【摘要】 目的:纯化毕赤酵母表达的Neuritin蛋白,通过观察其对PC12细胞和鸡胚背根神经节(dorsal root ganglion,DRG)的作用,研究其神经生物学功能。方法:采用镍离子金属螯合亲和层析法纯化Neuritin蛋白,Bradford法对纯化的Neuritin蛋白进行定量,然后加入到PC12细胞和DRG的培养液中,通过观察PC12细胞突起的生长情况和细胞形态的变化,研究其促进细胞突起生长的功能;通过观察鸡胚DRG的神经纤维生长情况,研究其促进DRG神经纤维生长活性;通过观察鸡胚背根神经节的裂解时间,研究其阻止细胞退化和凋亡的活性。结果:纯化后的Neuritin蛋白经SDS-PAGE电泳显示,获得11 kDa左右的唯一蛋白条带,即Neuritin蛋白;Bradford定量法计算出其浓度为490μg/ml。纯化的Neuritin蛋白加入到PC12细胞培养液中,PC12细胞长出突起,发生类神经元样改变;鸡胚DRG培养液中加入Neuritin蛋白后,长出神经纤维,裂解时间也延长,并与浓度呈正相关。结论:毕赤酵母表达的Neuritin蛋白得到有效纯化,纯化的Neuritin蛋白能促进细胞突起的生长,阻止细胞的退化和凋亡,延长细胞的存活时间,这为进一步研究neuritin的功能及作用机制奠定了良好的实验基础。更多还原

【Abstract】 Objective: To purify Neuritin protein from Pichia pastoris expression system of neuritin,and study its neurobiological function by observing its effects on PC12 cells and chicken embryonic dorsal root ganglion( DRG) neurons. Methods: Using Ni affinity chromatography to purify Neuritin protein,and Bradford method to quantify purified Neuritin protein,then added it into the culture medium of PC12 cells and chicken embryonic DRG neurons. The promoting neurite outgrowth on cells and cellular morphology change was detected by observing the neurite outgrowth of PC12 cells,and the activity of promoting nerve fibers growth was detected by observing the nerve fibers growth of DRG neurons. Then the activity of preventing cell degradation and apoptosis was detected by observing the pyrolysis time of DRG neurons. Results: The purified Neuritin protein was analyzed by SDS-PAGE,and showed only one band of protein about 11 kDa,that was Neuritin protein. Its concentration was 490 μg / ml through the Bradford quantitative method calculation. After adding purified Neuritin protein into PC12 cell culture medium,PC12 cells grew protrusions and neuron-like changes appeared; after adding Neuritin protein to chicken embryonic DRG neurons,DRG neurons grew the nerve fibers,its pyrolysis time also was prolonged,and which were positively correlated with the concentration. Conclusion: The Neuritin protein fromPichia pastoris expression system is purified successfully and it can promote the neurite outgrowth of cells,prevent cell degradation and apoptosis,and prolong cell survival time,which lays a good foundation for further study on the function and mechanism of neuritin.更多还原