小鼠动物模型中骨髓播散肿瘤细胞的检测方法

Detection methods for disseminated tumor cells in the bone marrow of mice

    摘要
  • 摘要:
      目的  探究小鼠骨髓中播散肿瘤细胞（disseminated tumor cells，DTCs ）的检测方法。
      方法  采用慢病毒感染的方法构建MDA-MB-231-GFP/Luc乳腺癌细胞系，将MDA-MB-231-GFP/Luc细胞经左心室接种于NOD-SCID小鼠体内，构建骨髓DTCs小鼠模型。采用荧光定量RT-qPCR、流式细胞计数、骨组织连续切片免疫荧光染色三种检测方法对小鼠骨髓DTCs进行定量和组织学定位研究。
      结果  荧光定量RT-qPCR法和流式细胞计数法的检测下限分别为22个和25个绿色荧光蛋白阳性（GFP+）细胞。骨组织连续切片免疫荧光染色法虽然不能定量骨髓DTCs数量，但可观察到GFP+ DTCs在骨组织中的分布，定位于成骨细胞或骨基质附近。
      结论  三种检测方法联合使用可满足小鼠骨转移研究动物实验中骨髓DTCs的定量和定位研究的需求，可为乳腺癌骨转移和骨髓中休眠癌细胞研究提供方法学支持。

     

    Abstract:
      Objective  To investigate the detection methods of disseminated tumor cells (DTCs) in the bone marrow of mice.
      Methods  The MDA-MB-231-GFP/Luc breast cancer cell line was constructed using lentiviral transfection. MDA-MB-231-GFP/Luc tumor cells were then inoculated into NOD-SCID mice via intracardiac injection in order to construct a mouse model with bone marrow DTCs. Real-time quantitative polymerase chain reaction (RT-qPCR), flow cytometry, and immunofluorescence staining on serial frozen sections of bone tissue were used to quantify DTCs and to observe the histological distribution of DTCs in the bone marrow.
      Results  The detection limits of the RT-qPCR and flow cytometry methods were 22 and 25 GFP+ cells, respectively. Immunofluorescence staining of serial frozen sections of bone tissue did not quantify bone marrow DTCs, but it showed that GFP+ DTCs were localized near osteoblasts or the bone matrix.
      Conclusions  The combination of these three detection methods meets the detection requirements for the quantification and localization of bone marrow DTCs in animal experiments, therefore providing methodological support for research on bone metastasis and dormancy of breast cancer cells in the bone marrow.

     

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