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目的建立增强型绿色荧光蛋白标记的腺样囊性癌肺高转移株(ACC-M-GFP)的原位肝肿瘤种植模型,探讨通过荧光成像观察肿瘤血管生成及侵袭转移的可行性和影响因素。方法2只 BALB/c nu/nu 裸小鼠上肢皮下注射10~7/ml ACC-M-GFP 细胞悬液0.2 ml。瘤结节直径接近0.5cm 时切取皮下肿块,分割成1 mm~3瘤块,采用隧道包埋法植入另外8例裸小鼠肝左叶。从第4周起,每隔2周同时采用整体荧光成像系统及体视荧光显微镜观察肿瘤生长转移及肿瘤血管生成情况;每次观察结束后随机处死裸鼠1只,取出肝、肺及邻近脏器,如纵隔淋巴结,行脏器荧光成像观察。肝肿瘤组织行连续冰冻切片,荧光显微镜下直接观察肿瘤血管生成。荧光成像结果与常规石蜡切片苏木素-伊红(HE)染色对照。结果8只裸鼠原位肝肿瘤均种植成功。采用整体荧光成像,肝肿瘤种植后第6～8周需通过皮瓣窗方能观察到肿瘤荧光;第8～10周透过皮肤可直接观察肿瘤荧光;第12～14周时肿瘤巨大,可观察到腹腔脏器转移荧光;第16—18周观察到肺转移及淋巴结转移。体视荧光显微镜下肝肿瘤种植后第4周可观察到肿瘤血管生成;第12～14周后,体视荧光显微镜下可观察到大量丰富的肿瘤血管形成,呈现为绿色背景下的树枝状黑色条纹。瘤组织冰冻切片后荧光显微镜观察发现肿瘤血管呈绿色背景下的黑色管状结构。结论采用荧光成像可实现对肝肿瘤生长浸润转移和肿瘤血管生成的实时动态观察。
Objective To establish an orthotopic liver tumor model of adenoid cystic carcinoma lung metastasis (ACC-M-GFP) labeled with enhanced green fluorescent protein (EGFP) and to explore the feasibility and influencing factors of tumor angiogenesis and invasion and metastasis by fluorescence imaging . Methods Two BALB / c nu / nu nude mice were subcutaneously injected with 0.2 ml of 10-7 / ml ACC-M-GFP cell suspension. Tumor nodules were cut into subcutaneous tumors at a diameter of 0.5 cm and cut into 1 mm to 3 nodules. Another 8 nude mice with left lobe of liver were implanted by tunnel embedding. From the fourth week onwards, the whole fluorescence imaging system and the stereoscopic fluorescence microscope were used to observe the tumor metastasis and tumor angiogenesis every two weeks. After the observation, one nude mouse was randomly sacrificed and the liver, lungs and adjacent organs were removed Such as mediastinal lymph nodes, line organ fluorescent imaging observation. Serial frozen sections of liver tumor tissue were observed under a fluorescence microscope for tumor angiogenesis. Fluorescence imaging results were compared with conventional paraffin-section hematoxylin-eosin (HE) staining. Results All nude mice were implanted with orthotopic liver tumors. Using whole-body fluorescence imaging, the tumor fluorescence can be observed through the window of the flap 6 to 8 weeks after the implantation of the liver tumor; the fluorescence of the tumor can be directly observed through the skin from the 8th to 10th week; the tumor is huge at the 12th to 14th week, Abdominal organs metastasized to fluorescence; lung metastases and lymph node metastases were observed at weeks 16-18. Tumor angiogenesis was observed in the fourth week after implantation of the liver tumor under the stereomicroscope of fluorescence microscope. After 12 to 14 weeks, a large number of tumorous vasculature were observed under the stereoscopic fluorescence microscope, showing a dendritic black color on a green background stripe. Frozen sections of tumor tissue were observed by fluorescence microscopy and the tumor vessels showed a black tubular structure with a green background. Conclusion Real-time dynamic observation of tumor invasion and metastasis and tumor angiogenesis can be achieved by fluorescence imaging.