Biological Effects of Low Power Ultrasound Wave Irradiation Microbubble Contrast Agent on Rat Endothelial Progenitor Cells

  • Dr Qiao Li, Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, China
  • Prof Zhigang Wang, Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, China
  • Prof Haitao Ran, Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, China
  • Dr Jianli Ren, Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, China
  • Dr Xingsheng Li, Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, China

    • Objective: To culture the rat endothelial progenitor cells (EPCs) in vitro. To investigate the effect of ultrasound microbubble contrast agent with different parameters to the rat EPCs.
    Methods: The mononuclear cell was isolated by percoll gradient centrifugation and cultured for 48h. Then the suspending cells were cultured in the lower glucose DMEM media ( with the supplement of 10% fetal bovine serum, 20ng/ml rat VEGF, 2ng/ml human bFGF, 20ng/ml human EGF). The attached cells were identified with immunohistochemistry and Dil-ae-LDL,FITC-UEA-1 fluorescence staining. EPCs were irradiated by various dosage ultrasound with the echo-contrast agent and immediately tested for cell viability by MTT colormetric assay and morphology by HE staining.
    Results: The attached EPCs were able to line up in the typical cord-like structure and formed clusters. Adherent cells showed CD34 , Flk-1,CD133 , VIII/vWF positive and took up Dil-ae-LDL, bound FITC-UEA-1 double positive fluorescence. When the microbubble concentration was 5%, the EPCs viability exposed to ultrasound (0.25 W/cm2×10 s and 0.5 W/cm2×5 s) did not significantly changed, but they decreased obviously in the groups: 0.5 W/cm2×10 s. 0.5 W/cm2×20 s. 0.5 W/cm2×40 s. 1.0 W/cm2×10 s. 2.0 W/cm2×10 s. In the same intensity level of 0.5 W/cm2 and sonication time of 10 s, EPCs viability decreased with the increased bubble concentration as compared with control group. Most of the cells were observed adhere to the cover glasses by inverted microscope in control group, only a few suspended living cells, but a large number of suspended dead cells were observed in the experiment group. Most of the attached EPCs differentiated wel. With the increase of the intensity of ultrasound , prolongation of sonication and concentration of microbubbles, the intercellular space enlarged, the cells became smaller and looked more round, showed some phenomenon as chromatin margination, karyopyknosis, nuclear fragmentation.
    Conclusion: EPCs could be isolated by density gradient centrifugation. The EPCs viability by microbubbles destruction is correlated with the intensity of ultrasound, prolongation of sonication and concentration of microbubbles.