摘要
目的利用小干扰RNA(siRNA)抑制CC趋化因子受体5(CCR5)的表达,以探讨CCR5 siRNA对类风湿关节炎(RA)大鼠滑膜细胞增殖与细胞周期的影响及其可能机制。方法采用胶原诱导法构建RA大鼠模型,分离和培养RA大鼠滑膜细胞,通过Lipofectamine 2000将CCR5 siRNA和NC siRNA载体转染入细胞以判断其转染效率。并将体外培养的滑膜细胞分为4组:RA组(RA大鼠滑膜细胞)、RA+NC组(加入脂质体包裹的NC siRNA)、RA+mock组(加入脂质体)、RA+siRNA组(加入脂质体包裹的CCR5 siRNA)。利用RNA干扰技术抑制RA大鼠滑膜细胞CCR5的表达后,再用qRT-PCR和Western Blot检测各组滑膜细胞CCR5、细胞周期相关基因(Cyclin D1、Cyclin E1、Cyclin B1)mRNA和蛋白表达情况;MTT检测各组滑膜细胞增殖情况;PI单染检测滑膜细胞细胞周期分布情况。结果与RA组比较,RA+siRNA组CCR5 mRNA表达水平(1.16±0.21)比(1.85±0.61)、CCR5蛋白水平(0.19±0.01)比(0.44±0.05)均明显降低(P<0.05);RA+siRNA组Cyclin D1、Cyclin E1、Cyclin B1 mRNA水平(1.32±0.01)、(1.23±0.26)、(1.33±0.05)分别较RA组(1.52±0.06)、(1.68±0.08)、(1.94±0.07)显著降低(P<0.05);RA+siRNA组Cyclin D1、Cyclin E1、Cyclin B1蛋白水平(0.88±0.03)、(1.46±0.13)、(1.56±0.13)分别较RA组(1.38±0.03)、(1.98±0.12)、(2.13±0.08)明显降低(P<0.05);与RA组比较,RA+siRNA组RA大鼠滑膜细胞G0/G1期比例(70.27±1.06)%较(59.26±0.59)%上升(P<0.05),S期比例(14.10±0.82)%较(23.94±1.71)%降低(P<0.05),并且滑膜细胞生长明显受抑,而RA+mock、RA+NC组无明显差别(P>0.05)。结论CCR5基因沉默可抑制滑膜细胞的生长,使G0/G1期滑膜细胞比例增加,S期细胞比例减少,还可通过调节细胞周期相关基因Cyclin D1、Cyclin E1、Cyclin B1的表达来调节细胞周期的分布,进而影响滑膜细胞的增殖。
Objective To explore the effect of CCR5 siRNA on the proliferation and cell cycle of synoviocytes in rats with rheumatoid arthritis(RA)and its possible mechanism by inhibiting the expression of CCR5 with small interfering RNA(siRNA).Methods An RA rat model was constructed by collagen induction method.Synoviocytes of the RA rats were isolated and cultured,in which CCR5 siRNA and negative control(NC)siRNA vectors were then transfected by Lipofectamine 2000 to determine the transfection efficiency.The synoviocytes cultured in vitro were divided into four groups:RA group(synoviocytes of RA rats),RA+NC group(with liposome-encapsulated NC siRNA),RA+mock group(with liposome),and RA+siRNA group(with liposome-encapsulated CCR5 siRNA).After the expression of CCR5 in the synoviocytes of RA rats was inhibited by RNA interference technology,q RT-PCR and Western blot were used to measure the mRNA and protein expression of CCR5 and cell cycle-related genes(Cyclin D1,Cyclin E1,and Cyclin B1)in each group.The proliferation and cell cycle distribution of synoviocytes in each group were determined by MTT and PI staining,respectively.Results Compared with the RA group,the RA+siRNA group showed significantly reduced CCR5 mRNA level(1.16±0.21 vs 1.85±0.61)and CCR5 protein level(0.19±0.01 vs 0.44±0.05)(P<0.05).The mRNA levels of Cyclin D1,Cyclin E1,and Cyclin B1 in the RA+siRNA group(1.32±0.01,1.23±0.26,and 1.33±0.05,respectively)were significantly decreased as compared with those in the RA group(1.52±0.06,1.68±0.08,and 1.94±0.07,respectively)(P<0.05).The protein levels of Cyclin D1,Cyclin E1,and Cyclin B1 in the RA+siRNA group(0.88±0.03,1.46±0.13,and 1.56±0.13,respectively)were significantly lower than those in the RA group(1.38±0.03,1.98±0.12,and 2.13±0.08,respectively)(P<0.05).Compared with the RA group,the RA+siRNA group showed an increased G0/G1 phase ratio of synoviocytes(70.27%±1.06%vs 59.26%±0.59%,P<0.05)and a decreased S phase ratio of synoviocytes(14.10%±0.82%vs 23.94%±1.71%,P<0.05),as well as significantl
作者
王友强
兰由玉
张为
姚娟
邹兴静
刘伟
WANG Youqiang;LAN Youyu;ZHANG Wei;YAO Juan;ZOU Xingjing;LIU Wei(Department of Laboratory Medicine,the Affiliated T.C.M Hospital of Southwest Medical University,Luzhou 646000,China;Department of Rheumatology and Immunology,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)
出处
《西南医科大学学报》
2022年第2期112-117,共6页
Journal of Southwest Medical University
基金
国家自然科学基金(81902169)
西南医科大学校级课题(2019ZQN038)。
