siRNA沉默ASIC1a基因对佐剂性关节炎大鼠关节软骨细胞凋亡的影响研究

目的: 探讨小分子干扰RNA(siRNA)技术诱导佐剂性关节炎(adjuvant-induced arthritis,AA)大鼠关节软骨细胞中ASIC1a表达沉默对细胞凋亡的影响。方法: 通过化学合成法合成特异性荧光短链ASIC1a siRNA-FAM,使用Lipofectamine 2000转染试剂盒将ASIC1a siRNA转染入关节软骨细胞,采用荧光显微镜、流式细胞术、实时荧光定量PCR(q-RT-PCR)及Western Blot法检测siRNA转染效率及其对ASIC1a mRNA和蛋白表达的抑制作用。同时采用Annexin-V /PI流式细胞术检测各组细胞凋亡情况。结果: ASIC1a siRNA能成功转入软骨细胞,转染后AA大鼠关节软骨细胞中ASIC1a mRNA表达显著低于对照组 (P<0.01),最大抑制率为85.4 %;Western Blot结果显示,转染特异性siRNA后ASIC1a蛋白表达明显低于对照组(P<0.01)。Annexin-V /PI流式细胞术结果表明,与模型组相比,siRNA-3转染引起ASIC1a表达沉默后AA大鼠软骨细胞凋亡明显减少。结论: siRNA介导的AA大鼠关节软骨细胞ASIC1a表达沉默模型是研究酸敏感离子通道对软骨细胞代谢影响的可靠模型,siRNA-3转染对胞外酸化刺激条件下AA大鼠关节软骨细胞凋亡的保护作用可能与其调节ASIC1a的表达有关。

Effects of ASIC1a gene silencing by siRNA on apoptosis of articular cartilage cells of adjuvant arthritis rats

AIM: To estimate the effects of ASIC1a gene silencing by siRNA on apoptosis in articular cartilage cells of adjuvant arthritis rats. METHODS: Lipofectamine 2000 transfection reagent was used to transfect the small interfering RNA(siRNA) with FAM in articular cartilage cells. The expression level of ASIC1a mRNA and protein were detected by fluorescence microscope,flow cytometry(FCM),real-time PCR and Western blotting, respectively. And the apoptotic cell dyed with Annexin-V and propidium iodide were measured with a flow cytometer. RESULTS: ASIC1a siRNA were transfected into articular cartilage cells successfully. The expression of ASIC1a mRNA and protein in transducted chondrocytes was lower than the control chondrocytes (P<0.01). The rate of apoptosis was lower in the ASIC1a-silenced cell than that in the model cell. CONCLUSION: The silence cells model of ASIC1a expression by siRNA in chondrocytes of adjuvant arthritis rats is the perfect model to observe the effect on metabolism of chondrocytes of the acid-sensing ion channels. Transfection siRNA-3 into articular cartilage cells protected from apoptosis by extracellular acid, which may be related with different expression levels of ASIC1a.