RNA Interference Mediated Silencing of Tissue Factor in Human Umbilical Vein Endothelial Cells

Objective: To construct to silence tissue factor (TF) expression in Human Um-bilical Vein Endothelial Cells (HUVECs) for providing evidence of the gene therapy and prevention of coagulation dysfunction in placental abruption (PA) neonates. Methods: Cultured HUVECs in vitro were divided into control group and PA group, each including non-prevention, scramble siRNA and TF-siRNA subgroups. pENTRTM/U6-shRNA/TF vector expressing TF were constructed and transfected into HUVECs. The mRNA expression of TF was tested with RT-PCR, and TF protein expression was detected with immunofluorescence staining. Results: Monolayer HUVECs with short-rod and short spindle shaped were adherent to the bottom, forming paving stone arrangement. The TF mRNA expression was significantly different between control group and PA group (P < 0.01) and among different subgroups (P < 0.01). In non-prevention subgroups, significant difference was observed in TF mRNA expression between control group and PA group (P < 0.05). Significant difference in TF mRNA expression was found in false-prevention subgroups (P < 0.05). The TF mRNA expression was markedly different among different subgroups in control (P < 0.01), and the similar result among different PA subgroups (P < 0.01). In both control and PA groups, the TF mRNA expression was the lowest after TF silencing. Immunofluorescence staining showed high TF expression in HUVECs in false-prevention subgroups. In scramble siRNA group, the TF protein expression reduced as compared to non-prevention group and reduced dramatically after TF silencing as compared to control. Conclusion: pENTRTM/U6-TF-shRNA is able to significantly inhibit the TF mRNA and protein expression in HUVECs from healthy neonates and PA neonates.