CAJ | 학술논문

目的：初步探讨IL-7联合IL-2对脐血CD4＋CD25-T细胞体外扩增的促进作用，建立稳定的体外培养扩增脐血CD4＋CD25-Tregs的培养体系，比较诱导扩增后的CD4＋CD25＋Tregs和自然分选的CD4＋CD25＋Tregs对PBMCs功能活性的影响。方法：采用免疫磁珠分选法分选出脐血CD4＋CD25-T细胞和CD4＋CD25＋T细胞；加入不同浓度的细胞因子IL-7结合适当浓度的IL-2作为诱导剂，分析IL-7体外诱导CD4＋CD25-T细胞增殖的有效性及最适浓度。我们利用流式细胞术检测体外扩增后的CD4＋CD25-T细胞表型变化；MTS法检测体外诱导扩增的CD4＋CD25＋Tregs及自然分选的CD4＋CD25＋Tregs分别对成人外周血中单个核细胞增殖的抑制作用；RT-PCR方法分析体外诱导扩增的CD4＋CD25＋Tregs 及自然分选的CD4＋CD25＋Tregs FOXP3基因、IL-10基因和TGF-β基因的cDNA表达的变化。结果：经3周体外培养、各组细胞均有明显的扩增。 IL-7诱导组的扩增最强。体外抑制试验显示体外扩增的Tregs对成人外周血中单个核细胞有明显的抑制作用，IL-7联合IL-2诱导CD4＋CD25-T细胞生成的CD4＋CD25＋Tregs具有较自然分选的CD4＋CD25＋Tregs稍弱的免疫抑制功能，其中IL-7浓度为4 ng／ml，IL-2浓度为2000 U／ml时诱导CD4＋CD25-T细胞生成的CD4＋CD25＋Tregs杀伤活性最强。结论：成功建立了CD4＋CD25＋Tregs的体外培养体系，联合应用IL-7、大剂量的IL-2和CD3／CD28单抗是体外诱导扩增CD4＋CD25-T细胞成为CD4＋CD25＋Tregs的优选方法，并且扩增倍数高、可持续高表达CD4及CD25细胞表型。
목적：초보탐토IL-7연합IL-2대제혈CD4＋CD25-T세포체외확증적촉진작용，건립은정적체외배양확증제혈CD4＋CD25-Tregs적배양체계，비교유도확증후적CD4＋CD25＋Tregs화자연분선적CD4＋CD25＋Tregs대PBMCs공능활성적영향。방법：채용면역자주분선법분선출제혈CD4＋CD25-T세포화CD4＋CD25＋T세포；가입불동농도적세포인자IL-7결합괄당농도적IL-2작위유도제，분석IL-7체외유도CD4＋CD25-T세포증식적유효성급최괄농도。아문이용류식세포술검측체외확증후적CD4＋CD25-T세포표형변화；MTS법검측체외유도확증적CD4＋CD25＋Tregs급자연분선적CD4＋CD25＋Tregs분별대성인외주혈중단개핵세포증식적억제작용；RT-PCR방법분석체외유도확증적CD4＋CD25＋Tregs 급자연분선적CD4＋CD25＋Tregs FOXP3기인、IL-10기인화TGF-β기인적cDNA표체적변화。결과：경3주체외배양、각조세포균유명현적확증。 IL-7유도조적확증최강。체외억제시험현시체외확증적Tregs대성인외주혈중단개핵세포유명현적억제작용，IL-7연합IL-2유도CD4＋CD25-T세포생성적CD4＋CD25＋Tregs구유교자연분선적CD4＋CD25＋Tregs초약적면역억제공능，기중IL-7농도위4 ng／ml，IL-2농도위2000 U／ml시유도CD4＋CD25-T세포생성적CD4＋CD25＋Tregs살상활성최강。결론：성공건립료CD4＋CD25＋Tregs적체외배양체계，연합응용IL-7、대제량적IL-2화CD3／CD28단항시체외유도확증CD4＋CD25-T세포성위CD4＋CD25＋Tregs적우선방법，병차확증배수고、가지속고표체CD4급CD25세포표형。
Objective:To explore the promoting effects of IL-7 and IL-2 on CD4+CD25-T cells proliferation in vitro and construct a stable culture system in vitro for CD 4+CD25+regulatory T cells from human umbilical cord blood.To compare the inhibiting effects between induced proliferated CD 4+CD25+Tregs and naturally isolated CD 4+CD25+Tregs on PBMCs functional activity.Methods:CD4+CD25-T cells and CD4+CD25+T cells were isolated from human umbilical cord blood mononuclear cells by magnetic activated cell sorting ( MACS) system and then expanded in vitro.Four different concentration levels of IL-7 combined with proper concentration of IL-2 were added as inducer and the efficiency and optimal concentration of IL-7 on inducing,CD4+CD25-T cells were analyzed via 4 different methods.Flow cytometry method was used to detect the changes of CD 4+CD25-T cells.The inhibitory effect of expanded CD 4+CD25+T cells on peripheral blood mononuclear cells (PBMCs) was tested by MTS.The expressions of Foxp3,IL-10 and TGF-βgenes in CD4+CD25+T cells were test by RT-PCR.Results:The CD4+CD25+T cells from each groups were expanded significantly after three weeks of culture.The results indicated that use of IL-7 combined with IL-2 resulted in the highest cell expansion comparing to the other groups.It was shown by the inhibitory test that the expanded CD 4+CD25+regulatory T cells could inhibit the proliferation of PBMCs ,but IL-7 induced CD4+CD25+regulatory T cells exerted weaker suppressor activity than natural regulatory T cells .Only IL-7 (4 ng/ml) and IL-2 (2 000 U/ml) induced CD4+CD25+regulatory T cells showed the strongest killing activity.Conclusion:We successfully expand CD4+CD25+regulatory T cells in vitro.The protocol is established in which the use of mAbCD 3/CD28 combined with IL-7 and IL-2 resulted in the highest cell expansion ,and intensely expressed cell phenotype of CD 4 and CD25.