小鼠动物模型中骨髓播散肿瘤细胞的检测方法

岳晓敏; 黄玉凡; 李晓青

doi:10.12354/j.issn.1000-8179.2023.20221304

小鼠动物模型中骨髓播散肿瘤细胞的检测方法

doi: 10.12354/j.issn.1000-8179.2023.20221304

天津医科大学肿瘤医院生物化学与分子生物学研究室，国家恶性肿瘤临床医学研究中心，乳腺癌防治教育部重点实验室，天津市肿瘤防治重点实验室，天津市恶性肿瘤临床医学研究中心（天津市300060）

基金项目: 本文课题受国家自然科学基金项目（编号：82273285）和天津市医学重点学科（专科）建设项目（(编号：TJYXZDXK-009A）资助

详细信息

作者简介:
岳晓敏：专业方向为乳腺癌转移基础研究

通讯作者:
李晓青　xqli@tmu.edu.cn

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出版历程
- 收稿日期: 2022-09-13
- 录用日期: 2023-01-06
- 修回日期: 2022-12-30

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

Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer, Key Laboratory of Breast Cancer Prevention and Therapy (Ministry of Education), Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China

Funds: This work was supported by Natural Science Foundation of China (No. 82273285) and Tianjin Key Medical Discipline (Specialty) Construction Project (No. TJYXZDXK-009A)

More Information

Corresponding author: Xiaoqing Li; E-mail: xqli@tmu.edu.cn

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。
- 乳腺癌 /
- 骨转移 /
- 骨髓 /
- 播散肿瘤细胞
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.
- breast cancer /
- bone metastasis /
- bone marrow /
- disseminated tumor cells (DTCs)

HTML全文

图 1 MDA-MB-231-GFP/Luc细胞的构建

A：GFP绿色荧光（镜下×100）；B：Luc发光

下载: 全尺寸图片幻灯片

图 2 小鼠骨髓DTCs动物模型的构建

A：小鼠骨髓DTCs动物模型；B：活体成像；C：X线、组织病理检测（H＆E、TRAP染色×400）

下载: 全尺寸图片幻灯片

图 3 荧光定量RT-qPCR法检测两组小鼠骨髓中的DTCs

A：扩增曲线；B：标准曲线；C：GFP⁺ DTCs数；**：P<0.01

下载: 全尺寸图片幻灯片

图 4 流式细胞计数法检测两组小鼠骨髓中的DTCs

A：流式图； B：GFP⁺ DTCs数

下载: 全尺寸图片幻灯片

图 5 免疫荧光染色法检测两组小鼠骨髓中的DTCs　（IFA×200）

箭头：GFP⁺ DTCs

下载: 全尺寸图片幻灯片

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