脂肪间充质干细胞及非甲基化胞嘧啶鸟嘌呤核苷酸对食物过敏幼鼠外周血中CD4+CD25+Treg的影响

Effects of adipose-derived stem cells and non-methylated CpG-oligodeoxynucleotides on peripheral blood CD4+CD25+ regulatory T cells in young mice with food allergy

摘要:

目的 观察脂肪间充质干细胞（ADSC）、非甲基化胞嘧啶鸟嘌呤核苷酸（CpG-ODN）对食物过敏幼鼠外周血中CD4⁺CD25⁺调节性T细胞（CD4⁺CD25⁺Treg）的表达水平及免疫干预作用。方法 将40只BALB/c雌性小鼠随机分为对照组、过敏组、ADSC治疗组（ADSC组）和非甲基化CpG-ODN治疗组（CpG-ODN组），每组10只；通过卵清蛋白（OVA）腹腔注射基础致敏和灌胃激发致敏建立小鼠食物过敏模型；对照组给予等量生理盐水替代；ADSC组在激发致敏前后两个时间点分别给予腹腔注射ADSC（1×10⁶个/只）；CpG-ODN组在每次灌胃激发前1 h给予腹腔注射非甲基化CpG-ODN（40 μg/只）溶液。模型成功建立后对各组小鼠进行过敏症状评分；酶联免疫吸附法检测各组小鼠血清中OVA-IgE水平；流式细胞仪检测小鼠外周血中CD4⁺CD25⁺Treg水平；取各组小鼠空肠行苏木精-伊红染色，进行病理分析。结果 过敏组过敏症状评分及血清中OVA-IgE水平均高于对照组（P < 0.05），ADSC组、CpG-ODN组过敏症状评分及血清中OVA-IgE水平比较差异均无统计学意义（P > 0.05），但均低于过敏组，高于对照组（P < 0.01）。过敏组外周血中CD4⁺CD25⁺Treg水平低于对照组（P < 0.05），ADSC组、CpG-ODN组外周血中CD4⁺CD25⁺Treg水平均高于过敏组（P < 0.05），且与对照组比较及两组间比较差异均无统计学意义（P > 0.05）。病理结果显示过敏组空肠黏膜层绒毛结构破坏、水肿，大量的嗜酸性粒细胞及淋巴细胞浸润；ADSC组和CpG-ODN组空肠黏膜层绒毛结构部分破坏、无明显水肿，有少量嗜酸性粒细胞及淋巴细胞浸润。结论 ADSC和非甲基化CpG-ODN干预对食物过敏均有一定的治疗效果；均可提高食物过敏幼鼠体内CD4⁺CD25⁺Treg水平，同时降低OVA-IgE的表达水平，与诱导免疫耐受有一定的关联作用，且两种干预效果相当，但具体作用机制仍需要进一步探究。

Abstract:

Objective To investigate the effects of adipose-derived stem cells (ADSC) and non-methylated CpG-oligodeoxynucleotides (CpG-ODN) on the expression of peripheral blood CD4⁺CD25⁺ regulatory T (Treg) cells in young mice with food allergy, as well as their immune intervention effects. Methods A total of 40 female BALB/c mice were randomly divided into control group, allergic group, ADSC treatment group, and CpG-ODN treatment group, with 10 mice in each group. A mouse model of food allergy was established by intraperitoneal injection and intragastric administration of ovalbumin (OVA) for sensitization and challenge. The mice in the control group were treated with normal saline at the same dose; the mice in the ADSC treatment group were given intraperitoneal injection of ADSC (1×10⁶ cells for each mouse) before and after OVA challenge, and those in the CpG-ODN treatment group were given intraperitoneal injection of non-methylated CpG-ODN solution (40 μg for each mouse) at 1 hour before challenge by gavage. The allergic symptom scores were determined for each group after model establishment. ELISA was used to measure the serum level of OVA-IgE. Flow cytometry was used to measure the percentage of peripheral blood CD4⁺CD25⁺ Treg cells. Hematoxylin and eosin staining was used for the pathological analysis of the jejunum. Results The allergic group had significantly higher allergic symptom scores and serum level of OVA-IgE than the control group (P < 0.05). There were no significant differences in the allergic symptom score and the serum level of OVA-IgE between the ADSC treatment group and the CpG-ODN treatment group (P > 0.05), but these two groups had significantly lower allergic symptom scores and serum level of OVA-IgE than the allergic group and significantly higher allergic symptom scores and serum level of OVA-IgE than the control group (P < 0.01). The allergic group had a significantly lower percentage of peripheral blood CD4⁺CD25⁺ Treg cells than the control group (P < 0.05). The ADSC treatment group and the CpG-ODN treatment group had a significantly higher percentage of peripheral blood CD4⁺CD25⁺ Treg cells than the allergic group (P < 0.05); there were no significant differences between these two groups or between them and the control group (P > 0.05). Pathological results showed structural damage and edema in the jejunal villi, a large number of eosinophils, and lymphocyte infiltration in the allergic group, while the ADSC treatment group and the CpG-ODN treatment group had less structural damage and edema in the jejunal villi, a lower number of eosinophils, and less lymphocyte infiltration. Conclusions ADSC and non-methylated CpG-ODN have a certain effect in the treatment of food allergy and can increase the percentage of peripheral blood CD4⁺CD25⁺ Treg cells and reduce the level of OVA-IgE. They may be associated with the induction of immune tolerance and these two treatment have comparable effects. Detailed mechanisms of action still need further investigation.