Objective: To investigate the effects of exogenous brain derived neurotrophic factor(BDNF) expression on in- jured spinal cord by being injected into the cord following its reconstruction with adenovirus. Method: A re...Objective: To investigate the effects of exogenous brain derived neurotrophic factor(BDNF) expression on in- jured spinal cord by being injected into the cord following its reconstruction with adenovirus. Method: A reliable rat model with injury of different gradation was established by using a weight-drop apparatus with a laser compression recorder. The replicate-defect recombinant adenoviral vector containing BDNF gene was transferred into the site of injured spinal cord by direct injection. The validity of gene transfer was verified with X-Gal staining. The morphological changes of the injured ax- on were studied quantitatively. The expression of BDNF mRNA, and immunocytochemical reactivity of BDNF and neurofila- ment(NF) in injured cord of rats were observed. Results: It was verified that the spinal coal could be effectively infected by injecting the adenoviral vector into the injured cord, and the reporter gene was expressed. The loss of axons reduced following the in vivo infection of adenoviral vector carrying exogenous BDNF gene after in injury, while more NF immunopositive ax- ons than that of normal spical cord were found. Conclusion: With in vivo transfer of adenovirus vector into the injured site, a certain extent of protection could be provided to the injured axons by increasing local expressions of exogenous DBNF, and renovation of the cytoskeleton in the injured neurons was facilitated. These double effects are both important in gene therapy of spinal cord injuries.展开更多
Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic ...Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.展开更多
利用 1株鹅源腺病毒 Y81G4 株 ,经鹅胚增殖后收获尿囊液 ,用差速离心法纯化病毒子并提取病毒基因组 DNA.病毒 DNA经 H ind 酶切后共产生 1 0个片段 ,分别回收各酶切片段 ,与经 H ind 单酶切的p UC1 8连接 ,获得 8个不同的重组质粒 .对...利用 1株鹅源腺病毒 Y81G4 株 ,经鹅胚增殖后收获尿囊液 ,用差速离心法纯化病毒子并提取病毒基因组 DNA.病毒 DNA经 H ind 酶切后共产生 1 0个片段 ,分别回收各酶切片段 ,与经 H ind 单酶切的p UC1 8连接 ,获得 8个不同的重组质粒 .对于未克隆到的两末端片段 ,经碱处理去除末端蛋白后 ,以平端和 H ind 粘端与经 Sma 和 H ind 双酶切的 p UC1 8连接 ,获得了重组质粒 p GAHC和 p GAHI.克隆的各酶切片段 ,通过酶切电泳和 Southern blotting结果鉴定 ,证明已分别克隆到该病毒基因组 DNA H ind 酶切片段 ,且各酶切片段大小之和约为 3 2 .展开更多
基金Supported by National Natural Science Fundation of China, No. 39670740
文摘Objective: To investigate the effects of exogenous brain derived neurotrophic factor(BDNF) expression on in- jured spinal cord by being injected into the cord following its reconstruction with adenovirus. Method: A reliable rat model with injury of different gradation was established by using a weight-drop apparatus with a laser compression recorder. The replicate-defect recombinant adenoviral vector containing BDNF gene was transferred into the site of injured spinal cord by direct injection. The validity of gene transfer was verified with X-Gal staining. The morphological changes of the injured ax- on were studied quantitatively. The expression of BDNF mRNA, and immunocytochemical reactivity of BDNF and neurofila- ment(NF) in injured cord of rats were observed. Results: It was verified that the spinal coal could be effectively infected by injecting the adenoviral vector into the injured cord, and the reporter gene was expressed. The loss of axons reduced following the in vivo infection of adenoviral vector carrying exogenous BDNF gene after in injury, while more NF immunopositive ax- ons than that of normal spical cord were found. Conclusion: With in vivo transfer of adenovirus vector into the injured site, a certain extent of protection could be provided to the injured axons by increasing local expressions of exogenous DBNF, and renovation of the cytoskeleton in the injured neurons was facilitated. These double effects are both important in gene therapy of spinal cord injuries.
文摘Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.
文摘利用 1株鹅源腺病毒 Y81G4 株 ,经鹅胚增殖后收获尿囊液 ,用差速离心法纯化病毒子并提取病毒基因组 DNA.病毒 DNA经 H ind 酶切后共产生 1 0个片段 ,分别回收各酶切片段 ,与经 H ind 单酶切的p UC1 8连接 ,获得 8个不同的重组质粒 .对于未克隆到的两末端片段 ,经碱处理去除末端蛋白后 ,以平端和 H ind 粘端与经 Sma 和 H ind 双酶切的 p UC1 8连接 ,获得了重组质粒 p GAHC和 p GAHI.克隆的各酶切片段 ,通过酶切电泳和 Southern blotting结果鉴定 ,证明已分别克隆到该病毒基因组 DNA H ind 酶切片段 ,且各酶切片段大小之和约为 3 2 .
