Impact of human umbilical cord mesenchymal stem cells combined with tacrolimus on T lymphocytes proliferation

Abstract

Objective To investigate the impact of human umbilical cord mesenchymal stem cell (hUC-MSC) combined with tacrolimus (FK506) on T lymphocytes proliferation in vitro. Methods The umbilical cord was obtained from healthy puerpera who underwent cesarean after full term pregnancy in Department of Obstetrics, the Third Affiliated Hospital of Sun Yat-sen University. The hUC-MSCs were derived by combined enzymatic digestion. According to the different processing, 6 groups were designed: hUC-MSC+ T lymphocytes (MSC) group, FK506+ T lymphocytes (FK506) group, hUC-MSC+ FK506+ T lymphocytes (combination) group, 24 h delay-combination (delay-combination) group, experimental control (control) group and blank control (blank) group. In MSC group, 1×104 hUC-MSCs were placed in 96-well plates and cleansed after 0.5 h of treatment with mitomycin-C. Then 1×105 human peripheral blood mononuclear cells (PBMC) were added by a hUC-MSC∶PBMC proportion of 1∶10. In FK506 group, 1×105 PBMCs were placed in 96-well plates and FK506 (final concentration 10 ng/ml) was added. In combination group, FK506 (final concentration 10 ng/ml) was added on the basis of MSC group. In delay-combination group, hUC-MSCs were cultured for 24 h and then FK506 (final concentration 10 ng/ml) was added. In control group, only 1×105 PBMCs were placed in 96-well plates. In blank group, 1×104 hUC-MSCs were placed in 96-well plates after treatment with mitomycin-C. Phytohaemagglutinin (PHA, final concentration 1 μg/ml) was added in every group, and was cultured for 3 d. The luminosity in each group was determined by chemiluminescence BrdU using multi-mode microplate reader and was compared among groups by one-way analysis of variance and LSD-t test. Results The luminosities of MSC group, FK506 group and control group were (6.56±0.91)×106 RLU/s, (2.07±0.38)×106 RLU/s, (9.53±0.72)×106 RLU/s respectively. The luminosities of MSC group and FK506 group were significantly lower than that of control group, indicating that both hUC-MSC and FK506 could obviously inhibit T lymphocytes proliferation induced by PHA (LSD-t=-6.06,-15.21; P<0.05). The luminosities of combination group and delay-combination group were (7.80±1.07)×106 RLU/s, (5.55±0.35)×106 RLU/s respectively. The luminosity of combination group was significantly higher than that of MSC group, while the luminosity of delay-combination group was significantly lower than that of MSC group. It indicated that when hUC-MSC and FK506 were added in co-culture system simultaneously, obvious antagonistic effect on T lymphocytes proliferation was observed in both groups, while FK506 were added in co-culture system 24 h later, obvious synergistic effect on T lymphocytes proliferation was observed in both groups (LSD-t=2.53,-2.06; P<0.05). Conclusions Single use of hUC-MSC or FK506 can either obviously inhibit T lymphocytes proliferation. Simultaneous use of hUC-MSC and FK506 results antagonistic effect on T lymphocytes proliferation. When FK506 is added 24 h later, synergistic effect is observed. Key words: Mesenchymal stem cells; Tacrolimus; T-lymphocytes; Immunosuppression; Cell proliferation