| Objectives 1 By subculturing Ad-BDDh FVIII-GFP and Ad-FIX-GFP transfected adipose stem cells,observe the expression of h FVIII and h FIX,and explore whether BDDh FVIII and h FIX can be stably expressed in ADSC.2 By passing BDDh FVIII through the in-vivo and the ex-vivo method were injected into hemophilia A mice to observe the expression of h FVIII in vivo,and explore whether ADSC can be used as a target cell carrier for HA gene therapy.Methods 1 Stable expression of BDDh FVIII in ADSC: The experiment was divided into 4 groups,P0 group: normal ADSC;P1 group: ADSC first transfected with AdBDDh FVIII-GFP;P2 group: the first generation after Ad-BDDh FVIII-GFP transfer;P3 group: the second generation after Ad-BDDh FVIII-GFP transfer.2 Expression of BDDh FVIII in vivo: Ad-GFP was transfected into ADSC(group A),Ad-BDDh FVIIIGFP-transfected ADSC(group B),Ad-BDDh FVIII-GFP(group C)and Ad-GFP(group D)were injected into hemophilia A mice by tail vein.Blood samples were collected at the first week before injection and the 1st,7th,14 th,21th and the 28 th day after injection,and liver tissue specimens were collected on the 28 th day after injection.3 Stable expression of h FIX in ADSC: the experiment is divided into 4 groups,group A: Ad-FIX-GFP transfected ADSC for the first time;Group B: the first generation after Ad-h FIX-GFP transfer;Group C: the second generation after Ad-h FIX-GFP transfer;Group D: normal ADSC.RT-PCR method was used to detect gene transcription in ADSC and liver,ELISA method was used to detect coagulation factor antigen in ADSC supernatant and plasma,and Western blot was used to detect coagulation factor protein in ADSC and liver.Results 1 The stable expression of BDDh FVI in ADSC: Compared with the P0 group,the BDDh FVIII gene expression bands were seen in the P1 group,P2 group and P3 group.h FVIII : Ag test results P1 group(140.260±2.290ng/m L),P2 group(118.140±21.050ng/m L),P3 group(107.730±4.530ng/m L)levels were higher than P0 group(80.020±0.210ng/m L),The difference is significant(P<0.05).Compared with the P0 group,the gray values of h FVIII protein expression were significantly higher in P1 group(0.630±0.070),P2 group(0.450±0.040),P3 group(0.250±0.020),and the difference was significant(P<0.05).2 BDDh FVIII expression in vivo: the increase of h FVIII:Ag can be seen in the plasma of group B and group C.The results of BDDh FVIII gene transcription and h FV protein in liver tissue showed that the results of group B and ?group C were positive.Compared with groups A and D,the difference is significant(P<0.05).3 Stable expression of h FIX in ADSC: Compared with group D,h FIX gene transcription can be detected in groups A,B and C.h FIX:Ag results showed that group A(81.620±8.820ng/m L),group B(52.496±3.246ng/m L),group C(47.407±4.002ng/m L)were significantly higher than group D(0.751±0.426ng/m L),The difference is significant(P<0.05).Compared with the group D,the gray values of h FIX protein expression was significantly higher in group A(0.680±0.100),group B(0.490±0.150),and group C(0.180±0.050),and the difference was significant(P<0.05).Conclusions 1 BDDh FVIII can be expressed stably in ADSC,and h FVIII can be detected in hemophilia A mice.Adenovirus-mediated hemophilia A gene therapy can be successfully expressed in animals.2 h FIX was expressed stably in ADSC transfected with Ad-h FIX-GFP.3 ADSC can serve as a target cell carrier for hemophilia gene therapy.Figure13;Table13;Reference 135... |
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