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1. 西南医科大学附属中医医院检验科,泸州,646000
2. 西南医科大学附属医院风湿免疫科,泸州,646000
纸质出版日期:2022,
网络出版日期:2022-3-20,
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王友强, 兰由玉, 张为, 等. CCR5 siRNA对类风湿关节炎大鼠滑膜细胞 增殖周期的影响[J]. 西南医科大学学报, 2022,45(2):112-117.
WANG Youqiang, LAN Youyu, ZHANG Wei, et al. Effect of CCR5 siRNA on proliferation and cell cycle of synoviocytes in rats with rheumatoid arthritis[J]. Journal of Southwest Medical University, 2022,45(2):112-117.
王友强, 兰由玉, 张为, 等. CCR5 siRNA对类风湿关节炎大鼠滑膜细胞 增殖周期的影响[J]. 西南医科大学学报, 2022,45(2):112-117. DOI: 10.3969/j.issn.2096-3351.2022.02.005.
WANG Youqiang, LAN Youyu, ZHANG Wei, et al. Effect of CCR5 siRNA on proliferation and cell cycle of synoviocytes in rats with rheumatoid arthritis[J]. Journal of Southwest Medical University, 2022,45(2):112-117. DOI: 10.3969/j.issn.2096-3351.2022.02.005.
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Cy⁃clin 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 rheu-matoid 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 effi-ciency. The synoviocytes cultured in vitro were divided into four groups: RA group(synoviocytes of RA rats),RA+NC group(with lipo-some-encapsulated NC siRNA),RA+mock group(with liposome),and RA+siRNA group(with liposome-encapsulated CCR5 siR-NA). After the expression of CCR5 in the synoviocytes of RA rats was inhibited by RNA interference technology,qRT-PCR and West-ern blot were used to measure the mRNA and protein expression of CCR5 and cell cycle-related genes(Cyclin D1,Cyclin E1,and Cy-clin B1)in each group. The proliferation and cell cycle distribution of synoviocytes in each group were determined by MTT and PI stain-ing,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 com-pared 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 significantly inhibited growth of synoviocytes, but there were no significant differences in the RA+mock and RA+NC groups(P > 0.05). Conclusion CCR5 gene silencing can inhib-it the growth of synoviocytes,manifesting as increasing the G0/G1 phase ratio of synoviocytes and decreasing the S phase ratio. The cell cycle distribution can be regulated by regulating the expression of cell cycle-related genes(Cyclin D1,Cyclin E1,and Cyclin B1),thus affecting the proliferation of synoviocytes.
类风湿关节炎CCR5基因沉默滑膜细胞细胞增殖细胞周期
Rheumatoid arthritisCCR5Gene silencingSynoviocytesCell proliferationCell cycle
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