Fcγ受體介導(dǎo)類風(fēng)濕關(guān)節(jié)炎中性粒細(xì)胞凋亡延遲的作用
發(fā)布時(shí)間：2018-09-12 19:58
【摘要】：目的:通過(guò)研究類風(fēng)濕關(guān)節(jié)炎(RA)患者關(guān)節(jié)滑液、外周血中性粒細(xì)胞(PMN)凋亡狀態(tài),觀察PMN表面Fcγ受體在凋亡過(guò)程中的變化,闡明PMN表面FcγR在RA患者PMN延遲凋亡中的作用。方法:本研究納入RA患者膝關(guān)節(jié)滑液22例、外周血22例,健康志愿者外周血11例。采用密度梯度離心法、冰純水裂紅法,分離、純化PMN。采用流式細(xì)胞鑒定PMN細(xì)胞純度,采用臺(tái)盼藍(lán)檢測(cè)細(xì)胞活力。分離出的PMN采用BD FITC AnnexinV-Apoptosis Detection Kit I染色后,使用流式細(xì)胞檢測(cè)PMN在4 h、8 h、12 h、24 h AnnexinV的陽(yáng)性率,以此說(shuō)明PMN的凋亡情況。采用50%RA膝關(guān)節(jié)滑液刺激健康志愿者外周血PMN后,流式細(xì)胞檢測(cè)4 h、8 h、12 h、24 h AnnexinV的陽(yáng)性率。采用CD16-APC、CD16b-PE、CD32-FITC、CD64-BV510分別標(biāo)記細(xì)胞表面FcγRIII、FcγRIIIb、FcγRII、FcγRI,流式細(xì)胞檢測(cè)RA患者膝關(guān)節(jié)滑液、外周血及健康志愿者外周血PMN表面FcγR(FcγRIII、FcγRIIIb、FcγRII、FcγRI)的表達(dá)。50%RA關(guān)節(jié)滑液與健康志愿者外周血、RA患者外周血來(lái)源的PMN共孵育5h,檢測(cè)PMN孵育前后FcγR表達(dá)差異。人口特征:性別比較采用頻數(shù)分布四格表-fisher確切概率法,年齡病程采用Mann-Whitney U非參數(shù)秩和檢驗(yàn);AnnexinV陽(yáng)性率采用Two way-ANOVA,組間采用Bonferroni分析;FcγR表達(dá)采用One way-ANOVA,組間采用Bonferroni分析。結(jié)果:(1)RA患者關(guān)節(jié)滑液組、RA患者外周血組及健康者外周血組在年齡和性別構(gòu)成比上無(wú)明顯差異,RA滑液組及RA外周血組在病程和疾病活動(dòng)度上無(wú)明顯差異。(2)與健康志愿者外周血相比,RA患者外周血中性粒細(xì)胞凋亡延遲;RA患者關(guān)節(jié)滑液與外周血中中性粒細(xì)胞凋亡無(wú)差異。(3)24 h(4 h、8 h、12 h和24 h)凋亡率:健康志愿者外周血PMN為49.2±8.5%、59.3±9.1%、66.5±8.3%和79.0±6.3%;RA患者外周血PMN為38.3±12.3%、44.0±4.5%、38.4±8.0%和51.4±8.7%;RA患者膝關(guān)節(jié)滑液PMN為36.9±7.7%、41.9±8.5%、42.9±8.7%和44.7±9.1%。(4)RA滑液能顯著抑制健康者外周血PMN凋亡(P0.01);RA滑液處理后,健康者外周血PMN凋亡率為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患者外周血中性粒細(xì)胞表面FcγRIIIb表達(dá)陽(yáng)性且僅為一群,FcγRs表達(dá)無(wú)差異(P0.05);與外周血相比,RA關(guān)節(jié)滑液中中性粒細(xì)胞FcγRI表達(dá)顯著增高(P0.05)。FcγRIIIb表達(dá)為FcγRIIIbhigh、FcγRIIIblow兩群,且FcγRIIIbhigh比例顯著低于FcγRIIIblow(P0.05)。(6)RA滑液能影響健康者、RA患者外周血中性粒細(xì)胞FcγRIIIb亞群的表達(dá):FcγRIIIblow群比例增加,FcγRIIIbhigh群比例減少(P0.05)。(7)RA滑液也可抑制健康者及RA患者外周血PMN FcγRII的表達(dá)(P0.05)。結(jié)論:(1)類風(fēng)濕關(guān)節(jié)炎患者關(guān)節(jié)滑液和外周血中中性粒細(xì)胞凋亡延遲;(2)類風(fēng)濕關(guān)節(jié)炎滑液可抑制中性粒細(xì)胞凋亡;(3)FcγR可能參與類風(fēng)濕關(guān)節(jié)炎中性粒細(xì)胞凋亡延遲通路。
[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 緯 receptor on PMN surface, and elucidate the role of PMN surface Fc 緯 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 緯 RIII,Fc 緯 RIIIb,Fc 緯 RII,Fc 緯 RI, flow cytometry was used to detect the synovial fluid of the knee joint in patients with RA. The expression of Fc 緯 R (Fc 緯 RIII,Fc 緯 RIIIb,Fc 緯 RII,Fc 緯 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 緯 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 鹵8.5 鹵9.1%, 66.5 鹵8.3% and 79.0 鹵6.3%, respectively. The PMN in peripheral blood of patients with RA was 38.3 鹵12.3% and 38.4 鹵8.0% and 51.4 鹵8.7%, respectively. The PMN of synovial fluid of knee joint in RA patients was 36.9 鹵7.7% and 42.9 鹵8.7% and 44.7 鹵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 鹵7.1% (4 h), 53.1 鹵6.8% (8 h), 58.7 鹵8.9% (12 h) and 65.9 鹵6.7% (24 h). (5) in healthy subjects and RA patients. The positive expression of Fc 緯 RIIIb on peripheral blood neutrophils was not different from that in peripheral blood (P0.05), and the expression of Fc 緯 RI 緯 RI in synovial fluid of RA joints was similar to that in peripheral blood (P0.05). The expression of FC 緯 RIIIb was significantly increased (P0.05). FC 緯 RIIIb was expressed in two groups of Fc 緯 RIIIbhigh,Fc 緯 RIIIblow. The proportion of Fc 緯 RIIIbhigh was significantly lower than that of Fc 緯 RIIIblow (P0.05). (6) RA synovial fluid could affect the expression of Fc 緯 RIIIb subsets of peripheral blood neutrophils in healthy controls (P0.05). The percentage of Fc 緯 RIIIb subsets in peripheral blood neutrophils increased and the percentage of FC RIIIbhigh 緯 groups decreased (P0.05). (7). RA synovial fluid also inhibited the expression of PMN Fc 緯 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 緯 R may be involved in delayed pathway of apoptosis of neutrophil in rheumatoid arthritis.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.22
本文編號(hào)：2240072
[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 緯 receptor on PMN surface, and elucidate the role of PMN surface Fc 緯 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 緯 RIII,Fc 緯 RIIIb,Fc 緯 RII,Fc 緯 RI, flow cytometry was used to detect the synovial fluid of the knee joint in patients with RA. The expression of Fc 緯 R (Fc 緯 RIII,Fc 緯 RIIIb,Fc 緯 RII,Fc 緯 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 緯 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 鹵8.5 鹵9.1%, 66.5 鹵8.3% and 79.0 鹵6.3%, respectively. The PMN in peripheral blood of patients with RA was 38.3 鹵12.3% and 38.4 鹵8.0% and 51.4 鹵8.7%, respectively. The PMN of synovial fluid of knee joint in RA patients was 36.9 鹵7.7% and 42.9 鹵8.7% and 44.7 鹵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 鹵7.1% (4 h), 53.1 鹵6.8% (8 h), 58.7 鹵8.9% (12 h) and 65.9 鹵6.7% (24 h). (5) in healthy subjects and RA patients. The positive expression of Fc 緯 RIIIb on peripheral blood neutrophils was not different from that in peripheral blood (P0.05), and the expression of Fc 緯 RI 緯 RI in synovial fluid of RA joints was similar to that in peripheral blood (P0.05). The expression of FC 緯 RIIIb was significantly increased (P0.05). FC 緯 RIIIb was expressed in two groups of Fc 緯 RIIIbhigh,Fc 緯 RIIIblow. The proportion of Fc 緯 RIIIbhigh was significantly lower than that of Fc 緯 RIIIblow (P0.05). (6) RA synovial fluid could affect the expression of Fc 緯 RIIIb subsets of peripheral blood neutrophils in healthy controls (P0.05). The percentage of Fc 緯 RIIIb subsets in peripheral blood neutrophils increased and the percentage of FC RIIIbhigh 緯 groups decreased (P0.05). (7). RA synovial fluid also inhibited the expression of PMN Fc 緯 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 緯 R may be involved in delayed pathway of apoptosis of neutrophil in rheumatoid arthritis.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.22
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