Background:Zinc-finger E-box-binding homeobox-1(ZEB1)is predominantly found in type-H vessels.However,the roles of ZEB1 and type-H vessels in steroid-i nduced osteonecrosis of the femoral head(SONFH)are unclear.Method...Background:Zinc-finger E-box-binding homeobox-1(ZEB1)is predominantly found in type-H vessels.However,the roles of ZEB1 and type-H vessels in steroid-i nduced osteonecrosis of the femoral head(SONFH)are unclear.Methods:Human femoral heads were collected to detect the expression of ZEB1and the levels of type-H vessels.Then,the SONFH model was developed by injecting C57BL/6 mice with lipopolysaccharide and methylprednisolone.Microcomputed tomography,angiography,double calcein labeling,immunofluorescence,immunohistochemistry,quantitative real-time polymerase chain reaction,and Western blotting were performed to detect the expression of ZEB1,the Wnt/β-catenin pathway,type-H vessels,and the extent to which ZEB1 mediates angiogenesis and osteogenesis.Human umbilical vein endothelial cells were also used to explore the relationship between ZEB1 and the Wnt/β-catenin pathway.Results:We found that ZEB1 expression and the formation of type-H vessels decreased in SONFH patients and in a mouse model.The number of vascular endothelial growth factors in the femoral heads also decreased.Moreover,the bone mineral density,trabecular number,mineral apposition rate,and expression of genes related to osteogenesis decreased.After ZEB1 knockdown,angiogenesis and osteogenesis decreased.However,the numbers of type-H vessels and the extent of angiogenesis and osteogenesis improved after activation of the Wnt/β-catenin pathway.Conclusions:The ZEB1 expression decreased in SONFH,causing a decrease in type-H vessel,and it mediated angiogenesis and osteogenesis by regulating the Wnt/β-catenin pathway,ultimately accelerating the process of SONFH.展开更多
Numerous studies have confirmed that vascular endothelial growth factor （VEGF） improves the function of neural cells following spinal cord injury （SCI）. However, some studies have also verified that VEGF cannot si...Numerous studies have confirmed that vascular endothelial growth factor （VEGF） improves the function of neural cells following spinal cord injury （SCI）. However, some studies have also verified that VEGF cannot significantly induce the increase in vascular density at or surrounding the lesion, and that VEGF therapy exacerbated secondary damage following SCI. Based on the dual effects of VEGF on SCI, we constructed the recombinant adeno-associated viruses （rAAV）-hVEGF165-IRES-human recombinant green fluorescent protein （hrGFP） （AAV-VEGF） and rAAV-IRES-hrGFP （AAV-GFP）. Our results suggested that rAAV expressed hVEGFles, and a low dose of VEGF relieved increased vascular permeability, improved microcirculation in the local spinal cord, lessened spinal cord edema, and decreased neuronal apoptosis. These results verified that the releasing effects of the rAAV virus vector had protective effects on the spinal cord.展开更多
BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration...BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE： To construct a non-pathogenic, recombinant adeno-associated virus （AAV） simultaneously expressing human vascular endothelial growth factor 165 （hVEGF165） and green fluorescent protein （GFP）. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS： AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS： The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC （the rep/cap-gene containing plasmid）, and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES： Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter （FACS） upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS： The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene. CONCLUSION： The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.展开更多
Objective： To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165（hVEGF165） and bone morphogenetic protein 7（hBMPT）, measure the virus titer and verify the recom...Objective： To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165（hVEGF165） and bone morphogenetic protein 7（hBMPT）, measure the virus titer and verify the recombination. Methods：The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid plRES was cut down and subcloned into ITR/ MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then, recombinant plasmid pAAV- hVEGF165-IRES-hBMP7, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells, and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results：Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5 ×10^11vp/ml, and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion：Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer, which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.展开更多
The weight-drop impact is widely used in making animal model of spinal cord injury(SCI).But there has not yet been an appropriate unit for the quantification of the impact.In this study,we compared the degrees of the ...The weight-drop impact is widely used in making animal model of spinal cord injury(SCI).But there has not yet been an appropriate unit for the quantification of the impact.In this study,we compared the degrees of the spinal cord injury caused by weight-drop impact with the same momentum but different combinations of drop weight and drop height,in order to test whether‘‘momentum’’is capable of being the unit for the quantification of weightdrop impact.Thirty adult rats were randomly allocated to three groups and were injured with 5 g–10 cm(group A),10 g–2.5 cm(group B)and 15 g–1.11 cm(group C)impacts with the same momentum to the spinal cord,respectively.Open-field locomotor function was evaluated using the Basso–Beattie–Bresnahan(BBB)locomotor rating scale.The percentage of spared tissue area(STA)at the epicenter,and 500,1000 and 1500 lm from the epicenter was calculated using serial sections stained by hematoxylin and eosin.As a result,the behavioral recovery(BBB scores)and the STA percentage were similar in group B and group C.However,the BBB score in group A was significantly lower than that in groups B and C at the same time point post injury.The STA percentage was significantly less and the lesion/cavity length was significantlygreater in group A than in groups B and C.These suggested that the 5 g–10 cm weight-drop impact,compared with the other two impacts with different weights and heights,caused a greater damage of the spinal cord when the momentum was the same.So,these impacts with the same momentum but different weights and drop heights cause different degrees of spinal cord injury.Momentum alone is inadequate to be the unit for the qualification of weightdrop impact and to be used to predict the extent of injury.展开更多
基金China Postdoctoral Science Foundation,Grant/Award Number:2021M692575Fundamental Research Funds for the Central Universities,Grant/Award Number:xzy022024016National Natural Science Foundation of China,Grant/Award Number:82002311。
文摘Background:Zinc-finger E-box-binding homeobox-1(ZEB1)is predominantly found in type-H vessels.However,the roles of ZEB1 and type-H vessels in steroid-i nduced osteonecrosis of the femoral head(SONFH)are unclear.Methods:Human femoral heads were collected to detect the expression of ZEB1and the levels of type-H vessels.Then,the SONFH model was developed by injecting C57BL/6 mice with lipopolysaccharide and methylprednisolone.Microcomputed tomography,angiography,double calcein labeling,immunofluorescence,immunohistochemistry,quantitative real-time polymerase chain reaction,and Western blotting were performed to detect the expression of ZEB1,the Wnt/β-catenin pathway,type-H vessels,and the extent to which ZEB1 mediates angiogenesis and osteogenesis.Human umbilical vein endothelial cells were also used to explore the relationship between ZEB1 and the Wnt/β-catenin pathway.Results:We found that ZEB1 expression and the formation of type-H vessels decreased in SONFH patients and in a mouse model.The number of vascular endothelial growth factors in the femoral heads also decreased.Moreover,the bone mineral density,trabecular number,mineral apposition rate,and expression of genes related to osteogenesis decreased.After ZEB1 knockdown,angiogenesis and osteogenesis decreased.However,the numbers of type-H vessels and the extent of angiogenesis and osteogenesis improved after activation of the Wnt/β-catenin pathway.Conclusions:The ZEB1 expression decreased in SONFH,causing a decrease in type-H vessel,and it mediated angiogenesis and osteogenesis by regulating the Wnt/β-catenin pathway,ultimately accelerating the process of SONFH.
基金the National Natural Science Foundation of China, No. 30772189
文摘Numerous studies have confirmed that vascular endothelial growth factor （VEGF） improves the function of neural cells following spinal cord injury （SCI）. However, some studies have also verified that VEGF cannot significantly induce the increase in vascular density at or surrounding the lesion, and that VEGF therapy exacerbated secondary damage following SCI. Based on the dual effects of VEGF on SCI, we constructed the recombinant adeno-associated viruses （rAAV）-hVEGF165-IRES-human recombinant green fluorescent protein （hrGFP） （AAV-VEGF） and rAAV-IRES-hrGFP （AAV-GFP）. Our results suggested that rAAV expressed hVEGFles, and a low dose of VEGF relieved increased vascular permeability, improved microcirculation in the local spinal cord, lessened spinal cord edema, and decreased neuronal apoptosis. These results verified that the releasing effects of the rAAV virus vector had protective effects on the spinal cord.
基金the National Natural Science Foundation of China, No. 30600624
文摘BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE： To construct a non-pathogenic, recombinant adeno-associated virus （AAV） simultaneously expressing human vascular endothelial growth factor 165 （hVEGF165） and green fluorescent protein （GFP）. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS： AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS： The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC （the rep/cap-gene containing plasmid）, and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES： Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter （FACS） upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS： The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene. CONCLUSION： The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.
基金National Natural Science Foundation of China(No.30600624)
文摘Objective： To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165（hVEGF165） and bone morphogenetic protein 7（hBMPT）, measure the virus titer and verify the recombination. Methods：The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid plRES was cut down and subcloned into ITR/ MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then, recombinant plasmid pAAV- hVEGF165-IRES-hBMP7, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells, and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results：Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5 ×10^11vp/ml, and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion：Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer, which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.
基金supported by the National Natural Science Foundation of China(81271340)
文摘The weight-drop impact is widely used in making animal model of spinal cord injury(SCI).But there has not yet been an appropriate unit for the quantification of the impact.In this study,we compared the degrees of the spinal cord injury caused by weight-drop impact with the same momentum but different combinations of drop weight and drop height,in order to test whether‘‘momentum’’is capable of being the unit for the quantification of weightdrop impact.Thirty adult rats were randomly allocated to three groups and were injured with 5 g–10 cm(group A),10 g–2.5 cm(group B)and 15 g–1.11 cm(group C)impacts with the same momentum to the spinal cord,respectively.Open-field locomotor function was evaluated using the Basso–Beattie–Bresnahan(BBB)locomotor rating scale.The percentage of spared tissue area(STA)at the epicenter,and 500,1000 and 1500 lm from the epicenter was calculated using serial sections stained by hematoxylin and eosin.As a result,the behavioral recovery(BBB scores)and the STA percentage were similar in group B and group C.However,the BBB score in group A was significantly lower than that in groups B and C at the same time point post injury.The STA percentage was significantly less and the lesion/cavity length was significantlygreater in group A than in groups B and C.These suggested that the 5 g–10 cm weight-drop impact,compared with the other two impacts with different weights and heights,caused a greater damage of the spinal cord when the momentum was the same.So,these impacts with the same momentum but different weights and drop heights cause different degrees of spinal cord injury.Momentum alone is inadequate to be the unit for the qualification of weightdrop impact and to be used to predict the extent of injury.
