电针足三里对小运鼠Ⅳ型超敏反应防治效应及免疫调节机制研究

发布时间：2018-05-03 03:00

本文选题：电针 + 足三里　；参考：《湖北中医药大学》2017年硕士论文

【摘要】：目的越来越多的研究表明,电针疗法有抗过敏和抗炎作用,但对于迟发性超敏反应(DTH)的作用机制不明。本研究旨在通过观察电针对小鼠DTH治疗效果,试探讨电针足三里防治Th1介导的DTH的效应机制。方法使用雌性C57BL/6小鼠,共32只,体重约为18-20g,将小鼠通过编号查询随机数字表随机分为对照组8只、模型组(OVA-DTH组)8只、电针治疗组(DTH+EA组)8只、假电针组(DTH+Sham组)8只共4组。测量小鼠足垫厚度,使用苏木精和伊红(HE)染色观察足垫炎性细胞浸润情况;酶联免疫吸附测定法(ELISA)检测血清OVA特异性抗体IgG和IgE水平,并检测足垫组织匀浆上清炎症细胞因子(IFN-γ,TNF-α,IL-4和IL-5)水平;流式细胞仪检测各组脾脏CD4~+IFN-γ+T细胞和CD4~+IL-4+T细胞数;蛋白质印迹法检测脾脏分离纯化的T细胞T-bet与GATA-3蛋白表达,并比较各组间的差异。结果通过组织形态学观察,与模型组相比,电针治疗组能明显减小OVA导致的小鼠足垫厚度(P0.05),假电针组与模型组相比无明显差异(P0.05);电针治疗组小鼠足垫组织真皮层炎性细胞浸润与聚集水平明显低于模型组(P0.01),假电针组与模型组相比无明显差异(P0.05);与模型组比较,电针治疗组可显著减轻OVA导致的小鼠足垫表皮层肿胀(P0.05)。通过酶联免疫吸附测定法(ELISA)检测,与模型组相比,电针治疗组可显著抑制血清OVA特异性抗体IgG水平和IgE水平(P0.01),假电针组与模型组相比无明显差异(P0.05);与模型组比较,电针治疗组小鼠足垫组织Th1类细胞因子水平(如IFN-γ和TNF-α)显著降低(IFN-γ:P0.05,TNF-α:P0.01),假电针组与模型组相比无明显差异(P0.05);对比模型组,电针治疗组和假电针组小鼠足垫组织Th2类细胞因子(IL-4,IL-5)水平无显著差异(P0.05)。通过流式细胞学的检测,与对照组比较,模型组中脾CD4~+IFN-γ+T细胞明显增加(P0.05),同时与对照组、模型组相比,电针治疗组脾CD4~+IFN-γ+T细胞数量明显降低,差异具有统计学意义(P0.01),假电针组与模型组相比无明显差异(P0.05);而对比对照组、模型组和电针治疗组三组间脾CD4~+IL-4+T细胞计数水平无明显差异(P0.05);依据脾CD4~+IFN-γ+T细胞和脾CD4~+IL-4+T细胞计数的变化水平,与对照组、模型组相比,电针治疗组的DTH小鼠脾Th1/Th2细胞比率(即脾CD4~+IFN-γ+T细胞数/脾CD4~+IL-4+T细胞数)降至正常值附近,且与模型组相比,差异具有统计学意义(P0.05)。通过蛋白质印迹法(Westen blot)检测,与对照组比较,模型组T-bet蛋白活性增强,而电针治疗组T-bet蛋白活性减弱,对GATA-3则无显著影响(P0.05);通过T-bet/GATA-3比率的统计分析,电针治疗组与模型组相比,电针治疗组比率明显低于模型组(P0.05),假电针组与模型组无明显差异(P0.05)。结论1.电针“足三里”对OVA诱导的小鼠DTH炎症具有明显的抑制作用。2.电针“足三里”可通过减少IFN-γ的转录因子T-bet蛋白的表达,重建Th1/Th2平衡,改善Th1介导的过敏性皮肤炎症。3.电针“足三里”可作为过敏性炎症和Th1介导的炎症反应的有效疗法。
[Abstract]:Objective more and more studies have shown that electroacupuncture therapy has anti allergic and anti inflammatory effects, but the mechanism of delayed hypersensitivity (DTH) is unknown. The purpose of this study was to observe the effect of Electroacupuncture on DTH in mice and to explore the effect mechanism of Zusanli on the prevention and treatment of Th1 mediated DTH by electroacupuncture. Methods 32 female C57BL/6 mice were used. About 18-20g, the mice were randomly divided into 8 control groups, 8 rats in model group (group OVA-DTH), 8 in group DTH+EA, 8 in group DTH+Sham (group DTH+Sham) in 4 groups. The foot pad thickness of mice was measured and the infiltration of foot pad inflammatory cells was observed with hematoxylin and eosin (HE) staining; enzyme linked immunosorbent assay ( ELISA) the levels of serum OVA specific antibody IgG and IgE were detected, and the levels of inflammatory cytokines (IFN-, TNF- a, IL-4 and IL-5) in the foot pad tissue homogenate were detected. The number of CD4~+IFN- gamma +T cells and CD4~+IL-4+T cells in each group was detected by flow cytometry; the expression of T cells and proteins in the splenic isolation and purification was detected by Western blot. Compared with the model group, the electroacupuncture group could significantly reduce the foot pad thickness (P0.05) caused by OVA in the electroacupuncture group compared with the model group, and there was no significant difference between the sham acupuncture group and the model group (P0.05), and the level of inflammatory cell infiltration and aggregation in the foot pad tissue of the electroacupuncture group was significantly lower than that of the model group (P0.01). There was no significant difference between the model group and the model group (P0.05). Compared with the model group, the electroacupuncture treatment group could significantly reduce the mouse foot pad epidermal swelling (P0.05) caused by OVA. The electroacupuncture treatment group could significantly inhibit the serum OVA specific antibody IgG level and IgE level (P0.01) by enzyme linked immunosorbent assay (ELISA), compared with the model group. There was no significant difference between the model group and the model group (P0.05). Compared with the model group, the level of Th1 cell factors (such as IFN- gamma and TNF- alpha) in the foot pad tissue of the electroacupuncture group decreased significantly (IFN-: P0.05, TNF- A: P0.01), and there was no significant difference (P0.05) in the sham acupuncture group compared with the model group (P0.05); the contrast model group, the electroacupuncture treatment group and the sham acupuncture group mice feet. There was no significant difference in the level of Th2 cell factor (IL-4, IL-5) in the padding tissue (P0.05). Compared with the control group, the CD4~+IFN- gamma +T cells in the model group were significantly increased by flow cytometry (P0.05). Compared with the control group, the number of IFN- gamma +T cells in the spleen CD4~+ IFN- +T cells in the electroacupuncture group was significantly lower than that in the model group. The difference was statistically significant (P0.01). There was no significant difference between the pseudo electroacupuncture group and the model group (P0.05), but there was no significant difference in the CD4~+IL-4+T cell count level between the three groups in the control group and the electroacupuncture group (P0.05), and the level of the CD4~+IFN- gamma +T cell and the spleen CD4~+IL-4+T cell count was compared with the control group and the model group, the DTH mouse spleen Th1/ in the electroacupuncture group. The ratio of Th2 cells (the number of spleen CD4~+IFN- +T cells / splenic CD4~+IL-4+T cells) dropped to the normal value, and compared with the model group, the difference was statistically significant (P0.05). Compared with the control group, the viability of T-bet protein in the model group was enhanced compared with the control group, but the activity of T-bet protein in the electroacupuncture group was weakened, and GATA-3 was less than that of the control group. There was no significant effect (P0.05). Through the statistical analysis of the T-bet/GATA-3 ratio, the ratio of electroacupuncture treatment group was significantly lower than that of model group (P0.05), and there was no significant difference between the electroacupuncture group and the model group (P0.05). Conclusion 1. electroacupuncture "Zusanli" had a significant inhibitory effect on the DTH inflammation induced by OVA in the OVA induced mice, ".2. acupuncture", "foot three" By reducing the expression of the IFN- gamma transcription factor T-bet protein, reconstructing the Th1/Th2 balance and improving the Th1 mediated allergic skin inflammation,.3. electroacupuncture "Zusanli" can be used as an effective therapy for allergic inflammation and Th1 mediated inflammatory reaction.

【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245.97

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