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【目的】比较研究婴儿双歧杆菌(Bifidobacterium infantis 13.085)对中国对虾原肌球蛋白致敏BALB/c小鼠预防与治疗过敏反应的差异,探究其对致敏小鼠Treg/Th17细胞平衡及相关细胞因子的影响。【方法】采用硫酸铵盐析及等电点沉淀法纯化中国对虾原肌球蛋白(TM),将中国对虾TM和弗氏佐剂混合液腹腔注射诱发BALB/c小鼠致敏,建立动物过敏模型。将实验小鼠随机分为正常对照组、治疗对照组、双歧杆菌治疗组、预防对照组和双歧杆菌预防组。观察分析小鼠过敏症状(腹泻、肺组织HE染色比较、称重法测定小鼠体重和脾脏脏器系数变化),采用ELISA测定小鼠血清中特异性IgE、IgG2a和组胺的含量,采用流式细胞术测定脾脏T淋巴细胞亚群(Treg、Th17)数量,采用荧光定量PCR测定脾脏中Treg型和Th17型细胞因子和转录因子的表达量。【结果】纯化得到中国对虾原肌球蛋白纯度为84.93%,得率为60.88%。体内试验表明,双歧杆菌治疗组和预防组相比于对照组,腹泻和过敏症状均有明显的缓解;不同时期的双歧杆菌干预均对过敏小鼠肺组织症状有明显的改善作用,且可降低过敏小鼠的脾脏脏器系数。第56天实验周期结束后发现,相比预防对照组和治疗对照组,双歧杆菌预防组和治疗组小鼠血清中特异性IgE和组胺含量显著降低(P<0.05),脾脏Treg/Th17比值显著升高(P<0.05),Th17型细胞因子IL-17A mRNA表达水平显著降低(P<0.01);双歧杆菌治疗组相对于治疗对照组,Treg型细胞因子CD25mRNA表达水平显著升高(P<0.01)。此外,双歧杆菌治疗组血清特异性IgE及IL-17A mRNA转录水平显著低于双歧杆菌预防组(P<0.05),而Treg/Th17比值及CD25 mRNA转录水平显著高于预防组(P<0.05)。【结论】双歧杆菌13.085能有效缓解小鼠过敏症状,且治疗免疫调控效果优于预防效果,其作用可能通过平衡Treg/Th17细胞亚群数量,促进Treg型细胞因子表达而抑制Th17型细胞因子分泌,从而阻断炎性抗体及组胺释放。
【Objective】 The aim of this study was to compare the effects of Bifidobacterium infantis 13.085 on the prevention and treatment of allergic reactions in pristinomycin-induced BALB / c mice and to investigate the effects of Bifidobacterium infantis 13.085 on the balance and correlation of Treg / Th17 cells in sensitized mice Effects of cytokines. 【Method】 Purification of tropomyosin from shrimp (Penaeus chinensis) by ammonium sulfate salting-out and isoelectric point precipitation method was used to sensitize BALB / c mice induced by intraperitoneal injection of mixture of Chinese shrimp TM and Freund’s adjuvant to establish animal allergy model. The experimental mice were randomly divided into normal control group, treatment control group, Bifidobacterium treatment group, prevention control group and Bifidobacterium prevention group. Observed and analyzed mouse allergy symptoms (diarrhea, lung tissue HE staining, weight determination of mouse body weight and spleen organ coefficient changes), the determination of serum specific IgE, IgG2a and histamine content by ELISA The number of T lymphocyte subsets (Treg, Th17) in spleen was determined by cytometry. The expression of Treg and Th17 cytokines and transcription factors in spleen were detected by real-time PCR. 【Result】 The purity of purified myosin of P. shrimp was 84.93% and the yield was 60.88%. In vivo tests showed that compared with the control group, the bifidobacterium treatment group and the prophylaxis group were significantly relieved of symptoms of diarrhea and allergy; the bifidobacteria intervention at different periods all had a significant improvement on the lung tissue symptoms in allergic mice, and Can reduce the allergic mouse spleen organ coefficient. At the end of the 56th experimental period, the serum levels of specific IgE and histamine in the Bifidobacterium prophylaxis and treatment groups were significantly decreased (P <0.05) compared with the prophylaxis and control groups, and the levels of Treg / Th17 (P <0.05), while the expression of Th17 cytokine IL-17A mRNA was significantly decreased (P <0.01). Compared with the control group, the expression of Treg cytokine CD25mRNA in Bifidobacterium group was significantly increased (P < P <0.01). In addition, the levels of serum specific IgE and IL-17A mRNA in Bifidobacterium treated group were significantly lower than those in Bifidobacterium prophylaxis group (P <0.05), while the Treg / Th17 ratio and CD25 mRNA level were significantly higher in the Bifidobacterium group than in the prophylaxis group (P < 0.05). 【Conclusion】 Bifidobacterium 13.085 can effectively relieve the allergic symptoms in mice, and the therapeutic effect of immunotherapy is better than that of prophylaxis. The effect of Bifidobacterium 13.085 may be through balancing the number of Treg / Th17 cell subsets, promoting the expression of Treg type cytokines and inhibiting the expression of Th17 type cytokines Secretion, thereby blocking the release of inflammatory antibodies and histamine.