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
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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-8mRNA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。
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.
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.
引文
[1]李春莳,孙英焕,杜明,等.凋亡与自噬的相互关系在类风湿关节炎中的研究进展[J].风湿病与关节炎,2017,6(10):65-68.LI Chunshi,SUN Yinghuan,DU Ming,et al.Advances in the study of the relationship between apoptosis and autophagy in rheumatoid arthritis[J].Rheumatism and Arthritis,2017,6(10):65-68.(in Chinese)
[2]DOROSHEVSKAYA A Y,KONDRATOVSKII P M,DUBIKOV A I,et al.Apoptosis regulator proteins:basis for the development of innovation strategies for the treatment of rheumatoid arthritis in patients of different age[J].Bull Exp Biol Med,2014,156(3):377-380.
[3]FAVALORO B,ALLOCATI N,GRAZIANO V,et al.Role of apoptosis in disease[J].Aging(Albany NY),2012,4(5):330-349.
[4]PETER M E,KRAMMERP H.Mechanisms of CD95(APO-1/Fas)-mediated apoptosis[J].Curr Opin Immunol,1998,10(5):545-551.
[5]BARTOK B,FIRESTEING S.Fibroblast-like synoviocytes:key effector cells in rheumatoid arthritis[J].Immunol Rev,2010,233(1):233-255.
[6]LIU H,POPE R M.The role of apoptosis in rheumatoid arthritis[J].Curr Opin Pharmacol,2003,3(3):317-322.
[7]KIM Y,SUH N,SPORN M,et al.An inducible pathway for degradation of FLIP protein sensitizes tumor cells to TRAIL-induced apoptosis[J].J Biol Chem,2002,277(25):22320-22329.
[8]曹永贺,刘健.类风湿性关节炎患者外周血CD4+T细胞存在凋亡缺陷[J].细胞与分子免疫学杂志,2015,31(5):682-685.CAO Yonghe,LIU Jian.Impaired apoptosis of peripheral blood CD4+T cells in patients with rheumatoid arthritis[J].Chinese Journal of Cellular and Molecular Immunology,2015,31(5):682-685.(in Chinese)
[9]余奇文,张勇,张冬青,等.s Fas、s Fas L及细胞因子在类风湿关节炎疾病中的意义[J].细胞与分子免疫学杂志,2001,17(2):141-142.YU Qiwen,ZHANG Yong,ZHANG Dongqing,et al.The role of s Fas,s Fas L and cytokines in the pathogeny of rheumatoid arthritis[J].Chinese Journal of Cellular and Molecular Immunology,2001,17(2):141-142.(in Chinese)
[10]张卓莉.类风湿关节炎诊断与治疗载入新里程[J/CD].中国医学前沿杂志(电子版),2011,3(2):39-42.ZHANG Zhuoli.Diagnosis and treatment of rheumatoid arthritis load milestone[J/CD].Chinese Journal of the Frontiers of Medical Science(Electronic Version),2011,3(2):39-42.(in Chinese)
[11]蔡敏敏,顾晓琼,高飞,等.分离外周血单个核细胞的条件优化[J].国际检验医学杂志,2016,37(1):1-2,5.CAI Minmin,GU Xiaoqiong,GAO Fei,et al.Optimization of condition on peripheral blood mononuclear cells separation[J].International Journal of Laboratory Medicine,2016,37(1):1-2,5.(in Chinese)
[12]张群燕,赵智明,姚茹冰,等.青藤碱抑制类风湿关节炎滑膜细胞增殖与诱导凋亡的研究[J].中华中医药学,2017,35(1):53-55.ZHANG Qunyan,ZHAO Zhiming,YAO Rubing,et al.Sinomenine suppresses proliferation and induces apoptosis of RA synovial cells[J].Chinese Archives of Traditional Chinese Medicine,2017,35(1):53-55.(in Chinese)
[13]林昌松,陈秀敏,刘清平,等.昆母汤对类风湿关节炎成纤维样滑膜细胞bax、bcl-2的影响[J].中药新药与临床药理,2014,25(6):656-659.LIN Changsong,CHEN Xiumin,LIU Qingping,et al.Effects of Kunmu decoction on Bax and Bcl-2expression in fibroblast-like synoviocytes of rheumatoid arthritis patients[J].Traditional Chinese Drug Research and Clinical Pharmacology,2014,25(6):656-659.(in Chinese)
[14]曹永贺,刘健.中医药调控类风湿关节炎细胞凋亡的研究[J].风湿病与关节炎,2014,3(8):49-52.CAO Yonghe,LIU Jian.Study on Chinese medicine regulating apoptosis of rheumatoid arthritis[J].Rheumatism and Arthritis,2014,3(8):49-52.(in Chinese)
[15]DU F,WANG L,ZHANG Y,et al.Role of GADD45 beta in the regulation of synovial fluid T cell apoptosis in rheumatoid arthritis[J].Clin Immunol,2008,128(2):238-247.
[16]陈占昆,吕厚山,王宁.PDCD5在类风湿关节炎成纤维样滑膜细胞凋亡中表达上调[J].中国生物化学与分子生物学报,2008,24(6):563-568.CHEN Zhankun,LYU Houshan,WANG Ning.Upregulation of PDCD5 in apoptosis of rheumatoid arthritis fibroblast-like synoviocytes[J].Chinese Journal of Biochemistry and Molecular Biology,2008,24(6):563-568.(in Chinese)
[17]王领,赵福涛.TRAIL及其受体在类风湿关节炎中研究新进展[J].医学综述,2016,22(3):447-450.WANG Ling,ZHAO Futao.Research progress of tumor necrosis factor related apoptosis inducing ligand and its receptors in rheumatoid arthritis[J].Medical Recapitulate,2016,22(3):447-450.(in Chinese)
[18]SIEGEL R M,FREDERIKSEN J K,ZACHARIAS DA,et al.Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations[J].Science,2000,288(5475):2354-2357.
[19]郦俊生,沙键,潘良,等.c-FLIP及突变p53蛋白表达与膀胱癌预后关系的研究[J].现代泌尿生殖肿瘤杂志,2010,2(2):101-103.LI Junsheng,SHA Jian,PAN Liang,et al.The correlation of c-FLIP and p53 protein expression and tumor prognosis in bladder cancer[J].Journal of Contemporary Urologic and Reproductive Oncology,2010,2(2):101-103.(in Chinese)
[20]JIN C H,CHAE S Y,KIM T H,et al.Effect of tumor necrosis factor-related apoptosis-inducing ligand on the reduction of joint inflammation in experimental rheumatoid arthritis[J].J Pharmacol Exp Ther,2010,332(3):858-865.
[21]孟明,梁红格,方皓,等.PI3K/Akt/m TOR通路与自噬在类风湿关节炎滑膜细胞增生中的意义[J].医学研究与教育,2013,30(5):69-74.MENG Ming,LIANG Hongge,FANG Hao,et al.The significance of PI3K/Akt/m TOR pathway and autophagy in the proliferation of synovial cells in rheumatoid arthritis[J].Medical Research and Education,2013,30(5):69-74.(in Chinese)
[22]SHANG L,WANG X.AMPK and m TOR coordinate the regulation of Ulk1 and mammalian autophagy initiation[J].Autophagy,2011,7(8):924-926.
[23]CHEN L C,CHUNG I C,HSUEH C,et al.The antiapoptotic protein,FLIP,is regulated by heterogeneous nuclear ribonucleoprotein K and correlates with poor overall survival of nasopharyngeal carcinoma patients[J].Cell Death Differ,2010,17(9):1463-1473.
[24]马田瑞,韩捷,陈海燕,等.类风湿关节炎患者外周血CD4+T细胞凋亡及其相关信号蛋白表达的检测和临床意义[J].同济大学学报(医学版),2011,32(5):36-40.MA Tianrui,HAN Jie,CHEN Haiyan,et al.Reduced apoptosis of CD4+T cells and expression of apoptosis-related signal proteins in patients with rheumatoid arthritis[J].Journal of Tongji University(Medical Science),2011,32(5):36-40.(in Chinese)
[2]DOROSHEVSKAYA A Y,KONDRATOVSKII P M,DUBIKOV A I,et al.Apoptosis regulator proteins:basis for the development of innovation strategies for the treatment of rheumatoid arthritis in patients of different age[J].Bull Exp Biol Med,2014,156(3):377-380.
[3]FAVALORO B,ALLOCATI N,GRAZIANO V,et al.Role of apoptosis in disease[J].Aging(Albany NY),2012,4(5):330-349.
[4]PETER M E,KRAMMERP H.Mechanisms of CD95(APO-1/Fas)-mediated apoptosis[J].Curr Opin Immunol,1998,10(5):545-551.
[5]BARTOK B,FIRESTEING S.Fibroblast-like synoviocytes:key effector cells in rheumatoid arthritis[J].Immunol Rev,2010,233(1):233-255.
[6]LIU H,POPE R M.The role of apoptosis in rheumatoid arthritis[J].Curr Opin Pharmacol,2003,3(3):317-322.
[7]KIM Y,SUH N,SPORN M,et al.An inducible pathway for degradation of FLIP protein sensitizes tumor cells to TRAIL-induced apoptosis[J].J Biol Chem,2002,277(25):22320-22329.
[8]曹永贺,刘健.类风湿性关节炎患者外周血CD4+T细胞存在凋亡缺陷[J].细胞与分子免疫学杂志,2015,31(5):682-685.CAO Yonghe,LIU Jian.Impaired apoptosis of peripheral blood CD4+T cells in patients with rheumatoid arthritis[J].Chinese Journal of Cellular and Molecular Immunology,2015,31(5):682-685.(in Chinese)
[9]余奇文,张勇,张冬青,等.s Fas、s Fas L及细胞因子在类风湿关节炎疾病中的意义[J].细胞与分子免疫学杂志,2001,17(2):141-142.YU Qiwen,ZHANG Yong,ZHANG Dongqing,et al.The role of s Fas,s Fas L and cytokines in the pathogeny of rheumatoid arthritis[J].Chinese Journal of Cellular and Molecular Immunology,2001,17(2):141-142.(in Chinese)
[10]张卓莉.类风湿关节炎诊断与治疗载入新里程[J/CD].中国医学前沿杂志(电子版),2011,3(2):39-42.ZHANG Zhuoli.Diagnosis and treatment of rheumatoid arthritis load milestone[J/CD].Chinese Journal of the Frontiers of Medical Science(Electronic Version),2011,3(2):39-42.(in Chinese)
[11]蔡敏敏,顾晓琼,高飞,等.分离外周血单个核细胞的条件优化[J].国际检验医学杂志,2016,37(1):1-2,5.CAI Minmin,GU Xiaoqiong,GAO Fei,et al.Optimization of condition on peripheral blood mononuclear cells separation[J].International Journal of Laboratory Medicine,2016,37(1):1-2,5.(in Chinese)
[12]张群燕,赵智明,姚茹冰,等.青藤碱抑制类风湿关节炎滑膜细胞增殖与诱导凋亡的研究[J].中华中医药学,2017,35(1):53-55.ZHANG Qunyan,ZHAO Zhiming,YAO Rubing,et al.Sinomenine suppresses proliferation and induces apoptosis of RA synovial cells[J].Chinese Archives of Traditional Chinese Medicine,2017,35(1):53-55.(in Chinese)
[13]林昌松,陈秀敏,刘清平,等.昆母汤对类风湿关节炎成纤维样滑膜细胞bax、bcl-2的影响[J].中药新药与临床药理,2014,25(6):656-659.LIN Changsong,CHEN Xiumin,LIU Qingping,et al.Effects of Kunmu decoction on Bax and Bcl-2expression in fibroblast-like synoviocytes of rheumatoid arthritis patients[J].Traditional Chinese Drug Research and Clinical Pharmacology,2014,25(6):656-659.(in Chinese)
[14]曹永贺,刘健.中医药调控类风湿关节炎细胞凋亡的研究[J].风湿病与关节炎,2014,3(8):49-52.CAO Yonghe,LIU Jian.Study on Chinese medicine regulating apoptosis of rheumatoid arthritis[J].Rheumatism and Arthritis,2014,3(8):49-52.(in Chinese)
[15]DU F,WANG L,ZHANG Y,et al.Role of GADD45 beta in the regulation of synovial fluid T cell apoptosis in rheumatoid arthritis[J].Clin Immunol,2008,128(2):238-247.
[16]陈占昆,吕厚山,王宁.PDCD5在类风湿关节炎成纤维样滑膜细胞凋亡中表达上调[J].中国生物化学与分子生物学报,2008,24(6):563-568.CHEN Zhankun,LYU Houshan,WANG Ning.Upregulation of PDCD5 in apoptosis of rheumatoid arthritis fibroblast-like synoviocytes[J].Chinese Journal of Biochemistry and Molecular Biology,2008,24(6):563-568.(in Chinese)
[17]王领,赵福涛.TRAIL及其受体在类风湿关节炎中研究新进展[J].医学综述,2016,22(3):447-450.WANG Ling,ZHAO Futao.Research progress of tumor necrosis factor related apoptosis inducing ligand and its receptors in rheumatoid arthritis[J].Medical Recapitulate,2016,22(3):447-450.(in Chinese)
[18]SIEGEL R M,FREDERIKSEN J K,ZACHARIAS DA,et al.Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations[J].Science,2000,288(5475):2354-2357.
[19]郦俊生,沙键,潘良,等.c-FLIP及突变p53蛋白表达与膀胱癌预后关系的研究[J].现代泌尿生殖肿瘤杂志,2010,2(2):101-103.LI Junsheng,SHA Jian,PAN Liang,et al.The correlation of c-FLIP and p53 protein expression and tumor prognosis in bladder cancer[J].Journal of Contemporary Urologic and Reproductive Oncology,2010,2(2):101-103.(in Chinese)
[20]JIN C H,CHAE S Y,KIM T H,et al.Effect of tumor necrosis factor-related apoptosis-inducing ligand on the reduction of joint inflammation in experimental rheumatoid arthritis[J].J Pharmacol Exp Ther,2010,332(3):858-865.
[21]孟明,梁红格,方皓,等.PI3K/Akt/m TOR通路与自噬在类风湿关节炎滑膜细胞增生中的意义[J].医学研究与教育,2013,30(5):69-74.MENG Ming,LIANG Hongge,FANG Hao,et al.The significance of PI3K/Akt/m TOR pathway and autophagy in the proliferation of synovial cells in rheumatoid arthritis[J].Medical Research and Education,2013,30(5):69-74.(in Chinese)
[22]SHANG L,WANG X.AMPK and m TOR coordinate the regulation of Ulk1 and mammalian autophagy initiation[J].Autophagy,2011,7(8):924-926.
[23]CHEN L C,CHUNG I C,HSUEH C,et al.The antiapoptotic protein,FLIP,is regulated by heterogeneous nuclear ribonucleoprotein K and correlates with poor overall survival of nasopharyngeal carcinoma patients[J].Cell Death Differ,2010,17(9):1463-1473.
[24]马田瑞,韩捷,陈海燕,等.类风湿关节炎患者外周血CD4+T细胞凋亡及其相关信号蛋白表达的检测和临床意义[J].同济大学学报(医学版),2011,32(5):36-40.MA Tianrui,HAN Jie,CHEN Haiyan,et al.Reduced apoptosis of CD4+T cells and expression of apoptosis-related signal proteins in patients with rheumatoid arthritis[J].Journal of Tongji University(Medical Science),2011,32(5):36-40.(in Chinese)