体外骨髓基质干细胞中腺病毒介导的胶质细胞源性神经营养因子基因的表达及其生物学活性(英文)  

Expression of adenovirus-mediated glial cell line-derived neurotrophic factor gene in bone marrow stromal cells in vitro and its biological activity

作　　者：彭松林[1] 方煌[1] 赵红卫[1] 蔡卫东[1] 黎逢峰[1] 胡宁[1] 陈安民[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,湖北省武汉市430030

出　　处：《中国临床康复》2005年第6期230-232,F003,共4页Chinese Journal of Clinical Rehabilitation

摘　　要：背景:骨髓基质干细胞(bonemarrowstromalcells,BMSCs)是外源性目的基因的良好靶细胞,在脊髓损伤的修复中具有良好的应用前景。目的:观察重组腺病毒介导的胶质细胞源性神经营养因子(glialcellline-derivedneurotrophicfactor,GDNF)基因在体外培养的骨髓基质干细胞中的表达,并探讨其生物学活性。设计:以细胞为研究对象,对照观察性研究。单位:一所大学医院骨科实验室。材料:实验于2004-03/06在华中科技大学同济医学院附属同济医院骨科实验室完成。SD大鼠24只,雌雄不限,体质量(180±20)g。干预:用重组腺病毒载体Adv-GDNF感染体外培养的BMSCs,并与脊髓背根神经节共培养。免疫荧光化学的方法检测BMSCs中的GDNF的表达,提取细胞总RNA进行RT-PCR扩增GDNF基因,应用ELISA方法检测其培养上清中的GDNF含量,并通过与脊髓背根神经节共培养观测GDNF的活性。主要观察指标:主要结局:①RT-PCR。②免疫荧光结果。③GDNF的体外活性。次要结局:①BMSCs的培养与鉴定。②ELISA检测蛋白表达与时间的关系。结果:免疫荧光显示Adv-GDNF感染BMSCs48h后即有GDNF的表达,体外培养的BMSCs经Adv-GDNF转染后有GDNF的转录,其培养上清应用ELISA方法分析,在感染24h后即有GDNF的表达,并可持续5～7d的高峰。Adv-GDNF感染的BMSCs的培养液上清可以?BACKGROUND: Bone marrow stromal cells(BMSCs) are the ideal gene target cells and will have a bright future in the gene therapy of spinal cord injury. OBJECTIVE : To detect the expression of glial cell line- derived neurotrophic factor(GDNF) gene after BMSCs were infected by adenovirus medialed GDNF(Adv GDNF) in vitro and to explore its biological activity. DESIGN:A randomized controlled trial study. SETTING:Laboratory of Orthopedic Department MATERIALS:The experiment was completed in the Laboratory of Orthopedic Department,Affiliated Tongji Hospital of Tongji Meidcal College,Huazhong University of Science and Technology.Twenty four SD rats of either gender,weighing(180± 20) g. INTERVENTIONS:BMSCs were infected by Adv GDNF in vitro and then cocultured with spinal cord dorsal root ganglion.The three methods,immunofluorescent chemistry,reverse transcriptase polymerase chain reaction(RT PCR) and enzyme linked immunosorbent assay(ELISA) were used to evaluate GDNF expression in the BMSCs.The biological activity of GDNF was observed by a phase contrast microscope. MAIN OUTCOME MEASURES:Primary outcomes:① RT PCR;② results of immunofluorescent chemical examination;③ biological activity of GDNF in vitro.Secondary outcomes:① culturing and identification of BMSCs;② time effect relationship of GDNF expression revealed by ELISA. RESULTS:Immunofluorescence displayed expression of GDNF in BMSCs 48 hours after Adv GNDF infection.RT PCR analysis demonstrated expression of GDNF mRNA 24 hours after Adv GNDF infection.ELISA confirmed the presence of GDNF in the liquid supernatant of BMSCs 24 hours after Adv GDNF infectionn and showed that GDNF was secreted.The supernatant can promote the neurite outgrowth in the rat dorsal root ganglion(DRG). CONCLUSION:It is demonstrated that BMSCs infected by Adv GDNF can express GDNF steadily and the expressed GDNF has the activity of promoting neurite outgrowth,which lays a foundation of the GDNF gene therapy for spinal cord injury.

关 键 词：腺病毒科 干细胞 脑源性神经营养因子 基因 转染 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]