Fcγ受体介导类风湿关节炎中性粒细胞凋亡延迟的作用

发布时间：2018-09-12 19:58

【摘要】：目的:通过研究类风湿关节炎(RA)患者关节滑液、外周血中性粒细胞(PMN)凋亡状态,观察PMN表面Fcγ受体在凋亡过程中的变化,阐明PMN表面FcγR在RA患者PMN延迟凋亡中的作用。方法:本研究纳入RA患者膝关节滑液22例、外周血22例,健康志愿者外周血11例。采用密度梯度离心法、冰纯水裂红法,分离、纯化PMN。采用流式细胞鉴定PMN细胞纯度,采用台盼蓝检测细胞活力。分离出的PMN采用BD FITC AnnexinV-Apoptosis Detection Kit I染色后,使用流式细胞检测PMN在4 h、8 h、12 h、24 h AnnexinV的阳性率,以此说明PMN的凋亡情况。采用50%RA膝关节滑液刺激健康志愿者外周血PMN后,流式细胞检测4 h、8 h、12 h、24 h AnnexinV的阳性率。采用CD16-APC、CD16b-PE、CD32-FITC、CD64-BV510分别标记细胞表面FcγRIII、FcγRIIIb、FcγRII、FcγRI,流式细胞检测RA患者膝关节滑液、外周血及健康志愿者外周血PMN表面FcγR(FcγRIII、FcγRIIIb、FcγRII、FcγRI)的表达。50%RA关节滑液与健康志愿者外周血、RA患者外周血来源的PMN共孵育5h,检测PMN孵育前后FcγR表达差异。人口特征:性别比较采用频数分布四格表-fisher确切概率法,年龄病程采用Mann-Whitney U非参数秩和检验;AnnexinV阳性率采用Two way-ANOVA,组间采用Bonferroni分析;FcγR表达采用One way-ANOVA,组间采用Bonferroni分析。结果:(1)RA患者关节滑液组、RA患者外周血组及健康者外周血组在年龄和性别构成比上无明显差异,RA滑液组及RA外周血组在病程和疾病活动度上无明显差异。(2)与健康志愿者外周血相比,RA患者外周血中性粒细胞凋亡延迟;RA患者关节滑液与外周血中中性粒细胞凋亡无差异。(3)24 h(4 h、8 h、12 h和24 h)凋亡率:健康志愿者外周血PMN为49.2±8.5%、59.3±9.1%、66.5±8.3%和79.0±6.3%;RA患者外周血PMN为38.3±12.3%、44.0±4.5%、38.4±8.0%和51.4±8.7%;RA患者膝关节滑液PMN为36.9±7.7%、41.9±8.5%、42.9±8.7%和44.7±9.1%。(4)RA滑液能显著抑制健康者外周血PMN凋亡(P0.01);RA滑液处理后,健康者外周血PMN凋亡率为51.7±7.1%(4 h)、53.1±6.8%(8 h)、58.7±8.9%(12 h)和65.9±6.7%(24 h)。(5)健康者及RA患者外周血中性粒细胞表面FcγRIIIb表达阳性且仅为一群,FcγRs表达无差异(P0.05);与外周血相比,RA关节滑液中中性粒细胞FcγRI表达显著增高(P0.05)。FcγRIIIb表达为FcγRIIIbhigh、FcγRIIIblow两群,且FcγRIIIbhigh比例显著低于FcγRIIIblow(P0.05)。(6)RA滑液能影响健康者、RA患者外周血中性粒细胞FcγRIIIb亚群的表达:FcγRIIIblow群比例增加,FcγRIIIbhigh群比例减少(P0.05)。(7)RA滑液也可抑制健康者及RA患者外周血PMN FcγRII的表达(P0.05)。结论:(1)类风湿关节炎患者关节滑液和外周血中中性粒细胞凋亡延迟;(2)类风湿关节炎滑液可抑制中性粒细胞凋亡;(3)FcγR可能参与类风湿关节炎中性粒细胞凋亡延迟通路。
[Abstract]:Aim: to investigate the apoptosis status of (PMN) in synovial fluid and peripheral blood of patients with rheumatoid arthritis (RA), observe the changes of Fc 纬 receptor on PMN surface, and elucidate the role of PMN surface Fc 纬 R in PMN delayed apoptosis in RA patients. Methods: 22 cases of synovial fluid of knee joint, 22 cases of peripheral blood and 11 cases of healthy volunteers were included in this study. PMN. was isolated and purified by density gradient centrifugation and ice water split red method. Flow cytometry was used to identify the purity of PMN cells and trypan blue was used to detect the viability of the cells. The PMN isolated was stained with BD FITC AnnexinV-Apoptosis Detection Kit I, and the positive rate of PMN was detected by flow cytometry at 4 h, 8 h, 12 h and 24 h AnnexinV, so as to explain the apoptosis of PMN. 50%RA synovial fluid was used to stimulate PMN in peripheral blood of healthy volunteers. Flow cytometry was used to detect the positive rate of AnnexinV for 4 h, 8 h, 12 h and 24 h. CD16-APC,CD16b-PE,CD32-FITC,CD64-BV510 labeled Fc 纬 RIII,Fc 纬 RIIIb,Fc 纬 RII,Fc 纬 RI, flow cytometry was used to detect the synovial fluid of the knee joint in patients with RA. The expression of Fc 纬 R (Fc 纬 RIII,Fc 纬 RIIIb,Fc 纬 RII,Fc 纬 RI on PMN surface of peripheral blood and healthy volunteers. 50% of RA was co-incubated with PMN from peripheral blood of healthy volunteers for 5 h. The difference of Fc 纬 R expression before and after PMN incubation was detected. Population characteristics: sex comparison was carried out by Four-grid table-Fisher exact probability method, age course by Mann-Whitney U nonparametric rank sum test and Annexin V positive rate by Bonferroni analysis between Two way-ANOVA, groups and Bonferroni analysis among One way-ANOVA, groups. Results: (1) there was no significant difference in age and sex composition ratio between peripheral blood group of RA patients and healthy controls in synovial fluid group of RA patients. There was no significant difference between RA synovial fluid group and RA peripheral blood group in course of disease and disease activity. (2) there was no significant difference between RA synovial fluid group and healthy group. There was no difference between synovial fluid and peripheral blood neutrophil apoptosis in patients with RA. (3) 24 h (4 h, 8 h, 12 h and 24 h) apoptosis rate: the PMN in peripheral blood of healthy volunteers was 49.2 卤8.5 卤9.1%, 66.5 卤8.3% and 79.0 卤6.3%, respectively. The PMN in peripheral blood of patients with RA was 38.3 卤12.3% and 38.4 卤8.0% and 51.4 卤8.7%, respectively. The PMN of synovial fluid of knee joint in RA patients was 36.9 卤7.7% and 42.9 卤8.7% and 44.7 卤9.1% respectively. (4) RA synovial fluid could significantly inhibit PMN apoptosis in peripheral blood of healthy subjects. The apoptosis rate of PMN in peripheral blood was 51.7 卤7.1% (4 h), 53.1 卤6.8% (8 h), 58.7 卤8.9% (12 h) and 65.9 卤6.7% (24 h). (5) in healthy subjects and RA patients. The positive expression of Fc 纬 RIIIb on peripheral blood neutrophils was not different from that in peripheral blood (P0.05), and the expression of Fc 纬 RI 纬 RI in synovial fluid of RA joints was similar to that in peripheral blood (P0.05). The expression of FC 纬 RIIIb was significantly increased (P0.05). FC 纬 RIIIb was expressed in two groups of Fc 纬 RIIIbhigh,Fc 纬 RIIIblow. The proportion of Fc 纬 RIIIbhigh was significantly lower than that of Fc 纬 RIIIblow (P0.05). (6) RA synovial fluid could affect the expression of Fc 纬 RIIIb subsets of peripheral blood neutrophils in healthy controls (P0.05). The percentage of Fc 纬 RIIIb subsets in peripheral blood neutrophils increased and the percentage of FC RIIIbhigh 纬 groups decreased (P0.05). (7). RA synovial fluid also inhibited the expression of PMN Fc 纬 RII in peripheral blood of healthy subjects and RA patients (P0.05). Conclusion: (1) delayed apoptosis of synovial fluid and peripheral blood neutrophils in patients with rheumatoid arthritis; (2) synovial fluid of rheumatoid arthritis can inhibit apoptosis of neutrophil; (3) Fc 纬 R may be involved in delayed pathway of apoptosis of neutrophil in rheumatoid arthritis.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22

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