Our study aimed to choose the better serotype between rAAV2 and rAAV2/1 in hDPCs. After that we constructed rAAV-hBMP-7 and transfected it into hDPCs to investigate effects of rAAV-hBMP-7 on differentiation of hDPCs.
Methods:
‡@Transfect rAAV2-EGFP and rAAV2/1-EGFP into hDPCs at different MOI (MOI=104, 105, 5x105); Oberve expressions of EGFP under fluorescence microscope and measure transfection efficiencies at 48h post transduction. Meanwhile proliferation of transfected cells was examined by MTT assay at days of 4, 6, 8, 10 and 12 for MOI=105.‡AConstruct the vector of rAAV-hBMP-7. ‡BExamine the production of hBMP-7 in hDPCs, which were transfected into rAAV-hBMP-7 at suitable MOI, at days of 7, 14, and 21.‡C ALP activity and mineralization nodule were evaluated by ALP kit and Alizarin Red staining, respectively. ‡DDSPP, OCN mRNA expressions were measured by real-time PCR at days of 7, 14, 21.
Results:
‡@We found EGFP expressed at 48h postgraduation, and their transduction efficiencies as follows: rAAV2-EGFP (42.33±3.42) % (MOI=104), (67.58±0.99) % (MOI=105), (73.46±1.36) %(MOI= 5x105), and rAAV2/1-EGFP was (13.63±0.91) % (MOI=104), (34.9±0.678) % (MOI= 105), (43.8±1.48)% (MOI=5x105), and MTT assay revealed there was no significant difference in the absorbance between tansfected and untransfected cells (P=0.193). ‡A rAAV2-hBMP-7 was constructed and obtained titers 5x1011 v.g/ml. ‡BWestern blot showed expression level of hBMP-7 peaked at 7 days post transduction and then declined with time, but still existed at 21. ‡C rAAV2-hBMP-7 transduced hDPCs showed an increase of ALPase activity in a time-independent manner (P<0.05). Alizarin Red staining indicated rAAV2-hBMP-7 group can induce the calcified nodules formation. ‡DDSPP and OCN mRNA expressions were greater in rAAV2-hBMP-7 transfected cells than non-transfected at days 7(P<0.01).
Conclusions:
Our result showed rAAV2 was more susceptible for hDPCs than rAAV2/1. After transfected rAAV2-hBMP-7 into hDPCs, hBMP-7 not only can be efficiently expressed, but also can induce differentiation of hDPCs.