HAP1与pro-BDNF胞吞关系的机制研究  

Roles of HAP1 in the Endocytosis of pro-BDNF

作　　者：陈锶[1] 肖波[1] 毕方方[1] 李艺[1] 冯莉[1] 龙莉莉[1] 黄清[1] 

机构地区：[1]中南大学湘雅医院神经内科,长沙410078

出　　处：《神经损伤与功能重建》2009年第3期185-189,共5页Neural Injury and Functional Reconstruction

摘　　要：目的:探讨HAP1与pro-BDNF胞吞的相关性和可能机制。方法:NGF诱导分化PC12细胞,利用脂质体lipofectamine2000将提取的荧光质粒HAP1A-CFP和(或)pro-BDNF-ds-red转染进入细胞,培养48h后在含有BDNF、p75NTR或sortilin抗体的DMEM/10%FCS中培养,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;利用培养的小鼠皮质神经元(正常型和HAP1基因敲除型)在生物素标记pro-BDNF的培养基中孵育60min,激光共聚焦显微镜观察皮质神经元免疫荧光的效果。结果:共转染HAP1A-CFP和pro-BDNF-ds-red质粒的细胞,以及pro-BDNF-ds-red荧光蛋白培养基孵育的转染HAP1A-CFP质粒的细胞,pro-BDNF与HAP1蛋白的共定位比例高达93%～97%;抗BDNF培养基孵育的共转染2种质粒的细胞,以及pro-BDNF-ds-red荧光蛋白+抗p75NTR/抗sortilin培养基孵育的转染HAP1A-CFP质粒的细胞,2种荧光蛋白的共定位比率下降近一半;pro-BDNF-ds-red荧光蛋白+抗BDNF培养基孵育的转染HAP1A-CFP质粒的细胞几乎未显示2种蛋白的共定位;正常新生小鼠皮质神经元内可见内吞的pro-BDNF免疫荧光,HAP1基因敲除小鼠的皮质神经元内未见。结论:pro-BDNF的胞吞必需HAP1的表达和参与。Objective： To investigate the roles of HAP1 in endocytosis of pro-BDNF and the underlying mech- anism. Methods： PC12 cells were differentiated by NGF and co-transfected with plasmid of HAPIA-CFP and （or） pro-BDNF-ds-red. Then the cells were incubated with either recombinant ds-red-labeled pro-BDNF, or in combination with sheep anti-BDNF antibodies, rabbit anti-p75NTR, or sheep anti-sortilin antibodies. 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 I were cultured and treated with biotin labeled pro-BDNF to trigger endocytosis. After 60 min, the cells were washed and fixed, followed by immunostaining and confocal imaging. Results： Co-transfected PC12 cells or cells transfected with HAP1A-CFP which were incubated with recombinant dsred-labeled pro-BDNF showed almost complete co-localization of HAP1 with pro-BDNF. The antibodies to BDNF, p75NTR, and sortilin abolished co-localization of HAP1 with pro-BDNF. In addition, the labeled pro-BDNF was detected in almost all HAP1＋/＋ , but not in HAP1-/- cortical neurons. Conclusion： HAP1 plays an important role in the endocytosis of pro-BDNF.

关 键 词：BDNF前体蛋白 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：R741[医药卫生—神经病学与精神病学] R741.02[医药卫生—临床医学]