Role of bone marrow mesenchymal stem cells in different concentrations on regulatory T cell/T-helper cell 17 balance in rats / 中华危重病急救医学

ABSTRACT

OBJECTIVE@#To explore the effects of bone marrow mesenchymal stem cells (BMSCs) in different concentrations on the balance of regulatory T cell/T-helper cell 17 (Treg/Th17).@*METHODS@#BMSCs were isolated from SPF grade male Wistar rats with age of 3 weeks old and weight of 50 g. BMSCs were cultured and identified when they were expanded to the 4th generation. CD4+ T lymphocytes were isolated from SPF grade male Wistar rat with age of 6 weeks old and weight of 200 g and assayed for cell purity by flow cytometry. BMSCs were divided into 0.5-fold concentration group, basal concentration group, 2-fold concentration group and 4-fold concentration group by their concentrations of 1×105/well, 2×105/well, 4×105/well and 8×105/well, which were cultured with CD4+ T lymphocytes for 72 hours, respectively. Then the proportion of Treg cells and Th17 cells in each group was detected by flow cytometry, and cytokines were detected by cytometric bead array.@*RESULTS@#The purities of BMSCs and CD4+ T lymphocytes were both higher than 95%. In the co-culture of BMSCs and CD4+ T lymphocytes, the proportions of Treg cells were statistically different among different concentration groups of BMSCs (F = 10.071, P = 0.001), in which BMSCs in 2-fold concentration group had the strongest ability to promote the Treg cells proliferation. The proportion of Treg cells in 2-fold concentration group was significantly higher than that in 0.5-fold concentration group, basal concentration group and 4-fold concentration group [(9.24±2.68)% vs. (3.87±0.38)%, (5.16±1.69)%, (3.86±0.36)%, all P < 0.01]. The level of interleukin-10 (IL-10) was lowest in 0.5-fold concentration group, and it was significantly lower than that in basal concentration group, 2-fold concentration group and 4-fold concentration group (ng/L 39.80±14.48 vs. 148.43±64.49, 156.40±59.27, 126.92±42.95, all P < 0.05). Transforming growth factor-β (TGF-β) was the highest in basal concentration group, and it was significantly higher than that in 0.5-fold concentration group, 2-fold concentration group and 4-fold concentration group [ng/L 3.17 (1.88, 5.74) vs. 0.71 (0.32, 1.38), 1.22 (0.47, 2.97), 0.52 (0.37, 1.23), all P < 0.05]. The proportions of Th17 cells were statistically different among the different concentration groups (F = 21.069, P = 0.000), with the highest proportion in basal concentration group which was significantly higher than that in 0.5-fold concentration group or 4-fold concentration group [(0.89±0.08)% vs. (0.64±0.15)%, (0.37±0.10)%, both P < 0.01], but no significant difference was found as compared with 2-fold concentration group [(0.83±0.06)%, P > 0.05]. However, the expressions of IL-17 and IL-6 were not different among the different concentration groups respectively (IL-17 χ2 = 0.550, P = 0.760; IL-6 χ2 = 0.010, P = 0.995).@*CONCLUSIONS@#BMSCs in moderate concentrations [(2-4)×105/well] could promote proliferation both in Treg cells and Th17 cells, but no change could be found in higher concentrations of BMSCs (8×105/well). However, the changes in related cytokines were not synchronized with Treg/Th17 cells.