Traumatic brain injury results in neuronal loss and glial scar formation.Replenishing neurons and eliminating the consequences of glial scar formation are essential for treating traumatic brain injury.Neuronal reprogr...Traumatic brain injury results in neuronal loss and glial scar formation.Replenishing neurons and eliminating the consequences of glial scar formation are essential for treating traumatic brain injury.Neuronal reprogramming is a promising strategy to convert glial scars to neural tissue.However,previous studies have reported inconsistent results.In this study,an AAV9P1 vector incorporating an astrocyte-targeting P1 peptide and glial fibrillary acidic protein promoter was used to achieve dual-targeting of astrocytes and the glial scar while minimizing off-target effects.The results demonstrate that AAV9P1 provides high selectivity of astrocytes and reactive astrocytes.Moreover,neuronal reprogramming was induced by downregulating the polypyrimidine tract-binding protein 1 gene via systemic administration of AAV9P1 in a mouse model of traumatic brain injury.In summary,this approach provides an improved gene delivery vehicle to study neuronal programming and evidence of its applications for traumatic brain injury.展开更多
目的获得能够过表达OPTN和TAU-P301-L基因的重组腺相关病毒,检测其在体外对293T细胞的感染,并且为阿尔茨海默氏病(AD)研究提供新思路。方法从含有目的基因的大肠杆菌中提取包涵目的基因的质粒,利用PCR扩增获得目的基因,目的基因OPTN构建...目的获得能够过表达OPTN和TAU-P301-L基因的重组腺相关病毒,检测其在体外对293T细胞的感染,并且为阿尔茨海默氏病(AD)研究提供新思路。方法从含有目的基因的大肠杆菌中提取包涵目的基因的质粒,利用PCR扩增获得目的基因,目的基因OPTN构建到pAAV-IRES-ZsGreen1载体,目的基因TAU-P301L-V5构建到pAAV-CMV-mkate-IRES载体。鉴定质粒阳性克隆并测序。重组质粒pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5分别与包装质粒pAAV-RC9、辅助质粒pHelper通过Lipofiter^(TM)2000共转染293T细胞,获得AAV9病毒。利用相同方法,不使用目的基因构建对应绿色和红色AAV9对照病毒。qPCR法测定重组腺相关病毒载体滴度,病毒载体感染293T细胞进行病毒有效性及安全性鉴定,Western blotting和RT-PCR进行蛋白表达的鉴定,MTT检测OPTN蛋白与TAU-P301-L蛋白对293T细胞活力的影响。结果测序结果显示成功构建pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5腺相关病毒载体,qPCR测得病毒滴度为1.0×10^(12) vg/ml,病毒载体感染293T细胞转染效率为33.6%和20.5%, Western blot和RT-PCR显示蛋白过量表达。MTT结果显示OPTN组挽救了TAU-P301-L组的细胞凋亡。结论成功构建过表达OPTN和TAU-P301-L基因的腺相关病毒。OPTN蛋白可以减弱TAU-P301-L蛋白的细胞毒性。展开更多
基金supported by the National Natural Science Foundation of China,No.82073783(to YY)the Natural Science Foundation of Beijing,No.7212160(to YY).
文摘Traumatic brain injury results in neuronal loss and glial scar formation.Replenishing neurons and eliminating the consequences of glial scar formation are essential for treating traumatic brain injury.Neuronal reprogramming is a promising strategy to convert glial scars to neural tissue.However,previous studies have reported inconsistent results.In this study,an AAV9P1 vector incorporating an astrocyte-targeting P1 peptide and glial fibrillary acidic protein promoter was used to achieve dual-targeting of astrocytes and the glial scar while minimizing off-target effects.The results demonstrate that AAV9P1 provides high selectivity of astrocytes and reactive astrocytes.Moreover,neuronal reprogramming was induced by downregulating the polypyrimidine tract-binding protein 1 gene via systemic administration of AAV9P1 in a mouse model of traumatic brain injury.In summary,this approach provides an improved gene delivery vehicle to study neuronal programming and evidence of its applications for traumatic brain injury.
文摘目的获得能够过表达OPTN和TAU-P301-L基因的重组腺相关病毒,检测其在体外对293T细胞的感染,并且为阿尔茨海默氏病(AD)研究提供新思路。方法从含有目的基因的大肠杆菌中提取包涵目的基因的质粒,利用PCR扩增获得目的基因,目的基因OPTN构建到pAAV-IRES-ZsGreen1载体,目的基因TAU-P301L-V5构建到pAAV-CMV-mkate-IRES载体。鉴定质粒阳性克隆并测序。重组质粒pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5分别与包装质粒pAAV-RC9、辅助质粒pHelper通过Lipofiter^(TM)2000共转染293T细胞,获得AAV9病毒。利用相同方法,不使用目的基因构建对应绿色和红色AAV9对照病毒。qPCR法测定重组腺相关病毒载体滴度,病毒载体感染293T细胞进行病毒有效性及安全性鉴定,Western blotting和RT-PCR进行蛋白表达的鉴定,MTT检测OPTN蛋白与TAU-P301-L蛋白对293T细胞活力的影响。结果测序结果显示成功构建pAAV-IRES-ZsGreen1-AAV9-OPTN和pAAV-CMV-mkate-IRES-AAV9-TAU-P301L-V5腺相关病毒载体,qPCR测得病毒滴度为1.0×10^(12) vg/ml,病毒载体感染293T细胞转染效率为33.6%和20.5%, Western blot和RT-PCR显示蛋白过量表达。MTT结果显示OPTN组挽救了TAU-P301-L组的细胞凋亡。结论成功构建过表达OPTN和TAU-P301-L基因的腺相关病毒。OPTN蛋白可以减弱TAU-P301-L蛋白的细胞毒性。
