CAJ | 학술논문

目的体外观察骨髓间充质干细胞(MSC)对原发免疫性血小板减少症(ITP)患者CD4+CD25-T细胞比例以及T细胞分泌IL-2、IFN-γ、IL-4、IL-10功能的影响.方法采用Ficoll分离骨髓单个核细胞,体外培养扩增MSC;采用Ficoll分离法和尼龙棉柱法获取正常人及ITP患者外周血T细胞,应用流式细胞术(FCM)检测T细胞中CD4+CD25T细胞比例;MSC经丝裂霉素(MMC)处理后分别以1×104、5× 104、2×105/孔接种于培养板,然后分别接种体外分离纯化的异体ITP患者及正常人T细胞,共培养5d后用FCM检测CD4CD25+T细胞比例,用ELISA法测定培养上清中IL-2、IFN-γ、IL-4、IL-10含量.结果 2×105/孔 MSC与ITP患者T细胞共培养5d后,CD4CD25+T细胞、CD4+CD257CD4+细胞均高于未加MSC的ITP患者T细胞[(4.56±0.70)％对(2.24+0.81)％,(9.91±1.18)％对(4.08±1.17)％],差异均有统计学意义(P＜0.05);与未加MSC的ITP患者T细胞组比较,培养上清中IL-2、IFN-γ浓度减低[(97.21±12.07)pg/ml对(280.47+ 17.33)pg/ml,(72.75+7.81)pg/ml对(129.33±16.34)pg/ml],IL-4、IL-10浓度升高[(37.98±4.05)pg/ml对(16.34±2.60)pg/ml,(113.77±5.68)pg/ml对(54.78±5.62)pg/ml].结论 MSC在体外可抑制ITP患者Th1细胞分泌IL-2、IFN-γ,促进Th2细胞分泌IL-4、IL-10,上调CD4+CD25+T细胞比例,进而诱导ITP患者免疫耐受形成.
목적 체외관찰골수간충질간세포(MSC)대원발면역성혈소판감소증(ITP)환자CD4+CD25-T세포비례이급T세포분비IL-2、IFN-γ、IL-4、IL-10공능적영향.방법 채용Ficoll분리골수단개핵세포,체외배양확증MSC;채용Ficoll분리법화니룡면주법획취정상인급ITP환자외주혈T세포,응용류식세포술(FCM)검측T세포중CD4+CD25T세포비례;MSC경사렬매소(MMC)처리후분별이1×104、5× 104、2×105/공접충우배양판,연후분별접충체외분리순화적이체ITP환자급정상인T세포,공배양5d후용FCM검측CD4CD25+T세포비례,용ELISA법측정배양상청중IL-2、IFN-γ、IL-4、IL-10함량.결과 2×105/공 MSC여ITP환자T세포공배양5d후,CD4CD25+T세포、CD4+CD257CD4+세포균고우미가MSC적ITP환자T세포[(4.56±0.70)％대(2.24+0.81)％,(9.91±1.18)％대(4.08±1.17)％],차이균유통계학의의(P＜0.05);여미가MSC적ITP환자T세포조비교,배양상청중IL-2、IFN-γ농도감저[(97.21±12.07)pg/ml대(280.47+ 17.33)pg/ml,(72.75+7.81)pg/ml대(129.33±16.34)pg/ml],IL-4、IL-10농도승고[(37.98±4.05)pg/ml대(16.34±2.60)pg/ml,(113.77±5.68)pg/ml대(54.78±5.62)pg/ml].결론 MSC재체외가억제ITP환자Th1세포분비IL-2、IFN-γ,촉진Th2세포분비IL-4、IL-10,상조CD4+CD25+T세포비례,진이유도ITP환자면역내수형성.
Objectives To analyze in vitro the effect of mesenchymal stem cells (MSCs) on secreting cytokines by T lymphocytes and ratio of CD4+CD25 T cells from patients with immune thrombocytopenia (ITP).Methods Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells.Allogeneic T lymphocytes of health adults and ITP patients were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column,and the ratio of CD4+CD25+T cells was detected by flow cytometry.Then the different amounts of 1 × 104,5 × 104,2 × 105 MSCs per well treated with mitomycin as stromal feeder layers were co-cultured with above-mentioned T lymphocytes,5 days after cocultivation,the ratio of CD4+CD25+ T cells was detected by flow cytometry and the levels of IL-2,IFN-γ,IL-4,IL-10 were measured by enzyme-linked immune sorbent assay (ELISA).Results After co-cultured with 2 × 105 MSCs for 5 days,the ratio of CD4CD25+ T cells and CD4+CD25+/CD4+ were significantly higher than of separate T lymphocytes in ITP patients [(4.56±0.70)％vs(2.24±0.81)％,(9.91+1.18)％ vs (4.08+1.17)％,respectively] (P＜0.05).To compare with separate T lymphocytes in ITP patients,the cytokine concentrations of IL-2 and IFN-γ from the culture supematants significantly reduced from (280.47+17.33) pg/ml to (97.21±12.07) pg/ml and from (129.33±16.34) pg/ml to (72.75+ 7.81) pg/ml,respectively.In contrast,the cytokine concentrations of IL-4 and IL-10 increased from (16.34±2.60) pg/ml to (37.98±4.05) pg/ml and from (54.78±5.62) pg/ml to (113.77±5.68) pg/ml,respectively.Conclusions MSCs significantly inhibited the cytokine levels of IL-2 and IFN-γ secreted by Th1 cells and promoted the releases of IL-4 and IL-10 by Th2 cells in ITP,thereby regulating the balance between Th1 and Th2 reaction,as well as up-regulating the expression of CD4+CD25+ T cells in vitro,then induced the immunologic tolerance of ITP.