沉默Annexin-A1对小胶质细胞BV-2生长和迁移能力的影响及其机制  

Effects of Annexin-A1 gene silencing induced by siRNA on the growth and migration of BV-2 cells and its possible mechanisms

作　　者：魏礼清[1] 刘璐[2] 丁钟欢 肖潇[1] 施静[2] 卢忠心[1] 罗振钊[1] 

机构地区：[1]武汉市中心医院检验科,430014 [2]华中科技大学同济医学院基础医学院神经生物学系,武汉430030

出　　处：《中华微生物学和免疫学杂志》2016年第3期207-212,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金面上项目（31171029）;武汉市中心医院博士基金（YB15804）

摘　　要：目的 探讨沉默Annexin-A1（Anxa1）基因对小胶质细胞系BV-2细胞生长、迁移能力的影响及其可能机制.方法 采用RT-PCR和Western blot法从基因和蛋白水平验证小干扰RNA（siRNA）对小胶质细胞系BV-2细胞内Anxa1的沉默效率,采用MTT法检测Anxa1 siRNA对BV-2细胞增殖的作用,膜联蛋白Ⅴ/碘化丙啶（AnnexinⅤ/PI）双染流式细胞术检测Anxa1 siRNA对BV-2细胞凋亡的影响,应用Transwell小室检测Anxa1 siRNA对BV-2细胞迁移能力的影响,Western blot法检测细胞周期和迁移相关信号通路蛋白的表达.结果 Anxa1 siRNA干扰组在24 h、48 h、72 h细胞生长抑制率分别为（16.9±2.1）%、（23.1±3.6）%和（42.4±1.7）%,与siRNA-NC组[分别为（1.35±0.5）%、（2.06±0.7）%和（8.65±0.9）%]比较,差异有统计学意义（P〈0.05）,沉默Anxa1对BV-2细胞的增殖发挥抑制作用;转染后48 h Anxa1 siRNA组细胞凋亡率为（18.4±2.1）%,明显高于siRNA-NC组（5.2±0.3）%和空白对照组（4.3±0.2）%;Anxa1 siRNA干扰组细胞迁移能力（28.7±5.2）明显低于siRNA-NC组（173.4±11.4,P〈0.01）,Anxa1 siRNA干扰可显著下调细胞周期蛋白Cyclin D1的表达水平和激活p38与JNK信号通路.结论 siRNA沉默Anxa1基因可抑制小胶质细胞的生长、迁移能力,分别通过下调Cyclin D1和激活p38与JNK信号通路来实现.Objective To investigate the effects of Annexin-A1 （ Anxa1 ） gene silencing induced by siRNA on the growth and migration of microglial BV-2 cells and its possible mechanisms.Methods A synthesized siRNA duplex targeting Anxa1 gene was transfected into BV-2 cells.The efficiency of siRNA-in-duced Anxa1 gene silencing was evaluated on both mRNA and protein levels by using reverse-transcription PCR and Western blot assay.MTT assay was performed to measure the proliferation of BV-2 cells with si-lenced expression of Anxa1 gene.Flow cytometry with Annexin V-FITC/PI double staining was used to de-tect the apoptosis rate of BV-2 cells.Transwell chambers were used to analyze the effects of siRNA-induced Anxa1 gene silencing on the migration of BV-2 cells.Western blot assay was performed to detect the expres-sion of signaling proteins related to cell cycle and migration.Results Compared with the siRNA negative control （ siRNA-NC） group, the inhibitory rates of siRNA-induced Anxa1 gene silencing on the proliferation of BV-2 cells were significantly increased at the time points of 24 h, 48 h and 72 h after intervention [（16.9 ±2.1）%, （23.1±3.6）%and （42.4±1.7）%vs （1.35±0.5）%, （2.06±0.7）% and （8.65±0.9）%, P〈0.05 ].The apoptosis rate of BV-2 cells transfected with Anxa1 siRNA was （18.4±2.1）%, which was significantly elevated as compared with that of the siRNA-NC group （5.2±0.3）%and control group （4.3±0.2）%.Cell migration of the Anxa1 siRNA transfected BV-2 cells was inhibited remarkably at 48 h as com-pared with that of the siRNA-NC group （28.7±5.2 vs 173.4±11.4, P〈0.01）.Moreover, the suppressed expression of Cyclin D1 protein and activation of p38 and JNK signaling pathways were induced by silenced expression of Anxa1 gene in BV-2 cells.Conclusion The growth and migration of BV-2 cells were signifi-cantly inhibited by silencing the expression of Anxa1 gene with siRNA, the possible mechanisms might be associated with the suppressed expression of Cyclin D1protein and

关 键 词：RNA干扰 Annexin-A1 BV-2细胞 细胞生长 迁移能力 Annexin-A1 

分 类 号：R741[医药卫生—神经病学与精神病学]