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Objective To explore the effect of stroma cell-derived factor receptor CXCR4 on the homing of the hematopoietic stem/progenitor cells in utero transplantation. Methods CD34~+ cells were collected by Ficoil density gradient centrifugation and MiniMACS and then stimulated for 48 h by SCF and IL-6 cytokines prior to transplantation. CD184(CXCR4) expressions and transmigrate rates of the CD34~+ cells were analysed by flow cytometer. Cells pre-treated with different treatment were transplanted into the abdominal cavity of the fetal BALB/c mouse in the pregnant days 13～14. Human CD45 cells as the marker of graft were detected by flow cytometry after 1 month the fetus born. Results Expression changes of CD184 on CD34~+ cells were from 9. 58%±1. 56% to 19. 32%±3. 64% after SCF and IL-6 stimulation. The CD34~+/CXCR4~(high) cells exhibited significant increases in SDF-1 mediated chemotaxis compared with the CD34~+/CXCR4~(low) cells. Transmigration of CD34~+ /CXCR4~(high) was inhibited by pretreatment with an-tiCXCR4mAb and PTX. The positive rates of human CD45 cells detected in the fetal mouse were significantly higher in the SCF and IL-6 pretreatment group. This effects were significantly abrogated after the addition of antiCXCR4mAb or PTX. Conclusion Up-regulation of CXCR4 expression may be useful for improving hematopoietic stem/progenitor cells homing in utero transplantation. This homing process is mediated and depends on the CXCR4 receptors. The signal transduction is mediated by PTX-sensitive Gi protein.
RESUMO
Objective To explore the effect of stroma cell-derived factor receptor CXCR4 on the homing of the hematopoietic stem/progenitor cells in utero transplantation. Methods CD34+ cells were collected by Ficoll density gradient centrifugation and MiniMACS and then stimulated for 48 h by SCF and IL-6 cytokines prior to transplantation. CD184(CXCR4) expressions and transmigrate rates of the CD34+ cells were analysed by flow cytometer. Cells pretreated with different treatment were transplanted into the abdominal cavity of the fetal BALB/c mouse in the pregnant days 13～14. Human CD45 cells as the marker of graft were detected by flow cytometry after 1 month the fetus born. Results Expression changes of CD184 on CD34+ cells were from 9.58%?1.56% to 19.32%?3.64% after SCF and IL-6 stimulation. The CD34+/CXCR4high cells exhibited significant increases in SDF-1 mediated chemotaxis compared with the CD34+/CXCR4low cells. Transmigration of CD34+/CXCR4high was inhibited by pretreatment with antiCXCR4mAb and PTX. The positive rates of human CD45 cells detected in the fetal mouse were significantly higher in the SCF and IL-6 pretreatment group. This effects were significantly abrogated after the addition of antiCXCR4mAbor PTX. Conclusion Up-regulation of CXCR4 expression may be useful for improving hematopoietic stem/progenitor cells homing in utero transplantation. This homing process is mediated and depends on the CXCR4 receptors. The signal transduction is mediated by PTX-sensitive Gi protein.
RESUMO
AIM: To investigate the use of therapy ultrasound to enhance nonviral gene delivery. METHODS: Endothelial cells (EC) and vascular smooth muscle cells (VSMC) were cultured in 6-well plates. Plasmid (pcDNA3.1/His/LacZ) with or without microbubbles at the different concentrations was transfected into the cells with the use of ultrasound for 1 min at 2 MHz, 1.8 mechanical index (MI). Additional controls included ultrasound alone, microbubble alone and microbubble plus plasmid. The rate of blue cells and the activities of ?-Gal were measured. In addition, cell viability was detected with different time from 1 to 30 min of ultrasound irradiation and the different concentrations of microbubbles. RESULTS: In the group of ultrasound with microbubble, the rate of blue cells and activity of ?-Gal markedly increased by 60% and 9-fold, respectively. Microbubbles at concentration of 10% led to the highest transfection effect. Ultrasoud exposure at 1 to 30 minute had no cell toxic effects, while microbubbles at the concentration of 50% had significant effect on cell survival. CONCLUSIONS: Albumin-coated microbubbles markedly enhance gene delivery by therapeutic ultrasound-mediated microbubble destruction, which can be used as a safe and practicality vectors in gene therapy.