海藻酸钠/乙二醇壳聚糖双网络水凝胶对小鼠调节性T细胞免疫功能的影响

Effects of alginate/glycol chitosan double-network hydrogel on immune response of regulatory T cells of mice

目的探讨海藻酸钠(Alg)/乙二醇壳聚糖(GC)双网络凝胶材料体外刺激对小鼠调节性T细胞(Treg)免疫功能的影响。方法制备Alg、GC单网络水凝胶材料及双网络(DN)水凝胶材料(Alg/GC),根据不同水凝胶材料将细胞分为Alg组、GC组、DN组及对照组。采用免疫磁珠分离法分选BALB/c小鼠脾脏CD4+CD25+Treg,应用流式细胞术分析Treg叉头翼状螺旋转录因子(Foxp3)及细胞毒性T细胞相关抗原(CTLA-4)在不同时效(刺激24h或48h)、不同培养环境[有无多黏菌素B(PMB)]下的表达水平,以确定后续细胞培养时间以及是否选用含PMB的培养基;观察Treg与效应T细胞共培养后T细胞增殖活性改变;采用酶联免疫吸附分析(ELISA)技术检测Treg分泌白细胞介素(IL)-10、转化生长因子(TGF)-β及效应T细胞分泌IL-2、IL-4与干扰素(IFN)-γ的水平。结果选定实验条件为刺激24h,并使用含PMB的培养基。Alg/GC双网络水凝胶及GC单网络水凝胶作用24h后,与对照组[CTLA-4 (47.73±5.35)%,TGF-β(558.75±48.58)pg/ml]比较,CD4+CD25+Treg中CTL A-4的表达水平[DN组(92.21±2.97)%,GC组(95.99±2.79)%]明显升高,TGF-β分泌量[DN组(905.03±73.04)pg/ml,GC组(957.06±138.70)pg/ml]明显增加,差异均有统计学意义(P<0.05),但各刺激组Foxp3表达水平差异无统计学意义(P>0.05)。Alg/GC双网络水凝胶及GC单网络水凝胶刺激后,与对照组[(42.04±1.35)%]相比,Treg对效应T细胞增殖活性的抑制作用[DN组(37.22±1.39)%,GC组(35.20±2.03)%]均明显增强,共培养后效应T细胞分泌IL-4/IFN-γ比值[DN组(2.30±0.22),GC组(2.57±0.23)]明显升高,与对照组(1.45±0.27)比较差异有统计学意义(P<0.05)。结论 Alg/GC双网络材料可显著增强Treg介导的免疫抑制功能,其成分中单网络材料GC水凝胶可能发挥了主要作用。

Objective To investigate the in vitro effect of alginate(Alg)/glycol chitosan(GC) double-network hydrogel on immune function of splenic regulatory T cells(Treg) of mice. Methods Single network hydrogel of Alg or GC and double-network(DN) gel material(Alg/GC) were prepared. Splenic CD4^+CD25^+Treg of BABL/c mice were isolated and purified by Magnetic Activated Cell Sorting(MACS) kit and further stimulated with different hydrogels for 24 hours and 48 hours with or without polymyxin B(PMB) exposure. Cells treated with Alg, or GC, or DN were divided into Alg group, GC group, DN group, respectively.Cells without hydrogens were served as control group. Expressions of the forkhead/winged helix transcription factor p3(Foxp3)and cytotoxic T cell-associated antigen 4(CTLA-4) on CD4^+CD25^+Treg were measured by flow cytometry. Their expression levels would be used to determine the conditions for further experiments. The CFSE kit was applied for assessing the proliferative activity of effector T cells(Teffs) after co-cultured with CD4^+CD25^+Treg. Enzyme linked immunosorbent assay(ELISA) was used to determine the cytokine levels including interleukin(IL)-10 and transforming growth factor(TGF)-β, IL-2, IL-4, and interferon(IFN)-γ. Results With or without PMB exposure, the expression of CTLA-4 and Foxp3 showed no significant difference under the stimulation of various hydrogels(P>0.05), including Alg, GC, and DN. Therefore, for further studies, cells were stimulated by different hydrogens in the presence of PMB for 24 hours. Cells without hydrogen simulation were served as control. Compared with the control group [(47.73±5.35)%], CTLA-4 expression was obviously up-regulated in both GC [(95.99±2.79)%] and DN[(92.21±2.97)%] hydrogel groups(P<0.05). In addition, TGF-β levels were markedly elevated in CD4^+CD25^+Treg stimulated with GC [(957.06±138.70)pg/ml] or DN [(905.03±73.04)pg/ml], compared with the level in the cells in the control group[(558.75±48.58)pg/ml, P<0.05]. However, no difference was detected in the expression of Foxp3 among different groups. In comparison with control group [proliferation(42.04±1.35)%, IL-4/IFN-γ(1.45±0.27)], Treg cells that were stimulated with GC or DN showed enhanced inhibitory effect on Teffs, including suppressed proliferation of Teffs [DN group(37.22±1.39)%, GC group(35.20±2.03)%] and increased ratio of IL-4/IFN-γ of Teffs [DN group(2.30±0.22), GC group(2.57±0.23)](P<0.05).Conclusion Alg/GC double-network hydrogel can greatly enhance the immunosuppressive response of Treg, which is dominantly seen with GC single network hydrogel.