Stable expression of human coagulation factor IX in mouse adipose-derived stem cells
Received:February 08, 2020  
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DOI:10. 3969 / j.issn.1005-4847. 2020. 04. 014
KeyWord:target gene; recombinant adenovirus; adipose-derived stem cell(ADSC)
李杰 华北理工大学附属医院血液科,河北 唐山
谢燕燕 华北理工大学附属医院血液科,河北 唐山
王馨 华北理工大学附属医院血液科,河北 唐山
王霖虹 华北理工大学附属医院血液科,河北 唐山
和红霞 华北理工大学附属医院血液科,河北 唐山
孙庆云 华北理工大学附属医院血液科,河北 唐山
晏亚辉 华北理工大学附属医院血液科,河北 唐山
闫振宇 华北理工大学附属医院血液科,河北 唐山
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      Objective To observe whether hFIX is stably expressed in adipose-derived stem cells (ADSCs) and to explore whether ADSCs can be used as a vector cell line for hemophilia gene therapy following transformation by recombinant adenoviruses carrying human coagulation factor IX ( hFIX) gene, F9. Methods ADSCs were isolated from C57BL/ 6 mice and cultured by tissue mass suspension. The third generation of ADSCs was transfected by recombinant adenovirus carrying F9 and GFP fluorescence marker. After transfection, the cells were subcultured again to the 4th and 5th passages. The level of fluorescence expressed by the cells was observed by fluorescence microscopy, the expression of F9 was detected by RT-PCR, and the protein expression of hFIX was detected by ELISA and western blotting. Results Fluorescence was observed after recombinant adenoviral transfer, and was still identified after cell passaging. RT-PCR revealed that all four groups of ADSCs could express internal reference GAPDH, but there was no expression of the target gene F9 in group A, and the expression of F9 could be detected in group D. Detection of hFIX:Ag by ELISA showed that the detection values of groups A (81. 62 ± 8. 82) ng / mL, B (52. 50 ± 3. 25) ng / mL, and C (47. 41 ± 4. 00) ng / mL were significantly higher than that of group D (0. 76 ± 0. 44) ng / mL, and there was a significant difference between the two groups (P< 0. 05). hFIX expression in the four groups of ADSCs was detected by western blotting. The result showed that the gray value of histone expression in groups A (0. 68 ± 0. 10), B (0. 49 ± 0. 15) and C was significantly higher than that in group D (0. 02 ± 0. 01), and the difference was statistically significant (P< 0. 05), while the gray value of histone expression in group A was significantly higher than that in group D (P< 0. 05). Conclusions The recombinant adenoviruses were transformed into ADSCs and cultured after passaging, and could still express high hFIX activity, thus demonstrating their potential as vector cells for hemophilia gene therapy.
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