新医学2026，Vol.57Issue(4)：390-401,12.DOI:10.12464/j.issn.0253-9802.2025-0409
过表达EP4基因的人CD34+细胞构建及EP4A对其在小鼠体内的归巢影响
Construction of human CD34+cells overexpressing the EP4 gene and preliminary observation of the impact of EP4A on their homing in mice
摘要
Abstract
Objective To construct a lentiviral vector for over-expressing the prostaglandin E receptor 4(EP4)gene,clone the exogenous EP4 gene into human CD34+cells,observe the homing of these EP4 gene over-expressing cells in mice,and preliminarily investigate the effect of the EP4-specific agonist(EP4A)on this homing.Methods ①An EP4-cDNA fragment was chemically synthesized and cloned into a GL107 plasmid to construct the GL107-EP4+plasmid.②The GL107-EP4+plasmid and a control GL107 plasmid were packaged into lentiviruses using 293T cells.③Human CD34+cells were infected with these lentiviruses.Infection efficiency was assessed via fluorescence microscopy and flow cytometry,while EP4 expression was detected by qRT-PCR and Western blot.④The effect of EP4A on the homing of EP4+-human-CD34+cells in mice and the determination of the appropriate observation time was observed using in vivo imaging.⑤Detect the expression of EP4 in the bone marrow of mice after human CD34+cells were transfused by immunohistochemistry after EP4A stimulation.⑥CXCR4 expression in the bone marrow cells of these mice was measured by qRT-PCR and Western blot.Result ①DNA sequencing confirmed the correct EP4-cDNA sequence in the GL107-EP4+plasmid.②The GL107-EP4+plasmid was successfully packaged into lentiviruses with a titer＞1×108TU/mL.③Fluorescence microscopy observed that the GFP fluorescence intensity of GL107-EP4+and GL107 lentivirus-infected human CD34+cells was enhanced compared to the background control(uninfected lentivirus human CD34+cells);flow cytometry detected the infection efficiency of GL107-EP4+and GL107 lentivirus as 28.06%and 66.76%,respectively.The expression of EP4 messenger RNA(mRNA)and protein in GL107-EP4+was higher than in background control and GL107 lentivirus groups(0.580±0.032 vs.0.256±0.027 vs.0.250±0.043,both P＜0.001).④Day 3 post-transplantation was identified as the appropriate observation time for the EP4A-enhanced homing effect(P＜0.05).⑤The relative expression level of EP4 in the bone marrow of the EP4A-stimulated group after transplantation is higher than that of the EP4+-Luc control group(0.164±0.004 vs.0.118±0.007,P=0.016).⑥Both CXCR4 mRNA and protein levels were higher in the EP4A-stimulated group(both P＜0.05).Conclusion The EP4 gene was successfully cloned into human CD34+cells,leading to EP4 protein over-expression.EP4A may increase the directional homing efficiency of human CD34+hematopoietic cells to the bone marrow of recipient mice by promoting the expression of CXCR4.关键词
人CD34+细胞/慢病毒载体/EP4 受体/EP4 受体激动剂/造血干细胞归巢Key words
Human CD34+cells/Lentiviral vector/EP4 receptor/EP4 receptor agonist/Hematopoietic stem cell homing引用本文复制引用
黄丽君,李萍,甄佳怡,陈惠珍,许多荣..过表达EP4基因的人CD34+细胞构建及EP4A对其在小鼠体内的归巢影响[J].新医学,2026,57(4):390-401,12.基金项目
国家自然科学基金(82070183) （82070183）
