Lin Yan, Li Yupin, Hu Chongkang, Zeng Lingchao, Liang Shiqian, Han Hua
Department of Pediatrics, Tangdu Hospital, Air Force Military Medical University, Xi'an 710038, China.
Department of Biochemistry and Molecular Biology, Air Force Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Dec;34(12):1057-1062.
Objective To study the role of mouse extracellular segment protein of signal-regulatory protein α gene (mSIRPα) of mice in tumor immune regulation by cloning mSIRPα, constructing its prokaryotic expression vector and achieving the soluble expression of SIRPα. Methods The mSIRPα gene was amplified from mouse lymph node and the prokaryotic expression vector of pET32a-SIRPα was further constructed. After the soluble expression of recombinant Trx-mSIRPα fusion protein containing thioredoxin (Trx) tag was achieved, mouse bone marrow-derived monocytes were cultured and induced to differentiate into macrophages. Then the macrophages were co-cultured with L1210 leukemia cells labeled with 5(6)-carboxyfluorescein diacetate succinimide ester (CFSE). Trx-mSIRPα protein and Trx control protein were added into the co-culture system. The role of recombinant protein in the macrophage phagocytosis of tumor cells was observed by immunofluorescence cytochemical staining and confocal microscopy. Results Purified soluble Trx-mSIRPα protein was obtained and it was showed that it could enhance the phagocytosis of macrophages in mouse L1210 leukemia cells in vitro phagocytosis experiments. Conclusion Prokaryotic expression of Trx-mSIRPα protein can effectively enhance the phagocytosis of macrophages in leukemia cells, thus playing the role of anti-tumor immunotherapy.
目的 通过克隆小鼠信号调节蛋白α基因（mSIRPα）的胞外段蛋白，构建其原核表达载体并实现SIRPα的可溶性表达，研究其在肿瘤免疫调节中的作用。方法 从小鼠淋巴结中扩增mSIRPα基因，进一步构建pET32a-SIRPα原核表达载体。在实现含硫氧还蛋白（Trx）标签的重组Trx-mSIRPα融合蛋白可溶性表达后，培养小鼠骨髓来源的单核细胞并诱导其分化为巨噬细胞。然后将巨噬细胞与用5(6)-羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）标记的L1210白血病细胞共培养。在共培养体系中加入Trx-mSIRPα蛋白和Trx对照蛋白。通过免疫荧光细胞化学染色和共聚焦显微镜观察重组蛋白在巨噬细胞吞噬肿瘤细胞中的作用。结果 获得了纯化的可溶性Trx-mSIRPα蛋白，体外吞噬实验表明其可增强巨噬细胞对小鼠L1210白血病细胞的吞噬作用。结论 Trx-mSIRPα蛋白的原核表达可有效增强巨噬细胞对白血病细胞的吞噬作用，从而发挥抗肿瘤免疫治疗作用。
