荧光标记中性粒细胞特异性敲除膜结合型前列腺素E2合酶-1小鼠的构建及其在静脉血栓形成中的应用

Construction of Fluorescent Labeled Neutrophil Specific Knockout Microsomal Prostaglandin E Synthase-1 Mice and Its Application in Venous Thrombosis

膜结合型前列腺素E2合酶-1(mPGES-1)是一个新型抗炎药靶,全身敲除mPGES-1不影响动脉血栓形成,抑制血管病理性重构。目的构建荧光(tdTomato)标记的mPGES-1中性粒细胞特异性敲除小鼠并鉴定其在静脉血栓形成中的应用。方法与结果人工合成基因打靶序列,包括mPGES-1同源序列、LoxP序列、Lox2272序列和tdTomato表达序列,利用CRISPR/Cas9技术将这段合成序列同源置换C57BL/6N小鼠的mPGES-1基因序列,制备转基因小鼠(mPGES-1fl/fl),将其与中性粒细胞特异性表达Cre重组酶的小鼠(Lyz-Cre+)杂交获得Lyz-Cre(+)mPGES-1fl/fl小鼠。向小鼠腹腔注射脂多糖诱导炎症,半定量PCR结果显示Lyz-Cre(+)mPGES-1fl/fl小鼠外周血白细胞mPGES-1表达缺失,荧光显微镜观察和流式细胞分析结果表明tdTomato成功标记Lyz-Cre(+)mPGES-1fl/fl小鼠中性粒细胞。通过限制血流诱导小鼠下腔静脉血栓,荧光标记的中性粒细胞参与静脉血栓形成。敲除中性粒细胞mPGES-1不影响基础状态血液PGE2水平,但显著抑制诱导静脉血栓相关的PGE2水平上调。结论成功构建报告基因tdTomato特异性标记的mPGES-1组织特异性敲除的转基因小鼠,利用此工具鼠进一步发现中性粒细胞参与静脉血栓形成,中性粒细胞mPGES-1介导静脉血栓形成相关的PGE2合成。

Microsomal prostaglandin E2 synthase-1(mPGES-1)is a novel anti-inflammatory drug target.Systemic knockout mPGES-1 does not affect arterial thrombosis and inhibit vascular remodeling.Objective To construct fluorescent(tdTomato)labeled neutrophil specific knockout mPGES-1 mice and identify their application in venous thrombosis.Methods and Results A sequence including mPGES-1 homologous sequence,LoxP sequence,Lox2272 sequence and tdTomato expression sequence was synthesized artificially,mPGES-1 fl/fl transgenic mice were constructed by CRISPR/Cas9 technology,which part of the mPGES-1 gene sequence of C57 BL/6 N mice was homologous replaced by the synthetic sequence.Then it was crossed with neutrophil-specific expression Cre recombinase mice(Lyz-Cre+)to breed Lyz-Cre(+)mPGES-1 fl/fl mice.Mice were intraperitoneally injected with lipopolysaccharide to induce inflammation,the semi-quantitative PCR results showed that the expression of mPGES-1 in peripheral blood leukocytes of Lyz-Cre(+)mPGES-1 fl/fl mice was absent,and fluorescence microscopy and flow cytometry results showed that td Tomato was successfully labeled neutrophils of Lyz-Cre(+)mPGES-1 fl/fl mice.Fluorescent labeled neutrophils were involved in venous thrombosis in mice induced by restriction of blood flow.Knockout of neutrophil mPGES-1 did not affect basal blood PGE2 levels,but significantly decreased the upregulation of PGE2 in thrombosis model.Conclusion mPGES-1 tissue specific knockout transgenic mice with reporter tdTomato were successfully constructed,and this tool was used to further discover that neutrophils were involved in venous thrombosis,and neutrophils mPGES-1 mediated PGE2 synthesis related to venous thrombosis.