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浙江大学学报（医学版）
浙江大学学报（医学版）  2018, Vol. 47 Issue (4): 381-388    DOI: 10.3785/j.issn.1008-9292.2018.08.09
原著     
类风湿关节炎患者外周血单个核细胞中c-FLIP与外源性凋亡途径的相关性分析
林美娜1(),许瑞元2,章涛3,张琳4,梅序桥2,*()
1. 漳州卫生职业学院医学技术系, 福建 漳州 363000
2. 福建医科大学附属漳州市医院检验科, 福建 漳州 363000
3. 福建医科大学基础医学免疫系, 福建 福州 350001
4. 武汉大学人民医院产科, 湖北 武汉 430060
Expression of c-FLIP in peripheral blood mononuclear cells of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway
LIN Meina1(),XU Ruiyuan2,ZHANG Tao3,ZHANG Lin4,MEI Xuqiao2,*()
1. School of Medical Technology, Zhangzhou Health Vocational College, Zhangzhou 363000, Fujian Province, China
2. Clinical Laboratory, Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000, Fujian Province, China
3. Department of Immunology, Fujian Medical University, Fuzhou 350001, China
4. Department of Obstetrics, Renmin Hospital of Wuhan University, Wuhan 430060, China
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摘要：

目的: 分析类风湿关节炎患者外周血单个核细胞（PBMC）中Fas相关死亡区样白介素1转换酶抑制蛋白（c-FLIP）与外源性凋亡途径相关蛋白死亡结构域蛋白（FADD）、caspase-8表达水平变化的相关性。方法: 选择2014年1月至2015年6月福建医科大学附属漳州市医院类风湿关节炎患者60例，按照类风湿关节炎疾病活动指数将患者分为低、中、高活动度组（分别为22、20和18例）。同时选择健康体检者25名作为健康对照组。采用实时定量RT-PCR和蛋白质印迹法分别检测PBMC中c-FLIP、FADD和caspase-8的mRNA和蛋白表达水平，并采用Pearson检验对c-FLIP与FADD、caspase-8表达的相关性进行分析。结果: 类风湿关节炎患者PBMC中c-FLIP、FADD和caspase-8的mRNA表达量均高于健康对照组（均P < 0.05）。其中，中活动度组PBMC中FADD和caspase-8的mRNA表达量高于低活动度组（均P < 0.05），c-FLIP的mRNA表达量与低活动度组相近（P>0.05）；高活动度组PBMC中c-FLIP的mRNA表达量高于中、低活动度组（均P < 0.05），caspase-8的mRNA表达量低于中、低活动度组（均P < 0.05），FADD的mRNA表达量高于低活动度组（P < 0.05）。类风湿关节炎患者PBMC中c-FLIP mRNA水平与FADD mRNA水平呈正相关（r=0.323，P < 0.05），与caspase-8 mRNA水平呈负相关（r=-1.104，P < 0.05）。中活动度组c-FLIP和FADD蛋白表达量高于健康对照组、低活动度组和高活动度组（P < 0.05或P < 0.01）；中、高活动度组caspase-8蛋白表达量高于健康对照组和低活动度组（P < 0.05或P < 0.01），且高活动度组caspase-8蛋白表达量高于中活动度组（P < 0.05）。结论: 类风湿关节炎患者PBMC中凋亡相关蛋白c-FLIP可能参与了外源性凋亡途径，并可为类风湿关节炎病情评估提供参考。
关键词： 关节炎, 类风湿/病理生理学;  单核细胞/代谢;  Fas相关死亡结构域蛋白质/代谢;  凋亡调节蛋白质类;  CASP8和FADD样细胞凋亡调节蛋白质/生理学;  细胞凋亡    
Abstract:

Objective: To investigate the expression of apoptosis related protein cellular Fas associated death domain like interleukin 1 converting enzyme inhibitory protein (c-FLIP) in peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway. Methods: Sixty patients with rheumatoid arthritis were collected from Zhangzhou Affiliated Hospital of Fujian Medical University during January 2014 and June 2015, including 22 patients with low activities (DAS28 < 3.2), 20 patients with middle activities (3.2 ≤ DAS28 ≤ 5.1), and 18 patients with high activities (DAS28>5.1). And 25 healthy controls were also collected. The mRNA and protein expression levels of c-FLIP and the extrinsic apoptotic pathway related proteins Fas-associated protein with death domain (FADD), caspase-8 in PBMCs were detected by real-time RT-PCR and Western blot, respectively. Correlations between c-FLIP and FADD, caspase-8 in PBMCs were analyzed by pearson test. Results: mRNA expression levels of c-FLIP, FADD and caspase-8 in PBMCs of patients with rheumatoid arthritis were all higher than those of healthy controls (all P < 0.05). mRNA expression levels of FADD and caspase-8 in patients with middle activities were significantly higher than those in patients with low activities (all P < 0.05), but the mRNA expression level of c-FLIP was not significantly higher than that in patients with low activities. mRNA expression level of c-FLIP in patients with high activities was higher than those in patients with middle or low activities (all P < 0.05), while the mRNA expression level of caspase-8 was lower than those in patients with middle or low activities (all P < 0.05). mRNA expression level of FADD in patients with high activities was higher than those in patients with low activities (P < 0.05). Pearson analysis showed that there was a positive correlation between c-FLIP and FADD mRNA expression (r=0.323, P < 0.05), and negative correlation between c-FLIP and caspase-8 mRNA expression (r=-1.104, P < 0.05). The protein expression levels of c-FLIP and FADD in patients with middle activities were significantly higher than those in control group and patients with low or high activities (P < 0.05 or 0.01). The protein expression levels of caspase-8 in patients with middle and high activities were significantly higher than those in control group and patients with low activities (P < 0.05 or P < 0.01), and the protein expression level of caspase-8 in patients with high activities was higher than that in patients with middle activities (P < 0.05). Conclusion: c-FLIP may be involved in the extrinsic apoptotic pathway in rheumatoid arthritis, and can provide reference for the evaluation of disease activities.
Key words: Arthritis, rheumatoid/physiopathology    Monocytes/metabolism    Fas-associated death domain protein/metabolism    Apoptosis regulatory proteins    CASP8 and FADD-like apoptosis regulating protein/physiology    Apoptosis
收稿日期: 2017-12-18 出版日期: 2018-12-04
:  R392.9  
基金资助: 福建省中青年教师教育科研项目(JAT160912);漳州卫生职业学院院本课题(ZYZ201607);漳州市级自然科学基金项目(ZZ2018J41)
通讯作者: 梅序桥     E-mail: lmn_0@163.com;qianyimei@126.com
作者简介: 林美娜(1979-), 女, 硕士, 讲师, 主要从事医学检验教学及免疫学研究; E-mail:lmn_0@163.com; https://orcid.org/0000-0002-4241-301X
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引用本文:

林美娜,许瑞元,章涛,张琳,梅序桥. 类风湿关节炎患者外周血单个核细胞中c-FLIP与外源性凋亡途径的相关性分析[J]. 浙江大学学报（医学版）, 2018, 47(4): 381-388.

LIN Meina,XU Ruiyuan,ZHANG Tao,ZHANG Lin,MEI Xuqiao. Expression of c-FLIP in peripheral blood mononuclear cells of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway. J Zhejiang Univ (Med Sci), 2018, 47(4): 381-388.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.08.09    或    http://www.zjujournals.com/med/CN/Y2018/V47/I4/381

引物名称 引物序列
c-FLIP 正向：3′-TTTACCACCCAGAGACACGC-5′
反向：5′-AGAACCTCTGCCTGCTGAAC-3′
FADD 正向：3′-GTGAAGACCCAGCAGGAAGC-5′
反向：5′-ACGCCACAGTGGTTGAGCAT-3′
caspase-8 正向：3′-TGATGAAGAGGCTCTGAGTAA-5′
反向：5′-TGGCAAAGTGACTGGATATA-3′
GAPDH 正向：3′-AATGAAGGGGTCATTGATGG-5′
反向：5′-AAGGTGAAGGTCGGAGTCAA-3′
表 1  实时定量RT-PCR引物序列
(${\bar x}$±s)
组别 n c-FLIP FADD caspase-8
c-FLIP：Fas相关死亡区样白介素1转换酶抑制蛋白；FADD：Fas相关死亡结构域蛋白.与健康对照组比较, *P＜0.05；与低活动度组比较, #P＜0.05；与中活动度组比较, △P＜0.05.
健康对照组 25 1.08±0.12 0.44±0.06 1.04±0.13
低活动度组 22 1.27±0.28* 0.70±0.16* 2.77±0.97*
中活动度组 20 1.32±0.34* 1.02±0.24*# 4.52±0.85*#
高活动度组 18 1.93±0.40*#△ 1.11±0.18*# 2.13±0.44*#△
表 2  各组c-FLIP、FADD和caspase-8 mRNA表达量比较
图 1  c-FLIP与FADD和caspase-8 mRNA水平的相关性分析
图 2  各组c-FLIP、FADD和caspase-8蛋白表达电泳图
(${\bar x}$±s)
组别 n c-FLIP FADD caspase-8
c-FLIP：Fas相关死亡区样白介素1转换酶抑制蛋白；FADD：Fas相关死亡结构域蛋白.与健康对照组比较，*P＜0.05, * *P＜0.01；与低活动组比较，#P＜0.05；与中活动组比较，△P＜0.05.
健康对照组 25 1.00±0.07 1.00±0.04 1.00±0.05
低活动度组 22 0.97±0.45 0.61±0.33 1.25±0.44
中活动度组 20 5.10±1.75**# 4.21±1.59*# 15.14±7.50**#
高活动度组 18 0.82±0.05△ 1.51±0.81*#△ 120.68±55.28**#△
表 3  各组c-FLIP、FADD和caspase-8蛋白表达量比较
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