[Expression of coagulation factor IX gene mediated by human source vector pHrnF9 in intestinal epithelial sw480 cells].

ABSTRACT

The objective of this study was to explore the feasibility of intestinal epithelial cell and human source vector used in gene therapy for hemophilia B. The intestinal epithelial sw480 cells were transfected with human source vector plasmid pHrnF9 which contained human coagulation factor IX gene. Transcription of its mRNA were measured by RT-PCR. The transfection efficiency were observed under fluorescence microscope. The expression of its protein and coagulant activities in the transfected sw480 cells were measured by ELISA and one-stage method. The results showed that the expression of hFIX mRNA could be detected after transfection. The transfection efficiency reached to the maximum at 48 hour. The hFIX protein amount was 11.3 +/- 0.23 ng/(10(6) cells.24 h) at 24 hours after transfection and reached to 29.34 +/- 1.00 ng/(10(6) cells.24 h) at 48 hours and decreased to 12.45 +/- 0.15 ng/(10(6) cells.24 hr) at 72 hours. Sw480 cells transfected with pHrnF9 were capable of producing hFIX with coagulant activity. The coagulant activity reached to (6.07 +/- 0.17)%/10(6) cells at 48 hours and decreased to 1.81 +/- 0.06%/10(6) cells at 72 hours. It is concluded that the sw480 cells transfected with pHrnF9 plasmid can express hFIX with coagulant activity, the intestinal epithelial cells may become target cells in the gene therapy for hemophilia B.