Preparation Of Acoustic Contrast Agent Targeted To Microangiopathy And Its Efficacy Of Adhesion To Targeted Cells

Objective1.To investigate the preparation methods of targeted antibody connecting to micro-bubbles, and to evaluate the impact of targeting on the physical properties of micro-bubbles.2. To establish human aortic endothelial cells (HAEC) model injured by glucose provide targeted cells so as to evaluating the adhesion efficacy of the targeted micro-bubbles, to achieve more data for early diagnosing diabetic microangiopathy (DMA).3.To evaluate the adhesion efficacy of the targeted micro-bubbles via the parallel flow chamber system, so as to providing some study data for ultrasonic diagnosis using targeted micro-bubbles.Methods1. The vascular cell adhesion molecular1(VCAM-1)monoclonal antibody was applied to connect micro-bubbles via biotin-streptavidin system, and the targeted micro-bubbles were evaluated by using indirect immunofluorenscence. The particle diameters of SonoVue micro-bubbles and targeted to VCAM-1micro-bubbles were tested by using Malvern ZEN3690. The ultrasonic enhancement of the micro-bubbles was observed by using ultrasonic diagnosis apparatus.2.All the HAEC in the study were subcultured for3-4generations. The HAEC were incubated in a37℃,5%CO2, incubator. We prepared six endothelial cell mediums which glucose concentrations were5.5mmol/1,7.7mmol/1,11.6mmol/1,17.7mmol/1,21.8mmol/1,30mmol/1. We incubated the HAEC for3hours,6hours,12hours,24hours in the different endothelial cell mediums. We marked the HAEC injured by glucose with FITC-antibody to VCAM-1and FITC-antibody to TM. Detected the positive rate of the HAEC by using BD FACSCanto Ⅱ Flow Cytometer. All the data were processed by the statistical. To select the ideal condition in which the cell model expressed damage molecular highest.3. The HAEC were incubated on a round cover slips in the medium which glucose concentration was16.7mmol/1for6hours. Took the cover slips into the parallel flow chamber system. Drove the micro-bubbles suspension over the HAEC. Detected the mount of micro-bubbles in the fields when the liquid speed changed. Calculated the adhesion rate, All data were processed by the statistical.Result1.SonoVue was successfully connected to targeted antibody. Indirect immunofluorenscence showed that the targeted micro-bubbles were positive. The diameter distribution of the targeted biotin-modified micro-bubbles was narrower than of normal SonoVue. There was little difference in ultrasonic enhancement between the targeted SonoVue and normal SonoVue.2. The VCAM-1expression of the HAEC was unimodal curve with the increasing glucose concentration, the peak concentration was16.7mmol/1. With the prolonged incubated time, the VCAM-1expression was also unimodal curve, the peak incubated time was6hours. The TM expression of the HAEC was similar to that of VCAM-1, but the peak concentration was16.7mmol/1, and the peak incubated time was3hours.3. The targeted micro-bubbles adhering to the HAEC injured glucose were more than normal micro-bubbles. There is little micro-bubbles adhering the normal HAEC. The micro-bubbles adhesion rate decreased with the increasing shear stress, but the targeted micro-bubbles adhesion rate fell more slowly than that of the normal micro-bubbles. There is significant difference between the groups.Conclusion l.The targeted ultrasound micro-bubbles is successfully prepared via biotin-streptavidin-biotin system. The processing factors affect on the diameter of the micro-bubbles, resulting little difference in ultrasonic enhancement.2. The HAEC model injured by glucose was successfully established. It provided some clues for investigation of diabetic angiopathy.3.The parallel flow chamber system could evaluate the adhesion of targeted microbubbles. It was ideal tool in testing preparation of targeted micro-bubbles. The targeted micro-bubbles could specifically bind to targeted cells. There was strong adhesion between them. Targeted micro-bubbles had its potential values when we need ultrasound specific diagnosis.