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.