PurposeTo develop a novel RGD microbubbles (RGD-MBs) and to evaluate the targeted binding effect with endothelial cells in vitro.Materials and MethodsThe RGD peptide was coated onto the microbubbles through biotin-avidin linkage including 10 μg/ml and 30 μg/ml groups. The microbubbles not carrying RGD peptide were obtained as negative control. Blocking studies were performed with pre-incubation of the cells with RGD peptide for 2 hours. The microbubbleswere characterized by Accusizer 780 and optical microscope. The binding specificity of RGD-MBs withανβ3-expressing mouse endothelial cells was determined with competitive inhibition experiments in vitro. The attachment study was performed using parallel plate flow chamber to investigate the dynamic adhesion on endothelial cells at various shear stresses.ResultsThe RGD-MBs had an average diameter of (4.09±0.07) μm. The binding RGD-MBs per cell were 2.98±0.35 for 10 μg/ml RGD and 1.78±0.23 for 30 μg/ml RGD. RGD-MBs binding to mouse endothelial cells decreased 54.64% and 67.00% in the presence of RGD peptide at a concentration of 10 μg/ml and 30 μg/ml respectively. When the shear stress was under 1.5 dyne/cm2, the accumulation rate was increased with the increase of shear stress (P<0.05). Accumulation rate reached the maximum (48.72±4.26) RGD-MBs/min at wall share stress of 1.5 dyne/cm2, and decreased as sheer stress >1.5 dyne/cm2 (P<0.05). Conclusion The RGD-MBs can specifically bind to endothelial cells, indicating its usefulness as ultrasonic molecular probe in monitoring integrinανβ3 expression during tumor angiogenesis, and is potentially valuable for in tumor early-staging and prognosis.

The ompA gene of Chlamyia psittaci in cows was amplified by PCR with primers designed based on those reported in GenBank.The amplified ompA gene was inserted into the bacterial plasmid vector pGEX-4T-1 and then transformed into E.coli BL21(DE3) with IPTG induction. The gene was derived from plasmid pMD18-T vector and then sequenced.It was demonstrated that this recombinant fusion protein of approximately 68kD in molecular mass was highly expressed in inclusion body and more pure proteins would be produced after purification.The fusion protein specifically reacted with positive sera of bovine Chlamydia as demonstrated by Western blotting. These results indicate that this recombinant fusion protein shows good reactivity and could be used to develop the diagnostic kit for bovine Chlamydia and genetic engineering vaccine.