神经营养素3基因转染神经干细胞及其表达(英文)

Expression of neural stem cells transfected by neurenergen-3 gene

背景:应用包括神经营养素3的神经营养因子促进周围神经再生是当今周围神经再生研究的热点之一,但是临床应用上缺乏安全、有效的给药途径。基因转染技术为神经营养因子的临床应用提供了新的思路和途径。目的:用神经营养素3基因修饰神经干细胞,并观察在转染后的神经干细胞内的表达情况。设计:完全随机试验。单位:广西医科大学第一附属医院创伤骨科手外科和华中科技大学同济医学院附属协和医院手外科。材料:实验选用健康SD大鼠3只,鼠龄4个月,雌雄不拘,由华中科技大学同济医学院动物房提供。用于转染神经干细胞的重组腺病毒表达载体由华中科技大学同济医学院协和医院骨科实验室构建,浓度为0.15g/L。方法:实验于2002-12/2004-03在华中科技大学同济医学院协和医院骨科实验室和同济医院中心实验室完成。采用阳离子脂质体介入法,将含绿色荧光蛋白(GFP)基因的重组质粒pAd-神经营养素3转染原代培养的神经干细胞,转染72h后采用荧光倒置显微镜观察绿色荧光蛋白在神经干细胞上的表达,测定转染率。采用免疫细胞化学染色法和反转录-聚合酶链反应检测转染72h,1,5周后神经营养素3基因在神经干细胞中的表达和转录情况。主要观察指标:神经营养素3基因在转染后的神经干细胞内的表达情况。结果:①重组质粒pAd-神经营养素3转染神经干细胞:转染72h绿色荧光蛋白在部分神经干细胞上有表达,转染率为40%。5周后仍可见绿色荧光蛋白的表达。②神经营养素3基因在神经干细胞中的转录与表达:转染72h,1,5周后神经干细胞中均有神经营养素3mRNA的转录,转染5周后仍有神经营养素3mRNA的表达。结论:神经营养素3基因以阳离子脂质体为载体,通过腺病毒的介导,可有效的转染培养的神经干细胞并长期表达。

BACKGROUND： It is one of hot topics for the application of neurotrophic factors including neurenergen-3 to promote peripheral neural regeneration nowadays; however, clinical application is restricted to safety and effective administration route. Gene transfection brings a novel thinking and pathway for neurotrophic factors used in clinic. OBJECTIVE： To observe the expression of neural stem cells modified by neurenergen-3 gene after transfection. DESIGN： Completely randomized study. SETTING： Department of Traumatic Orthopaedics and Hand Surgery, the First Affiliated Hospital, Guangxi Medical University; Department of Hand Surgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： Three healthy SD rats of four months old and either gender were selected from Animal Center, Tongji Medical College, Huazhong University of Science and Technology. Recombinant adenoviral expressing vector for transfection of neural stem cells was constructed in Laboratory of Orthopaedics, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, and the concentration was 0.15 g/L. METHODS： The experiment was carried out in the Orthopaedic Laboratory and Central Laboratory, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology from December 2002 to March 2004. Recombinant plasmid pAD-neurenergen-3 containing with green fluorescent protein （GFP） gene was transfectd into primarily cultured neural stem cells by using cationic liposome interventional method. At 72 hours after transfection, fluorescent inverted microscope was used to observe GFP expression in neural stem cells, and transfection efficiency was measured simultaneously. Expression and transcription of neurenergen-3 gene in neural stem cells were detected at 72 hours, 1 and 5 weeks after transfection by using immunocytochemical stain and reverse transcription polymerase chain reaction （RT-PCR）. MAIN OUTCOME MEASURES： Expression of neurenergen-3 gent in transfected neural stem cells. RESULTS： （1） Neural stem cells transfected by recombinant plasmid pAD-neurenergen-3： GFP expressed on partial neural stem cells at 72 hours after transfection, and the transfection efficiency was 40%. Five weeks later, GFP expression was still observed. （2） Transcription and expression of neurenergen-3 gene in neural stem cells： Transcription of neurenergen-3 mRNA was observed in neural stem cells at 72 hours, 1 and 5 weeks after transfection, and expression of neurenergen-3 rnRNA was still observed in 5 weeks after transfection. CONCLUSION： As the carder of cationic liposome, neurenergen-3 gene can effectively transfect, culture and long-term express neural stem cells via the introduction of adenoviruses.