Experimental Study On Adenoviral Mediated B Hemophilia Gene Therapy

Objectives 1 To investigate the expression of human coagulation factor IX（hFIX）gene in adipose-derived stem cells（ADSCs）of SD rats by transfecting recombinant adenoviral vector（Ad-hFIX-GFP）carrying human coagulation factor IX（hFIX）into ADSCs.2 The ADSCs of C57BL/6j mice transfected with Ad-hFIX-GFP were injected into hemophilia B（HB）mice via the tail vein to observe the expression of hFIX gene in HB mice and to explore whether ADSCs can be used as a target cell vectors for HB gene therapy.Methods 1 Stable expression of hFIX gene in ADSCs of SD rats:The experiment was divided into F9 groups:Ad-hFIX-GFP was transfected into ADSCs for the first time;F9₁group:Ad-hFIX-GFP was transfected into ADSCs for 1 passage after passage;F9₂ group:Ad-hFIX-GFP was transfected into ADSCs for 2 passages after passage;F9₃ group:Ad-hFIX-GFP was transfected into ADSCs for 3 passages after passage;unloaded group:Ad-GFP was transfected into ADSCs;blank group:normal ADSCs.2 hFIX expression in HB mice:The experiment was divided into HB₁group:Ad-hFIX-GFP transfected C57BL/6j mice ADSCs;HB₂ group:Ad-GFP transfected C57BL/6j mice ADSCs;HB₃group:normal saline,and the three groups of cells were injected into HB mice via the tail vein,respectively,and blood samples were collected 1 week before injection and on days 1,7,14,21,and 28 after injection,and liver and lung tissue samples were collected on day 28after injection.3 RT-PCR was used to detect the expression of hFIX gene in ADSCs of SD rats after transfection and in the liver and lung of HB rats,Western blot was used to detect the expression of hFIX protein in ADSCs of SD rats after transfection and in the liver and lung of HB rats,and ELISA was used to detect the expression of hFIX antigen in the supernatant of ADSCs of SD rats after transfection and in the plasma of HB rats.Results 1 The stable expression of hFIX gene in ADSCs of SD rats:RT-PCR showed that hFIX gene expression bands were observed in F9,F9₁,F9₂ and F9₃ groups,but not in unloaded and blank group（P<0.05）.The gray values of hFIX protein expression in the WB test results were higher in the F9（0.906±0.042）,F9₁（0.619±0.015）,F9₂（0.496±0.031）,and F9₃（0.288±0.060）groups than in the unloaded（0.182±0.017）and blank（0.169±0.027）groups,and the differences were significant（P<0.05）.The results of ELISA showed that the h Fâ…¨expression levels in the F9（59.268±1.443 ng/ml）,F9₁（44.822±1.645 ng/ml）,F9₂（37.083±1.886 ng/ml）,and F9₃（20.848±1.798 ng/ml）groups were higher than those in the unloaded（0.622±0.105 ng/ml）and blank（0.611±0.128 ng/ml）groups,and the differences were significant（P<0.05）.2 Expression results of Ad-hFIX-GFP in HB mice:The results of hFIX gene expression and hFIX protein in liver tissue showed that the results of HB₁group was positive,and the results of HB₂ and HB₃ group were negative,with significant differences among the three groups（P<0.05）.The results of hFIX gene expression and hFIX protein in lung tissue showed that the results of HB₁,HB₂ and HB₃ group were negative,and there was no significant difference among the three groups（P>0.05）.Compared with the HB₂ and HB₃ group,the increased level of hFIX antigen in the plasma of the HB₁ group could be seen by ELISA.Conclusions 1 Ad-hFIX-GFP transfection of SD rat ADSCs can stably express hFIX gene.2 Ad-hFIX-GFP can secrete hFIX in HB mice,and adenovirus-mediated hemophilia B gene therapy can be expressed in animals.3 ADSCs can be used as one of the target cell vectors for HB gene therapy.Figure 10;Table 13;Reference 103...