Nogo-B与Clusterin结合对细胞凋亡的影响

神经轴突生长抑制因子Nogo—B在体分布广泛，提示其除了具有抑制中枢神经系统轴突再生作用外，可能还扮演其他重要的功能角色。该研究为探讨Nogo-B下游新的结合分子及其功能开展相应研究。通过设计诱饵蛋白筛选人脑cDNA文库、免疫共沉淀方法，寻找Nogo-B下游结合分子：通过流式细胞术，检测结合对于细胞凋亡的影响：通过绿色荧光蛋白标记和免疫组织化学方法，探讨Nogo-B诱导细胞凋亡的机制。结果提示，Clusterin除了与Nogo-66功能域在酵母双杂交系统中存在结合，与Nogo—B在哺乳细胞中也能发生结合。过表达Nogo-B可明显诱导HEK293细胞凋亡，与Clusterin共表达可下调早期细胞凋亡率，但后期Nogo—B可通过调节Clusterin由胞浆到胞核转位，进一步诱导细胞凋亡进程。该研究首次提出Nogo—B与Clusterin之间存在结合，且结合参与了Nogo-B诱导的细胞凋亡进程。

Interaction of Nogo-B and Clusterin Functions in Cell Apoptosis

Including inhibition of neurite outgrowth in CNS, neurite outgrowth inhibitor family members may play more essential functional roles in vivo, because of their widely location in tissues and difference expression variance with certain live affairs such as development and cancer. In this research, we want to find new downstream interaction molecules with Nogo-B, and try to seek the function of iteraction of these two molecules in vivo. Bait pro- tein plasmid was constructed to screen human brain cDNA library and immuno- coprecipitation was used to find new downstream interacted molecules with Nogo-B. Flow Cytometer test was carried out to analyse function of the inter- action in cell apoptosis. Combined methods of green fluorescence protein tagging and immunochemistry were used to find mechanism ofapoptosis induced by Nogo-B. Results suggest that Nogo-66 domain and Clusterin may interact with each other in yeast two-hybrid system. Interaction of Nogo-B and clusterin in mammalian cell could even been identified. Nogo-B could induce HEK293 cell apotosis obviously. Although co-expression of Nogo-B and Clusterin could moderately reduce the rate of HEK293 cell apoptosis in early stage, co-expression couldn＇t resist the apoptosis induced by Nogo-B. Moreover, Nogo-B might induce cell apoptosis by modulate Clusterin translocation from cell plasma to nuclei during apoptosis process. Totoally speaking, from this research, we firstly found the interaction of Nogo-B and Clusterin, which might play an important role in apoptosis process induced by Nogo-B.