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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: To establish a stable and reproducible Th1 / Th2 imbalanced animal model with certain characteristics is an important basis for studying the mechanism of Th1 / Th2 imbalance. Objective: To analyze the imbalance of Th1 / Th2 in Balb / C mouse spleen cells induced by keyhole limpet hemocyanin. Design: Randomized controlled exploratory experiment. Unit: Basic Medical College of Hebei Medical University in Hebei Province Forensic Laboratory. MATERIALS: The experiment was performed at the Forensic Science Laboratory of Hebei Province, School of Basic Medicine, Hebei Medical University from September 2005 to February 2007. Balb / c mice were selected as experimental animals, which met animal ethical requirements. Methods: ① Balb / C mice were immunized with keyhole limpet hemocyanin + complete Freund’s adjuvant, splenocytes were isolated and the peak of cytokine secretion was measured. The experiment was divided into 3 groups: Keyhole limpet hemocyanin 6.25, 12.5 , 25mg / kg group. The experiment of different doses and times of immunization were divided into 7 groups, which were keyhole limpet hemocyanin 6.25,12.5,25mg / kg group, 6.25,12.5,25mg / kg secondary immunization group and control group respectively. MAIN OUTCOME MEASURES: Th1 cytokine interferon-γ, interleukin-2, interleukin-12p40 and Th2-type cytokines interleukin-4, interleukin 5 secretion and serum Th1 antibody IgG2a and Th2 antibody IgG1 levels. Results: ① keyhole limpet hemocyanin immunization mice splenomegaly, splenic cell count increased. ② The restimulation of alloantigen in vitro increased the concentration of 4 cytokines in the spleen cell culture supernatant of the immunized mice, in which the secretion of interleukin 2 increased significantly at 24h and peaked at 48h; the levels of interleukin-4, interferon-γ And interleukin 5 slightly increased at 24h, then gradually increased, peaked at 96h. The content of interleukin 12p40 in the culture supernatant at each time point was lower than that in the control group. ③ The single or secondary immunization of different doses could cause the increase of four cytokines to different extents, and the ratio of interleukin 4 / γ-interferon increased, among which 12.5 mg / kg keyhole limpet hemocyanin secondary cytokines secretion The levels were significantly higher than those in other groups (P <0.01). ④ Keyhole limpet hemocyanin immunized mice serum IgG1, IgG2a levels increased to varying degrees, including IgG1 increased more significantly. Conclusion: Keyhole limpet hemocyanin plus complete Freund ’s adjuvant can induce Th2 predominant reaction in Balb / C mice spleen cells.