人嗅黏膜来源间充质干细胞对实验性自身免疫性脑脊髓炎小鼠的治疗作用与机制

Mechanisms and Therapeutic Effects of Human Olfactory Mucosa-Derived Mesenchymal Stem Cells on Experimental Autoimmune Encephalomyelitis in Mice

[目的]研究人嗅黏膜来源的间充质干细胞(OMSC)对实验性自身免疫性脑脊髓炎(EAE)小鼠的治疗作用及机制.[方法]经鼻内镜局麻下取健康捐献者嗅区黏膜上皮,消化、传代后获取嗅黏膜间充质干细胞,培养至第5代,进行干细胞表面分子鉴定、多向诱导分化及染色.以髓鞘少突胶质细胞糖蛋白(MOG35-55)和百日咳毒素(PT)诱发C57小鼠EAE模型,记录小鼠神经功能评分(10分法).造模后第16天(发病高峰期)将已发病小鼠随机分为治疗组和对照组,分别进行经尾静脉OMSC和等体积PBS治疗.造模后第24天经内眦静脉取血,CBA法检测小鼠血浆IL-10、IL-17、IFN-γ、IL-6水平.取脊髓,HE和LFB染色观察损伤灶大小.取健康捐献者外周血淋巴细胞与OMSC共培养,2 d后比较单独培养组和共培养组分泌IFN-γ的CD4+T淋巴细胞(Th1细胞)比例;加入IDO、COX抑制剂干预,观察2 d后各组Th1细胞比例.[结果]OMSC表面分子表达符合间充质干细胞特性并具有多向分化潜能.MOG35-55及PT成功诱导EAE模型,小鼠出现不同程度的神经功能损伤,OMSC治疗后,治疗组小鼠发病严重程度较对照组减轻(P=0.002),且治疗组血清IFN-γ浓度低于对照组(P=0.032),IL-10、IL-17、IL-6浓度差异无统计学差异.HE、LFB染色切片分别显示OMSC治疗组炎症因子浸润程度较对照组减轻,脱髓鞘区域范围也较对照组减小.人外周血淋巴细胞与OMSC共培养2 d后,Th1细胞比例较淋巴细胞单独培养组降低(P=0.001).IDO抑制剂组Th1细胞比例高于共培养组(P=0.01),与单独培养组的差异无统计学意义;COX抑制剂组Th1细胞比例与共培养组相比无统计学差异.[结论]OMSC可能通过IDO通路调控Th1淋巴细胞比例,从而抑制实验性自身免疫性脑脊髓炎小鼠的脱髓鞘损伤反应.本研究为临床治疗多发性硬化疾病提供了新的思路.

【Objective】To study the mechanisms and therapeutic effects of human olfactory mucosa-derived mesenchymal stem cells(OMSC)on experimental autoimmune encephalomyelitis(EAE)in mice.【Methods】Under local anesthesia by using nasal endoscopy,olfactory epithelia of healthy donors were obtained,digested and cultured up to the 5th passage.OMSC were identified,differentiated and stained.EAE models were induced in C57 female mice by myelin oligodendrocyte glycoprotein(MOG35-55)and pertussis toxin(PT).Neurological function was documented daily.On day 16 after immunization(peak of incidence),the mice were divided randomly into two groups and treated with OMSC and PBS via tail vein injection respectively.On day 24 after immunization,blood was collected from angular vein and levels of IL-10,IL-17,IFN-γand IL-6 were determined by cytometric beads array(CBA).The size of the spinal cord lesion in mice was observed and measured by using HE and LFB staining.Peripheral blood lymphocytes(PBL)of healthy donors were obtained and then co-cultured with OMSC.The proportions of CD4+T cells secreting IFN-γ(Th1 cells)in lymphocyte group and co-culture group were compared after 2 days of cultivation.Adding IDO or COX pathway inhibitor to co-culture group and cultivating for 2 days,we observed and compared the proportions of Th1 cells in lymphocyte group,co-culture group and inhibitor treatment group respectively.【Results】OMSC exhibited certain mesenchymal stem cell-like characteristics with respect to expression of stem cell surface markers and multilineage differentiation potentials.After induced by MOG35-55 and PT,EAE models showed different levels of neurological damage.Compared with those in PBS treatment group,in OMSC treatment group,the severity of neural dysfunction in mice was significantly reduced(P=0.002),the level of IFN-γin serum was lower(P=0.032),but no significant differences in the levels of IL-10,IL-17 and IL-6 were found between two groups.HE and LFB staining revealed that the inflammatory infiltration and demyelinating areas in OMSC treatment group were less than those in PBS treatment group.The proportion of Th1 cells was lower in co-culture group than that in lymphocyte group(P=0.001),higher in IDO inhibitor group than that in co-culture group(P=0.01),but no significant difference was found between IDO inhibitor group and lymphocyte group or between COX inhibitor group and co-culture group.【Conclusions】OMSC may regulate the proportion of Th1 lymphocytes through IDO pathway so as to inhibit the demyelinating injuries of EAE in mice.This study provides a new idea for the clinical treatment of multiple sclerosis.