To investigate the impact of human umbilical cord mesenchymal stem cell (hUC-MSC) combined with tacrolimus (FK506) on T lymphocytes proliferation in vitro.

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.

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).

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.