Expression Of Adenovirus-mediated B-domain-deleted Human Coagulation Factor ? In ADSCs Of SD Rats

Objectives A recombinant adenoviral vector containing B-domain-deleted human coagulant factor ?(BDDhF?)was infected with SD rat adipose tissue-derived stem cells(ADSCs)in vitro.Explore the possibility of ADSCs as target cells for hemophilia A gene therapy.Methods 1 Cell culture and identification: Under sterile conditions,the adipose tissue of the inguinal region of SD rats was taken by collagenase digestion and the ADSCs were isolated and cultured.The cell growth curve was detected by MTT method after P3generation;the cell surface marker was detected by flow cytometry;.The Osteogenic/adipogenic inducer detects the multipotential differentiation potential of stem cells.2 The experimental group: group A was infected with ADSCs by Ad-BDDhF?-GFP,group B was infected with ADSCs by Ad-GFP,group C was normal cultured with ADSCs group;3 The cell growth of the three groups was detected by CCK-8 method.The expression of F? antigen in the supernatant was detected by ELISA.The expression of BDDhF? gene and protein in the three groups after infection was detected by RT-PCR and Western blot.Results 1 The P3 cells isolated and cultured were arranged in a spiral or spindle shape.The growth curve of the P3 cells was inverted and S-shaped.The flow cytology detected P29 cells CD29(99.05%)and CD90(97.82%).CD44(92.41%)was positively expressed and CD45(8.38%)was negatively expressed.After 14 days of adipogenic induction,Oil Red O staining was positive.After 7 days of osteogenic induction,ALP staining was dark blue,and after osteogenic induction for 30 days,alizarin red Dyeing suggests mineralized nodules.2 Recombinant adenovirus vector Ad-BDDhF?-GFP infected with ADSCs showed green fluorescence at 24 h,and the fluorescence expression intensity was the strongest at 72 h.The best MOI was 350.3 After CCK-8 detection,the three groups still showed an inverted "S" curve growth.4 The ELISA results showed that after three groups of ADSCs were infected for 72 hours,The expression of hF?Ag in group A(140.26±2.29)was significantly higher than that in group B(99.95±1.05)and group C(80.02±0.21),the difference was statistically significant(P<0.05).5 RT-PCR results showed that BDDhF?gene expression was observed in group A after 72 hours of infection in ADSCs,and no BDDhF? gene expression in group B and C.6 Western blot results showed that after three groups of ADSCs were infected for 72 hours,Western blot analysis showed that the expression of hF? protein in group A(1.10±0.57)was significantly higher than that in group B(0.60±0.20)and group C(0.33±0.12).The difference was statistically significant(P<0.05).Conclusions 1 The human factor ? gene vector deleted by the B region of adenovirus can successfully infect ADSC in SD rats.2 The infected cells can effectively express human coagulation factor F?.Figure 9;Table 4;Reference 61...