CAJ | 학술논문

目的建立一种稳定且高效的不成熟树突状细胞(imDC)体外培养方法,探讨细胞外信号调节激酶(ERK)1/2信号传导通路抑制剂GW5074对imDC体外诱导同种初始性CD4+T淋巴细胞分化为调节性T淋巴细胞(Treg)的影响.方法从健康成人外周血单个核细胞(PBMC)中分离、培养成熟树突状细胞(mDC)和imDC,并对mDC和imDC的免疫表型和功能进行鉴定.取新生儿脐静脉血分离初始性CD4+T淋巴细胞.实验分为5组:(1)空白对照组为单纯培养的初始性CD4+T淋巴细胞,不做任何处理;(2)阳性对照组将imDC与初始性CD4+T淋巴细胞以1∶10的细胞比例混合培养;(3)低浓度GW5074组;(4)中浓度GW5074组;(5)高浓度GW5074组.后3组在阳性对照组基础上,分别加入终浓度为8、24和40μmol/L的GW5074.培养5 d后,用流式细胞仪检测初始性CD4+T淋巴细胞转化为Treg细胞的转化率.结果 imDC呈CD1a高表达,CD80和CD83低表达;mDC呈CD1a低表达,CD80和CD83高表达.imDC和mDC的刺激指数分别为1.12±0.03、2.85±0.07.空白对照组,阳性对照组,低、中及高浓度GW5074组的CD4+CD25+Treg转化率分别为(5.81±1.36)%、(35.73±2.07)%、(22.53±2.11)%、(11.55±1.73)%和(4.97±1.83)%,除空白对照组与高浓度GW5074组间的差异无统计学意义(P＞0.05)外,其余各组之间两两比较,差异均有统计学意义(P＜0.01).结论通过应用重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素4联合诱导人外周血PBMC可获得高纯度imDC,ERK1/2信号传导通路在诱导免疫耐受中发挥作用,GW5074可抑制初始性CD4+T淋巴细胞向Treg转化.
목적 건립일충은정차고효적불성숙수돌상세포(imDC)체외배양방법,탐토세포외신호조절격매(ERK)1/2신호전도통로억제제GW5074대imDC체외유도동충초시성CD4+T림파세포분화위조절성T림파세포(Treg)적영향.방법 종건강성인외주혈단개핵세포(PBMC)중분리、배양성숙수돌상세포(mDC)화imDC,병대mDC화imDC적면역표형화공능진행감정.취신생인제정맥혈분리초시성CD4+T림파세포.실험분위5조:(1)공백대조조위단순배양적초시성CD4+T림파세포,불주임하처리;(2)양성대조조장imDC여초시성CD4+T림파세포이1∶10적세포비례혼합배양;(3)저농도GW5074조;(4)중농도GW5074조;(5)고농도GW5074조.후3조재양성대조조기출상,분별가입종농도위8、24화40μmol/L적GW5074.배양5 d후,용류식세포의검측초시성CD4+T림파세포전화위Treg세포적전화솔.결과 imDC정CD1a고표체,CD80화CD83저표체;mDC정CD1a저표체,CD80화CD83고표체.imDC화mDC적자격지수분별위1.12±0.03、2.85±0.07.공백대조조,양성대조조,저、중급고농도GW5074조적CD4+CD25+Treg전화솔분별위(5.81±1.36)%、(35.73±2.07)%、(22.53±2.11)%、(11.55±1.73)%화(4.97±1.83)%,제공백대조조여고농도GW5074조간적차이무통계학의의(P＞0.05)외,기여각조지간량량비교,차이균유통계학의의(P＜0.01).결론 통과응용중조인립세포-거서세포집락자격인자화중조인백세포개소4연합유도인외주혈PBMC가획득고순도imDC,ERK1/2신호전도통로재유도면역내수중발휘작용,GW5074가억제초시성CD4+T림파세포향Treg전화.
Objective To establish a stable and efficient method of culturing imDCs in vitro,and to explore the effect of GW5074, which blocks ERK1/2 signal pathway in the process of imnature dentritic cells (imDCs) on inducing differentiation of the na(i)ve allogeneic CD4+ T cells into Treg cells in vitro. Methods The imDCs and mature DCs (mDCs) were isolated and cultured from the peripheral blood mononuclear cells (PBMC) derived from a healthy adult male volunteer, and they were identified by cell morphology, cell surface marker and cell functions respectively. Na(i)ve CD4+ T cells were isolated from newborn umbilical vein blood and were divided into 5 groups to be cultured: (1) Blank control group: Na(i)ve CD4+ T cells were cultured alone;(2) Positive control group: The irrDCs were Middle-concentration GW5074 group;(5) High-concentration GW5074 group. In the last three groups, imDCs and na(i)ve CD4+ T cells were co-cultured, the same as the positive control group, but these groups were added by GW5074 dilution at the concentrations of 8, 24, and 40μmol/Lrespectively. After co-culture for 5 days, the transformation ratio from naive CD4+T cells to Treg T cells was detected by flow cytometry. Results On the surface of imDCs, there was stronger pression of CD1a, but weaker expression of CD80 and CD83. On the contrary, on the surface of mDCs, there was weaker expression of CD1a, but stronger expression of CD80 and CD83. The stimulation index in imDCs group and mDCs group was 1.12±0.03 and 2.85±0. 07 respectively. The transformation ratio of Treg T cells in blank control group, positive control group, low-concentration GW5074 group, middle-concentration GW5074 group and high-concentration GW5074 group was (5. 81±1.36)%, (35.73±2.07)%, (22.53±2.11)%, (11.55±1.73)%, and (4.97±1.83)%respectively. One-way ANOVA analysis revealed that there was no significant difference between high-concentration GW5074 group and blank control group, P＞0. 05, but significant difference between the remaining groups, P＜0.01. Conclusion High purity of imDCs can be obtained from PBMC by induction with rhGM-CSF and rhIL-4. ERK1/2 signal pathway plays a role in inducing the immune tolerance. GW5074 can inhibit differentiation of na(i)ve CD4+ T cells into Treg T cells.