水流动力学注射方法建立肝内胆管癌动物模型

One mouse model of intrahepatic cholangiocarcinoma by hydrodynamic injection of sleeping beauty vectors

目的 建立一种小鼠肝内胆管癌动物模型。方法 采用水流动力学注射方法将表达Notch1基因胞内区活性形式pT3-EF1a-Notch1分子的胞内结构域（NICD1）转座子和表达myr-蛋白激酶B（Akt）基因转座子质粒溶液通过小鼠尾静脉注射至肝脏。注射生理盐水作为对照。注射4周后处死，病理学检查肿瘤发生情况，免疫组织化学、免疫荧光检测细胞角蛋白19（CK19）及标签蛋白流感病毒血凝素蛋白表面抗原决定簇衍生的蛋白标签序列（HA）标记的Akt表达。结果 实验组小鼠均形成肿瘤，成癌率为100%（14/14）；病理学检测为胆管细胞性肝癌，免疫组织化学检测CK19，14例中有9例强阳性表达（64.3%），证实瘤细胞是胆管细胞性肝癌。同样，在瘤细胞中检测到HA强阳性表达率为42.9%（6/14）；免疫荧光双标记CK19和HA升高。 结论 水流动力学注射方法成功构建肝内胆管癌小鼠模型且诱癌时间短，方法简单，重复性好。

Objective To establish one mouse model of intrahepatic cholangiocarcinoma （ICC） for liver cancer reseach.Methods The transposon vectors of sleeping beauty were constructed harboring the activated protein kinase B （myr-Akt） or the activated Notch1 intracellular domain （NICD1）, respectively. These vectors were hydrodynamic injected through tail veil. Mice injected with saline was used as the control. Mice were sacrificed around 4 weeks. Tumors were featured by macro-and micro-histological observation. Immunohistological staining and fluorescence staing were used for cytokeratin （CK）19 and Akt cellular expression detected by the sequence Tag of hemagglutinin surface antigen determinant of influenza virus （HA）-tag. Results Compared with the control, mice in the experimental group progressed to tumor with the rate of 100% （14/14）. The features of macro-and micro-histological observation indicate the formation of intraheptic cholangiocarcinoma. High percentage （64.3%） of CK19-positive staining in the malignant cells of liver confirmed the tumorigenesis of ICC. Similarly, high rate of HA-positive（42.9%） expression was observed in malignant cells. Colocalization of CK19 and HA staining by immunofluorescence staining also verify high expression.Conclusion We provide one method to generate ICC murine model with the advantages of easy-manupilation and time-saving and recapitulation.