双光子显微镜技术下胶质瘤-壁细胞在体多荧光示踪小鼠模型的建立及应用  

Establishment and utilization of a living genetically engineered mouse model for examination of fluorescently labeled glioma cells and mural cells using two⁃photon microscopy

作　　者：马铖延 杨星九 史旭东[1,2,3,4] 高苒 MA Chengyan;YANG Xingjiu;SHI Xudong;GAO Ran(Institute of Laboratory Animal Science,Chinese Academy of Medical Science(CAMS),Comparative Medicine Center,Peking Union Medical College(PUMC),Beijing 100021,China;National Human Disease Animal Model Resource Center,Beijing 100021,China;NHC Key Laboratory of Human Disease Comparative Medicine,Beijing 100021,China;Beijing Engineering Research Center for Experimental Animal Models of Human Critical Diseases,Beijing 100021,China)

机构地区：[1]中国医学科学院医学实验动物研究所北京协和医学院比较医学中心,北京100021 [2]国家人类疾病动物模型资源库,北京100021 [3]国家卫生健康委员会比较医学重点实验室,北京100021 [4]北京市人类重大疾病实验动物模型工程技术研究中心,北京100021

出　　处：《中国实验动物学报》2024年第6期702-711,共10页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家重点研发计划(2022YFF0710700)。

摘　　要：目的建立双光子显微镜下可视化胶质瘤、壁细胞和血管的活体自发荧光的基因小鼠模型并进行评价。方法以PDGFRβ-Cre^(+/-):Rosa26-tdTomato+/-基因工程小鼠为观察壁细胞和血管结构的载体,接种GL261-CFP小鼠胶质瘤细胞并对颅骨进行透明化,通过双光子显微镜动态跟踪观察胶质瘤增殖侵袭过程中血管与壁细胞的动态变化。结果通过基因鉴定证实PDGFRβ-Cre^(+/-):Rosa26-tdTomato+/-基因工程小鼠成功繁育。对比C57BL/6小鼠,基因工程小鼠的形态外观和繁殖等无显著性差异,组织苏木素-伊红(HE)染色切片分析显示脏器发育无异常。Tamoxifen诱导下基因工程小鼠的Cre重组酶活性至第7天作用完全。接种GL261-CFP后观察到胶质瘤增殖侵袭的动态过程及肿瘤内血管形态结构紊乱、游离壁细胞增多。结论成功构建了荧光可视化壁细胞的基因工程小鼠,分别利用异硫氰酸荧光素-葡聚糖标记血管和青色荧光标记肿瘤细胞,使用玻璃圆片与固定环替代小鼠颅骨,实现了活体状态下长期稳定地动态跟踪小鼠接种脑肿瘤后血管及血管支持细胞的形态结构变化,为研究脑胶质瘤提供了病理可视化的动物模型。Objective This study was performed to develop and assess a genetically engineered mouse model for visualizing in vivo fluorescence of glioma cells,mural cells,and blood vessels using two⁃photon microscopy.Methods PDGFRβ⁃Cre^(+/-):Rosa26⁃tdTomato+/-genetically engineered mice underwent skull clearance and were injected with GL261⁃CFP.This was performed to study the dynamic alterations in blood vessels and mural cells during the progression and invasion of glioma using two⁃photon microscopy.Results PDGFRβ⁃Cre^(+/-):Rosa26⁃tdTomato+/-mice were successfully bred and subjected to hematoxylin⁃eosin section analysis of functional organ tissues.The mice exhibited no discernible differences from C57BL/6 mice in terms of appearance and morphology.Cre recombinase activity was fully induced following tamoxifen treatment on day 7.Subsequent GL261⁃CFP inoculation demonstrated the dynamic progression of glioma proliferation and invasion,as well as vascular abnormalities and increased mural cell detachment within the tumor.Conclusions Genetically engineered mice expressing fluorescent mural cells were successfully bred.Blood vessels labeled with fluorescein isothiocyanate⁃dextran and blue fluorescent tumor cells were utilized.Glass discs and fixed rings were employed to replace the skulls of the mice.This allowed for the tracking of morphological and structural changes in blood vessels and vascular supporting cells following the development of brain tumors in vivo over an extended period.This model offers a valuable tool for studying brain diseases through pathological visualization.

关 键 词：双光子显微镜 胶质瘤 壁细胞 基因工程小鼠 动物模型 

分 类 号：Q95-33[生物学—动物学]