CAJ | 학술논문

目的探讨利妥昔单抗对原发免疫性血小板减少症(ITP)患者树突细胞(DC)功能的影响,探讨其治疗机制.方法取小剂量利妥昔单抗治疗有效的ITP患者治疗前后的外周血单个核细胞(PBMC)与重组人粒-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素4(rhIL-4)于37℃、5％CO2条件下共孵育诱导培养DC；第5天加入肿瘤坏死因子-α(TNF-α)继续培养48 h,获得成熟DC.倒置显微镜下观察DC形态；流式细胞术检测DC表型；ELISA法检测DC培养上清人白细胞介素12(IL-12p70)和转化生长因子-β1(TGF-β1)的浓度；噻唑蓝(MTT)法检测DC刺激自体T淋巴细胞的增殖能力.结果 ①治疗后DC较治疗前DC细胞膜缺乏典型的树枝状突起、体积较小、核多居中规则；②治疗后DC HLA-DR、CD80、CD83和CD86表达水平[(56.37±3.95)％、(36.41±2.82)％、( 30.45±4.61)％和(41.98±4.17)％]明显低于治疗前[(73.71±7.61)％、(55.14±7.30)％、(80.91±7.09)％和(59.03±3.43)％](P值均＜0.05),IL-12p70水平[(50.17±14.52)％]低于治疗前[(66.87±4.29)％],TGF-β1水平[(9.70±0.31)％]高于治疗前[(2.70±0.36)％](P值均＜0.05)；③治疗后DC诱导的T细胞增殖指数较治疗前明显减低.结论小剂量利妥昔单抗治疗后ITP患者DC表型及分泌IL-12p70的能力明显降低、分泌TGF-β1能力增高、对自体T细胞增殖的刺激能力减低.小剂量利妥昔单抗下调DC的免疫活性可能是其治疗ITP的机制之一.
목적 탐토리타석단항대원발면역성혈소판감소증(ITP)환자수돌세포(DC)공능적영향,탐토기치료궤제.방법 취소제량리타석단항치료유효적ITP환자치료전후적외주혈단개핵세포(PBMC)여중조인립-거서세포집락자격인자(rhGM-CSF)、중조인백세포개소4(rhIL-4)우37℃、5％CO2조건하공부육유도배양DC；제5천가입종류배사인자-α(TNF-α)계속배양48 h,획득성숙DC.도치현미경하관찰DC형태；류식세포술검측DC표형；ELISA법검측DC배양상청인백세포개소12(IL-12p70)화전화생장인자-β1(TGF-β1)적농도；새서람(MTT)법검측DC자격자체T림파세포적증식능력.결과 ①치료후DC교치료전DC세포막결핍전형적수지상돌기、체적교소、핵다거중규칙；②치료후DC HLA-DR、CD80、CD83화CD86표체수평[(56.37±3.95)％、(36.41±2.82)％、( 30.45±4.61)％화(41.98±4.17)％]명현저우치료전[(73.71±7.61)％、(55.14±7.30)％、(80.91±7.09)％화(59.03±3.43)％](P치균＜0.05),IL-12p70수평[(50.17±14.52)％]저우치료전[(66.87±4.29)％],TGF-β1수평[(9.70±0.31)％]고우치료전[(2.70±0.36)％](P치균＜0.05)；③치료후DC유도적T세포증식지수교치료전명현감저.결론소제량리타석단항치료후ITP환자DC표형급분비IL-12p70적능력명현강저、분비TGF-β1능력증고、대자체T세포증식적자격능력감저.소제량리타석단항하조DC적면역활성가능시기치료ITP적궤제지일.
Objective To explore the changes of surface antigen and function of rituximab on dendritic cells derived from patients with Primary immune thrombocytopenia(ITP) to further understand the effective mechanism of immunotherapy.Methods The peripheral blood mononuelear cells (PBMCs) were isolated from remission patients with ITP befoe and after low-dose rituximab infusion,and the PMNCs were stimulated for 5 days by rhGM-CSF and rhlL-4 in 5％ CO2 air at 37 ℃ incubator.Then all of DCs were cultured with TNF-α for 48 hours.The morphology of DCs was monitored under inverted microscope daily,and the surface antigens of the DCs were analysed by flow cytometry,meanwhile the levels of IL-12p70 and TGF-β1 in supernatants were detected by ELISA,mix lymphocyte reaction was performed by MTT assay.Results ①Rituximab-treated-DCs showed no obvious tree-like protruding compared with untreated-DCs.The former cells were small and most of nucleus were centric.②The expressions of HLA-DR,CD80,CD83 and CD86 on rituximab-treated-DCs [56.37 ± 3.95 ) ％,( 36.41 ± 2.82) ％,(30.45 ± 4.61 ) ％ and (41.98 ± 4.17 ) ％,respectively]were significantly lower than those untreated-DCs [(73.71 ± 7.61 )％,(55.14 ± 7.30)％,(80.91 ± 7.09) ％ and (59.03 ± 3.43 ) ％,respectively]( all P ＜ 0.05 ),the concentration of IL-12p70 was significantly lower,[( 66.87 ± 4.29 ) ％ vs ( 50.17 ± 14.52 ) ％],while that of TGF-β1 [( 9.70 ±0.31)％]higher than the untreated-DCs [(2.70 ±0.36)％](P ＜0.05).(③The abilities to activate Tcells proliferation of rituximab-treated-DCs reduced compared with untreated-DCs.Conclusion The surface antigen of ITP-DCs and the concentration of IL-12p70 reduced after the low-dose rituximab infusion.The abilities to activate T cells proliferation reduced while the concentration of TGF-β1 increased. Rituximabmay achieve its therapeutic effect on ITP by downregulating the immunoreactivity of DCs.