北京大学学报(医学版) ›› 2021, Vol. 53 ›› Issue (2): 235-239. doi: 10.19723/j.issn.1671-167X.2021.02.001

• 论著 •    下一篇

基因沉默肽基精氨酸脱亚胺酶4的表达对胶原诱导关节炎小鼠肺间质病变的影响

赵凯,常志芳,王志华,庞春艳(),王永福()   

  1. 内蒙古科技大学包头医学院第一附属医院风湿免疫科,内蒙古自治区自体免疫学重点实验室, 内蒙古包头 014010
  • 收稿日期:2019-04-22 出版日期:2021-04-18 发布日期:2021-04-21
  • 通讯作者: 庞春艳,王永福 E-mail:pchy_fighting@163.com;wyf5168@hotmail.com
  • 基金资助:
    国家自然科学基金(81560270)

Therapeutic effect of gene silencing peptidyl arginine deaminase 4 on pulmonary interstitial lesions induced by collagen-induced arthritis mice

ZHAO Kai,CHANG Zhi-fang,WANG Zhi-hua,PANG Chun-yan(),WANG Yong-fu()   

  1. Department of Rheumatism and Immunology, the First Affiliated Hospital of Baotou Medical College, Inner Monolia University of Secience and Technology, Inner Mongolia Autoimmunology Key Laboratory, Baotou 014010, Inner Mongolia, China
  • Received:2019-04-22 Online:2021-04-18 Published:2021-04-21
  • Contact: Chun-yan PANG,Yong-fu WANG E-mail:pchy_fighting@163.com;wyf5168@hotmail.com
  • Supported by:
    National Nature Science Foundation of China(81560270)

摘要:

目的: 研究基因沉默肽基精氨酸脱亚胺酶 4 (peptidyl arginine deaminase 4,PAD4)的表达对胶原诱导的关节炎(collagen-induced arthritis,CIA)小鼠肺间质病变的治疗作用及可能机制。方法: DBA/1小鼠建立CIA模型,尾静脉注射PAD4-siRNA表达载体制备的病毒液,每周1次,共8次。处死小鼠,实时荧光定量PCR(qRT-PCR)法检测肺中PAD4 mRNA的表达水平;免疫组织化学法检测PAD4蛋白的表达;取脾组织进行细胞培养,流式细胞术检测Tfh细胞和Tfr细胞比例的变化;HE染色观察肺病理学的改变。结果: (1)与空白组比较,模型组小鼠肺组织PAD4 mRNA的表达水平增加,差异有统计学意义(P<0.05), PAD4-siRNA治疗后CIA小鼠肺组织中PAD4 mRNA的表达水平较模型组和阴性对照组明显减少,差异有统计学意义(P<0.05);(2)空白组小鼠肺组织红色荧光较少,而模型组和阴性对照组小鼠肺组织的炎细胞浸润区和气管周围可见较多的红色荧光,PAD4-siRNA治疗后3组的红色荧光明显减少;(3)与空白组比较,模型组脾细胞中Tfh细胞比例升高,差异有统计学意义(P<0.05),PAD4-siRNA治疗后CIA小鼠脾细胞中Tfh细胞比例较模型组和阴性对照组明显降低,差异有统计学意义(P<0.05);与空白组比较,模型组小鼠脾细胞中Tfr细胞比例略有降低,但差异无统计学意义,PAD4-siRNA治疗后小鼠脾细胞中Tfr细胞比例升高,但只有PAD4-siRNA2组与模型组和阴性对照组比较差异有统计学意义(P < 0.05);(4)模型组脾细胞中Tfh/Tfr值升高,与空白组比较差异有统计学意义(P < 0.05);PAD4-siRNA治疗后3组Tfh/Tfr值均下降,差异有统计学意义(P < 0.05);(5)与空白组比较,模型组小鼠肺组织的肺泡壁增厚,炎性细胞浸润增加,PAD4-siRNA治疗后CIA小鼠的肺组织破坏及炎性浸润减少,纤维化程度减轻。结论: 基因沉默PAD4的表达可以降低Tfh细胞的比例,升高Tfr细胞的比例,逆转Tfh/Tfr值,减轻肺组织的间质病变程度和炎症浸润程度。

关键词: 类风湿关节炎, 肽基精氨酸脱亚胺酶4, 基因沉默, 滤泡辅助性T细胞, 滤泡调节性T细胞

Abstract:

Objective: To investigate the therapeutic effect of gene silencing peptidyl arginine deaminase 4 (PAD4) on pulmonary interstitial lesions induced by collagen-induced arthritis (CIA) mice, and possible mechanisms. Methods: A CIA mouse model was established in DBA/1 mice, followed by a tail vein injection of the virus solution prepared by the PAD4-siRNA expression vector once a week for 8 times. The mice were sacrificed at the end of the experiment. The expression of PAD4 mRNA in lungs was detected by real-time quantitative PCR (qRT-PCR). The expression of PAD4 protein was detected by tissue immunohistochemistry. Cell culture was performed by spleen tissue. Flow cytometry changes in the ratio of Tfh cells to Tfr cells were examined; lung staining was performed in the lungs to observe changes in lung pathology. Results: (1) Compared with the blank group, the expression of PAD4 mRNA in the lung tissue of the model group increased, the difference was statistically significant (P< 0.05). PAD4 mRNA in the lung tissue of the CIA mice after PAD4-siRNA treatment. The expression level was significantly lower than that of the model group and the negative control group, and the difference was statistically significant (P<0.05). (2) Red fluorescence was less in the lung tissue of the blank group, while more red fluorescence was observed in the inflammatory cell infiltration area and trachea around the lung tissue of the model group and the negative control group, and the red fluorescence of the three groups after PAD4-siRNA treatment was significantly reduced; (3) Compared with the blank group, the proportion of Tfh cells in the model group increased, the difference was statistically significant (P < 0.05), the proportion of Tfh cells in spleen cells of the CIA mice after PAD4-siRNA treatment was significantly lower than that of the model group and the negative control group, the difference was statistically significant (P < 0.05); compared with the blank group, in the mouse spleen cells in the model group the proportion of Tfr cells was slightly decreased, but the difference was not statistically signifi-cant. The proportion of Tfr cells in the spleen cells of the mice increased after PAD4-siRNA treatment, but the difference was statistically significant only in the PAD4-siRNA2 group compared with the model group and the negative control group (P<0.05); (4) The proportion of Tfh/Tfr in the spleen cells of the model group was increased, compared with the blank group, the difference was statistically significant (P<0.05); the ratio of Tfh/Tfr in the three groups after PAD4-siRNA treatment all decreased, the difference was statistically significant (P<0.05); (5) Compared with the blank group, the alveolar wall of the lung tissue of the model group was thickened, the inflammatory cell infiltration was increased, and the lung tissue destruction and inflammatory infiltration of the CIA mice were decreased after PAD4-siRNA treatment. The degree of reduction was reduced. Conclusion: Gene silencing of PAD4 can reduce the proportion of Tfh cells, increase the proportion of Tfr cells, reverse the proportion of Tfh/Tfr, and reduce the degree of interstitial lesions and inflammatory infiltration of lung tissue.

Key words: Rheumatoid arthritis, Peptidyl arginine deaminase 4, Gene silencing, Follicular helper T cells, Follicular regulatory T cell

中图分类号: 

  • R593.22

图1

小鼠肺中PAD4蛋白的表达"

图2

小鼠脾细胞中Tfh细胞的比例"

图3

小鼠脾细胞中Tfr细胞的比例"

图4

小鼠脾细胞中Tfh/Tfr值的变化"

图5

小鼠肺的组织病理学改变"

[1] Haridas V, Shetty P, Sarathkumar E, et al. Reciprocal regulation of pro-inflammatory Annexin A2 and anti-inflammatory Annexin A1 in the pathogenesis of rheumatoid arthritis[J]. Mol Biol Rep, 2019,46(1):83-95.
doi: 10.1007/s11033-018-4448-5 pmid: 30426384
[2] Yuzhalin AE, Gordon-Weeks AN, Tognoli ML, et al. Colorectal cancer liver metastatic growth depends on PAD4-driver citrullination of the extracellular matrix[J]. Nat Commun, 2018,9(1):4783.
pmid: 30429478
[3] 郭靖, 钱龙, 李向培, 等. 类风湿关节炎患者外周血单个核细胞肽酰基精氨酸脱亚氨酶4表达及组蛋白甲基化水平[J]. 中华内科杂志, 2013,52(11):928-931.
[4] Thanapati S, Ganu M, Giri P, et al. Impaired NK cell functiona-lity and increased TNF-α production as biomarkers of chronic chikungunya arthritis and rheumatoid arthritis[J]. Hum Immunol, 2017,78(4):370-374.
pmid: 28213049
[5] 王建. Th细胞在类风湿关节炎发病作用的研究进展[J]. 临床与病理杂志, 2015,35(2):263-266.
[6] Xu B, Li J, Wu C, et al. CXCL10 and TRAIL are upregulated by TXNDC5 in rheumatoid arthritis fibroblast-like synoviocytes[J]. J Rheumatol, 2018,45(3):335-340.
doi: 10.3899/jrheum.170170 pmid: 29247155
[7] 刘倩, 杨宇, 陈立, 等. 血小板源性生长因子及其受体在类风湿性关节炎大鼠肺组织的表达及与风湿肺关系的探讨[J]. 中国医科大学学报, 2002,31(1):11-14.
[8] Darrah E, Giles JT, Davis RL, et al. Autoantibodies to peptidylarginine deiminase 2 are associated with less severe disease in rheumatoid arthritis[J]. Front Immunol, 2018,9:2696.
doi: 10.3389/fimmu.2018.02696 pmid: 30515171
[9] Apa I, Saliba D, Ponzoni M, et al. TFH-derived dopamine accelelates productive synapses in germinal centres[J]. Nature, 2017,547(7663):318-323.
doi: 10.1038/nature23013 pmid: 28700579
[10] 邹晓月, 熊御云, 张龙锋, 等. 类风湿关节炎患者外周血滤泡辅助性T淋巴细胞百分率的变化及意义[J]. 重庆医学, 2017,46(35):4920-4922.
[11] Wang X, Yang C, Xu F, et al. Imbalance of circulating Tfr/Tfh ratio in patients with rheumatoid arthritis[J]. Clin Exp Med, 2019,19(1):55-64.
[12] Hou S, Clement RL, Diallo A, et al. FoxP3 and Ezh2 regulate Tfr cell suppressive function and transcriptional program[J]. J Exp Med, 2019,216(3):605-620.
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