Weng Wen-Jun, Pan Li, Fang Jian-Pei, Xu Lv-Hong
Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120,Guangdong Province, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Oct;21(5):1266-70. doi: 10.7534/j.issn.1009-2137.2013.05.036.
This study was purposed to compare the effect of 3 different cell components for expanding CD4(+) CD25(+) Treg in vitro, and identify their immunosuppressive function. CD4(+) T cells, CD4(+) CD25(-)T cells and CD4(+) CD25(+)T cells were isolated from mouse splenocytes by MACS and then expanded in vitro. Phenotype of the T cell lines and expression of the FOXP3 was determined by flow cytometry. The inhibitory effect of expanded CD4(+) CD25(+) T cells on CD4(+) CD25(-)T cells was tested by MLR method. The results showed that the Treg cells from all the three groups were expanded significantly after culture for 2 weeks. In the CD4(+) T cells group, the proliferation rate was (77.8 ± 5.32) folds with a percentage of Treg cells increasing from (6.61 ± 1.00)% to (15.33 ± 1.31)%. The proliferation rate in the CD4(+) CD25(-) T cells group was (95.20 ± 7.67) folds, with the percentage of CD4(+) CD25(+) T cells raising from (0.37 ± 0.13)% to (9.84 ± 0.98)%. The proliferation rate in the CD4(+) CD25(+) T cells group was (41.20 ± 6.92) folds, the proportion of Treg cells decreased from (86.75 ± 1.25)% to (85.32 ± 1.62)%, and the expression of Foxp3 decreased from (76.92 ± 1.72)% to (75.33 ± 2.11)% during the culture, there were not significant differences in the cell purity and the expression of Foxp3, compared with pre-amplification. The inhibitory test showed that the expanded CD4(+) CD25(+) T cells could inhibit the proliferation of CD4(+) CD25(-) T cells in vitro in a cell dose-dependent manner. It is concluded that the amplification of CD4(+) CD25(+) Treg cells is successful in vitro, especially in the CD4(+) CD25(+) T cells group, the cell purity and Foxp3 gene is not obviously changes after amplification.
本研究旨在比较3种不同细胞成分在体外扩增CD4(+) CD25(+)调节性T细胞（Treg）的效果，并鉴定其免疫抑制功能。通过磁珠分选法（MACS）从小鼠脾细胞中分离出CD4(+) T细胞、CD4(+) CD25(-)T细胞和CD4(+) CD25(+)T细胞，然后进行体外扩增。采用流式细胞术检测T细胞系的表型及叉头框蛋白P3（FOXP3）的表达。通过混合淋巴细胞反应（MLR）法检测扩增后的CD4(+) CD25(+) T细胞对CD4(+) CD25(-)T细胞的抑制作用。结果显示，培养2周后，三组的Treg细胞均显著扩增。在CD4(+) T细胞组中，增殖率为（77.8 ± 5.32）倍，Treg细胞百分比从（6.61 ± 1.00）%增至（15.33 ± 1.31）%。CD4(+) CD25(-) T细胞组的增殖率为（95.20 ± 7.67）倍，CD4(+) CD25(+) T细胞百分比从（0.37 ± 0.13）%升至（9.84 ± 0.98）%。CD4(+) CD25(+) T细胞组的增殖率为（41.20 ± 6.92）倍，培养过程中Treg细胞比例从（86.75 ± 1.25）%降至（85.32 ± 1.62）%，Foxp3表达从（76.92 ± 1.72）%降至（75.33 ± 2.11）%，与扩增前相比，细胞纯度和Foxp3表达无显著差异。抑制试验表明，扩增后的CD4(+) CD25(+) T细胞可在体外以细胞剂量依赖的方式抑制CD4(+) CD25(-) T细胞的增殖。结论是，CD4(+) CD25(+) Treg细胞在体外扩增成功，尤其是在CD4(+) CD25(+) T细胞组中，扩增后细胞纯度和Foxp3基因无明显变化。
