内皮细胞条件性敲除EMCN小鼠模型建立及肿瘤肺转移对比分析  

Establishment of endothelial conditional EMCN-knockout mouse model and comparative analysis of tumor lung metastasis

作　　者：张国新 高苒 ZHANG Guoxin;GAO Ran(National Human Disease Animal Model Resource Center,NHC Key Laboratory of Human Disease Comparative Medicine,Beijing 100021 China;Beijing Engineering Research Center for Experimental Animal Models of Human Critical Disease,Beijing 100021;Institute of Laboratory Animal Sciences Chinese Academy of Medical Science(CAMS),Comparative Medicine Center,Peking Union Medical College(PUMC),Beijing 100021)

机构地区：[1]国家人类疾病动物模型资源库,国家卫生健康委员会人类疾病比较医学重点实验室,北京100021 [2]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 [3]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

出　　处：《中国实验动物学报》2022年第2期185-190,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家重点研发计划(2021YFF0702801)。

摘　　要：目的建立内皮细胞条件性敲除EMCN基因小鼠模型,探讨C57BL/6小鼠与内皮细胞条件性敲除EMCN小鼠模型体内肿瘤肺转移存在的差异。方法将内皮细胞特异性Tek-CreERT2小鼠与EMCN^(flox/flox)小鼠杂交,将子代雄性EMCNflox/wt/Tek-CreERT2+与雌性EMCN^(flox/flox)小鼠交配,取得内皮细胞特异性敲除EMCN基因小鼠(EMCN^(flox/flox)/Tek-CreERT2+,EMCN^(ecko))。通过在DNA和蛋白水平验证基因敲除准确性及效率,提取小鼠基因组DNA后,PCR扩增检测Tek-CreERT2及FLOX位点,Tamoxifen诱导后,Western Blot检测组织中EMCN蛋白的表达。对正常C57BL/6小鼠与EMCN^(ecko)小鼠表型进行观察及脏器HE染色。为了证实EMCN敲除对肿瘤转移的影响,将LLC细胞尾静脉注射C57BL/6与EMCN^(ecko)小鼠,2周后解剖取肺检测肺转移情况,并对2组小鼠的肺转移结果统计对比分析。将LLC细胞皮下注射C57BL/6与EMCN^(ecko)小鼠,2周后手术切除皮下肿瘤,分别在术后2、3周观察肺转移情况,将两组肺组织进行HE染色,并对两组小鼠的肺转移结果统计对比分析。结果从DNA和蛋白水平证实EMCN^(flox/flox)/Tek-creERT2^(+)小鼠成功构建。小鼠组织HE切片分析显示基因敲除小鼠脏器发育无异常。尾静脉注射LLC及皮下注射LLC后切除肿瘤的C57BL/6和EMCN^(ecko)小鼠均可以发生肺转移,与C57BL/6小鼠相比,内皮细胞EMCN的缺失显著促进了小鼠肺转移。结论成功获得内皮细胞EMCN条件性敲除小鼠,内皮细胞EMCN敲除小鼠肿瘤肺转移的能力显著优于正常C57BL/6小鼠,预期为肿瘤肺转移动物模型各项研究提供快速发生肺转移的动物模型。Objective To establish an endothelial conditional EMCN-knockout mouse model and explore the differences between tumor lung metastases in these and C57BL/6 mice.Methods Endothelial-cell-specific Tek-creERT2 mice were mated with EMCN^(flox/flox)mice.Male EMCN^(flox/flox)/Tek-creERT2^(+)mice were crossed with female EMCN^(flox/flox)mice to acquire endothelial cell-specific EMCN-knockout mice(EMCN^(flox/flox)/Tek-creERT2^(+),EMCN^(ecko)).To verify the gene knockout accuracy and efficiency at the DNA and protein level,the genomic DNA of mice was extracted,and the Tek-creERT2 and Flox sites were detected by PCR amplification.After tamoxifen induction,expression of the EMCN protein was detected on Western Blot.The phenotypes of normal C57BL/6 mice and EMCN^(ecko)mice were observed,and the organs were stained with hematoxylin and eosin(HE).To confirm the effect of EMCN knockout on tumor metastasis,(Lewis lung carcinoma,LLC)LLC cells were intravenously injected into C57BL/6 and EMCN^(ecko)mice.After 2 weeks,the lungs were dissected to detect metastasis,and the result for the two groups of mice were compared and analyzed.LLC cells were subcutaneously injected into C57BL/6 and EMCN^(ecko)mice.The subcutaneous tumors were removed 2 weeks later,and lung metastasis was observed 2 and 3 weeks after the operation.The lung tissues were stained with HE,and the metastasis result of the two groups were compared and analyzed.Results We confirmed the successful construction of EMCN^(flox/flox)/Tek-creERT2^(+)mice at the gene and protein level.Preliminary phenotypic analysis showed no abnormal organ development in the gene-knockout mice.C57BL/6 and EMCN^(ecko)mice given intravenous injection and tumor resection after subcutaneous injection can develop lung metastases.Compared with C57BL/6 mice,the deletion of endothelial cell EMCN expression significantly promoted lung metastasis.Conclusions Endothelial-cell-specific EMCN-knockout mice were successfully obtained.The frequency of lung metastasis in endothelial cell EMCN-knockout mice wa

关 键 词：EMCN基因 内皮细胞 Tek-CreERT2/EMCN^(flox/flox) 肺转移 

分 类 号：Q95-33[生物学—动物学]