条件性血管瘤转基因小鼠模型的建立

Establishment and identification of conditional transgenic mouse model with hemangioma

目的:采用条件性转基因策略构建小鼠血管瘤动物模型,并对其表型进行鉴定。方法:构建血管内皮细胞特异性表达启动子Tie2驱动的鼠多瘤病毒中间T基因(PyMT)表达质粒(pTie2-PyMT),采用DNA显微注射方法,将血管内皮特异性表达的PyMT目的基因导入供体C57BL/6J小鼠的雄原核中,再移植到假孕鼠的输卵管中,产生转基因首建鼠。PCR方法检测目的基因整合情况,检查转基因鼠基因型,观察转基因鼠表型,对于转基因鼠出现的新生物进行组织学及免疫组织化学检测。采用Graph Pad Prism 5.0软件包对实验数据进行统计学分析。结果:经测序分析证实,pTie2-PyMT质粒中PyMT、Tie2启动子和Tie2增强子序列等组成元件被正确克隆、连接,且阅读框正确。出生小鼠基因型经PCR鉴定证实,阳性的转基因小鼠均出现血管瘤样新生物表型。血管瘤样新生物主要表达部位在小鼠的耳、舌、皮肤、黏膜、肝等部位,组织学检查证实为血管瘤样病变,免疫组织化学方法证实新生物的内皮细胞表达Py MT蛋白及血管内皮标志物CD31。结论:Tie2启动子驱动下的PyMT基因可以在小鼠体内诱发血管瘤,该模型鼠可用于血管瘤发病机制的研究。条件控制下的转基因技术是一种有效的建立血管瘤动物模型的方法。

Objective： To develop an animal model of infantile hemangioma by applying conditional transgenic mice. Methods ： pTie2-PyMT,a plasmid of Tie2 promoter-driven polyomavirus middle T （PyMT） gene,was constructed. Endothelial specific PyMT gene was transported to male pronucleus of donor C57BL/6Jmice by DNA micro-injection,and the fertilized eggs were transplanted to receptor mice. The founder mice were developed from the surviving fertilized eggs. The gene integration was checked by PCR,and phe-notypes of transgenic mice were examined. The neoplasms of transgenic mice were detected by histological method. GraphPad Prism 5.0 software package was used for statistical analysis. Results： The PyMT, Tie2 promoter and Tie2 enhancer were cloned and integrated exactly in pTie2-PyMT plasmid according to sequencing analysis. All the newborn positive transgenic mice had hemangioma phenotypes. The hemangioma-like neoplasms were identified by HE in the ear, tongue, skin surface, mucosa and liver tissue in the transgenic- mice, and immunohistology staining showed positive of PyMT and CD31 in endothelial cells. Conclusions： PyMT gene driven by Tie2 promoter can induce hemangioma in mice. Thus, the transgenic mice can be applied as a model system for studying the mechanism of infantile hemangioma. Conditional transgenic technology is an ideal method to develop hemangioma animal model.