尼古丁对BV-2小胶质细胞α7烟碱型乙酰胆碱受体、小胶质细胞P2X4受体表达和脑源性神经营养因子释放的影响

Effect of nicotine on the expression of α7 nicotine acetylcholine receptor, P2X4 receptor and brain derived neurotrophic factor in BV-2 cells

目的观察尼古丁对BV-2小胶质α7烟碱型乙酰胆碱受体（α7nicotine acetyleholine receptor，α7nAChR）、小胶质细胞P2X4受体（P2X4receptor，P2X4R）表达和脑源性神经营养因子（brain derived neurotrophic factor,BDNF）释放的影响，以探讨小胶质细胞参与尼古丁所致痛觉过敏的可能分子机制。方法①BV-2小胶质细胞接种在含12mm圆形盖玻片无菌12孔板，待细胞处于对数生长期，完全随机分为2组：α7nAChR组和P2X4R组。免疫荧光标记检测α7nAChR和P2X4R在BV-2小胶质细胞上的表达。②当12孔板中BV-2细胞处于对数生长期，完全随机分为4组：尼古丁实验组（N组），尼古丁终浓度为100μm／L；无血清培养基培养的空白对照组（C组）；尼古丁拮抗剂组（NM组），10nmol／L甲基牛扁碱（methyllycaconitine，MLA）预孵育30min，改用含尼古丁的无血清培养基培养；单纯拮抗剂组（M组），10nmol／LMLA预孵育30min，改用无血清培养基培养。培养72h后收集各组细胞。应用实时荧光定量PCR（real-timequantitativePCR，RT-qPCR）检测α7nAChRmRNA和P2X4RmRNA表达量的变化，Westernblot检测α7nAChR和P2X4R蛋白表达量的变化。③当12孔板中细胞处于对数生长期，完全随机分为4组：正常对照组（Control组）、尼古丁预处理＋DMEM组（Nicotine＋DMEM组）、尼古丁预处理＋激动剂组（Nieotine＋ATP组），尼古丁预处理＋拮抗剂组（Nicotine＋5-BDBD组）。其中激动剂和拮抗剂由DMEM无血清培养基稀释，在尼古丁处理72h后加入，各组总体积保持一致，24h后ELISA检测培养液中BDNF释放量。结果①免疫荧光标记结果显示BV-2细胞存在α7nAChR和P2X4R的阳性表达。②RT-qPCR结果显示尼古丁可上调BV-2细胞α7nAChRmRNA和P2X4RmRNA的表达，α7nAChR特异性拮抗剂MLA可抑制α7nAChRmRNA和P2X4RmRNA表达的上调；Westernblot结果显示尼古丁处理可使BV-2细胞α7nAChR和P2X4R蛋白的表达上调，α7nAChR特异性拮抗剂MLA可抑制α7nAChR和P2X4R蛋白表达的上调。③ELISA检测培养液中BDNF含量结果显示：Nieofine＋ATP组较Nieofine＋DMEM组和Control组显著增多（P〈0．05），Nicotine＋DMEM组较Control组增多（P〈0．05）；Nicotine＋5-BDBD组较Nicotine＋DMEM组和Control组显著减少（P〈0．05）.结论尼古丁可能通过α7nAChR上调小胶质细胞上P2X4R的表达进而通过BDNF的释放引起痛觉过敏的产生。

Objective To observe the expression of α7 nicotine acetylcholine receptor （α7nAChR）, P2X4 receptor（P2X4R）and brain derived neurotrophic factor （BDNF） in BV-2 cells after nicotine treatment, and to investigate the molecular mechanism of the involvement of microglia in the nicotine induced pain hypersensitivity. Methods ① BV-2 mieroglial cells were seeded on 12 hole plate that containing 12 mm circular sterile coverslips, the cells in the logarithmic phase were randomly divided into 2 groups： α7nAChR group and P2X4R group. Expression of α7nAChR and P2X4R in BV-2 cells was detected by immunofluorescence staining. ② When BV-2 ceils of 12 well plate in the logarithm growth phase, the eells were randomly divided into 4 groups： nicotine treatment group（Group N）, nicotine at a final eoncentration of 100 μmol/L, serum free medium was used as a control group （Group C）, nicotine antagonists group（Group NM）, pretreatment ceils with methyllycaconitine（MLA） 30 min at a final concentration of 10 nmol/L, then use nicotine treatment cells, simple antagonist group （group M）, pretreatment cells with MLA 30 min at a final concentration of 10 nmol/L, then use serum free medium treatment cells, after 72 h, real-time quantitative PCR（RT-qPCR） was used to detect the expression of ot7nAChR mRNA and P2X4R mRNA, and the expression of α7nAChR and P2X4R protein was detected by Western blot. ③ When BV-2 cells of 12 well plate in the logarithm growth phase, the cells were randomly divided into 4 groups： normal control group （Control group） , nicotine pretreatment＋DMEM group （Nicotine＋DMEM group）,nieotine pretreatment＋ agonist group（Nicotine＋ATP group） , nicotine pretreatmem＋antagonist group（Nicotine＋5-BDBD group）. Agonist and antagonist diluted by DMEM medium without serum. After nicotine treatment 72 h add agonist and antagonist. BDNF released in eultured solution were detected by ELISA after 24 h. Results ① The expression of α7nAChR and P2X4R was founded in BV-2 cells by immunofluorescence staining. ② RT-qPCR results showed that nicotine can up-regulate the expression of α7nAChR mRNA and P2X4R mRNA in BV-2 ceils, and this up-regulation could be inhibited by MLA, a specific antagonist of α7nAChR. Western blot results showed that nicotine can up-regulate the expression of α7nAChR and P2X4R protein in BV-2 cells, and this up-regulation could be inhibited by MLA. ③ ELISA results showed that the content of BDNF in the culture media, the Nicotine＋ATP group was significantly increased compared with the Control group and the Nieotine＋DMEM group, the Nicotine＋DMEM group was increased significantly compared with the Control group, the Nicotine＋5-BDBD group was lower than that in the Nicotine＋DMEM group and the control group. Conclusions Nicotine may increase the expression of P2X4R through α7nAChR, and then through the release of BDNF to cause hypersensitivity.