骨髓间充质干细胞浓度调节Treg/Th17表达的研究

Study on the regulation of Treg/Th17 expression by the concentration of bone marrow mesenchymal stem cells

目的探讨骨髓间充质干细胞(BMMSCs)浓度对调节性T细胞(Treg)/辅助性T细胞17(Th17)表达的影响。方法贴壁法提取大鼠BMMSCs并传代培养至P4代,油红O染色法及钙盐染色法测定P3代成脂及成骨诱导能力。倒置显微镜下标记CD29、CD90、CD45荧光抗体。磁珠法分选大鼠CD4^(+)T细胞,流式细胞仪测定细胞纯度。将CD4^(+)T细胞与浓度不同的BMMSCs(0.5倍、1倍、2倍、4倍浓度组)共培养3 d,流式细胞仪测定Treg、Th17增值比例,流式微球分析法检测转化生长因子-β(TGF-β)及白介素(IL-6、IL-10、IL-17)水平。结果本研究P4代BMMSCs形态均匀,且呈梭形紧密排列,成脂诱导后镜下呈现橘红色脂肪小滴,成骨诱导后镜下呈现黑钙盐结节。CD90、CD29于BMMSCs表面阳性表达,CD45弱表达。磁珠法分选出CD4^(+)T细胞约(5.75±0.36)×10^(6)个/ml,细胞纯度>95%。共培养后,在Treg增殖比例上,2倍>1倍>0.5倍>4倍浓度组,2倍浓度组与其他各组比较,差异有统计学意义(P<0.05)。在Th17增殖比例上,1倍>2倍>0.5倍>4倍浓度组,0.5倍浓度组与各组组间比较,4倍浓度组与1倍、2倍浓度组比较,差异有统计学意义(P<0.05)。在TGF-β水平上,1倍>2倍>4倍>0.5倍浓度组,1倍浓度组与0.5倍、4倍浓度组组间比较,差异有统计学意义(P<0.05)。在IL-10水平上,2倍>1倍>4倍>0.5倍浓度组,0.5倍浓度组与其他各组比较,差异有统计学意义(P<0.05)。在IL-6、IL-17水平上,各组组间比较,差异无统计学意义(P>0.05)。结论BMMSCs具多向分化潜能,CD4^(+)T细胞分化成Treg、Th17的增殖比例受到BMMSCs浓度的影响,较高浓度时增殖效应最强,高浓度时抑制增殖,两者增殖比例变化与相关因子表达水平并不同步。

Objective To investigate the effect of the concentration of bone marrow mesenchymal stem cells(BMMSCs)on the expression of regulatory T cells(Treg)/T helper 17 cells(Th17).Methods Rat BMMSCs were extracted by adherence culture method and subcultured to P4 generation,and the lipogenic and osteogenic induction potential of P3 generation was determined by oil red O staining and calcium salt staining.CD29,CD90 and CD45 fluorescent antibodies were labeled under inverted microscope.Rat CD4^(+)T cells were sorted by magnetic bead method,and cell purity was determined by flow cytometry.CD4^(+)T cells were co-cultured with BMMSCs of different concentrations(0.5×,1×,2×,and 4×)group for 3 d.Treg and Th17 value-added ratios were determined by flow cytometry,and the levels of transforming growth factor-β(TGF-β)and interleukin(IL-6,IL-10,IL-17)were measured by cytometric bead array.Results In this study,BMMSCs of P4 generation were uniform in morphology and closely arranged in a shuttle shape,showing small fat droplets in tangerine microscopically after lipogenic induction and black calcium salt nodules microscopically after osteogenic induction.CD90 and CD29 were positively expressed on the surface of BMMSCs and CD45 was weakly expressed.CD4^(+)T cells were sorted out by magnetic bead method at about(5.75±0.36)×10^(6) cells/ml,with cell purity>95%.After co-culture,the ratio of Treg proliferation was:2×>1×>0.5×>4×concentration group,with the 2×concentration group significantly different from all other concentration groups,with statistically significant differences(P<0.05).The ratio of Th17 proliferation was:1×>2×>0.5×>4×concentration group,with the 0.5×concentration group significantly different from all other concentration groups,and the 4×concentration group significantly different from 1×and 2×concentration groups,with statistically significant differences(P<0.05).The level of TGF-β was:1×>2×>4×>0.5×concentration group,with the 1×concentration group significantly different from 0.5×and 4×concentration groups,with statistically significant differences(P<0.05).The level of IL-10 was:2×>1×>4×>0.5×concentration group,with the 0.5×concentration group different from all other concentration groups,with statistically significant differences(P<0.05).There were no statistically significant differences in the levels of IL-6 and IL-17 among each group(P>0.05).Conclusion BMMSCs have multi-lineage differentiation potential,and the proliferation ratio of CD4^(+)T cells differentiated into Treg and Th17 is affected by the concentration of BMMSCs,with the strongest proliferative effect at relatively high concentrations and inhibition of proliferation at higher concentrations,and the changes in the proliferation ratios of Treg and Th17 are not synchronized with the expression levels of related factors.