人神经营养素-3受体TrkC基因重组腺病毒载体的表达和生物学活性检测  

EXPRESSION AND BIOLOGICAL ACTIVITY ANALYSIS OF RECOMBINANT ADENOVIRUS EXPRESSION VECTOR FOR RECEPTOR TrkC OF NEUROTROPHIN-3

作　　者：王俊梅[1] 曾园山[1] 黄文林[2] 熊轶[1] 王延华[3] 

机构地区：[1]中山大学中山医学院组织学胚胎学教研室神经科学研究室 [2]中山大学肿瘤防治中心,广州510080 [3]昆明医学院神经研究所,昆明650031

出　　处：《解剖学报》2006年第5期520-524,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(30270700);广东省社会发展攻关项目(2003C33808);高等学校博士学科点专项科研基金(2004558068)

摘　　要：目的探讨人神经营养素-3受体TrkC基因重组腺病毒载体(Adeno-TrkC)在神经干细胞内的表达及其对神经干细胞的生物活性作用。方法用RT-PCR检测Adeno_TrkC感染的293细胞的TrkCmRNA含量。用免疫细胞化学和Westernblotting检测Adeno-TrkC感染的神经干细胞表达TrkC受体蛋白。在体外培养条件下,观察人神经营养素-3(NT-3)对感染了Adeno-TrkC的神经干细胞分化为神经元样细胞和星形胶质样细胞的影响。结果在Adeno-TrkC感染的293细胞和神经干细胞中检测到TrkCmRNA和TrkC受体蛋白,且表达产物和其配体NT-3结合后能使体外培养的神经干细胞更多地分化为神经元样细胞,分化率达到55.2%,均高于其他对照组。结论Adeno_TrkC能在神经干细胞内表达TrkC受体蛋白,这种TrkC受体蛋白具有促进神经干细胞分化为神经元样细胞活性的作用,这为进一步应用NT-3和TrkC联合进行基因治疗中枢神经损伤研究奠定了基础。Objective To detect the expression and the biological activity of a recombinant adenovirus expression vector carrying human neurotrophin-3 （NT-3） receptor TrkC gene （Adeno-TrkC） in neural stem cells. Methods The expression of TrkC mRNA in 293 cells infected with Adeno-TrkC was detected by RT-PCR, and the expression of TrkC protein in neural stem cells infected by Adeno-TrkC was detected with immunocytochemistry and Western blotting. The effects of human neurotrophin-3 （ NT- 3） on the neural stem cells infected by Adeno-TrkC differentiating into neuron-like cells and astrocyte-like cells in vitro were observed. Results The transcription of TrkC mRNA and the expression of TrkC protein was detected in 293 cells and neural stem cells infected by Adeno-TrkC, This kind of TrkC was able to make more neural stem cells differenting into neuron-like cells in vitro with its ligand NT-3 and the percentage of neuron-like cell＇s differentiation was 55.2% which was higher than that of other control groups. Conclusion TrkC protein is expressed in neural stem cells transfected with Adeno-TrkC and is able to make neural stem cells differenting into neuron-like cells. The present study may provide the basis on gene therapy of central nervous injury using further NT-3 and TrkC.

关 键 词：TRKC 重组腺病毒 基因表达 神经干细胞 分化 WESTERN BLOTTING 

分 类 号：R338.21[医药卫生—人体生理学]