食欲素A对阿尔茨海默病细胞模型存活率的影响及作用机制

Protective role and mechanism of orexin A in cell viability of Alzheimer＇s disease cell model PC12

目的探讨食欲素A对阿尔茨海默病（AD）细胞模型存活率的影响及机制。方法分别以0、10、20、30、40、50μmol／Lβ-淀粉样蛋白25-35（Aβ25-35）处理PCI2细胞24h，筛选合适的Aβ25-35，浓度用于构建AD细胞模型。建模成功后实验分为3部分：（1）分别加入0（对照组）、0．01、0．1、1、2μmol／L食欲素A预处理细胞24h，然后以30μmol／LAβ25-35处理细胞24h，通过四唑盐（MTT）比色法测定细胞存活率，确定食欲素A适宜干预浓度。（2）采用倒置相差显微镜对对照组、30μmol／LAβ25-35，处理组、0．01μmol／L食欲素预处理组细胞形态进行观察。（3）观察细胞加入食欲素A受体抑制剂SB408124预处理2h后细胞存活率的变化。结果（1）30μmol／LAβ25-35为构建AD细胞模型的最适浓度。不同浓度食欲素A预处理AD细胞模型后，各组细胞存活率差异有统计学意义（F=27．120，P=-0．000），0．01μmol／L食欲素A为适宜干预浓度。（2）30μmol／LAβ25-35处理组部分细胞突起断离，胞体受损。0．01μmol／L食欲素A预处理组细胞突起断离，胞体受损较30μmol／LA1325-35处理组程度减轻。（3）设对照组细胞存活率为100％，加入食欲素A受体抑制剂SB408124后，细胞存活率为（109．10±0．36）％，较0．01μmol/L食欲素A处理组细胞存活率（117．24±2．72）％明显降低，差异有统计学意义（m0．05）。结论食欲素A可通过与受体结合提高AD细胞模型的存活率．可能成为AD防治的一个重要靶点。

Objective To study the role and mechanism of orexin A in cell viability of Alzheimer＇s disease （AD） cell model PC12. Methods PC 12 cells were treated with 0, 10, 20, 30, 40 and 50 μmol/L Aβ25-35 for 24 h, and then, cell viability was measured by MTT to confirm which concentration was the suitable one to establish the AD cell models. （1） AD cell models were treated with 0, 0.01, 0.1, 1 and 2 μmol/L orexin A for 24 h, and then, 30 μmol/L Aβ25~.35 was added for 24 h; MTT assay was used to determine the cell viability to conform the suitable concentration of orexin A. （2） Inverted phase contrast microscope was employed to observe the morphology changes of PC 12 cells from the control group, 30 p, mol/L Aβ25-35 treatment group, and 0.01 μmol/L orexin A＋30 μmol/L A^25-35 treatment group. （3） The PC12 cells were given pretreatment of orexin A receptor inhibitor SB408124 for 2 h, and cell viability was detected. Results （1） Aβ25~35 at concentration 30 μmol/L was the suitable one to establish the AD cell models; after being pretreated with different concentrations of orexin A, the cell viability showed significant differences （F=27.120, P--0.000）, and 0.01 μmol/L orexin A was the suitable concentration. （2） Some of the cells from the 30 p＋mol/L Aβ25-35 treatment group had breaking-off of protuberance and damaged soma; cells from 0.01 p＋mol/L orexin A＋30 txmol/L Aβ25~35 treatment group had breaking-off of protuberance, and the degree of damaged soma was eased as compared with that in the 30 ixmol/L Aβ25-35 treatment group. （3） If the cell viability of the control group was 100%, cell viability oforexin A receptor inhibitor group was 109.10%±0.36%, which was significantly decreased as compared with that in the 0.01 mol/L orexin A pretreated group （117.24%±2.72%, P〈0.05）. Conclusion Orexin A can improve the cell viability via combination of its specific receptor; orexin A and its specific receptor may be new targets for prevention and cure of AD.