Abstract
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.
Highlights
Placental abruption (PA) refers to the complete or partial separation of a normal placenta from the wall of the uterus before delivery
tissue factor (TF) expression occurs at the initiation of coagulation, and inhibition of TF expression has the possibility to reduce the risk for coagulation dysfunction in placental abruption (PA) neonates
In non-transfection subgroups, the TF mRNA expression was significantly different between control group and PA group (t = 11.71, P < 0.05); in scramble shRNA transfection subgroups, the TF mRNA expression was significantly different between control group and PA group (t = 21.112, P < 0.05); in TF shRNA transfection subgroups, the TF mRNA expression was comparable between control group and PA group (t = 0.023, P > 0.05)
Summary
Placental abruption (PA) refers to the complete or partial separation of a normal placenta from the wall of the uterus before delivery. PA is a major cause of bleeding but an important cause of coagulation dysfunction in neonates after birth in the Department of Obstetrics [1] [2]. PA may cause stillbirth, premature delivery, neonatal asphyxia, neonatal anemia and coagulation disorders. Previous studies reported that the incidence of PA is 0.49% - 1.8% [3] [4]. Studies have shown that a large amount of tissue factor (TF) is released from the placental villi and decidua in PA and forms TF-VII complex with fetal F-VII. TF expression occurs at the initiation of coagulation, and inhibition of TF expression has the possibility to reduce the risk for coagulation dysfunction in PA neonates. Our findings may provide a new way for the clinical therapy and prevention of coagulation dysfunction in PA neonates
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