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口服免疫耐受预防实验性自身免疫性葡萄膜视网膜炎中细胞因子网络的研究

Study on the cytokine network in the prevention of experimental autoimmune uveoretinitis by oral tolerance

摘要:

目的 观察口服免疫耐受预防大鼠实验性自身免疫性葡萄膜视网膜炎(EAU)过程中血清以及脾细胞培养上清液中的Th1类细胞因子干扰素(IFN)-γ、白细胞介素(IL)-2和Th2类细胞因子IL-4,IL-10表达水平.方法 Lewis大鼠72只随机分为EAU组、口服视网膜S抗原10、100 μg组和1、10 mg组、对照组,每组各12只大鼠.其中,EAU组用视网膜S抗原50 μg和弗氏完全佐剂免疫诱导EAU模型,口服10、100 gg组和1、10 mg组,分别插管喂饲视网膜S抗原10、100 μg.1、10 mg和1 mg胰蛋白酶抑制剂的混合液1 ml.隔日1次,共7次,然后按上述方法诱导EAU;对照组插管喂饲磷酸盐缓冲液和1 mg胰蛋白酶抑制剂的混合液1 ml,隔日1次,共7次,然后诱导EAU,观察每组大鼠眼部EAU的临床表现.在EAU高峰期取大鼠眼球,进行病理分级.同时取大鼠血清,分离脾细胞,培养后取上清液,用酶联免疫吸附试验(ELISA)检测血清以及脾细胞培养上清液中IFN-γ、IL-γ、IL-4和IL-10细胞因子的水平.结果 100 μg、1 mg组血清中Th1类细胞因子IFN-γ、IL-2浓度降低,Th2类细胞因子IL-4、IL-10的浓度增高.与EAU组和对照组比较,差异均有统计学意义(F=51.9,68.8,35.7,7.5,P<0.01);10μg、10 mg组血清中Th1、Th2类细胞因子与EAU组和空白对照组比较,差异无统计学意义.与EAU组和空白对照组相比,100μg、1 mg组大鼠脾细胞培养上清液中Th1类细胞因子IFN-γ和IL-2的浓度降低,Th2类细胞因子IL-4、IL-10的浓度增高,与EAU组和对照组比较,差异均有统计学意义(F=57.1,15.6,33.1,167.7,P<0.01),10 μg、10 mg组大鼠脾细胞培养上清液血清中Th1、Th2类细胞因子与EAU组和空白对照组比较,差异无统计学意义.结论 口服免疫耐受预防EAU过程中,Th1类细胞因子IFN-γ和IL-2的表达降低,而Th2类细胞因子IL-4、IL-10的表达增高.口服过高或过低剂量抗原不能预防EAU,细胞因子的表达水平也无明显改变.提示细胞因子在口服免疫耐受预防EAU的过程中起到重要作用.

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Objective To observe the interferon-γ(IFN-γ),interhukin-2(IL-2)levels of Th1 cytokine and IL-4、IL-10 levels of Th2 cytokine in serum and culture supernatants of splenic cells of the rats in the prevention of experimentaI autoimmune uveoretinitis(EAU)by oral tolerance. Methods 72 Lewis rats were randomly divided into EAU group,oral tolerance group(which including 10 μg、100 μg、1 mg、10 mg of S antigen group respectively)and control group,12 rats in each group.The animal model of EAU was induced by immunization with S antigen(50 μg)and Freund's complete adjuvant.Oral tolerance 10 μg、100μg、1 mg and 10 mg group were fed with 1 ml mixture of 10 μg、100μg、l mg、10 mg S antigen and 1 mg trypsin inhibitor respectively by intuhation,once the other day,totally 7 times,and then induced EAU according to above methods;control group was fed with 1 ml mixture of phosphate buffered saline and 1 mg trypsin inhibitor,once the other day,totally 7 times,and then induced EAU.The clinical manifestation of EAU in the eye were recorded,the eyeballs were enucleated at the peak of EAU,followed by pathological grading.Meanwhile the serum was colleced;splentic cells were separated and cultured to collect the supernatant.Cytokine levels of IFN-γ,IL-2,IL-4 and IL-10 in serum.cultured supernatant of splenic cells were determined by enzyme-linked immunosorbent assay(ELISA). Results Compared with EAU and control group,the levels of IFN-γ and IL-2(Th1 eytokine)in the serum in 100 μg and 1 mg group were decreased while the levels of IL-4 and IL-10(Th2 eytokine)were inereased,the diffefences were statistically significant(F=51.9,68.8,35.7,7.5,P<0.01).Compared the levels of Th1 and Th2 cytokines in the serum in 10 μg,10 mg group with EAU and control group.the differences were not statistically significant.In 100 gg、1 mg group,the levels of IFN-γ and IL-2(Th1 cytokine)in the culture supernatant of splenic cells were decreased while the Ievels of IL-4 and IL-10(Th2 cytokine)were increased,compared with EAU and control group,the differences were statistically significant(F=57.1,15.6,33.1,167.7,P<0.01).Compared the levels of Th1 and Th2 eytokine in the culture supernatant of splenic cells in 10 μg、10 mg groups with EAU and control group,the difference are not statistically significant. Conclusions In the process to prevent EAU by oral intake,the levels of IFN-γ and IL-2 (Th1 cytokine)were decrease while the levels of IL-4 and IL-10(Th2 cytokine).Oral administration with tOO high or low dose of the antigen can not prevent EAU as well as the cytokine levels do not change obviously.Cytokines has played an important role in the prevention of EAU.

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