低剂量甲氨蝶呤下调巨噬细胞iNOS表达抑制肝脏炎性损伤

发布时间：2018-05-28 05:00

本文选题：炎症 + 免疫调节　；参考：《华中科技大学》2011年硕士论文

【摘要】：目的:本文拟通过不同剂量甲氨蝶呤在小鼠炎症模型中对一氧化氮的作用来探讨低剂量甲氨蝶呤对免疫炎症的影响。方法:1.用conA尾静脉注射昆明小鼠,各组分别用不同浓度的MTX处理,48小时取脾细胞RNA,检测IL-2、Fox-P3的表达;2、用LPS刺激昆明小鼠腹腔巨噬细胞,各组分别用不同浓度的MTX处理,48小时后检测培养上清NO含量,提取巨噬细胞RNA,检测IL-2、Fox-P3的表达;3、建立conA所致小鼠急性肝炎模型,各组用不同浓度的MTX处理,6小时后检测外周血转氨酶活性;4、建立DH5α大肠杆菌所致小鼠感染模型,各组用不同浓度的MTX处理,6小时后取肝脏研磨培养,24小时后观察菌落生长情况;5、用CFSE标记BALB/C小鼠脾脏T细胞后尾静脉注入其他BALB/C体内,并用ConA和不同浓度的MTX处理,48小时后取脾脏流式细胞术检测CFSE标记的T细胞增值情况。6、清除小鼠肝脏巨噬细胞(kupffer cells),36小时后建立conA所致小鼠急性肝炎模型,6小时后眼球取血,测血清中转氨酶活性;7、建立conA所致小鼠急性肝炎模型,用不同浓度的MTX尾静脉注射,6小时候分离肝脏Kupper cells,RT-PCR检测iNOS的表达。结果:1、低剂量的MTX(0.1ug/ml)可以下调IL-2的表达和上调Fox-P3的表达;2、LPS刺激小鼠腹腔巨噬细胞后,低剂量的MTX更能有效地抑制巨噬细胞NO的产生;3、在小鼠肝炎模型中,谷丙转氨酶活性升高,而低剂量MTX处理组转氨酶活性降低,MTX剂量加大后转氨酶活性有所提升。4、在小鼠感染模型中,低剂量的MTX促进了细菌在肝脏的生长,而随着剂量的加大,这种促进作用有所减弱;5、低剂量的MTX没有看到明显的抑制T细胞增值作用,而相对高剂量的MTX产生了一定的抑制作用。6、未清除肝脏Kupper cells组转氨酶较清除组明显升高;7、低剂量的MTX作用后所取的Kupper cells中iNOS表达较其余组明显降低。结论:MTX剂量的不同其生物学作用也不同,相对于高剂量主要表现出细胞毒性,低剂量的MTX则表现出一定的免疫抑制作用,其通过抑制巨噬细胞中iNOS的表达从而降低在炎症刺激下巨噬细胞中NO的产生,达到其抗炎作用。而在感染模型中,由于其对免疫功能的抑制,又会使得感染加重。
[Abstract]:Aim: to investigate the effect of low dose methotrexate on immune inflammation in mouse inflammatory model by using different doses of methotrexate. Method 1: 1. Kunming mice were injected with conA tail vein. Spleen cells were treated with different concentrations of MTX for 48 hours. The expression of IL-2 and Fox-P3 was detected. The macrophages of Kunming mice were stimulated by LPS. Each group was treated with different concentrations of MTX for 48 hours to detect the content of no in culture supernatant, to extract macrophage RNAs, to detect the expression of IL-2 Fox-P3, and to establish a mouse model of acute hepatitis induced by conA. After treated with different concentrations of MTX for 6 hours, the activity of transaminase in peripheral blood of each group was detected and the infection model of mice induced by DH5 伪 Escherichia coli was established. Each group was treated with different concentrations of MTX for 6 hours. After 24 hours of liver grinding culture, the colony growth was observed. The spleen T cells of BALB/C mice were labeled with CFSE and injected into the tail vein of other BALB/C. After 48 hours of treatment with ConA and different concentrations of MTX, splenic T cell proliferation was detected by flow cytometry (FCM). The proliferation of T cells labeled by CFSE was detected by flow cytometry. After 36 hours of clearance of murine hepatic macrophages, the acute hepatitis model induced by conA was established, and the blood was taken from the eyeball after 6 hours. The activity of transaminase in serum was measured to establish mouse model of acute hepatitis induced by conA. The expression of iNOS was detected by RT-PCR of liver Kupper cells at 6 hours after different concentrations of MTX were injected into caudal vein. Results: low dose of MTX 0.1 g / ml could down-regulate the expression of IL-2 and up-regulate the expression of Fox-P3. The low dose of MTX could effectively inhibit the production of no in mouse peritoneal macrophages, and the activity of glutamate-pyruvate aminotransferase increased in mouse hepatitis model. In low dose MTX group, the activity of transaminase increased with the increase of the dose of MTX. In the model of mouse infection, low dose of MTX promoted the growth of bacteria in liver, but with the increase of dose, the activity of transaminase increased. This effect was weakened, and low dose of MTX did not show a significant inhibition of T cell proliferation. However, the relative high dose of MTX produced a certain inhibitory effect. The transaminase expression in the liver Kupper cells group was significantly higher than that in the clearance group, and the iNOS expression in the Kupper cells of the low dose MTX group was significantly lower than that in the other groups. Conclusion the biological effects of different doses of MTX are different. Compared with the high dose, the cytotoxicity is mainly observed, while the low dose of MTX has a certain immunosuppressive effect. By inhibiting the expression of iNOS in macrophages, it can reduce the production of no in macrophages stimulated by inflammation and achieve its anti-inflammatory effect. But in the infection model, because of its inhibition to the immune function, will make the infection aggravation.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【共引文献】