动物造模 z-bio 2022-11-19 789

　　Objective To transfect C57BL/6 mouse adipose-derived mesenchymal stem cells with recombinant adenovirus carrying human coagulation factor IX (hFIX) gene (F9). Vector cells for hemophilia gene therapy.

　　Methods The inguinal fat of C57BL/6 mice was harvested, and ADSCs were isolated and cultured according to the tissue block suspension method and subcultured. The third generation ADSCs were transfected with recombinant adenovirus carrying hFIX gene and containing GFP fluorescent marker, and then again after transfection. Cells were subcultured to passages 4 and 5. The amount of fluorescence carried by cells was observed under a fluorescence microscope, the expression of F9 gene was detected by RT-PCR, the supernatant of cells was detected by ELISA and the expression protein of target gene in cells was detected by Western Blot.

　　Results (1) After transfection of recombinant adenovirus, fluorescence expression was observed under the fluorescence microscope, and the cells still had fluorescence after passage. (2) RT-PCR results showed that all four groups of ADSCs could express the internal reference GAPDH gene fragment, the expression of target gene F9 could be detected in groups A, B and C, and the expression of F9 was 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) The expression of hFIX protein in four groups of cells was detected by Western Blot, and the results showed 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) were significantly higher than those in D The gray value of histone expression was (0.02±0.01), and the difference was significant (P<0.05).

　　Conclusions After transfecting ADSCs with recombinant adenovirus, the cells can still express high hFIX activity after subculture and can be used as carrier cells for gene therapy of hemophilia.