人凝血因子IX基因,脂肪干细胞 z-bo 2020-10-22 397

　　Objective To transfect the recombinant adenovirus carrying human coagulation factor IX (hFIX) gene (F9) into C57BL/6 mouse adipose-derived stem cell (ADSC), and observe whether F9 is in ADSC after subculture To explore whether ADSC can be used as a carrier cell for gene therapy of hemophilia.

　　Methods The fat in the groin area of C57BL/6 mice was taken, and the mouse ADSCs were isolated and cultured according to the tissue block suspension method and subcultured. The third-generation ADSCs were transfected with the recombinant adenovirus carrying the hFIX gene and containing the GFP fluorescent marker. Proceed to subculture to the fourth and fifth passages. Observe the number of cells carrying fluorescence under a fluorescence microscope, RT-PCR to detect F9 gene expression, ELISA method to detect cell supernatant and Western Blot to detect the target gene expression protein in the cell.

　　Results (1) Fluorescence expression can be seen under a fluorescence microscope after recombinant adenovirus transfection, and the cells still have fluorescence after passage. (2) RT-PCR results showed that the four groups of ADSCs can express the internal reference GAPDH gene fragments, the expression of the target gene F9 can be detected in groups A, B, and C, and the expression of F9 is not detected in group D. (3) ELISA method to detect coagulation factor IX antigen (hFIX:Ag) The results showed: group A (81.62±8.82) ng/mL, group B (52.50±3.25) ng/mL, group C (47.41±4.00) ng/mL It was significantly higher than the detection value of group D (0.76±0.44) ng/mL, and the difference was significant (P<0.05). (4) Western Blot method detects the expression of hFIX protein in the four groups of cells, and the results show that the gray value of protein expression in group A (0.68±0.10), group B (0.49±0.15), and group C (0.18±0.05) are significantly higher than those in group D. The gray value of histone expression (0.02±0.01), the difference is significant (P<0.05).

　　Conclusion After the recombinant adenovirus is transfected into ADSC, it can still express high hFIX activity after passage and culture, and can be used as a carrier cell for gene therapy of hemophilia.