摘要
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ASIC1asiRNA-FAM,使用Lipo-fectamine 2000转染试剂盒将ASIC1asiRNA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WesternBlot法检测siRNA转染效率及其对ASIC1amRNA和蛋白表达的抑制作用。同时采用An-nexin-V/PI流式细胞术检测各组细胞凋亡情况。结果:ASIC1asiRNA能成功转入软骨细胞,转染后AA大鼠关节软骨细胞中ASIC1amRNA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的表达有关。
To estimate the effects of ASICla gene silencing by siRNA on apoptosis in articular cartilage cells of adjuvant arthritis rats. METHODS： Lipofectamine 2000 transfection re- agent was used to transfect the small interfering RNA（siRNA） with FAM in articular cartilage ceils. The expression level of ASICla mRNA and protein were detected by fluorescence micro- scope,flow cytometry（FCM） ,real-time PCR and Western blotting, respectively. And the apop- totic cell dyed with Annexin-V and propidium i- odide were measured with a flow cytometer. RE- SULTS： ASICla siRNA were transfected into ar- ticular cartilage cells successfully. The expres- sion of ASICla mRNA and protein in transduct- ed chondrocytes was lower than the controlchondrocytes （P〈0.01）. The rate of apoptosis was lower in the ASICla-silenced cell than that in the model cell. CONCLUSION： The silence cells model of ASICla expression by siRNA in chondrocytes of adjuvant arthritis rats is the per- fect model to observe the effect on metabolism of chondrocytes of the acid-sensing ion channels. Transfection siRNA-3 into articular cartilage ce ac ls protected from apoptosis by extracellular d, which may be related with different ex- pression levels of ASICla.
出处
《中国临床药理学与治疗学》
CAS
CSCD
2012年第3期256-262,共7页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
国家自然科学基金(30873080)
