调节性T细胞促进球囊损伤颈动脉内皮化的作用及机制

目的 探讨调节性T细胞（Treg细胞）促进球囊损伤颈动脉内皮化的作用及机制。 方法 使用Treg细胞分选试剂盒分选大鼠脾脏Treg细胞；构建大鼠颈动脉损伤模型；模型制作成功后分为对照组（尾静脉注射相同体积的生理盐水）、血管内皮生长因子（VEGF）组（尾静脉注射VEGF， 20 μmol/kg）和Treg细胞组（尾静脉注射1×105个Treg细胞）。HE染色检测内皮化情况；ELISA和免疫组织化学法检测血清中白细胞介素（IL）-10、转化生长因子-β（TGF-β）、IL-1β和肿瘤坏死因子α（TNF-α）的含量；流式细胞术检测单核细胞、T细胞和内皮祖细胞（EPC）的比例。 结果 组织学染色结果显示，对照组未见内皮细胞层的形成，Treg细胞组可见较多内皮细胞覆盖在颈动脉内层；免疫组织化学结果显示，对照组与Treg细胞组IL-10、TGF-β、IL-1β和TNF-α蛋白表达差异均有统计学意义（t=8.252，P<0.01；t=3.254，P<0.05；t=6.237，P<0.01；t=7.529，P<0.01）。ELISA结果显示，对照组IL-10、TGF-β、IL-1β和TNF-α的含量分别为（17.38±2.595）μg/L、（4.750±1.549）μg/L、（11.65±1.908）μg/L和（1.163±0.3333）μg/L；Treg细胞组的含量分别为（58.43±6.060）μg/L、（14.17±2.250）μg/L、（1.550±0.3819）μg/L和（0.2100±0.06938）μg/L，差异有显著性（t=6.170，P<0.01；t=3.558，P<0.01；t=5.191，P<0.01；t=2.800，P<0.05）；流式细胞术的结果显示，对照组CD34+VEGFR-2+EPC比例为（0.2838±0.01975）%，Treg细胞组为（0.5667±0.05993）%，差异有显著性（t=4.483，P<0.01）；IL-10阻断组EPC比例为（0.4807±0.03067）%，相对于Treg细胞组，差异不具有统计学意义（t=1.278，P>0.05）；TGF-β阻断组EPC比例为(0.3082±0.02291)%，相对于Treg细胞组，差异有显著性（t=4.029，P<0.01）。 结论 Treg细胞通过诱导EPC动员，促进球囊损伤颈动脉的快速内皮化。

Role and mechanism of regulatory T cells in promoting endothelialization of carotid artery after balloon injury

Objective To investigate the role and mechanism of regulatory T cells (Treg cell) in promoting endothelialization of carotid artery after balloon injury. Methods Treg cells were isolated from rat spleen by Treg cell sorting kit and carotid artery injury model was established. The rats were divided into three groups: control group (the same volume of normal saline), vascular endothelial growth factor (VEGF) group (intravenous injection of VEGF，20 μmol/kg) and Treg cell group (1×105 Treg cells injected via tail vein). HE staining and immunohistochemistry were used to detect endothelialization. The levels of interleukin (IL)-10, transforming growth factor β(TGF-β), IL-1β and tumor necrosis factor α (TNF-α)in serum were detected by ELISA. Flow cytometry was used to detect the proportion of monocytes, T cells and endothelial progenitor cells (EPC). Results Histological staining showed that no endothelial cells were observed in the control group, and more endothelial cells were found in the carotid intima in the Treg cell group. The result of immunohistochemistry showed that the expression of IL-10,TGF-β, IL-1 β and TNF-α protein in the control group and the Treg cell group were significantly different (t=8.252, P<0.01; t=3.254, P<0.05; t=6.237, P<0.01; t=7.529, P<0.01). ELISA result showed that the contents of IL-10, TGF-β, IL-1β and TNF-α in the control group were (17.38±2.595) μg/L, (4.750±1.549) μg/L, (11.65±1.908) μg/L and (1.163±0.3333)μg/L, respectively, while those in the Treg cell group were (58.43±6.060) μg/L, (14.17±2.250) μg/L, (1.550±0.3819) μg/L and (0.2100±0.06938) μg/L, with significant differences (t=6.170, P<0.01; t=3.558, P<0.01; t=5.191, P<0.01; t=2.800, P<0.05). The result of flow cytometry showed that the ratio of CD34+VEGFR-2+EPC in control group was (0.2838±0.01975)%, and that in Treg cell group was (0.5667±0.05993)% (t=4.483, P<0.01).The proportion of EPC in IL-10 blocking group was 0.4807±0.03067, compared with the Treg cell group, there was no significant difference (t=1.278, P>0.05).The proportion of EPC in TGF-β blocking group was(0.3082±0.02291)%, compared with the Treg cell group, the difference was significant (t=4.029, P<0.01). Conclusion Treg cells promote the rapid endothelialization of carotid artery after balloon injury by inducing EPC mobilization.