In this study, we examined the effects of neuroglobin gene （Ngb） transfection into SH-SY5Y cells, using ultrasound-targeted microbubble destruction （UTMD）, on cobalt chloride-induced hypoxia. With an ultrasound in...In this study, we examined the effects of neuroglobin gene （Ngb） transfection into SH-SY5Y cells, using ultrasound-targeted microbubble destruction （UTMD）, on cobalt chloride-induced hypoxia. With an ultrasound intensity of 0.8 W/cm2, a 60-second exposure duration, 50% duty cycle, and 20% microbubble concentration, pAcGFP1-C1-Ngb-transfected cells exhibited the highest cell viability and transfection efficiency. The efficiency of plasmid delivery was significantly higher with UTMD than transfection with plasmid alone, transfection with plasmid using microbubbles, or transfection of plasmid by ultrasound. In addition, during cobalt chloride-induced hypoxia, caspase-3 activity in pAcGFP1-C1-Ngb-transfected cells was significantly lower than in untransfected cells. Ngb protein and mRNA expression were significantly higher in cells transfected by UTMD than in cells transfected with the other methods. These results demonstrate that UTMD can very efficiently mediate exogenous gene delivery, and that Ngb overexpression protects cells against cobalt chloride-induced hypoxia.展开更多
Background:Ultrasound-triggered microbubble destruction(UTMD) is a widely used noninvasive technology in both military and civilian medicine,which could enhance radiosensitivity of various tumors.However,little inform...Background:Ultrasound-triggered microbubble destruction(UTMD) is a widely used noninvasive technology in both military and civilian medicine,which could enhance radiosensitivity of various tumors.However,little information is available regarding the effects of UTMD on radiotherapy for glioblastoma or the underlying mechanism.This study aimed to delineate the effect of UTMD on the radiosensitivity of glioblastoma and the potential involvement of autophagy.Methods:GL261,U251 cells and orthotopic glioblastoma-bearing mice were treated with ionizing radiation(IR) or IR plus UTMD.Autophagy was observed by confocal microscopy and transmission electron microscopy.Western blotting and immunofluorescence analysis were used to detect progesterone receptor membrane component 1(PGRMC1),light chain 3 beta 2(LC3B2) and sequestosome 1(SQSTM1/p62) levels.Lentiviral vectors or siRNAs transfection,and fluorescent probes staining were used to explore the underlying mechanism.Results:UTMD enhanced the radiosensitivity of glioblastoma in vitro and in vivo(P<0.01).UTMD inhibited autophagic flux by disrupting autophagosome-lysosome fusion without impairing lysosomal function or autophagosome synthesis in IR-treated glioblastoma cells.Suppression of autophagy by 3-methyladenine,bafilomycin A1 or ATG5 siRNA had no significant effect on UTMD-induced radiosensitization in glioblastoma cells(P<0.05).Similar results were found when autophagy was induced by rapamycin or ATG5 overexpression(P>0.05).Furthermore,UTMD inhibited PGRMC1expression and binding with LC3B2 in IR-exposed glioblastoma cells(P<0.01).PGRMC1 inhibitor AG-205 or PGRMC1siRNA pretreatment enhanced UTMD-induced LC3B2 and p62 accumulation in IR-exposed glioblastoma cells,thereby promoting UTMD-mediated radiosensitization(P<0.05).Moreover,PGRMC1 overexpression abolished UTMD-caused blockade of autophagic degradation,subsequently inhibiting UTMD-induced radiosensitization of glioblastoma cells.Finally,compared with IR plus UTMD group,PGRMC1 overexpression significantly increased tumor size [(3.8±1.1) mm^(2)vs.(8.0±1.9) mm^(2),P<0.05] and decreased survival time [(67.2±2.6) d vs.(40.0±1.2) d,P=0.0026] in glioblastoma-bearing mice.Conclusions:UTMD enhanced the radiosensitivity of glioblastoma partially by disrupting PGRMC1-mediated autophagy.展开更多
Background:Ultrasound-targeted microbubble destruction(UTMD)induces cellular inflow of drugs at low intensity,while high intensity eradicates tumor vessels.Since vascular endothelial growth factor receptor 2(VEGFR2)is...Background:Ultrasound-targeted microbubble destruction(UTMD)induces cellular inflow of drugs at low intensity,while high intensity eradicates tumor vessels.Since vascular endothelial growth factor receptor 2(VEGFR2)is highly expressed in pancreatic ductal adenocarcinoma(PDAC),VEGFR2-targeted microbubble(MB)might additionally increase the tissue specificity of drugs and thus improve antitumor effects.In addition,fixing the dual pulse intensity could maximize MB properties.This study evaluated the one-off(experiment 1)and cumulative(experiment 2)treatment effect of UTMD by regulating the dual pulse output applied to PDAC using VEGFR2-targeted MB.Methods:C57BL/6 mice inoculated with Pan-02 cells were allocated to five groups:VEGFR2-targeted MB+gemcitabine(GEM),VEGFR2-targeted MB,non-targeted MB+GEM,GEM,and control groups.After injection of GEM or GEM and either VEGFR2-targeted or non-targeted MB,UTMD was applied for several minutes at low intensity followed by high intensity application.In experiment 1,mice were treated by the protocol described above and then euthanized immediately or at the tumor diameter doubling time(TDT).In experiment 2,the same protocol was repeated weekly and mice were euthanized at TDT regardless of protocol completion.Histological analysis by CD31 and VEGFR2 staining provided microvascular density(MVD)and VEGFR2 expression along vessels(VEGFR2v)or intra/peripheral cells(VEGFR2c).Results:In experiment 1,TDT was significantly longer in the VEGFR2-targeted MB+GEM group compared to the non-targeted MB+GEM,GEM,and control groups,while the VEGFR2-targeted MB group showed no statistical significance.MVD and VEGFR2v in the immediate euthanasia was significantly lower in the VEGFR2-targeted MB+GEM and VEGFR2-targeted MB groups than other conditions.In experiment 2,the VEGFR2-targeted MB+GEM group produced significantly longer TDT than the GEM or control groups,whereas the VEGFR2-targeted MB group showed no significant difference.Histology revealed significantly reduced VEGFR2v and VEGFR2c in the VEGFR2-targeted and non-targeted MB+GEM groups,while only VEGFR2v was significantly less in the VEGFR2-targeted MB group.Conclusions:UTMD-mediated GEM therapy with the dual pulse application using VEGFR2-targeted MB substantially suppresses PDCA growth.展开更多
This study aimed to examine the preparation of cationic lipid microbubble（CLM）,and evaluate its physical and chemical properties and toxicity,measure the gene transfection efficiency by ultrasound triggered microbob...This study aimed to examine the preparation of cationic lipid microbubble（CLM）,and evaluate its physical and chemical properties and toxicity,measure the gene transfection efficiency by ultrasound triggered microbobble destruction（UTMD） in combination with CLM.The CLM was prepared by the method of the thin film hydration,and its morphology was observed under the electron microscopy at 1st,3rd,7th,10th,and 14th day after preparation,respectively.The size,Zeta potential and stability of CLM were tested.The acute toxicity of CLM was assessed.The green fluorescent protein gene（EGFP） transfection efficiency was evaluated.The experiment grouping was as follows：naked plasmid group（P group）,ultrasonic irradiation plus naked plasmid group（P-US group）,naked plasmid plus CLM group（P-CLM group）,naked plasmid plus ultrasound and CLM group（UTMD group）.The expression of EGFP was detected by fluorescent microscopy and flow cytometry.The results showed that CLMs were spherical in shape,with the similar size and good distribution degree under the light and electron microscopies.The size of CLMs was varied from 250.4±88.3 to 399.0±99.8 nm and the Zeta potential of CLMs from 18.80±4.97 to 20.1±3.1 mV.The EGFP expression was the strongest in the UTMD group,followed by the P-CLM group,P-US group and P group.Flow cytometry results were consistent with those of fluorescent microscopy.The transfection efficiency was substantially increased in the P-US group,P-CLM group and UTMD group as compared with that in the P group,almost 7 times,10 times and 30 times higher than that in the P group respectively.It is suggested that CLMs prepared by the method of thin film hydration are uniform in diameter,and proved non-toxic.UTMD combined with CLM can significantly increase the transfection efficiency of EGFP to targeted cells.展开更多
Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthes...Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix(ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications.However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fi brosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process.Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.展开更多
文摘In this study, we examined the effects of neuroglobin gene （Ngb） transfection into SH-SY5Y cells, using ultrasound-targeted microbubble destruction （UTMD）, on cobalt chloride-induced hypoxia. With an ultrasound intensity of 0.8 W/cm2, a 60-second exposure duration, 50% duty cycle, and 20% microbubble concentration, pAcGFP1-C1-Ngb-transfected cells exhibited the highest cell viability and transfection efficiency. The efficiency of plasmid delivery was significantly higher with UTMD than transfection with plasmid alone, transfection with plasmid using microbubbles, or transfection of plasmid by ultrasound. In addition, during cobalt chloride-induced hypoxia, caspase-3 activity in pAcGFP1-C1-Ngb-transfected cells was significantly lower than in untransfected cells. Ngb protein and mRNA expression were significantly higher in cells transfected by UTMD than in cells transfected with the other methods. These results demonstrate that UTMD can very efficiently mediate exogenous gene delivery, and that Ngb overexpression protects cells against cobalt chloride-induced hypoxia.
基金supported by the National Natural Science Foundation of China (82073544 and 81971774)the Chongqing Talent Project (CQYC2019)the Chongqing Chief Expert Program in Medicine (CQYC2018)。
文摘Background:Ultrasound-triggered microbubble destruction(UTMD) is a widely used noninvasive technology in both military and civilian medicine,which could enhance radiosensitivity of various tumors.However,little information is available regarding the effects of UTMD on radiotherapy for glioblastoma or the underlying mechanism.This study aimed to delineate the effect of UTMD on the radiosensitivity of glioblastoma and the potential involvement of autophagy.Methods:GL261,U251 cells and orthotopic glioblastoma-bearing mice were treated with ionizing radiation(IR) or IR plus UTMD.Autophagy was observed by confocal microscopy and transmission electron microscopy.Western blotting and immunofluorescence analysis were used to detect progesterone receptor membrane component 1(PGRMC1),light chain 3 beta 2(LC3B2) and sequestosome 1(SQSTM1/p62) levels.Lentiviral vectors or siRNAs transfection,and fluorescent probes staining were used to explore the underlying mechanism.Results:UTMD enhanced the radiosensitivity of glioblastoma in vitro and in vivo(P<0.01).UTMD inhibited autophagic flux by disrupting autophagosome-lysosome fusion without impairing lysosomal function or autophagosome synthesis in IR-treated glioblastoma cells.Suppression of autophagy by 3-methyladenine,bafilomycin A1 or ATG5 siRNA had no significant effect on UTMD-induced radiosensitization in glioblastoma cells(P<0.05).Similar results were found when autophagy was induced by rapamycin or ATG5 overexpression(P>0.05).Furthermore,UTMD inhibited PGRMC1expression and binding with LC3B2 in IR-exposed glioblastoma cells(P<0.01).PGRMC1 inhibitor AG-205 or PGRMC1siRNA pretreatment enhanced UTMD-induced LC3B2 and p62 accumulation in IR-exposed glioblastoma cells,thereby promoting UTMD-mediated radiosensitization(P<0.05).Moreover,PGRMC1 overexpression abolished UTMD-caused blockade of autophagic degradation,subsequently inhibiting UTMD-induced radiosensitization of glioblastoma cells.Finally,compared with IR plus UTMD group,PGRMC1 overexpression significantly increased tumor size [(3.8±1.1) mm^(2)vs.(8.0±1.9) mm^(2),P<0.05] and decreased survival time [(67.2±2.6) d vs.(40.0±1.2) d,P=0.0026] in glioblastoma-bearing mice.Conclusions:UTMD enhanced the radiosensitivity of glioblastoma partially by disrupting PGRMC1-mediated autophagy.
基金This work was supported(in part)by a grant from Kudo Academic Foundation(Support for the academic researcher,2017).
文摘Background:Ultrasound-targeted microbubble destruction(UTMD)induces cellular inflow of drugs at low intensity,while high intensity eradicates tumor vessels.Since vascular endothelial growth factor receptor 2(VEGFR2)is highly expressed in pancreatic ductal adenocarcinoma(PDAC),VEGFR2-targeted microbubble(MB)might additionally increase the tissue specificity of drugs and thus improve antitumor effects.In addition,fixing the dual pulse intensity could maximize MB properties.This study evaluated the one-off(experiment 1)and cumulative(experiment 2)treatment effect of UTMD by regulating the dual pulse output applied to PDAC using VEGFR2-targeted MB.Methods:C57BL/6 mice inoculated with Pan-02 cells were allocated to five groups:VEGFR2-targeted MB+gemcitabine(GEM),VEGFR2-targeted MB,non-targeted MB+GEM,GEM,and control groups.After injection of GEM or GEM and either VEGFR2-targeted or non-targeted MB,UTMD was applied for several minutes at low intensity followed by high intensity application.In experiment 1,mice were treated by the protocol described above and then euthanized immediately or at the tumor diameter doubling time(TDT).In experiment 2,the same protocol was repeated weekly and mice were euthanized at TDT regardless of protocol completion.Histological analysis by CD31 and VEGFR2 staining provided microvascular density(MVD)and VEGFR2 expression along vessels(VEGFR2v)or intra/peripheral cells(VEGFR2c).Results:In experiment 1,TDT was significantly longer in the VEGFR2-targeted MB+GEM group compared to the non-targeted MB+GEM,GEM,and control groups,while the VEGFR2-targeted MB group showed no statistical significance.MVD and VEGFR2v in the immediate euthanasia was significantly lower in the VEGFR2-targeted MB+GEM and VEGFR2-targeted MB groups than other conditions.In experiment 2,the VEGFR2-targeted MB+GEM group produced significantly longer TDT than the GEM or control groups,whereas the VEGFR2-targeted MB group showed no significant difference.Histology revealed significantly reduced VEGFR2v and VEGFR2c in the VEGFR2-targeted and non-targeted MB+GEM groups,while only VEGFR2v was significantly less in the VEGFR2-targeted MB group.Conclusions:UTMD-mediated GEM therapy with the dual pulse application using VEGFR2-targeted MB substantially suppresses PDCA growth.
基金supported by a grant from National Natural Sciences Foundation of China (No. 81071280)
文摘This study aimed to examine the preparation of cationic lipid microbubble（CLM）,and evaluate its physical and chemical properties and toxicity,measure the gene transfection efficiency by ultrasound triggered microbobble destruction（UTMD） in combination with CLM.The CLM was prepared by the method of the thin film hydration,and its morphology was observed under the electron microscopy at 1st,3rd,7th,10th,and 14th day after preparation,respectively.The size,Zeta potential and stability of CLM were tested.The acute toxicity of CLM was assessed.The green fluorescent protein gene（EGFP） transfection efficiency was evaluated.The experiment grouping was as follows：naked plasmid group（P group）,ultrasonic irradiation plus naked plasmid group（P-US group）,naked plasmid plus CLM group（P-CLM group）,naked plasmid plus ultrasound and CLM group（UTMD group）.The expression of EGFP was detected by fluorescent microscopy and flow cytometry.The results showed that CLMs were spherical in shape,with the similar size and good distribution degree under the light and electron microscopies.The size of CLMs was varied from 250.4±88.3 to 399.0±99.8 nm and the Zeta potential of CLMs from 18.80±4.97 to 20.1±3.1 mV.The EGFP expression was the strongest in the UTMD group,followed by the P-CLM group,P-US group and P group.Flow cytometry results were consistent with those of fluorescent microscopy.The transfection efficiency was substantially increased in the P-US group,P-CLM group and UTMD group as compared with that in the P group,almost 7 times,10 times and 30 times higher than that in the P group respectively.It is suggested that CLMs prepared by the method of thin film hydration are uniform in diameter,and proved non-toxic.UTMD combined with CLM can significantly increase the transfection efficiency of EGFP to targeted cells.
文摘Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix(ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications.However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fi brosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process.Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.
