髓系特异性SOCS3基因敲除小鼠的构建及应用

Construction and application of myeloid-specific SOCS3 gene knockout mice

目的:通过Cre-loxP基因敲除系统特异性敲除髓系SOCS3基因,构建早期髓系来源抑制细胞(eMDSCs)高浸润荷瘤鼠模型。方法:通过将SOCS3fl/-小鼠与Lyz2-Cre小鼠杂交繁育,获得髓系特异性SOCS3基因敲除小鼠,PCR法鉴定小鼠基因型,Western印迹验证基因敲除效果,流式细胞术检测髓系特异性SOCS3基因敲除小鼠骨髓中eMDSCs比例及其对T细胞的抑制作用,并在该小鼠基础上分别构建乳腺癌、肺癌和黑色素瘤3种eMDSCs高浸润荷瘤鼠模型,流式细胞术检测肿瘤组织中eMDSCs浸润情况。结果:PCR鉴定和Western印迹检测证实髓系特异性SOCS3基因敲除小鼠构建成功,流式细胞术结果表明髓系SOCS3基因敲除小鼠骨髓中eMDSCs比例显著升高(t=17.94,P<0.001),且该群eMDSCs抑制T细胞增殖(t=14.21,P<0.001)、促进T细胞凋亡(t=13.53,P<0.001)。在黑色素瘤、乳腺癌、肺癌3种髓系特异性SOCS3基因敲除荷瘤鼠的肿瘤组织中eMDSCs数量显著增加(t=24.14、24.56、14.93,均P<0.001)。结论:通过髓系特异性SOCS3敲除可成功构建eMDSCs高浸润荷瘤鼠模型。

Objective:To construct mouse models of cancer with high infiltration of eMDSCs through myeloid-specific SOCS3 gene knockout mice based on the Cre-loxP system.Methods:SOCS3fl/fl mice were crossed with Lyz2-Cre mice to generate myeloid-specific SOCS3 gene knockout mice.The genotypes of the offspring were identified by PCR,and the knockout effect was verified by Western blotting.The proportion of eMDSCs in the bone marrow of myeloid-specific SOCS3 gene knockout mice and their inhibitory effection T cells were detected by flow cytometry.Based on the myeloid-specific SOCS3 gene knockout mice,three kinds of tumor-bearing mouse model s include breast cancer,lung cancer,and melanoma with high infiltration of eMDSCs were constructed,and the infiltration of eMDSCs in tumors was detected by flow cytometry.Results:PCR results and Western blotting analysis confirmed that myeloid-specific SOCS3 gene knockout mice were successfully constructed.Flow cytometry results demonstrated that the proportion of eMDSCs in the bone marrow of myeloid-specific SOCS3 gene knockout mice was significantly increased(t=17.94,P<0.001),and the eMDSCs significantly inhibited T cell proliferation(t=14.21,P<0.001)and promoted T cell apoptosis(t=13.53,P<0.001).The number of eMDSCs was significantly increased in tumor tissue of three types of myeloid-specific SOCS3 gene knockout tumor-bearing mice with melanoma,breast cancer,and lung cancer(t=24.14,24.56,14.93,all P<0.001).Conclusion:Mouse models of cancer with high infiltration of eMDSCs are successfully constructed by specifically knocking out the SOCS3 gene of mouse myeloid cells.