钥孔戚血蓝蛋白诱导Balb/C小鼠脾细胞Th1/Th2失衡(英文)

Keyhole limpet hemocyanin induced Th1/Th2 imbalance in splenocytes of Balb/C mice

背景:建立具有一定特征的、稳定的、可重复性强的Th1/Th2失衡动物模型是研究Th1/Th2失衡机制的重要基础。目的:分析钥孔戚血蓝蛋白诱导Balb/C小鼠脾细胞Th1/Th2失衡的特点。设计:随机对照探索性实验。单位:河北医科大学基础医学院河北省法医学实验室。材料:实验于2005-09/2007-02在河北医科大学基础医学院河北省法医学实验室完成。实验选用Balb/c小鼠,对动物处置方法符合动物伦理学要求。方法:①以钥孔戚血蓝蛋白+完全弗氏佐剂免疫Balb/C小鼠,分离脾细胞,细胞因子分泌高峰点测定实验分为3组,分别为钥孔戚血蓝蛋白6.25,12.5,25mg/kg组;不同免疫剂量和免疫次数实验分为7组,分别为钥孔戚血蓝蛋白6.25,12.5,25mg/kg组,6.25,12.5,25mg/kg二次免疫组及对照组。主要观察指标:以酶联免疫吸附法检测小鼠脾细胞培养上清中Th1型细胞因子γ-干扰素、白细胞介素2、白细胞介素12p40及Th2型细胞因子白细胞介素4、白细胞介素5的分泌量及血清中Th1型抗体IgG2a和Th2型抗体IgG1水平。结果:①钥孔戚血蓝蛋白免疫使小鼠出现脾肿大,脾细胞计数增高。②体外同种抗原再刺激使免疫小鼠脾细胞培养上清中4种细胞因子浓度增高,其中白细胞介素2分泌量于24h即明显增高,48h达高峰;白细胞介素4、γ-干扰素和白细胞介素5在24h轻度增高,以后逐渐上升,96h达高峰。各时间点培养上清中白细胞介素12p40含量均较低,与对照组无明显差异。③不同剂量单次或二次免疫均可致4种细胞因子不同程度增高,白细胞介素4/γ-干扰素比值增高,其中12.5mg/kg钥孔戚血蓝蛋白二次免疫组细胞因子分泌量明显高于其他各组(P<0.01)。④钥孔戚血蓝蛋白免疫小鼠血清IgG1,IgG2a水平发生不同程度增高,其中IgG1增高更为明显。结论:钥孔戚血蓝蛋白加完全弗氏佐剂可诱导Balb/C小鼠脾细胞发生Th2型优势反应。

BACKGROUND： Establishing a characteristic, stable and repeatable model of Th1/Th2 imbalance in animals, is the key of studying the mechanism of Th1/Th2 imbalance. OBJECTIVE： To observe the characteristics of Th 1/Th2 imbalance in splenocytes derived from Balb/C mice immunized by keyhole limpet hemocyanin （KLH）. DESIGN： A randomized control exploratory experiment. SETTING： Hebei Provincial Forensic Laboratory, Institute of Basic Medicine, Hebei Medical University. MATERIALS： The experiment was carried out in the Hebei Provincial Forensic Laboratory, Institute of Basic Medicine, Hebei Medical University from September 2005 to January 2007. Balb/C mice were adopted in this study, and all the disposals were in accordance with the guidance of animal ethics. METHODS： Balb/C mice were immunized with KLH emulsified in complete Freund＇s adjuvant （CFA）, splenocytes were acquired, and the peak of cytokine secretion was determined in 3 groups： KLH groups of 6.25 mg/kg, 12.5 mg/kg and 25 mg/kg. According to the immunizing dose and immunizing frequency, mice were divided into 7 groups： KLH groups of 6.25 mg/kg, 12.5 mg/kg and 25 mg/kg, secondary immunity groups of 6.25 mg/kg, 12.5 mg/kg and 25 mg/kg, as well as control group. According to the determined levels of IgG1 and IgG2a in blood serum, mice were divided into KLH group of 6.25 mg/kg and control group. MAIN OUTCOME MEASURES： Mice splenocytes supernatant was detected with enzyme linked immunosorbent assay （ELISA） for the production of Th1 cytokines interferon （IFN）- γ, intedeukin （IL）-2, IL-12 p40 and Th2 cytokines IL-4 and IL-5. The levels of Th1 antibody IgG2a and Th2 antibody IgG1 in blood serum were also detected by ELISA. RESULTS： The spleen derived from KLH-immunized mice appeared hypertrophy, and the number of splenocytes was manifold. Splenocytes restimulated with KLH in vitro produced much more IFN- γ, IL-2, IL-4, IL-5, but not IL-12p40. IL-2 secretion was obviously elevated after incubated for 24 hours and achieved pinnacle at 48 hours; Productions of IL-4, IL-5 and IFN- γ were elevated after 24 hours, and increased gradually to 96 hours; IL-12p40 production was very low at every time point. Using different doses of KLH immunity once or twice, could all lead to the elevated productions of IL-2, IL-4, IL-5, 1FN- γ, and the elevation of IL-4/IFN- γ ratio, but the secondary immunity group of 6.25 mg/kg KLH showed obviously higher levels than other groups （P 〈 0.01）. The level of KLH specific antibody IgG2a and IgG1, especially IgG 1 was elevated in blood serum of KLH-immunized mice. CONCLUSION- Balb/C mice immunized with KLH emulsified in CFA can induce a Th2 predominant imbalance in splenocytes.