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To transfect the recombinant adenovirus gene plasmid of biotinylated co-expressed red fluorescent protein and bone morphogenetic protein-2 (pAd-RFP / BMP-2) into rat bone marrow mesenchymal stem cells (BMSCs) using ultrasound targeting microbubble destruction localization technique (UTMD), and observe the expression of BMP-2 gene.
The BMSCs were extracted, isolated and purified from rat. The third generation BMSCs were randomly divided into five groups: I: BMSCs. II: ultrasound+microbubble+BMSCs. III:ultrasound +BMSCs. IV:microbubble+BMSCs. V:ultrasound+microbubble+BMSCs+pAd-RFP/BMP-2. BMSCs were transfected in vitro by ultrasound irradiation at 1MHz, 0.5W / cm2, 20% duty cycle, irradiated for 30s, cell density = 1 × 105 / well, cells /microbubbles = 1/25, pAd-RFP / BMP -2 MOI = 25. After transfection the expression of red fluorescent protein was observed by fluorescence inverted microscope at 24h, 48h and 72h. After 72 hours, the expression of BMP-2 in BMSCs and the transfection efficiency were detected by laser confocal microscopy and flow cytometry. The activity of BMSCs was measured by Cell Counting Kit-8(CCK8). The mRNA and protein expression of pAd-RFP / BMP-2 and the expression of protein in BMSCs were detected by RT-PCR and Western blot on the 14th day after transfection.
The expression of red fluorescent protein in BMSCs was the highest at 72h after transfection. The expression of fluorescent protein in group V was 80% by laser confocal microscopy. Flow cytometry was used to detect the expression of pAd-RFP / BMP-2 in group V, the transfection rate was 90%. Compared with the transfected group, there was no significant difference in the activity of CCK8 (P> 0.05). On the 14th day after transfection, the expression of BMP-2 mRNA was highest in group V(7.43 ± 1.00) by RT-PCR and Western blotting showed that the content of BMP-2 protein was increased only in group V (12.46 ± 0.90).
UTMD can improve the transfection efficiency and expression level of exogenous pAd-RFP / BMP-2 recombinant gene in rat BMSCs, and this method has no effect on the activity of BMSCs. Thus, UTMD is expected to be a new approach for Gene therapy.