Chen Qian, Wang Jia-Zheng, Liu Hong, Wu Yan, Fan Ming
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2000;32(2):121-125.
In order to deliver brain-derived neurotrophic factor(BDNF) and neurotrophin-3(NT3) into CNS to prevent or reduce degeneration of CNS neurons in human neurodegenerative diseases and neuron injuries, recombinant adenoviruses Ad-BDNF and Ad-NT3 were constructed. BDNF and NT3 genes were inserted into an E1-substituted adenovirus shuttle plasmid, respectively, and were driven by the human cytomegalovirus immediate-early gene promoter/enhancer. After cotransfection into 293 cells with the adenovirus plasmid pJM17, the recombinant adenovirus Ad-BDNF and Ad-NT3 were propagated in 293 cells via homologous recombination. After two rounds of CsCl centrifugation, the human recombinant adenovirus Ad-BDNF and Ad-NT3 were obtained with titer of 1x10(12) and 8x10(11) pfu/ml, respectively. To examine the expression of BDNF and NT3, HeLa cells were infected with Ad-BDNF and Ad-NT3, respectively. RT-PCR, Western blot and ELISA analysis results showed that BDNF and NT3 genes could be transcribed and translated in Ad-BDNF and Ad-NT3 infected HeLa cells. And the culture media containing 10% conditioned medium of Ad-BDNF and Ad-NT3 infected Hela cells could induce the neurite outgrowth from E8 dorsal root ganglion neurons.
为了将脑源性神经营养因子（BDNF）和神经营养素-3（NT3）导入中枢神经系统（CNS），以预防或减少人类神经退行性疾病中CNS神经元的退化和神经元损伤，构建了重组腺病毒Ad-BDNF和Ad-NT3。BDNF和NT3基因分别插入E1缺失的腺病毒穿梭质粒中，并由人巨细胞病毒立即早期基因启动子/增强子驱动。将腺病毒质粒pJM17与上述质粒共转染到293细胞中后，重组腺病毒Ad-BDNF和Ad-NT3通过同源重组在293细胞中增殖。经过两轮氯化铯离心后，获得了人重组腺病毒Ad-BDNF和Ad-NT3，其滴度分别为1×10¹²和8×10¹¹ pfu/ml。为检测BDNF和NT3的表达，分别用Ad-BDNF和Ad-NT3感染HeLa细胞。逆转录-聚合酶链反应（RT-PCR）、蛋白质免疫印迹法（Western blot）和酶联免疫吸附测定（ELISA）分析结果表明，BDNF和NT3基因在Ad-BDNF和Ad-NT3感染的HeLa细胞中能够转录和翻译。并且，含有10% Ad-BDNF和Ad-NT3感染的Hela细胞条件培养基的培养液能够诱导E8背根神经节神经元长出神经突。
