In vitro study on blocking HUVEC from apoptosis by transfecting siRNA targeting cytoplasmic domain of tissue factor / 中华器官移植杂志

ABSTRACT

Objective To investigate the effect of small interfering RNA (siRNA) targeting cytoplasmic domain of tissue factor on apoptosis of vascular endothelial cells. Methods Specific siRNA targeting cytoplasmic domain of tissue factor were designed, and synthetic oligos were inserted into plasmid DNA. The siRNA constructs were transfected into human umbilical vascular endothelial cells (HUVEC) with liposome. The HUVEC were transfected with the constructs encoding siRNA Ⅰ, siRNA Ⅱ and pcDNA~(TM)6.2 GW/-miR plasmid separately. The transfected HUVEC were mixed with CD8~+ T lymphocytes. The apoptotic rate of tranfected HUVEC mixed with lymphocytes was analyzed by flow cytometry. Magnetic beads were used to measure PT of the supematant in the mixed lymphocytes culture. Results The siRNA constructs were confirmed by DNA sequence analysis. The apoptotic rate of HUVEC transfected with siRNA Ⅰ and Ⅱ plasmids was decreased significantly as compared with the empty control group (P<0.01). The apoptosis rate of HUVEC transfected with siRNA Ⅰ plasmid was lower than that of HUVEC transfected with siRNA Ⅱ plasmid (P<0.05). APTT of the culture supernatants in the three transfection groups was lower in the control groups (P <0.05), but there was significant difference among the three transfection groups. Conclusion The siRNA targeting cytoplasmic domain of tissue factor were successfully constructed, siRNA can protect HUVEC, and reduce the apoptotic rate of endothelial cells in mixed lymphocyte reaction without influencing the coagulation function.