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
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
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）
