亨廷顿蛋白相关蛋白1与脑源性神经营养因子的胞吞关系的机制研究  

Huntingtin associated protein 1 is involved with the endocytosis of mature brain derived neurotrophic factor

作　　者：陈锶[1] 肖波[1] 毕方方[1] 冯莉[1] 

机构地区：[1]中南大学湘雅医院神经内科,湖南省长沙市410008

出　　处：《国际神经病学神经外科学杂志》2011年第1期19-23,共5页Journal of International Neurology and Neurosurgery

摘　　要：目的探讨亨廷顿蛋白相关蛋白1(HAP1)与脑源性神经营养因子(mBDNF)胞吞的相关性和可能的机制。方法神经营养因子(NGF)诱导分化PC 1 2细胞,将荧光质粒HAP1A-CFP和(或)mBDNF-ds-red转染进入细胞,培养4 8 h后在含有BDNF或p7 5NTR抗体的培养基中继续培养,激光共聚焦显微镜观察荧光的表达情况及其在细胞中的定位;利用小鼠皮层神经元(正常型和HAP1基因敲除型)在生物素标记mBDNF的培养基中孵育6 0 m in,激光共聚焦显微镜观察皮层神经元免疫荧光的效果。结果共转染HAP1A-CFP和mBDNF-ds-red质粒的细胞,2种荧光蛋白存在部分共定位3 4%。共转染的细胞在抗BDNF培养基孵育下,或者单转染HAP1A-CFP质粒的细胞在mBDNF-ds-red荧光蛋白+抗BD-NF/抗p7 5NTR培养基孵育下,2种荧光蛋白几乎没有共定位现象。单转染HAP1A-CFP质粒的细胞在mBDNF-ds-red荧光蛋白培养基孵育下,mBDNF与HAP1蛋白的共定位比例高达9 3%。正常新生小鼠皮层神经元内可见内吞的mBDNF免疫荧光,HAP1基因敲除小鼠的皮层神经元内未见。结论 mBDNF的胞吞必需HAP1的表达和参与。Objective To investigate the roles of Huntingtin associated protein 1（HAP1） in the endocytosis of mature brain derived neurotrophic factor（mBDNF） and the underlying mechanism.Methods PC12 cells were differentiated by NGF and co-transfected with plasmid of HAP1A-CFP and（or） mBDNF-ds-red.The cells were incubated with either recombinant ds-red-labeled mBDNF,or in combination of sheep anti-BDNF antibodies or rabbit anti-p75NTR.The expression of fluorescence and its intercellular location were determined by laser scanning confocal microscope.Cortical neurons from HAP1＋/＋ and HAP1-/-mice at postnatal day 1 were cultured and treated with biotin labeled mBDNF to trigger endocytosis.After 60 minutes,the cells were washed and fixed,followed by immunostaining and confocal imaging.Results Co-transfected PC12 cells expressed partly co-localization of HAP1 with mBDNF.Cells transfected with HAP1A-CFP which were incubated with recombinant ds-red-labeled mBDNF showed almost complete co-localization of HAP1 with mBDNF.The antibodies to BDNF and p75NTR abolished co-internalization of HAP1 with mBDNF.In addition,the labeled mBDNF was detected in almost all HAP1＋/＋,but not in HAP1-/-cortical neurons.Conclusions HAP1 plays an important role in the endocytosis of mBDNF.

关 键 词：脑源性神经营养因子 亨廷顿蛋白相关蛋白1 转染 荧光质粒 激光共聚焦显微镜 

分 类 号：R742.3[医药卫生—神经病学与精神病学]