Ni Jiong, Wang Pei-jun
Department of Medical Imaging, Tongji Hospital, Tongji University, Shanghai 200065, China.
Zhonghua Yi Xue Za Zhi. 2012 Nov 20;92(43):3085-8.
To investigate the effect of silencing P2X7 receptor (P2X7R) by RNA interference on microglial phagocytosis of amyloid-β (Aβ) protein and to explore its possible mechanism.
The small interfering RNA (siRNA) targeting the P2X7R gene was identified. The microglial cells activated by Aβ1-42 were infected with the Lipofectamine-siP2X7R and it was designated as siP2X7R group. Microglia infected with Lipofectamine-siNC was designated as siNC group and non-infected cells as con group. The levels of P2X7R mRNA were detected by real-time PCR and the P2X7R protein was determined by Western blotting. The levels of IL-1β and TNF-α were measured by ELISA. The microglial phagocytosis of Aβ1-42 was observed by ELISA and immunocytochemistry staining.
Detected by the Real-time PCR, the expression level of P2X7R mRNA of siP2X7R group decreased significantly versus siNC and con groups (P<0.05). The lowered expression of P2X7R protein detected by Western blotting was consistent with Real-time PCR. After RNA interference silencing P2X7R, the levels of IL-1β and TNF-α detected by ELISA in siP2X7R group less than those in con, siNC groups, significantly (P<0.05). In con, siNC and siP2X7R groups respectively, the levels of Aβ1-42 in supernatant were (423±20) pg/ml, (417±16) pg/ml, (296±30) pg/ml and the levels of Aβ1-42 in the microglia were (190±37) pg/ml, (187±39) pg/ml, (322±26) pg/ml. Compared to siNC and con groups, in siP2X7R group the levels of Aβ1-42 in supernatant decreased (P<0.05) and the levels of Aβ1-42 in the microglia increased (P<0.05). Aβ1-42 immunofluorescence staining showed that the red fluorescent products were seen in the cytoplasm of most microglias in siP2X7R group, but in con or siNC groups in only few microglias these products were depicted.
The silence expression of P2X7R by RNA interference effectively decreases the levels of IL-1β and TNF-α released by microglia and promotes microglia to phagocytose Aβ. P2X7R could be used as an effective therapeutic target for RNA interference treatment of Alzheimer's disease.
探讨RNA干扰沉默P2X7受体（P2X7R）对小胶质细胞吞噬β淀粉样蛋白（Aβ）的影响，并探讨其可能机制。
筛选出靶向P2X7R基因的小干扰RNA（siRNA）。用Lipofectamine-siP2X7R感染经Aβ1-42激活的小胶质细胞，设为siP2X7R组。用Lipofectamine-siNC感染的小胶质细胞设为siNC组，未感染细胞设为对照组。采用实时荧光定量PCR检测P2X7R mRNA水平，蛋白质免疫印迹法检测P2X7R蛋白水平。采用酶联免疫吸附测定法（ELISA）检测白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）水平。采用ELISA和免疫细胞化学染色观察小胶质细胞对Aβ1-42的吞噬情况。
实时荧光定量PCR检测显示，siP2X7R组P2X7R mRNA表达水平较siNC组和对照组显著降低（P<0.05）。蛋白质免疫印迹法检测显示P2X7R蛋白表达降低，与实时荧光定量PCR结果一致。RNA干扰沉默P2X7R后，siP2X7R组ELISA检测的IL-1β和TNF-α水平低于对照组和siNC组，差异有统计学意义（P<0.05）。对照组、siNC组和siP2X7R组上清液中Aβ1-42水平分别为（423±20）pg/ml、（417±16）pg/ml、（296±30）pg/ml，小胶质细胞中Aβ1-42水平分别为（190±37）pg/ml、（187±39）pg/ml、（322±26）pg/ml。与siNC组和对照组相比，siP2X7R组上清液中Aβ1-42水平降低（P<0.05），小胶质细胞中Aβ1-42水平升高（P<0.05）。Aβ1-42免疫荧光染色显示，siP2XGR组大多数小胶质细胞胞质内可见红色荧光产物，而对照组和siNC组仅少数小胶质细胞有此产物。
RNA干扰沉默P2X7R表达可有效降低小胶质细胞释放的IL-1β和TNF-α水平，促进小胶质细胞吞噬Aβ。P2X7R可作为RNA干扰治疗阿尔茨海默病的有效治疗靶点。
