论文部分内容阅读
目的:观察HIV-1 gp41融合多肽(FP)能否影响CD3抗体激活的调节性T细胞(Treg)。方法:用免疫磁珠分离小鼠脾脏CD4+CD25+的Treg、CD4+CD25-的效应性T细胞(Teff),丝裂霉素C处理小鼠脾细胞获得APC。通过CCK-8法和羧基荧光素双乙酸盐琥珀酰亚胺酯(CFSE)标记检测FP对CD3抗体刺激的Teff增殖效应分析其对Treg抑制功能的影响;用ELISA检测FP对Treg激活后IL-10分泌的影响;用激光共聚焦显微镜观察细胞表面FP与TCR的分布。结果:通过CCK-8法和流式细胞术分析发现,在CD3抗体刺激下Teff细胞有显著增殖效应;当Treg和Teff混合培养时,Treg能够明显抑制共培养的Teff增殖;当加入25μg/mL FP后,Treg+Teff组与Teff组细胞增殖没有明显变化;当FP浓度为5μg/mL时,Treg+Teff组比Teff组增殖显著降低。Treg未活化时IL-10分泌较低,经过CD3抗体刺激后其IL-10分泌显著增多,而当FP浓度为25μg/mL时IL-10显著下降,5μg/mL FP对IL-10分泌无显著影响。通过激光共聚焦显微镜检测发现Treg未活化时,T细胞受体(TCR)在细胞表面均匀分布,同时未见FP与TCR共分布;当Treg在CD3抗体刺激下,TCR活化形成半月形,且FP与活化的TCR在细胞表面共分布。结论:25μg/mL FP对Treg体外抑制功能具有显著抑制作用,而5μg/mL FP不影响Treg的抑制功能,可能与其抑制Treg细胞IL-10分泌以及阻断APC在Treg细胞跨膜区域活化信号的递呈有关。
Objective: To investigate whether HIV-1 gp41 fusion polypeptide (FP) can affect CD3 antibody-activated regulatory T cells (Tregs). Methods: Treg of CD4 + CD25 + and Teff of CD4 + CD25- were isolated by immunomagnetic beads. APC was obtained from mouse spleen cells treated with mitomycin C. The effects of FP on the proliferation of Treg stimulated by CD3 antibody were analyzed by CCK-8 assay and carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling assay. The effect of FP on Treg inhibition was assayed by ELISA. IL- -10 secretion. The distribution of FP and TCR on the cell surface was observed by laser confocal microscopy. Results: The results of flow cytometry and CCK-8 showed that Teff cells had a significant proliferative effect when stimulated with CD3 antibody. When Treg and Teff were mixed, Treg significantly inhibited the proliferation of Teff cells. When 25μg / mL After FP, Treg + Teff group and Teff group had no significant changes in cell proliferation; When FP concentration was 5μg / mL, Treg + Teff group than Teff group proliferation was significantly reduced. IL-10 secretion was significantly decreased when Tregs were not activated, and IL-10 secretion was significantly increased after stimulation with CD3 antibody. IL-10 was significantly decreased when FP concentration was 25μg / mL, while IL-10 secretion was no significant influences. When Tregs were not activated, TCRs were evenly distributed on the cell surface by confocal laser scanning microscopy, and no co-distribution of FPs and TCRs was observed. When Tregs were stimulated by CD3 antibody, TCRs formed a half-moon shape, and FPs Co-distribution with activated TCRs on the cell surface. CONCLUSION: 25μg / mL FP can significantly inhibit Treg cell proliferation in vitro, while 5μg / mL FP did not affect Treg suppression, which may be related to its inhibitory effect on IL-10 secretion in Treg cells and block the activation of APC in the transmembrane region of Treg cells Presented related.