SPK1基因转染脂肪间充质干细胞对实验性自身免疫性脑脊髓炎小鼠的治疗效果及对辅助性T细胞17/调节性T细胞平衡的影响

Therapeutic Effect of SPK1 Gene Transfected Adipose Derived Mesenchymal Stem Cells on Experimental Autoimmune Encephalomyelitis Mice and Its Effect on T Helper Cell 17/Regulatory T Cells Balance

目的考察SPK1基因转染脂肪间充质干细胞(ADMSC)对实验性自身免疫性脑脊髓炎(EAE)小鼠的治疗效果及对辅助性T细胞17/调节性T细胞(Th17/Treg)平衡的影响。方法采用髓鞘少突胶质细胞糖蛋白35-55对小鼠进行EAE诱导,将44只小鼠随机分为4组:空白对照组(NC组)、模型组(EAE组)、ADMSC组和ADMSC-鞘氨醇激酶1(SPK1)组。在免疫后40 d时,对小鼠进行脑与脊髓的病理学观察、脑组织Th17/Treg相关炎症指标检测、脑和脊髓组织中白细胞介素17和叉头框蛋白P3蛋白表达检测以及脾脏免疫细胞的流式细胞术分析。结果免疫后40 d时,EAE组小鼠脑和脊髓组织发生严重炎性细胞浸润髓鞘脱失严重,ADMSC组与ADMSC-SPK1组脱髓鞘和轴突损伤得到改善;与EAE组比较,ADMSC组和ADMSC-SPK1组的脑组织IL-17A(Z=1.021,P=0.017;Z=1.193,P=0.009)、肿瘤坏死因子-α(TNF-α)(Z=2.540,P=0.004;Z=3.005,P=0.006),与ADMSC组比较,ADMSC-SPK1组的脑组织IL-17A与TNF-α水平显著降低(Z=1.223,P=0.011;Z=2.364,P=0.010);小鼠脑和脊髓组织中IL-17A在ADMSC组的表达水平显著降低(t=10.323,P=0.013;t=7.422,P=0.008),且ADMSC-SPK1组显著低于ADMSC组(t=14.244,P=0.017;t=16.865,P=0.006);Foxp3在ADMSC组的表达水平显著升高(t=14.544,P=0.008;t=9.420,P=0.002),且ADMSC-SPK1组显著高于ADMSC组(t=9.654,P=0.005;t=11.535,P=0.009);ADMSC-SPK1组小鼠脾脏中的IL-17+IFN-γ+T细胞比例降低,CD25+Foxp3+Treg细胞比例升高。结论SPK1转染ADMSC对EAE小鼠具有理想的治疗效果,且该效果优于未经SPK1转染的ADMSC,其机制可能与ADMSC-SPK1能显著降低EAE小鼠Th17/Treg细胞比率并减少相关炎症细胞因子的释放有关。

Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg)cells balance.Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected.Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(Z=1.021,P=0.017;Z=1.193,P=0.009)and tumor necrosis factor-α(TNF-α)(Z=2.540,P=0.004;Z=3.005,P=0.006).The expression level of IL-17A in mouse brain and spinal cord tissues was significantly reduced in the ADMSC group(t=10.323,P=0.013;t=7.422,P=0.008),and it was significantly lower in the ADMSC-SPK1 group than in the ADMSC group(t=14.244,P=0.017;t=16.865,P=0.006).The expression level of Foxp3 in the ADMSC group was significantly increased(t=14.544,P=0.008;t=9.420,P=0.002),and it was significantly higher in the ADMSC-SPK1 group than ADMSC group(t=9.654,P=0.005;t=11.535,P=0.009).The proportion of IL-17+IFN-γ+T cells in spleen decreased in ADMSC-SPK1 group,while that of CD25+Foxp3+Treg cells increased.Conclusions ADMSC transfected with SPK1 has an ideal therapeutic effect on EAE mice,and the effect is superior to ADMSC without SPK1 transfection.The mechanism may be that ADMSC-SPK1 can markedly reduce the Th17/Treg cell ratio and decrease the release of related inflammatory cytokines in EAE mice.