摘要
目的 研究自然杀伤(NK)细胞及其CD38阳性亚群在类风湿关节炎(RA)患者外周血、滑液、滑膜组织中的分布特征以及CD38分子对RA患者外周血和滑液淋巴细胞功能调节机制。方法 流式细胞仪检测健康人外周血和RA患者配对的外周血、膝关节滑液中NK、CD38亚群及NK细胞表面趋化因子受体(CXCR3、CCR5)、颗粒酶B、穿孔素的表达。体外培养RA患者外周血和滑液淋巴细胞,流式细胞仪测定NK细胞颗粒酶B、穿孔素的表达,ELISA法测定培养上清TNF-α、IL-6的浓度。脂质体CD38-siRNA转染RA患者外周血淋巴细胞,Western blot法检测刺激CD38和CD38-siRNA沉默对RA外周血淋巴细胞内磷酯酶C-γ1(PLC-γ1)蛋白表达水平的影响。结果 RA患者外周血中NK细胞高表达趋化因子受体CCR5和CXCR3,与正常健康人群差异有统计学意义(P <0. 05),两种趋化因子受体主要表达于CD38+NK细胞亚群。体外培养实验,RA滑液淋巴细胞上颗粒酶B和穿孔素的表达水平均显著高于RA外周血淋巴细胞,两者间差异有统计学意义(P均<0. 05);IL-2+IB4联合刺激组和IB4组滑液与外周血淋巴细胞培养上清中IL-6和TNF-α的分泌量高于空白对照组(P <0. 05)。CD38-siRNA干扰处理RA外周血淋巴细胞后,PLC-γ1条带的灰度值低于空白对照组(P <0. 05)。结论 RA患者炎症部位(滑液、滑膜)均存在NK细胞异常浸润。CD38分子参与调节NK细胞分泌和杀伤两种功能。
Objective To study the distribution of natural killer(NK)cells and the CD38 positive subpopulations in different tissues in rheumatoid arthritis(RA)patients and the mechanism of CD38 agonist.Methods The levels of NK cells,CD38 positive subpopulation and NK cell surface chemokine receptors(CXCR3,CCR5),as well as granzyme B and perforin were detected by flow cytometry in peripheral blood and knee synovial fluid.The isolated cells were then cultured in vitro and divided into 3 groups,namely blank control group,IB4 stimulation group(Anti-CD38 mAb),IL-2 and IB4 co-stimulation group.And the concentration of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in culture supernatants was determined by enzyme linked immunosorbent assay(ELISA).Liposome CD38-siRNA was transfected into peripheral blood lymphocytes of RA patients,and transfection efficiency was detected by reverse transcription-polymerase chain reaction(RT-PCR).The effect of CD38 and CD38-siRNA silencing on the expression of phospholipase C-gamma 1(PLC-γ1)protein in the peripheral blood lymphocytes of RA was detected by Western blot.Results The expression of CCR5 and CXCR3 in NK cells in peripercal NK cells of RA was significantly higher than that in normal healthy subjects(P<0.05).The two chemokine receptors(CXCR3,CCR5)were mainly expressed in CD38+NK cell subsets.The levels of granzyme B and perforin in synovial lymphocytes cells were significantly higher than those in peripheral blood lymphocytes(P<0.05).The secretion of IL-6 and TNF-αin synovial and peripheral blood lymphocytes cells existed significant difference only in IL-2 and IB4 co-stimulation group(P<0.05).The levels of PLC-γ1 protein in the peripheral blood lymphocytes of RA patients was significantly decreased than that in the blank control group after the treatment with CD38-siRNA(P<0.05).Conclusions The synovial NK cells are more lethal than the peripheral NK cells in the RA patients.CD38 might mediate the phosphorylation of intracellular proteins via the Protain kinase C(PKC)pathway.Blocking CD38 molecules can reduce the phosphorylation level of intracellular proteins.CD38 maybe involved in the mechanism of chemotaxis and killing capability of NK cells.
作者
潘文萍
王庆
王晓慧
王秀花
岳龙涛
李树锋
常晓天
Pan Wenping;Wang Qing;Wang Xiaohui;Wang Xiuhua;Yue Longtao;Li Shufeng;Chang Xiaotian(Department of Rheumatology,Qianfoshan Hospital Affiliated to Shandong University,Jinan250014,China;Department of Rheumatology,the Second People's Hospital of Rizhao,Rizhao276807,China;Medical Research Center,Qianfoshan Hospital Affiliated to Shandong University,Jinan250014,China;Department of Orthopedics,Qianfoshan Hospital Affiliated to Shandong University,Jinan250014,China)
出处
《中国医师杂志》
CAS
2019年第9期1330-1335,共6页
Journal of Chinese Physician
基金
山东省中医药管理局科技发展计划项目(2011-191)~~
