| Objectives:1.Prepare Annexin V targeted microbubbles and evaluate their physical and chemical properties.2.Annexin V-targeted microbubbles specifically recognized apoptotic CAL-62 cells induced by microwave ablation in vitro.3.Annexin V targeted microbubbles specifically identified apoptotic thyroid tumor tissue induced by microwave ablation in vitro.Methods:1.DPPC,MPEG200,DPPA,DSPE-PEG2000 were thoroughly mixed and evenly in a certain molar ratio and then packed into 3mL vials.After freeze-drying according to a specific freeze-drying process,lysozyme solution was added and perfluoropropane(C3F8)was used to replace the internal air.The vial was fully oscillated with a mechanical shaker to obtain ordinary microbubbles(MBs).DSPE-PEG2000 was replaced with DSPE-PEG200-Biotin to prepare biotinylated microbubbles at the same way.The microbubbles were conjugated with antibodies through the biotin-avidin system.After the ordinary microbubbles and biotinylated microbubbles prepared above were centrifuged and washed,streptavidin,biotinylated Annexin V and FITC-labeled secondary antibodies were added in sequence,and incubated on ice in the dark environment,Annexin V microbubbles(AVMBs)were obtained after centrifugation.Observed the morphology and size of MBs and AVMBs under ordinary light microscope and fluorescence microscope respectively.The particle size distribution range and Zeta potential of AVMBs were measured by particle size analyzer and Malvern surface potentiometer.2.Cultivated CAL-62 cells,and induced apoptosis by microwave ablation,and verified them by flow cytometry and TUNEL method;select cells with similar growth conditions and design experiment for target group exploration in vitro.group A:apoptotic cells+ MBs,group B:normal cells+ AVMBs,group C:apoptotic cells+AVMBs.After DiI staining,the three groups of cells were fully incubated with microbubbles,and their targeting was detected by fluorescence microscopy.Image analysis software IPP was used for offline analysis and SPSS software package was used for statistical analysis of microbubbles bound to cells.3.Established some human thyroid undifferentiated carcinoma CAL-62 nude mouse models and induced tumor apoptosis by microwave ablation.Flow cytometry and TUNEL method were used for verification.Designed experiments and conducted in vitro target exploration in groups,group a:apoptotic tissue+MBs,group b:antibody presaturation+AVMBs,group c:apoptotic tissue+AVMBs.After the tissue and microbubbles have been fully incubated,the fluorescence microscopy was used to detect the targeting.The image analysis software IPP was used for offline analysis and the SPSS software package was used to statistically analyze the microbubbles bound to the tissues.Results:1.The ordinary microbubbles and Annexin V microbubbles were successfully prepared.MBs and AVMBs were round,with uniform distribution,consistent size,and stable morphology under the optical microscope.MBs were not visualized under the fluorescence microscope,but AVMBs had the same size and uniformly distributed floral ring structure with green fluorescence.The average particle size of AVMBs was(0.78 ± 0.06)μm,and the Zeta potential was(-22.7±1.8)mV.2.After microwave ablation of CAL-62 cells,apoptosis was confirmed by flow cytometry and TUNEL detection.Therefore,samples of apoptotic cells were successfully obtained.Under the microscope,a large number of green fluorescent microbubbles can be seen on the surface of group C cells,but there is almost no stable accumulation of microbubbles on the surface of cells in groups A and B.According to SPSS22.0 statistical analysis,"A-C" groups and "B-C" groups were U test,P value were both<0.05,the difference was statistically significant.It showed that AVMBs can be combined with apoptotic CAL-62 cells stably in vitro3.Some nude mouse model of human thyroid undifferentiated carcinoma were successfully established.Microwave ablation induced tumor apoptosis,which was verified by flow cytometry and TUNEL detection.In group c,targeted microbubbles were stably bound on apoptotic tumor tissues,while individual microbubbles adhered in only a few fields in groups a and b.According to IPP image analysis and SPSS22.0 statistics,the P value of Wilcoxon rank sum test of "a-c" groups and "b-c" groups were less than 0.05,the difference was statistically significant.It showed that AVMBs can stably and effectively label apoptotic CAL-62 cells in vitro.Conclusion:1.Nano-sized ultrasound microbubbles with uniform size,uniform distribution and good stability were prepared.AVMBs were prepared by biotin-avidin method,which provided a contrast agents for subsequent research such as targeted identification of apoptotic cells.2.AVMBs could stably and efficiently recognized apoptotic CAL-62 cells in vitro.3.AVMBs to identified apoptotic solid tumor tissues in vitro,and provides molecular imaging detection methods for tumor apoptotic states induced by low-energy microwave ablation. |
