用RNA干扰沉默P2X7受体抑制小胶质细胞释放炎性介质的探讨

摘要

Objective To observe the effect of silencing P2X7 receptor (P2X7R) by RNA interference on microglial releasing interleukin 1β (IL-1β),tumor necrosis factor-α (TNF-α) and nitric oxide (NO).Methods The small interfering RNA (siRNA) targeting P2X7R gene was observed.The primary microglial cells activated by amyloid-β (Aβ-42) were infected with the lipofectaminesiRNA.The groups were designated as Aβ-1-42,Aβ1-42/siNC,Aβ1-42/siP2X7R and blank control groups according to the different stimulus.After RNA interference for 48 hours,the microglial cells were collected.The survival rate of microglia was detected by CCK-8.The levels of P2X7R mRNA and protein were detected by Real-time PCR and Western blotting respectively.The microglial morphology and the P2X7R immune response were observed by immunocytochemistry staining.Then levels of IL-1β,TNF-α and NO in the supernatantwere measured.Results The Aβ1-42 and siRNA had no effects on the survival rate of microglia.After RNA interference silencing P2X7R,the expression levels of P2X7R mRNA and protein in Aβ1-42/siP2X7R group were decreased significantly,and in this group,the microglial activity and P2X7R immune response were all reduced.In Aβ1-42,Aβ1-42/siNC,Aβ1-42 /siP2X7R and blank control groups,the supernatant levels of IL-1β were (52.54±3.21) μg L,(54.94±2.54) μg/L,(28.70±3.58) μg/L,(24.55±4.34) μg/L,respectively and the supernatant levels of TNF-α were (64.40±4.80) μg/L,(60.94±1.63) μg/L,(25.69±3.13) μg/l,(19.21±1.97)μg/L,respectively.As compared with Aβ1-42 and Aβ-42/siNC groups,the levels of IL 1β and TNF-α in Aβ1-42 /siP2X7R group was decreased significantly (P＜ 0.05),and IL-1β level had no significant difference between Aβ1-42/siP2X7R and blank control groups.The same result was observed in the level of NOin the supernatant.The levels of NO were (1.33±0.19) μmol/L,(4.49±0.28) μmol/L,(4.78±0.33) μmol/L and (1.07±0.36) μmol/L in Aβ1-42/siP2X7R,Aβ1-42,Aβ1-42 /siNC and blank control groups respectively.Conclusions The silencing expression of P2X7 R by RNA interference effectively decreases the levels of IL-1β,TNF-α and NO released by microglia.P2X7 R can be used as an effective therapeutic target for RNA interference treatment of Alzheimer's disease.%目的 观察RNA干扰(RNAi)沉默小胶质细胞内的P2X7受体(P2X7R)对小胶质细胞释放炎性介质白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和一氧化氮(NO)的影响. 方法 构建靶向P2X7R基因的小干扰RNA(siRNA).以脂质体为转染剂将siRNA转染到经β淀粉样蛋白(Aβ1-42)激活的原代培养的小胶质细胞内.根据刺激物不同分为Aβ1-42、Aβ1-42/siNC、Aβ1-42/siP2X7R 及空白对照组.处理48 h后,收集各组小胶质细胞,用CCK-8检测细胞存活率,用Real-Time PCR和Western blot检测P2X7R的mRNA和蛋白表达水平,用免疫细胞化学染色观察细胞形态及P2X7R免疫反应强度;收集各组细胞上清液测定IL-1β、TNF-α和NO浓度. 结果 Aβ1-42和siRNA对小胶质细胞存活率没有影响,与空白对照组比较差异无统计学意义.RNA干扰后Aβ1-42/siP2X7R组P2X7R的mRNA和蛋白表达均显著下降,免疫细胞化学染色该组小胶质细胞活性和P2X7R免疫反应强度均降低.沉默P2X7R后,Aβ1-42、Aβ1-42/siNC、Aβ1-42/siP2X7R及空白对照组细胞上清液IL-1β浓度分别为(52.54±3.21)μg/L、(54.94±2.54) μg/L、(28.70±3.58)μg/L和(24.55±4.34) μg/L;TNF-α浓度分别为(64.40±4.80)μg/L、(60.94±1.63)μg/L、(25.69±3.13)μg/L和(19.21±1.97)μg/L.Aβ1-42/siP2X7R组细胞上清液IL-1β及TNF-α浓度均低于Aβ1-42和Aβ1-42/siNC组(P＜0.05),与空白对照组比较差异无统计学意义.Aβ1-42/siP2X7R组细胞上清液NO浓度为(1.33±0.19)μmol/L,低于Aβ1-42组(4.49±0.28) μmol/L和Aβ1-42/siNC组(4.78±0.33) μmol/L,与空白对照组(1.07±0.36) μmol/L比较差异无统计学意义. 结论 RNA干扰可有效沉默小胶质细胞内的P2X7R,抑制小胶质细胞释放炎性介质IL-1β、TNF-α和NO.P2X7R有望成为RNA干扰治疗阿尔茨海默病的一个新靶点.