| Backgrounds and objectivesCerebral thrombosis and cerebral embolism is a common disease with high incidence and high morbidity in the elderly population, which seriously affects the health of the elderly group. The prevention of cerebral thrombosis or cerebral embolism is important for decreasing the incidence of cerebrovascular disease or disability.Recent studies show that the high expression of tissue factor (TF) induced by shear stress on the blood vessel wall is one of the major factors to trigger vascular thrombosis. Interfering with TF expression in endothelial cells is important for preventing or delaying thrombosis induced by shear stress and reducing the incidence of thrombosis. Triplex- forming oligodexinucleotide (TFO) is designed and synthesized in this subject, which can block the shear stress responsive eliment(SSRE) in TF gene promoter, thereby block the expression of TF, decrease thrombosis reduced by shear stress. But the study found that the absorption rate of the cells to TFO was low and the inhibitive efficiency of the TF expression was not obvious. Present study has indicated that ultrasonic microbubble under the action of ultrasound irradiation can improve gene transfection efficiency. We use the combined effect of the ultrasonic microbubble technology and the ultrasonic irradiation, then observe the efficiency of TF inhibition by TFO.In this research, it has been observed that TFO absorption and inhibitive effect of TF expression in endothelial cell in vitro and in vivo by constructing the lipid ultrasonic microbubble with TFO and using ultrasound irradiation. The objective is to verify whether the combined effect of ultrasonic microbubble technology and ultrasonic irradiation enhances the transfection effects of TFO and inhibites the expression of TF. This study provides experimental basis for further developing the TFO as new drugs that protects endothelial cells and inhibits internal carotid artery thrombosis. It offers a novel therapeutic approach for preventing and treating thrombosis.Methods1. Lipid ultrasonic microbubble with TFO was prepared. Its shape was observed by the light microscope. Its fluorescence labeling was also observed by the fluorescence microscope. The particle size and diameter, the surface potential was detected by Coulter events-per-unit-time meter, Zetasizer 3000 respectively.2. It was observed that the TFO absorption in endothelial cells and its inhibitive effects of TF expression by the combined use of the ultrasonic microbubble technology and the ultrasonic irradiation in vitro study. The ECV304 cells were randomly devided into four groups. Blank contro1 group(SSRE), which was only dealed with the shear stress for 6 hours; TFO+ultrasonic irradiation (TFO+U) group, where 60μl of TFO (the final concentration was 0.2μmol/L) was added to the cell and 30s of ultrasonic irradiation was provided simultaneously; TFO-lipid microbubble (TFO-M) group, where 60μl of lipid microbubble with TFO (the final concentration was 0.2μmol/L) was added to the cell; TFO-lipid microbubble+ultrasonic irradiation (TFO-M+U) group, where 60μl of lipid microbubble with TFO (the final concentration was 0.2μmol/L) was added to the cell and 30s of ultrasonic irradiation was provided simultaneously. The ECV304 cells in three experimental groups were subjected to fluid shear of 12 dyn/cm for 6 hours at 24 hours after they were dealed with TFO as the different condition respectively. Then the TFO absorption rate in ECV304 cells was observed by fluorescent microscopy. The expression of the tissue factor was detected by immunofluorescencal method and reverse ranscriptase polymerase chain reaction (RT-PCR).3. It was observed that the inhibitive effects of TF by using the ultrasonic microbubble technology and the ultrasonic irradiation simultaneously in vivo study. The lipid ultrasonic microbubble with TFO centrifuged at low speed just before experiment. It was diluted with 0.9% NaCl to 1.0mg ? mL - 1 and injected into rat tail vein as 0.5mg ? kg– 1 at 0.5 hour before animal model was made. The SD rats were randomly devided into four groups. Blank contro1 group(SSRE): The rats were euthanized after the model was made successfully; TFO+ ultrasonic irradiation (TFO+U) group; The rats were injected into the same volume of the TFO mixture diluted to 1.0 mg ? mL - 1 with 0.9% NaCl. The carotid of rats were exposed to ultrasound with ultrasonic frequency of 347KHz, intensity of 2.4MPa immediately , and the time were all 2min; TFO-lipid microbubble (TFO-M) group: The rats were injected into the same volume of the lipid ultrasonic microbubble with TFO; TFO-lipid microbubble+ultrasonic irradiation (TFO-M+U) group: The rats were injected into the same volume of the lipid ultrasonic microbubble with TFO. The carotid of rats were exposed to ultrasound with the same irradiation parameters as TFO+U group. Then the carotid stenosis model of rats were prepared. With different conditions, the expression of TF in endothelial cell of carotid artery was detected by immunofluorescencal method.Results1. The lipid ultrasonic microbubble with TFO was prepared successfully. The appearance of the lipid ultrasonic microbubble with FITC-TFO was pale yellow suspension. It showed round, smooth surface, even size, light dense under light microscope. Microbubble concentration was about 7×109/ml. Its surface showed bright yellow-green fluorescence under the fluorescence microscope. But the lipid microbubbles without FITC-TFO are not visible. This study demonstrated that FITC-TFO had been successfully packaged on the microbubble lipid membrane. The particle size and diameter analysis showed that the intensity mean was 2092.8nm, the volume mean was 2114.2nm and the average mean was 2166.9nm. The surface potential analysis showed -46.0±1.6Mm. The lipid microbubbles with FITC-TFO were particles with negative charge.2. The TFO absorption in endothelial cells and its inhibitive effects of TF expression by the combined use of the ultrasonic microbubble technology and the ultrasonic irradiation in vitro study.1) The cell absorption rate of TFO was detected by fluorescence microscopy. The green fluorescence was seen in ECV304 cells within FITC-TFO. There were visible green fluorescent in three groups. The positive cells increased significantly in TFO-M+U group comparing with the TFO-M group and the TFO+U group. The green fluorescence in the TFO-M group and the TFO+U group was weak and mainly distributed in the cytoplasm. The TFO-M+U group showed brighter green fluorescent. The image analysis revealed that the transfection efficiency of the TFO in TFO-M+U group (38.83%±6.52%) was higher than that of the TFO-M group (9.50%±2.88%) and the TFO+U group (12.66%±3.01%,P<0.01), which there was significant difference between the last two groups.2) The expression of TF protein was detected by the immunofluorescence method. TF protein presented in red fluorescent fine particles. They were seen in ECV304 cells of SS group, mainly in the cytoplasm and membrane area, a small amount in the nucleus. Compared to the SS group, the intensity of red fluorescence TF protein in the TFO+U group, the TFO-M group and the TFO-M+U group decreased; but the intensity in the TFO-M+U group was significantly lower than that in the TFO+U group and in the TFO-M group. The image analysis revealed that the content of TF protein in the TFO+U group(36.83±8.34), the TFO-M group(40.77±9.40)and the TFO-M+U group(13.98±6.39) decreased (P<0.01) comparing with the SSRE group(74.00±16.67). The gray value in the TFO-M+U group was significantly lower than that in the TFO+U group and in the TFO-M group (P<0.01) without significant difference between the last two groups.3) The expression of TF mRNA was detected by the RT-PCR. The amplified bands of TF were obvious in SSRE group. Compared with it, the content of amplified band in the TFO+U group, the TFO-M group and the TFO-M+U group decreased; the value in the TFO-M+U group was significantly lower than that in the TFO+U group and in the TFO-M group. The image analysis revealed that the content of TF mRNA in the TFO+U group(0.36±0.07), the TFO-M group(0.38±0.07) and the TFO-M+U group(0.11±0.02) decreased significantly (P<0.01) compared with the SSRE group(0.71±0.08). The value in the TFO-M+U group was significantly lower than that in the TFO+U group and that in the TFO-M group (P<0.01), but there was no significant difference between the TFO+U group and the TFO-M group.3. It was observed that the inhibitive effects of TF expression induced by shear stress in endothelial cell of rat carotid artery by the combined use of ultrasonic microbubble technology and ultrasonic irradiation. The animal model of carotid stenosis was prepared successfully. Under different conditions, the expression of TF protein in endothelial cell of carotid artery was detected by the immunofluorescence metho d. The number of positive cells and the degree of staining were significant in SS group. Compared with the SSRE group, the amount of red fluorescence in the TFO+U group, the TFO-M group and the TFO-M+U group decreased; the amount in the TFO-M+U group was significantly lower than that in the TFO+U group and that in the TFO-M group. Image analysis revealed that the content of TF protein in the TFO+U group(51.22±5.69), the TFO-M group(55.22±6.47)and the TFO-M+U group(20.59±4.38)decreased significantly(P<0.01) compared with the SSRE group(71.78±7.10). The value in the TFO-M+U group was also significantly lower than that in the TFO+U group and in the TFO-M group (P<0.01) without significant difference between the last two groups.Conclusion1. The lipid ultrasonic microbubble with TFO was maded successfully. Their surface had negative charge. The particle size fit for intravascular injection. The lipid ultrasonic microbubble as a carrier to carry TFO is feasible. The lipid ultrasonic microbubble with TFO was prepared successfully.2. The decline of the TF expression was related to the increasing of TFO in ECV304 cells in vitro. With the increased in TFO transfection efficiency, the TF expression decreased. That suggested TFO was effective for inhibiting TF expression induced by shear stress in endothelial cell. The combined use of the ultrasonic microbubble technology and the ultrasonic irradiation can significantly increase the absorption rate of cell to TFO and strengthen the inhibition of TF expression subsequently.3. The study on the animal model of carotid stenosis had further proved the inhibition of TF expression. The TF expression induced by shear stress in endothelial cells of carotid artery could be inhibited by TFO. The combined use of the ultrasonic microbubble technology and the ultrasonic irradiation can promote the inhibitive effects. It is favorable for preventing the thrombosis induced by shear stress.
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