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目的探讨人脐带间充质干细胞(hUC-MSC)和他克莫司(FK506)联用在体外对T淋巴细胞增殖的影响。方法脐带来自中山大学附属第三医院产科足月妊娠的健康剖宫产产妇,采用酶联合消化法分离hUC-MSC。根据处理方式不同分为hUC-MSC+T淋巴细胞组(MSC组)、FK506+T淋巴细胞组(FK506组)、hUC-MSC+FK506+T淋巴细胞组(联合组)、24 h延迟给药联合组(延迟联合组)、实验对照组(对照组)、空白对照组(空白组)。MSC组取1×104个hUC-MSC接种于96孔板,丝裂霉素C处理0.5 h后洗净,hUC-MSC:外周血单个核细胞(PBMC)按1∶10加入1×105个PBMC;FK506组取1×105个PBMC接种于96孔板,加入终浓度为10 ng/ml的FK506;联合组在MSC组基础上加入终浓度为10 ng/ml的FK506;延迟联合组为MSC组培养24 h再加入终浓度为10 ng/ml的FK506;对照组仅接种1×105个PBMC;空白组为丝裂霉素C处理后的1×104个hUC-MSC接种96孔板。各组均加入终浓度1μg/ml的植物血凝素(PHA),培养3 d,采用BrdU化学发光法,应用多功能酶标仪检测各组的发光度。各组发光度的比较采用单因素方差分析和LSD-t检验。结果 MSC组发光度为(6.56±0.91)×106RLU/s,FK506组为(2.07±0.38)×106RLU/s,对照组为(9.53±0.72)×106RLU/s,MSC组和FK506组发光度明显低于与对照组,显示hUC-MSC和FK506均能明显抑制PHA刺激的T淋巴细胞增殖(LSD-t=-6.06,-15.21;P<0.05)。联合组发光度为(7.80±1.07)×106RLU/s,延迟联合组为(5.55±0.35)×106RLU/s,联合组发光度较MSC组明显升高,延迟联合组较MSC组明显降低,显示hUC-MSC和FK506同时加入共培养系统时,对于T淋巴细胞增殖的抑制作用,两者表现为明显拮抗效应,而延迟24 h加入共培养系统时,两者表现为明显的协同效应(LSD-t=2.53,-2.06;P<0.05)。结论单独使用hUC-MSC或FK506均能明显抑制T淋巴细胞增殖;两者联合应用时表现为拮抗效应;当延迟24 h加入FK506则表现为协同效应。
Objective To investigate the effects of human umbilical cord mesenchymal stem cells (hUC-MSCs) and tacrolimus (FK506) on the proliferation of T lymphocytes in vitro. Methods The umbilical cords were from healthy cesarean section women who gave full-term pregnancy obstetrics and gynecology at the Third Affiliated Hospital of Sun Yat-sen University. HUC-MSCs were isolated by enzymatic digestion. According to the different treatment methods, hUC-MSC + T lymphocyte group (MSC group), FK506 + T lymphocyte group (FK506 group), hUC-MSC + FK506 + T lymphocyte group Combination group (delayed combination group), experimental control group (control group), blank control group (blank group). 1 × 104 hUC-MSCs were seeded in 96-well plates in MSC group and 0.5 h after mitomycin C treatment. HUC-MSC: Peripheral blood mononuclear cells (PBMC) were added 1 × 10 5 PBMCs 1:10 ; FK506 group, 1 × 105 PBMCs were seeded in 96-well plates and added with FK506 at a final concentration of 10 ng / ml; the combination group was added with FK506 at a final concentration of 10 ng / ml on the basis of MSC group; Cultured for 24 h and then added with FK506 at the final concentration of 10 ng / ml. The control group was inoculated with only 1 × 105 PBMCs. The blank group was treated with 1 × 104 hUC-MSCs treated with mitomycin C and seeded in 96-well plates. Phytohemagglutinin (PHA) at a final concentration of 1μg / ml was added into each group and cultured for 3 days. BrdU chemiluminescence was used to detect the luminescence of each group. The luminosity of each group was compared using one-way ANOVA and LSD-t test. Results The luminosity of MSC group and FK506 group was (6.56 ± 0.91) × 106 RLU / s, (2.07 ± 0.38) × 106 RLU / s in the group of FK506 and (9.53 ± 0.72) × 106 RLU / Lower than the control group, hUC-MSCs and FK506 both showed significant inhibition of PHA-stimulated T lymphocyte proliferation (LSD-t = -6.06, -15.21; P <0.05). The luminosity of the combination group was (7.80 ± 1.07) × 106RLU / s and that of the delayed combination group was (5.55 ± 0.35) × 106RLU / s, and the luminescence intensity of the combination group was significantly higher than that of the MSC group When both hUC-MSCs and FK506 were added into the co-culture system, their inhibitory effects on the proliferation of T lymphocytes showed obvious antagonistic effects. However, when hUC-MSCs and FK506 were added into the co-culture system for 24 h, the two showed obvious synergistic effects (LSD- t = 2.53, -2.06; P <0.05). Conclusion Both hUC-MSCs and FK506 alone can significantly inhibit the proliferation of T lymphocytes. The combination of these two drugs showed antagonistic effects. When FK506 was added for 24 h, the synergistic effect was observed.