵袭性腺泡腺癌结构，伴腺体结构紊乱及基膜破坏；乳腺癌病理学特征表现为浸润性导管癌，伴导管上皮异型增生及间质淋巴细胞浸润。免疫组织化学检测证实前列腺及乳腺的肿瘤组织中Pten与p53蛋白完全失表达，验证了前列腺和乳腺特异性基因敲除效应。免疫组织化学检测也证实了Pbsn蛋白特异性表达于前列腺腺泡上皮、卵泡及乳腺导管上皮细胞，而心脏、肝脏及肾脏中未见表达。结论： Pbsn-iCre在雌性乳腺中存在功能性表达， Pbsn-iCre所诱导的Pten/Trp53同时缺失可驱动雄鼠自发形成前列腺癌、雌鼠自发形成乳腺癌。

关键词: 转基因小鼠自发性肿瘤模型, Pbsn-iCre, Pten/Trp53双敲除, 前列腺癌, 乳腺癌

Abstract:

Background and purpose: Prostate cancer and breast cancer are highly prevalent malignant tumors, and there occurrence and development are related to the tumor suppressor genes phosphatase and tensin homolog deleted on chremosome ten (Pten) and the transformation related protein 53 gene (Trp53). The loss of function of Trp53 is closely related. The simultaneous loss of the two can accelerate the malignant progression of tumors and induce therapeutic resistance. The gene-edited spontaneous tumor model of mice based on the Cre-loxP system is a key tool for studying the mechanism of cancer. Studies have shown that prostate-specific promoter (probasin, Pbsn)-driven iCre recombinase (Pbsn-iCre) can induce spontaneous prostate cancer in male mice, but its role in female breast cancer and transgender expression characteristics have not yet been clarified. In this study, we constructed Pten^fl/fl;Trp53^fl/fl; Pbsn-iCre⁺ transgenic mouse model which was designed to explore its spontaneous tumor phenotype in prostate cancer and breast cancer, and to verify the expression characteristics of Pbsn in breast tissue. Methods: The Pten^fl/fl;Trp53^fl/fl; Pbsn-iCre⁺ mouse model was established using Cre-loxP system by hybridization and continuous backcross screening with Pten^fl/fl mouse, Trp53^fl/fl mouse, and Pbsn-iCre⁺ mouse (Ethical No.: FUSCC-IACUC-2025115). Pten, Trp53 and Pbsn-iCre genotypes were verified by polymerase chain reaction and agarose gel electrophoresis. The incidence of tumor in transgenic mice was monitored, and the histopathological characteristics of tumor were evaluated by hematoxylin-eosin staining. The protein levels of Pten and p53 in prostate and breast tumor tissues were analyzed by immunohistochemistry, and the distributions of Pbsn in breast, prostate, ovary, heart, liver and kidney were detected. Results: Pten^{fl/ fl};Trp53^fl/fl;Pbsn-iCre⁺ male mouse developed spontaneous prostate tumor at age of 5 month, and female mouse developed spontaneous breast tumor at age of 6 months. The pathological manifestations of prostate cancer were invasive acinar adenocarcinoma structure with glandular structure disorder and basement membrane destruction. The pathological manifestations of breast cancer were invasive ductal carcinoma with ductal epithelial dysplasia and interstitial lymphocyte infiltration. Immunohistochemistry confirmed the complete deletion of Pten and p53 proteins in prostate and breast tumor tissues, which verified the prostate and mammary gland specific gene knockout effect. Immunohistochemistry also confirmed that Pbsn protein was specifically expressed in prostate acinar epithelial cells, ovarian tissue, and mammary duct epithelial cells, but not in heart, liver and kidney. Conclusion: Pbsn-iCre is functionally expressed in female mammary glands, and the simultaneous loss of Pten/Trp53 induced by Pbsn-iCre may drive the development of prostate cancer in male and breast cancer in female mouse.

Key words: Transgenic mouse spontaneous tumor model, Pbsn-iCre, Pten/Trp53 double deletion, Prostate cancer, Breast cancer

中图分类号:

吴佳辰, 何丽娜, 汤鑫茹, 唐爽. Pten^fl/fl;Trp53^fl/fl;Pbsn-iCre⁺基因编辑小鼠自发形成前列腺癌与乳腺癌的模型构建研究[J]. 中国癌症杂志, 2025, 35(8): 769-775.

WU Jiachen, HE Lina, TANG Xinru, TANG Shuang. The research on construction of the spontaneous prostate tumor and breast cancer model of Pten^fl/fl;Trp53^fl/fl;Pbsn-iCre⁺ transgenic mouse[J]. China Oncology, 2025, 35(8): 769-775.

图/表 4

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Gene primer	Sequence (5’-3’)	Product size/bp
Pten
Forward	CAAGCACTCTGCGAACTGAG	WT: 156 Targeted: 328
Reverse	AAGTTTTTGAAGGCAAGATGC	WT: 156 Targeted: 328
Trp53
Forward	AGCACATAGGAGGCAGAGAC	WT: 288 Targeted: 370
Reverse	CACAAAAACAGGTTAAACCCAG	WT: 288 Targeted: 370
Pbsn-iCre
5’-forward	GGGCAGTCTGGTACTTCCAAGCT	WT: 0 Targeted: 453
5’-reverse	TGGACAATGCCCAATGCCTG	WT: 0 Targeted: 453
WT-forward	CAGCAAAACCTGGCTGTGGATC	WT: 412 Targeted: 0
WT-reverse	ATGAGCCACCATGTGGGTGTC	WT: 412 Targeted: 0