| Objective:To prepare the IL-8 monoclonal antibody-targeted ultrasonic contrast agent and to evaluate its adhesion-targeting property and investigate the role of IL-8 in early myocardial infarction (MI) by targeting in vitro, providing a new imaging basis for the early clinical diagnosis and treatment of acute myocardial infarction.Methods:1. Establishment of rabbit model of myocardial infarction:Thirty adult Japanese white rabbit (male or female) weighed 2.5 to 3.0 kg were randomized into two groups: Groups A (MI group, in which rabbit anterior descending coronary artery was ligated) and B (sham operation group, in which sutures only passed through myocardium without ligation). ECG monitoring was performed all the way intraoperatively, and pre-, intra- and postoperative changes in ECG were recorded for both groups. Routine echocardiography was performed 6 h and one week postoperatively, respectively, and ventricular wall motions were recorded. Venous blood was collected for both groups of rabbits 0.5 and 6 h pre- and postoperatively, respectively, and the IL-8 level was detected following centrifugation. At the end of the experiment, rabbit hearts were removed and stained histopathologically to provide the basis for further determination of acute MI in rabbits.2. Monitoring of expression of IL-8 in the injured myocardial tissue:The rabbi t model of MI was established successfully. Pathological paraffin blocks of my ocardial cells were taken from rabbits in Groups A (MI) and B (sham operatio n) and stained immunohistochemically to verify the expression level of IL-8 in the cell with MI.3. Preparation and identification of the targeted ultrasound contrast agent:IL-8 monoclonal antibody was coupled onto the surface of the SonoVue microbubble with SPDP crosslinker by covalent coupling. The size, morphology and dispersity of the microbubble were observed microscopically, and the slide agglutination test and the immunofluorescent staining were performed to confirm whether the coupling was successful.4. Culture and injury of myocardial cells:Rat myocardial cells (H9C2) were cultured in the DMEM+10%FBS medium and the passage was obtained until the cell confluence was up to more than 90%. These cells were transferred into four 35 mm culture dishes and divided into two groups as follows:control group (a/b-labeled high glucose medium) and experimental group (c/d-labeled low glucose medium). The experimental group was placed in an oxygen-free pure nitrogen bag, and myocardial cells of both groups were observed morphologically under inverted microscope after 6 h.5. Role of self-made IL-8 monoclonal antibody-targeted ultrasonic contrast agent in myocardial cells:Self-made targeted ultrasound microbubble suspension was added in the control group and the experimental group, while the uncoupled microbubble suspension was added in the control group. After division into four groups, aggregation effect of myocardial cells in the four culture dishes on self-made targeted ultrasound contrast agent was observed under inverted microscope. Numbers of myocardial cells and microbubbles adhered were counted at high magnification, and a quantitative analysis was conducted on the interaction between them by calculating the ratio of microbubble to myocardial cell.Results:1. Establishment of rabbit model of MI:Both two-dimensional ultrasonography (USG) and ECG showed no abnormity in either group of rabbits preoperatively. (I) ECG: Slightly increased heart rate (HR) and ST elevation were observed in Group A; slightly increased HR and no change in ST elevation were observed in Group B. (?) In Group A, USG showed decreased range of ventricular wall motion compared with that before operation or in the sham operation group. In Group B (sham operation), no abnormal ventricular wall motion was observed compared with preoperatively. (?) Serological detection showed that:serum IL-8 level markedly higher in Group A than in Group B or preoperatively (P<0.05), and the IL-8 level gradually increased as injury time prolonged; no significant change in serum IL-8 level was observed in Group B and postoperatively. (IV) Pathologic results showed that, in Group A, myocardial tissues in the area supplied by the anterior descending artery developed MI while no cell with MI was observed in Group B.2. Detection of IL-8 by immunohistochemical staining showed that:(i) in Group A, nuclei of injured myocardial cells were hyacinthine and cell surfaces were brown; and (ii) in Group B, nuclei of myocardial cells were also hyacinthine, but cell surfaces were unstained.3. Uncoupled common SonoVue microbubbles were homogeneously distributed, regular and scattered. Levels of both coupled IL-8 monoclonal antibody microbubble suspension and SonoVue microbubbles in the control group slightly decreased over time, but no distinct change in size, morphology and properties was observed basically compared with uncoupled microbubbles.4. (I) Slide agglutination test:Self-made targeted ultrasound microbubble suspension was mixed with second antibody, and agglutination between them was observed under inverted microscope; in the control group, uncoupled microbubble suspension was mixed with second antibody, and no agglutination was observed under inverted microscope. No agglutination was observed under inverted microscope after mixture of either contrast agent with glacial acetic acid. (?) Immunofluorescent staining: Fluorescence was observed on the surface of the self-made targeted ultrasound microbubble suspension, whereas no fluorescence was observed on the surface of the microbubble of the uncoupled contrast agent in the control group.5. Observation of culture and injury of myocardial cells under inverted microscope: Normal myocardial cells were well-grown, plump and various in shape (round, fusiform and subulate), with peripheral halo and strong refractivity; injured myocardial cells were shrunk, with partial membranolysis, increased cytoplasmic granules, destroyed intercellular coupling, foot process effacement and decreased refractivity.6. Observation of the interaction between ultrasound microbubbles and myocardial cells:Adhesion of self-made targeted ultrasound microbubble suspension to both injured myocardial cells and deformed myocardial cells at early stage of injury markedly increased compared with the control group (P<0.05); meanwhile, such adhesion also markedly increased compared with that to normal myocardial cells (P<0.05), and the number of microbubbles adhered increased gradually as injury severity increased.Conclusion:1. By ligating the left anterior descending artery of the rabbit, a rabbit model of MI is established successfully, providing an animal model for further immunohisto-chemistry.2. The serological surveillance shows that:IL-8 can be detected in serum during early myocardial infarction, and secretion of IL-8 gradually increases as the injury is aggravated; IL-8 also causes neutrophils to play a chemotactic role in aggravating the inflammatory response and is highly expressed in serum.3. Immunofluorescent staining of pathological sections shows high expression of IL-8 on the surface of the injured myocardial tissue, thus we select the IL-8 monoclonal antibody as the targeted antibody and its coupling onto the surface of the SonoVue microbubble can provide a new method for early detection of cells with MI.4. Both slide agglutination test and immunofluorescent staining confirm that the IL-8 monoclonal antibody has been successfully coupled onto the surface of the SonoVue microbubble and that coupled microbubbles have basically similar concentration, size, morphology and properties to uncoupled microbubbles, suggesting that no distinct change is observed in physical and biological properties of the ultrasound contrast agent prepared by covalent coupling. Therefore, it can be used as an ultrasound contrast agent functioning in vivo.5. Hypoxia and aglycaemia can injure myocardial cells and induce them to secret a quantity of IL-8, providing an experimental model for in vitro cell experiments.6. Targeting in vitro shows that a number of self-made targeted ultrasound microbubbles adhere to the surface of the injured myocardial cell, suggesting covalent coupling of IL-8 monoclonal antibody onto the surface of the SonoVue microbubble is still immunocompetent.7. Targeting in vitro shows that parts of uncoupled SonoVue microbubbles adhere to the surface of the injured myocardial cell, but its binding action is markedly lower than that of self-made targeted ultrasound microbubble suspension, suggesting that binding of the IL-8 monoclonal antibody-targeted ultrasonic contrast agent to target cells is efficient and specific.8. By using IL-8 monoclonal antibody-targeted microbubbles to react with specific antigen and antibody on the surface of the injured myocardial cell, targeting in vitro demonstrates that:IL-8 can be generated in early MI; it can be highly expressed as the injury aggravates; IL-8 monoclonal antibody can specifically bind to IL-8 and plays a certain role in alleviation of MI. Therefore, successful preparation of IL-8 monoclonal antibody-targeted ultrasonic contrast agent is highly valuable in early diagnosis of MI. |
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