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

中国临床药理学与治疗学 ›› 2012, Vol. 17 ›› Issue (3): 256-262.

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

江晟¹, 陈飞虎¹, 陈寸知², 荣超¹, 胡伟³, 吴繁荣¹, 葛金芳¹, 唐杰¹

¹安徽医科大学药学院,合肥 230032,安徽;
²诺丁汉大学生物科学院, 诺丁汉LE12 5RD,美国;
³安徽医科大学第二附属医院,合肥 230032,安徽

收稿日期:2011-12-09 修回日期:2012-02-16 出版日期:2012-03-26 发布日期:2012-04-20
通讯作者: 陈飞虎,男,博士,教授,博士生导师,研究方向:临床药理学,抗炎免疫药理学。Tel: 0551-5161115, E-mail: cfhchina@sohu.com
作者简介:江晟,女,硕士, 研究方向:分子药理学。Tel: 13505613489, E-mail: jiangsheng0923@163.com
基金资助:
国家自然科学基金(30873080)

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

JIANG Sheng¹, CHEN Fei-hu¹, CHEN Cun-zhi², RONG Chao¹, HU Wei³, WU Fan-rong¹, GE Jing-fang¹, TANG Jie¹

¹School of Pharmacy, Anhui Medical University, Hefei 230032, Anhui, China;
²School of Biosciences, University of Nottingham, Nottingham LE12 5RD, USA;
³the Second Hospital Affiliated to Anhui Medical University, Hefei 230601, Anhui, China

Received:2011-12-09 Revised:2012-02-16 Online:2012-03-26 Published:2012-04-20

摘要/Abstract

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。

关键词: 关节软骨细胞, 酸敏感离子通道(ASICs), 阳离子脂质体, 表达沉默, 细胞凋亡

Abstract: 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.

Key words: Articular cartilage cells, Acid-sensing ion channels (ASICs), Liposomes, Gene silence, Cell apoptosis

中图分类号:

R965.2

江晟, 陈飞虎, 陈寸知, 荣超, 胡伟, 吴繁荣, 葛金芳, 唐杰. 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[J]. 中国临床药理学与治疗学, 2012, 17(3): 256-262.

JIANG Sheng, CHEN Fei-hu, CHEN Cun-zhi, RONG Chao, HU Wei, WU Fan-rong, GE Jing-fang, TANG Jie. Effects of ASIC1a gene silencing by siRNA on apoptosis of articular cartilage cells of adjuvant arthritis rats[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2012, 17(3): 256-262.

参考文献

[1] Jasti J, Furukawa H, Gonzales EB, et al. Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH[J]. Nature, 2007, 449(7160): 316-323.
[2] Xiong ZG, Chu XP, Simon RP. Acid sensing ion channels--novel therapeutic targets for ischemic brain injury[J]. Front Biosci, 2007, 12: 1376-1386.
[3] Yuan FL, Hu W, Lu WG, et al. Targrting interleukin-21 in rheumatoid arthritis[J]. Mol Biol Rep, 2011, 38(3): 1717-1721.
[4] 袁凤来,陈飞虎,李霞,等. 大鼠关节软骨酸敏感离子通道的表达[J]. 安徽医科大学学报,2007, 42(5): 513-516.
[5] 袁凤来,陈飞虎,黄学应,等. 酸敏感离子通道在大鼠佐剂性关节炎关节软骨中的表达[J]. 中华风湿病学杂志,2008, 12(5): 321-324.
[6] 陈飞虎,袁凤来,李霞,等. 几种抗类风湿性关节炎药物对佐剂性关节炎大鼠关节软骨细胞酸敏感离子通道的表达影响及作用[J]. 中国临床药理学与治疗学,2008, 13 (2) :131-137.
[7] 张腾跃,陈飞虎,王念,等. 酸敏感离子通道阻断剂Amiloride对AA大鼠关节软骨细胞凋亡抑制作用的初步研究[J]. 中国临床药理学与治疗学,2010, 15 (1) : 41 -46.
[8] 王颖,蔡刚祥,吴亮. 靶向Survivin基因siRNA诱导肝癌细胞凋亡的实验[J]. 医药导报,2006, 25(7): 648-651.
[9] Liechti LA, Berneche S, Bargeton B, et al. A combined computational and functional approach identifies new residues involved in PH-dependent gating of ASIC1a[J]. J Biol Chem, 2010, 285(21): 16315-16329.
[10] 袁凤来,陈飞虎,陆伟国,等. 酸敏感离子通道阻断剂阿米洛利对佐剂性关节炎大鼠关节软骨损伤的影响[J]. 中国药理学通报,2009, 25(7): 895-900.
[11] Lopes RB, Gangeswaran R,McNeish IA,et al. Expression of the IAP protein family is dysregulated in pancreatic cancer cells and is important for resistance to chemotherapy[J]. Int J Cancer, 2007, 120(11): 2344-2352.
[12] Agrawal N, Dasaradhi PV, Mohmmed A, et al. RNA interference: Biology, mechanism and applications[J]. Microbiol Mol Biol Rev, 2003, 67(4): 657-685.
[13] 朱有凯,林汉良,马怡晖,等. RNA干扰致基因表达沉默细胞模型的建立[J]. 解剖学研究,2005, 27(3): 190-193.
[14] 邹颖刚,薛惠荣,崔满华. 靶向卵巢癌HIF-1a基因siRNA真核表达载体的构建及鉴定[J]. 中国实验诊断学,2010, 14(8):1265-1266.
[15] Yang XH,Sladek TL,Liu X,et al. Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin and etoposide-induced apoptosis[J]. Cancer Res, 2001, 61(1): 348-354.
[16] Yuan FL, Chen FH, Lu WG, et al. Inhibition of acid-sensing ion channels in articular chondrocytes by amiloride attenuates articular cartiage destruction in rats with adjuvant arthritis[J]. Inflamm Res, 2010, 59(11): 939-947.
[17] Yuan FL, Chen FH, Lu WG, et al. Acid-sensing ion channel 1a mediates acid-induced increases in intracellular calcium in rat articular chondrocytes[J]. Mol Cell Biochem, 2010, 340(1/2): 153-159.[18] Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells[J]. Proc Natl Acad Sci USA, 2002, 99(9): 6047-6052.