ABSTRACT
The aim of the present study was to explore the gene transfection efficiency of Tat peptide/plasmid DNA/ liposome (TDL) compound combined with ultrasound-targeted microbubble destruction (UTMD) in human umbilical vein endothelial cell (HUVEC). Tat peptide, plasmid DNA (pIRES2-EGFP-HGF) and Lipofectamine 2000 were used to prepare the TDL compound. Microbubbles were prepared using mechanic vibration. The expression of the report gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The viability of HUVEC was measured by MTT assay. mRNA and protein of HGF was analyzed by reverse transcription-polymerase chain reaction and Western Blot. The intensity of green fluorescence and the gene transfection efficiency of TDL compound + microbubbles + ultrasound group were higher than those of other groups, and no significantly different viability was found between TDL compound + microbubbles + ultrasound group and the other groups. The HGF mRNA and HGF protein of TDL compound + microbubbles + ultrasound group were higher than those of other groups. Our finding demonstrated that UTMD could enhance the transfection efficiency of TDL compound without obvious effects on the cell viability of HUVEC, suggesting that the combination of UTMD and TDL compound might be a useful tool for the gene therapy of ischemic heart disease.
Subject(s)
Endothelium, Vascular/metabolism , Genetic Vectors , Hepatocyte Growth Factor/biosynthesis , Sonication/methods , Transfection/methods , Cell Survival , Cells, Cultured , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Gene Expression , Gene Products, tat/genetics , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Hepatocyte Growth Factor/genetics , Humans , Liposomes , Microbubbles , Microscopy, Fluorescence/methods , Plasmids , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
OBJECTIVE: The objective of this study was to noninvasively evaluate the severity of renal ischemia-reperfusion (I-R) injury in rabbits with microbubbles targeted to activated neutrophils [phosphatidylserine-conjugated surfactant perfluoropropane-filled microbubbles (SPMB-PS)]. METHODS: Microbubbles targeted to activated neutrophils (SPMB-PS) were prepared by conjugating phosphatidylserine (PS) to self-assembling surfactant perfluoropropane-filled microbubbles (SPMB). Flow cytometry was performed to assess the presence of PS in SPMB. A renal I-R injury model was established in 18 rabbits for contrast-enhanced ultrasonography. Examination of ultrasonography with SPMB-PS and SPMB was performed on 12 rabbits before and after I-R injury. The time-intensity curve (TIC) was generated from a selected region of interest. Another six rabbits with renal I-R injury underwent contrast-enhanced ultrasonography for 15 min after intravenous injection of SPMB-PS. The renal tissues were immediately excised for immunohistochemical staining and myeloperoxidase (MPO) activity analysis. The correlation between MPO activity and echo intensity (VI) was analyzed. RESULTS: Flow cytometry demonstrated that PS was located on the surface of SPMB. TIC showed that the time at which the maximum VI was reached and the time needed for the microbubbles to wash out were the same in the normal kidneys injected with SPMB-PS or SPMB, while there was an obvious delay in emptying time with SPMB-PS compared with SPMB after I-R injury. Fifteen minutes after the injection of SPMB-PS and SPMB, VI was not remarkably different (P>.05) in the normal kidneys, while it was significantly higher (P<.01) in the I-R-injured kidneys. There was a strong correlation between MPO activity and VI 15 min after the injection of SPMB-PS (r=.933, P<.01). Immunohistochemistry showed that most of the inflammatory cells in the I-R-injured kidneys were neutrophils. CONCLUSION: A delayed emptying phenomenon was observed during contrast-enhanced ultrasonography in the I-R-injured kidneys, with SPMB-PS targeted to activated neutrophils. Therefore, contrast-enhanced ultrasonography with SPMB-PS may noninvasively evaluate the severity of ischemia-reperfusion injury to the kidneys.
Subject(s)
Image Enhancement/methods , Kidney Diseases/diagnostic imaging , Kidney Diseases/pathology , Reperfusion Injury/diagnostic imaging , Reperfusion Injury/pathology , Animals , Contrast Media , Disease Models, Animal , Evaluation Studies as Topic , Flow Cytometry , Immunohistochemistry , Microbubbles , Neutrophil Infiltration , Peroxidase/metabolism , Rabbits , Renal Circulation/physiology , Sensitivity and Specificity , UltrasonographyABSTRACT
OBJECTIVE: This study aimed to explore the relationship between integrated backscatter (IBS) and mitochondria in arrested myocardium. METHODS: Twelve open-chest dogs were randomly divided into two groups: one group with cardiac arrest in systole and the other with cardiac arrest in diastole. IBS images at parasternal papillary muscle short-axis view were stored at different time frames (0, 30, and 60 min after cardiac arrest). The values of ultrasonic IBS were obtained using the acoustic densitometry technique. After ultrasound examination, tissue samples of corresponding times were harvested and observed under the transmission electron microscope. The microscopic images were analyzed using a computer imaging analysis system to obtain the stereological parameters of mitochondria. The correlation between IBS and the stereological parameters was analyzed. RESULTS: After cardiac arrest, swollen mitochondria with electron-lucent matrix could be observed in some myofibers following the progression of time. The alteration of IBS correlated well with that of mitochondrial stereological parameters, such as volume density (Vv), surface density (Sv), average volume (v), average surface area (s), and specific surface (Rsv, ratio between surface and volume). CONCLUSION: Mitochondria might be an important scatterer in the myocardium for IBS.
