鼠CD300e多克隆抗体的制备及其在肝再生模型中的组织定位鉴定

Preparation and characterization of a rabbit anti-mouse CD300e polyclonal antibody and its tissue localization in a mouse liver regeneration model

摘要

目的 制备并鉴定兔源抗鼠CD300e多克隆抗体, 探讨其在小鼠肝再生模型中识别和定位CD300e⁺单核/巨噬细胞的应用价值。方法 设计并合成小鼠CD300e胞外区+Fc tag重组蛋白, 抗原免疫新西兰大白兔后采血分离血清, 间接ELISA测定血清抗体效价, 流式细胞术验证抗体有效性。通过生信分析挖掘CD300e⁺单核/巨噬细胞肝内分布特点, 使用流式细胞术和免疫荧光染色探索CD300e⁺细胞在小鼠肝切除后的肝内浸润程度的改变。结果 终末免疫兔血清抗体的ELISA效价>1.6×10⁵, 部分肝切除后小鼠受损肝叶内CD300e⁺巨噬细胞浸润增加。结论 本研究成功制备了兔源抗鼠CD300e多克隆抗体, 并在小鼠受损肝叶内的血管周围鉴定出了CD300e⁺巨噬细胞, 为后续进一步挖掘其促进肝再生的作用奠定基础。

Abstract

Objective To prepare and characterize a rabbit anti-mouse CD300e polyclonal antibody, and to evaluate its application value in identifying and localizing CD300e⁺ monocytes/macrophages in a mouse liver regeneration model. Methods A recombinant protein consisting of the extracellular domain of mouse CD300e fused with an Fc tag was designed and synthesized as the immunogen. New Zealand white rabbits were immunized, followed by blood collection and serum isolation. Antibody titers were determined by indirect ELISA, and antibody validity was verified by flow cytometry. Bioinformatic analyses were performed to explore the intrahepatic distribution characteristics of CD300e⁺ monocytes/macrophages. Flow cytometry and immunofluorescence staining were used to assess changes in the hepatic infiltration of these cells after partial hepatectomy in mice. Results The indirect ELISA titer of terminal rabbit serum exceeded 1.6×10⁵. Following partial hepatectomy, an increased accumulation of CD300e⁺ macrophages was observed in the injured liver lobes. Conclusion A rabbit anti-mouse CD300e polyclonal antibody is successfully prepared in this study. CD300e⁺ macrophages are identified in perivascular regions of injured liver lobes in mice, providing a foundational tool for further mechanistic studies on their role in promoting liver regeneration.

关键词

CD300e / 巨噬细胞 / 肝切除 / 肝再生 / 多克隆抗体 / 转录组测序 / 空间转录组测序 / 门静脉高压症

Key words

CD300e / macrophage / hepatectomy / liver regeneration / polyclonal antibody / RNA-seq / spatial transcriptomics / portal hypertension

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基金资助

国家自然科学基金面上项目(82471809)