类风湿关节炎患者PBMC中凋亡相关蛋白Bcl-2与c-FLIP的表达及其意义
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摘要
目的探讨类风湿关节炎(RA)患者外周血单个核细胞(PBMC)中凋亡相关蛋白Bcl-2与c-FLIP的表达水平及其临床意义。方法 60例类风湿关节炎患者外周血标本按RA疾病活动指数(DAS28)分为低度活动期(DAS28 <3. 2)、中度活动期(DAS28 3. 2～5. 1)与高度活动期(DAS28> 5. 1),以同期25例健康体检者外周血作为健康对照组(N组)。实时荧光定量PCR法检测RA患者PBMC中Bcl-2、c-FLIP、caspase-8凋亡相关蛋白的mRNA表达水平;蛋白免疫印迹法检测PBMC中cFLIP、caspase-8蛋白表达水平。结果 Bcl-2的低活动组mRNA相对表达量为1. 02±0. 35,高于健康对照组(0. 32±0. 18,P<0. 05),而中、高活动组分别为0. 08±0. 01,0. 05±0. 02,低于健康对照组(均P <0. 05)。c-FLIP的低、中、高三个活动组mRNA相对表达量分别为1. 27±0. 28,1. 32±0. 34,1. 93±0. 40,均高于健康对照组(1. 08±0. 12,均P <0. 05); caspase-8的低、中、高组mRNA相对表达量分别为2. 77±0. 97,4. 52±0. 85,2. 13±0. 44,也均高于健康对照组(均P <0. 05)。中活动组cFLIP蛋白表达明显高于健康对照组(P <0. 05),而低、高活动组与对照组差异无统计学意义(P> 0. 05);中、高活动组caspase-8蛋白表达明显高于健康对照组(P <0. 05),而低活动组表达差异无统计学意义(P> 0. 05)。结论 PBMC中凋亡相关蛋白c-FLIP mRNA表达水平随着RA疾病的进程增高,而Bcl-2只有低活动组有明显增高,此可为类风湿关节炎发病机制的后续研究提供参考。
Objective To investigate the expression and its significance of apoptosis-related proteins Bcl-2,c-FLIP and caspase-8 in peripheral blood mononuclear cells of patients with rheumatoid arthritis( RA). Methods The blood samples of 60 patients with rheumatoid arthritis were divided into low activity( DAS28 < 3. 2) group,moderate activity( 3. 2-5. 1 DAS28) group and high activity( DAS28 > 5. 1) group according to the RA disease activity index( DAS28). The blood of 25 normal controls for health check-up were used as normal control group. The mRNA expression of apoptosis related proteins Bcl-2,c-FLIP and caspase-8 was detected by realtime PCR,and the protein levels of c-FLIP and caspase-8 were detected by Western blot. Results The mRNA expression of Bcl-2 in low activity group was higher than in normal control group( P < 0. 05),but it was lower in moderate activity group and high activity group( P < 0. 05). The relative mRNA expression of c-FLIP in low activity group,moderate activity group and high activity group were higher than that in normal control group respectively( P < 0. 05). The expression level of Caspase-8 mRNA was higher in low activity group,moderate activity group and high activity group than in normal control group( P < 0. 05). The protein level of c-FLIP in moderate activity group was significantly higher than that in control group( P < 0. 05),but there was no difference between low activity group and normal control group. The protein levels of Caspase-8 in moderate activity group and high activity group were significantly higher than that in normal control group( P < 0. 05),but there was no difference between low activity group and normal control group. Conclusion In peripheral blood mononuclear cells of patients with RA,the relative mRNA expression level of c-FLIP is increased with the progress of the disease,while Bcl-2 mRNA increases only in low activity patients compared with normal controls,but decreases in moderate and high activity patients. It provides reference for the further study of the pathogenesis of rheumatoid arthritis.
Objective To investigate the expression and its significance of apoptosis-related proteins Bcl-2,c-FLIP and caspase-8 in peripheral blood mononuclear cells of patients with rheumatoid arthritis( RA). Methods The blood samples of 60 patients with rheumatoid arthritis were divided into low activity( DAS28 < 3. 2) group,moderate activity( 3. 2-5. 1 DAS28) group and high activity( DAS28 > 5. 1) group according to the RA disease activity index( DAS28). The blood of 25 normal controls for health check-up were used as normal control group. The mRNA expression of apoptosis related proteins Bcl-2,c-FLIP and caspase-8 was detected by realtime PCR,and the protein levels of c-FLIP and caspase-8 were detected by Western blot. Results The mRNA expression of Bcl-2 in low activity group was higher than in normal control group( P < 0. 05),but it was lower in moderate activity group and high activity group( P < 0. 05). The relative mRNA expression of c-FLIP in low activity group,moderate activity group and high activity group were higher than that in normal control group respectively( P < 0. 05). The expression level of Caspase-8 mRNA was higher in low activity group,moderate activity group and high activity group than in normal control group( P < 0. 05). The protein level of c-FLIP in moderate activity group was significantly higher than that in control group( P < 0. 05),but there was no difference between low activity group and normal control group. The protein levels of Caspase-8 in moderate activity group and high activity group were significantly higher than that in normal control group( P < 0. 05),but there was no difference between low activity group and normal control group. Conclusion In peripheral blood mononuclear cells of patients with RA,the relative mRNA expression level of c-FLIP is increased with the progress of the disease,while Bcl-2 mRNA increases only in low activity patients compared with normal controls,but decreases in moderate and high activity patients. It provides reference for the further study of the pathogenesis of rheumatoid arthritis.
引文
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[11] Song W,Song C,Chen Y,et al. Polysaccharide-induced apoptosis in H22 cells through G2/M arrest and BCL2/BAX caspaseactivated Fas pathway[J]. Cell Mol Biol(Noisy-le-grand),2015,61:88.
[12]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.
[13] Nakajima T,Aono H,Hasunuma T,et al. Apoptosis and functional Fas antigen in rheumatoid arthritis synoviocytes[J]. Arthritis Rheum,1995,38:485-491.
[14]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.
[15] Isomaki P,Soderstrom KO,Punnonen J,et al. Expression of bcl-2 in rheumatoid arthritis[J]. Br J Rheumatol,1996,35(7):611-619.
[16] Krueger A,Baumann S,Krammer PH,et al. FLICE-Inhibitory proteins:regulators of death receptor mediated apoptosis[J]. J Mol Cell Biol,2001,21(24):8247-8254.
[17] Bartok B,Firestein GS. Fibroblst-like synoviocytes:key effectors cells in reheumatoid arthritis[J]. Immunol Rev,2010,23(1):233-255.
[18]吴凤霞,杨明辉,刘宁涛,等. Fas相关死亡区样白介素1转换酶抑制蛋白在类风湿关节炎患者滑膜组织中的表达[J].第四军医大学学报,2008,29(21):1987-1989.
[2]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.
[3]陈瑞林,黄文辉,黄成辉,等. PPAR-γ在类风湿关节炎患者外周血单个核细胞中的表达及与疾病活动度的关系[J].实用临床医药杂志,2015,19(7):65-67.
[4]张卓莉.类风湿关节炎诊断与治疗载入新里程[J].中国医学前沿杂志:电子版,2011,3(2):39-42.
[5]马芳,尹林,熊华伟,等.分血时间对外周血单个核细胞蛋白质表达谱的影响[J].实用医学杂志,2012,28(7):1058-1061.
[6] Korczowska I. Rheumatoid arthritis susceptibility genes:an overview[J]. World J Orthop,2014,5(4):544-549.
[7] Cha HS,Bae EK,Ahn JK,et al. Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblastlike synoviocytes treated with hydrogen peroxide[J]. Exp Mol Med,2010,42(6):428-436.
[8] Favaloro B,Allocati N,Grazinao V,et al. Role of apoptosis in disease[J]. Aging(Albany NY),2012,4(5):330-349.
[9]张慧,郑纪容,郭潮. miR-630调控BCL2抑制肺癌细胞的生长[J].中国热带医学,2018,18(1):22-27.
[10] Conti A,Majorini MT,Fontanella E,et al. Lemur tyrosine kinase2(LMTK2)is a determinant of cell sensitivity to apoptosis by regulating the levels of the BCL2 family members[J]. Cancer Lett,2017,389:59.
[11] Song W,Song C,Chen Y,et al. Polysaccharide-induced apoptosis in H22 cells through G2/M arrest and BCL2/BAX caspaseactivated Fas pathway[J]. Cell Mol Biol(Noisy-le-grand),2015,61:88.
[12]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.
[13] Nakajima T,Aono H,Hasunuma T,et al. Apoptosis and functional Fas antigen in rheumatoid arthritis synoviocytes[J]. Arthritis Rheum,1995,38:485-491.
[14]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.
[15] Isomaki P,Soderstrom KO,Punnonen J,et al. Expression of bcl-2 in rheumatoid arthritis[J]. Br J Rheumatol,1996,35(7):611-619.
[16] Krueger A,Baumann S,Krammer PH,et al. FLICE-Inhibitory proteins:regulators of death receptor mediated apoptosis[J]. J Mol Cell Biol,2001,21(24):8247-8254.
[17] Bartok B,Firestein GS. Fibroblst-like synoviocytes:key effectors cells in reheumatoid arthritis[J]. Immunol Rev,2010,23(1):233-255.
[18]吴凤霞,杨明辉,刘宁涛,等. Fas相关死亡区样白介素1转换酶抑制蛋白在类风湿关节炎患者滑膜组织中的表达[J].第四军医大学学报,2008,29(21):1987-1989.