| Background and ObjectiveVascular endothelial injury and endothelial "inflammatory" are commonly found in coronary heart disease,hypertension and diabetes,the three major killers which are endangering human health and life.At present,it is still absence of a convenient, non-invasive and specific approach of direct evaluation of the vascular endothelial inflammation.Contrast-enhanced ultrasound(CEU) is one kind of technique that can markedly improve the resolution,sensitivity and specificity of ultrasonic diagnosis by using the ultrasound contrast agent to enhance the scattering echo.Targeted ultrasound contrast imaging is a new development approach of detecting and imaging the micro pathological change of the body tissues and organs at molecular levels,combine ultrasonic imaging technology and targeted ultrasound contrast agent.Targeted ultrasound contrast imaging is the leading subject,making a leaping forward in ultrasonic diagnosis and treatment accompanying with the clinical application and practice.Cell adhesion molecule of endothelial cells and leukocytes mediating inflammation was expression on the process of inflammation,through the development of targeted ultrasound contrast agent that targeted to cell adhesion molecules,target Molecular imaging of Inflammation site was by applied targeted ultrasound contrast.it is expected to be early evaluation and diagnosis of various diseases on the molecular level.P-selectin begin to be stored and expressed on the surface of endothelial cells within a few minutes after inflammatory stimulation.It will be able to evaluate vascular endothelial inflammation of the heart,kidneys and other vital organs of the body through the development of targeted microbubbles to aim directly at P-selectin.The application of targeted ultrasound microbubbles with anti P-selectin monoclone antibody in evaluating vascular inflammation has been acquired a success in the experiment by foreign scholars.However,it is still only the initial stage of the study,as there are many problems to be resolved.The adoption of "complex of streptavidin/biotin " constructed of specific targeted ultrasound microbubbles is an internationally favored way and there is no concerned research in the literature in internal yet.The specificity and efficacy of targeted ultrasound microbubbles are the basics which determine molecular image of targeted ultrasound.The efficacy of targeted ultrasound microbubbles can be evaluated by vivo and vitro methods.In recent years, fluorescence microscope and flow cytometry have been used to evaluate the binding rate of specific targeted ultrasound microbubbles qualitatively and quantitatively. Fluorescence in vitro can display construction of targeted ultrasound microbubbles directly.To qualitatively and quantitatively investigate the reliability of antibody-combination of the targeted ultrasound microbubbles,which carded the anti-mouse P-selectin monoclone antibody(MB-BSBp) via biotin - avidin - biotin (BSB) ligand bridging technology,by Fluorometric Method In vitro.Methods and Results 1.Preparation and fluorescence labeling of common liposome microbubbles(MB):①Weighed a certain proportion of lipid materials and auxiliary materials of DPPC, PEG-4000 and F-188 in containers.Then added distilled water and mixed symmetrically.Put perfluoropropane through saturation solution and got the liposme microbubbles(MB) packed with perfluoropropane by ultrasound activation and Ultrasonic vibration to a milky white liquid.Finally the liposme microbubbles were part-installed into ampoules,refrigerated and conserved.②Weighed a certain proportion of lipid materials,auxiliary materials of DPPC,PEG-4000,F-188 and DiI lipid(lipophilic fluorescent dye) in containers,fluorescence lipid microbubbles (MB-DiI) were prepared by using the same method,under dark and cold storage. Common liposome microbubbles were measured by Coulter counter.The average particle size of them were 2.83 um and the concentration were about 1.5×10~9/ml respectively.Under fluorescence microscope:common liposome microbubbles labeled by DiI were excitated a bright red fluorescence.2.Construction and fluorescence labeling of targeted ultrasound microbubbles with anti P-selectin monoclone antibody(MB-BSBP):weighed a certain proportion of lipid materials and auxiliary materials of DSPE-PEG2000-Biotin,DPPC,PEG-4000 and F-188 in containers.Then added distilled water and mixed symmetrically.Put perfluoropropane through the saturation solution and got the Biotin-PEG-Microbubbles(MB-B) packed with perfluoropropane by ultrasound activation and Ultrasonic vibration to a milky white liquid.Purificated the MB-B. Streptavidin and Biotin anti-mouse P-selectin monoclone antibody were joined in the MB-B successively,by using biotin - avidin - biotin(BSB) ligand bridging technology, streptavidin- Biotin- PEG- Microbubbles(MB-BS) and anti-mouse P-selectin monoclone antybody-biotin-streptavidin-biotin-PEG-microbubles(MB-BSB) were prepared.Preparation of FITC-streptavidin- Biotin- PEG- Microbubbles(MB-BS-FITC) by incubating The MB-B with the streptavidin-FITC.MB-BSBp were marked with DTAF antibody as second antibody(antibody of anti-P-selectin monoclonal antibody.The MB-BSBP were measured by Coulter counter,the average particle size of them were 2.73um um,the concentration were about 7.1×10~8 / ml respectively.Under fluorescence microscope:MB-BSBp were finer distributed evenly.MB-BSBp labeled by DTAF fluorescence second antibody were excitated a bright green fluorescence. MB-BS-FITC were also excitated a bright green fluorescence.3.Identification of targeted ultrasound microbubbles with anti P-selectin monoclone antibody(MB-BSBP):①Different parts of MB-BSBp were marked with various fluoresent markers,and the fluorescence intensity of microbubbles graded from 0,1,2 to 3 were evaluated among groupes of marked MB-BSBp and group of common lipid ultrasound microbubbles(MB)as control.After incubating with FITC- avidin,the biotinylated lipid microbubbles(MB-B) under Fluorescence microscopy displayed bright green fluorescence(grade 3),but MB had not any fluorescence(grade 0).MB, MB-B,MB-BS,and MB-BSBp were marked with DTAF-second antibody(antibody of anti-P-selectin monoclonal antibody) at two concentrationes(1:4 and 1:16 dilution). Bright green fluorescence(grade 3) were observed in MB - BSBp in both concentrations,faint green fluoresence(grade 1) in MB-B and MB-BS only in concentration of 1:4 dilution,but no fluorescence in MB in either concentration(grade 0).②Took streptavidin-FITC and DTAF fluorescence second antibody as fluorescence probe,marking MB-B and MB-BSBP respectively.Took 5×10~7 MB-B respectively, one tube as a control,another tube of MB-B was incubated with FITC-streptavidin,then washed,purified microbubbles,and using flow cytometry to analysise the green fluorescent intensity of microbubbles;Took 5×10~7 MB-B,MB-BS and MB-BSBP, incubated with equivalence DTAF-second antibody,using flow cytometry analysis the binding rate of each group of microbubble with DTAF-second antibody.The experiments were repeated three times.Flow cytometry(FCM) found that:the Fluorescence curve of MB-BS-FITC was clearly shifted to right,showing that the successful preparation of the MB-BS.The binding rate of MB-BSBP with DTAF-second antibody was(59.66±2.30)%,significantly higher than that of MB-BS and MB-B(F=130.329,P＜0.001).The fluorescence intensity of MB-B and MB-BS was no difference(P=0.187),demonstrating that the successful construction of targeted ultrasound microbubbles with anti P-selectin monoclone antibody(MB-BSBP)。Conclusion1.Anti P-selectin monoclonal antibody via streptavidin bridge was effectively incorporated to the surface of the biotinylated lipid ultrasound microbubbles,targeted ultrasound microbubbles with anti P-selectin monoclone antibody were successfully constructed.2.Fluorometric method in vitro is a effective and simple method for investigating the reliability of the ligands -combination of the targeted ultrasound microbubbles.
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