巨噬细胞中FBXW7基因缺失对小鼠黑色素瘤肺转移的影响

doi:10.3785/j.issn.1008-9292.2017.04.01

浙江大学学报（医学版）

2017, Vol. 46

Issue (2): 111-117 DOI: 10.3785/j.issn.1008-9292.2017.04.01

专题报道

巨噬细胞中FBXW7基因缺失对小鼠黑色素瘤肺转移的影响

张玮1,2(

),来利华3,王青青3,*(

)

1. 杭州市第一人民医院肿瘤科, 浙江杭州 310006
2. 杭州市肿瘤医院肿瘤化疗科, 浙江杭州 310002
3. 浙江大学免疫学研究所, 浙江杭州 310058

Effects of myeloid specific deficiency of FBXW7 on lung metastasis of murine melanoma

ZHANG Wei1,2(

),LAI Lihua3,WANG Qingqing3,*(

)

1. Department of Oncology, Hangzhou First People's Hospital, Hangzhou 310006, China
2. Department of Chemotherapy, Hangzhou cancer Hospltal, Hangzhou 310002, China
3. Institute of Immunology, Zhejiang University, Hangzhou 310058, China

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摘要：

目的：探究巨噬细胞中E3泛素连接酶FBXW7基因的表达对小鼠B16F10黑色素瘤肺转移的影响及可能的机制。方法：构建巨噬细胞中缺失FBXW7基因的条件性基因敲除小鼠。将FBXW7^flox/flox小鼠与带有Lysozyme M-Cre的转基因小鼠杂交繁殖，通过提取尾巴基因组DNA进行PCR鉴定，建立髓系细胞选择性FBXW7基因缺失的小鼠即Lysm⁺FBXW7^f/f小鼠（实验组），同窝生的Lysm^-FBXW7^f/f小鼠作为对照组。尾静脉注射小鼠黑色素瘤细胞系B16F10至两组小鼠，14 d后处死小鼠，计数肺部肿瘤的转移灶，称量肺部的重量。将肺组织消化成单细胞悬液后，流式细胞术检测免疫细胞亚群。结果：构建的Lysm-Cre-FBXW7^f/f小鼠腹腔巨噬细胞和骨髓来源巨噬细胞中，FBXW7基因成功敲除。与对照组比较，实验组肺肿瘤转移灶减少（P < 0.05），脾脏中巨噬细胞、树突状细胞、粒细胞的比例均无变化（均P > 0.05），肺组织中的转移相关巨噬细胞比例降低（P < 0.05），而居住型巨噬细胞比例增加（P < 0.05）。结论：巨噬细胞FBXW7基因缺失能够抑制小鼠黑色素瘤肺部转移，其机制可能与转移部位的转移相关巨噬细胞比例降低有关；FBXW7可能参与调控转移相关巨噬细胞的趋化及功能。

关键词： 巨噬细胞; 黑色素瘤; 肺肿瘤/继发性; 肿瘤转移; 细胞运动; 泛素蛋白连接酶复合物; 基因缺失

Abstract:

Objective: To investigate the effects of myeloid-specific deficiency of FBXW7 on lung metastasis of murine B16F10 melanoma and its mechanisms. Methods: Mice carrying the floxed allele of FBXW7 and lysozyme M-Cre were used for generation of mice with myeloid cell-specific deletion of FBXW7. Mouse genotypes were examined by genomic DNA PCR. B16F10 cells in PBS were injected into the tail vein of Lysm^-FBXW7^f/f and Lysm⁺FBXW7^f/f mice. After 14 d, the mice were sacrificed, and the lungs were removed and weighed. B16F10 tumor colonies in the lungs were counted. The myeloid cells were analyzed by flow cytometry. Results: Myeloid-specific deficiency of FBXW7 mice were generated successfully, as FBXW7 expressions in peritoneal macrophages and bone marrow-derived macrophages (BMDMs) of Lysm⁺FBXW7^f/f mice were knockdown. Flow cytometry results showed the deletion of FBXW7 in myeloid lineages did not affect the development of myeloid immune cell subsets. Metastasis was reduced in Lysm⁺FBXW7^f/f mice compared with control mice. The number of tumor colonies was 165±42, 122±12 respectively. The proportion of metastasis-associated macrophages (MAM) in the lungs of Lysm⁺FBXW7^f/f mice was reduced [(23.15±7.59)% vs (13.13±2.26)%], while the proportion of resident macrophages was increased [(5.426±0.42)% vs (10.42±1.90)%]. The proportion of myeloid-derived suppressor cells in the lung showed no difference between Lysm^-FBXW7^f/f and Lysm⁺FBXW7^f/f mice. Conclusion: Myeloid-specific deficiency of FBXW7 can inhibit lung metastasis of B16F10 melanoma in mice, and the mechanism may be associated with regulation of MAM in the metastatic tumor lesions.

Key words: Macrophages Melanoma Lung neoplasms/secondary Neoplasm metastasis Cell movement Ubiquitin-protein ligase complexes Gene deletion

收稿日期: 2017-01-04 出版日期: 2017-10-31

基金资助: 国家自然科学基金（81373115）

通讯作者: 王青青 E-mail: zhangwei329@hotmail.com;wqq@zju.edu.cn

作者简介: 张玮(1980—), 女, 硕士, 主治医师, 主要从事肿瘤复发转移相关机制的研究; E-mail:zhangwei329@hotmail.com; http://orcid.org/0000-0003-0687-123X

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引用本文:

张玮,来利华,王青青. 巨噬细胞中FBXW7基因缺失对小鼠黑色素瘤肺转移的影响[J]. 浙江大学学报（医学版）, 2017, 46(2): 111-117.

ZHANG Wei,LAI Lihua,WANG Qingqing. Effects of myeloid specific deficiency of FBXW7 on lung metastasis of murine melanoma. J Zhejiang Univ (Med Sci), 2017, 46(2): 111-117.

链接本文:

http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2017.04.01 或 http://www.zjujournals.com/xueshu/med/CN/Y2017/V46/I2/111

图 1 建立Lysm+FBXW7f/f小鼠技术路线图

图 2 Lysm+FBXW7f/f小鼠基因组PCR结果

图 3 两组脾脏中髓系细胞流式细胞术检测结果

表 1 两组脾脏中髓系细胞比例比较

图 4 髓系细胞特异性FBXW7基因缺失小鼠黑色素瘤肺转移灶大体变化

表 2 两组肺部黑色素瘤转移灶数及肺质量比较

图 5 髓系细胞特异性FBXW7基因缺失黑色素瘤小鼠肺部髓系细胞亚群变化

表 3 两组肺组织髓系来源的巨噬细胞比例比较

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