FTY720对EAE小鼠脑组织MMP-9表达影响
发布时间：2018-09-02 07:34
【摘要】：目的：观察FTY720(fingolimod,芬戈莫德)对实验性自身免疫性脑脊髓炎(experimental autoimmune eneephalomyelitis,EAE)小鼠脑组织基质金属蛋白酶-9（matrix metalloproteinase-9,MMP-9）的mRNA及蛋白表达的影响,并比较不同剂量FTY720的干预对MMP-9表达影响差异。 方法：按照随机的原则将120只雌性C57BL/6小鼠分为6组，每组20只。正常对照组；CFA组；EAE组；10mg/kg FTY720干预EAE组；3mg/kg FTY720干预EAE组；1mg/kg FTY720干预EAE组。构建EAE小鼠模型，免疫第14天，给予不同剂量FTY720干预，相应的正常对照组、CFA组、EAE组给予生理盐水。于FTY720干预后第30天处死所有小鼠。免疫组化技术对脑组织中的MMP-9蛋白表达进行检测。实时荧光定量PCR技术检测MMP-9的mRNA表达。 结果： 1.各组小鼠的临床表现观察:⑴正常组、CFA组无小鼠发病。⑵与EAE组比较,1mg/kg干预组能减轻临床症状、减少体重下降、延长潜伏期(p0.05),对发病率无影响(P0.05);10mg/kg干预组、3mg/kg干预组能减轻临床症状、减少体重下降、延长潜伏期、降低发病率(p0.05)。⑶与1mg/kg干预组比较,10mg/kg干预组、3mg/kg干预组能减轻临床症状、减少体重下降、延长潜伏期，差异有统计学意义(p0.05)。 2.各组小鼠脑组织的病理结果观察:⑴HE染色显示正常组、CFA组小鼠侧脑室周围白质无炎性病灶。⑵与EAE组比较,10mg/kg干预组、3mg/kg干预组、1mg/kg干预组炎性病灶数减少，差异有统计学意义(p0.05)。⑶与1mg/kg干预组比较,10mg/kg干预组、3mg/kg干预组炎性病灶数减少(p0.05)。⑷LFB染色显示正常组、CFA组小鼠侧脑室周围白质无髓鞘脱失,EAE组有较多典型脱髓鞘病灶,10mg/kg干预组、3mg/kg干预组、1mg/kg干预组中仅少数脱髓鞘病灶。 3.各组小鼠脑白质免疫组化观察结果:⑴与正常组、CFA组比较,EAE组MMP-9蛋白的表达水平明显升高，差异有统计学意义(p0.05)。⑵与EAE组比较,10mg/kg干预组、3mg/kg干预组、1mg/kg干预组MMP-9蛋白的表达水平降低，差异有统计学意义(p0.05)。⑶与1mg/kg干预组比较,10mg/kg干预组、3mg/kg干预组MMP-9蛋白的表达水平降低，差异有统计学意义(p0.05)。 4.各组小鼠脑组织MMP-9的PCR定量检测：⑴与正常组、CFA组比较,EAE组MMP-9的mRNA表达水平明显升高，差异有统计学意义(p0.05)。⑵与EAE组比较,10mg/kg干预组、3mg/kg干预组、1mg/kg干预组MMP-9的mRNA表达水平降低，差异有统计学意义(p0.05)。⑶与1mg/kg干预组比较,10mg/kg干预组、3mg/kg干预组MMP-9的mRNA表达水平降低，差异有统计学意义(p0.05)。 5.FTY720干预剂量与神经功能评分呈负相关(r=-0.779, p=0.000)，，与MMP-9蛋白表达呈负相关(r=-0.759, p=0.000)，与MMP-9mRNA表达呈负相关(r=-0.801, p=0.000)，均具有统计学意义。 结论： 1.FTY720对EAE小鼠治疗作用可能在一定程度上呈剂量相关性。 2.FTY720对EAE小鼠的作用机制之一可能是通过下调脑组织中MMP-9的mRNA及蛋白表达水平,进而保护血脑屏障，从而减轻EAE小鼠脑组织的炎性反应。
[Abstract]:Aim: to observe the effect of FTY720 (fingolimod, Fengomod) on the expression of mRNA and protein of matrix metalloproteinase-9 (matrix metalloproteinase-9,MMP-9) in brain tissue of mice with experimental autoimmune encephalomyelitis (experimental autoimmune eneephalomyelitis,EAE), and to compare the effects of different doses of FTY720 on the expression of MMP-9. Methods: 120 female C57BL/6 mice were randomly divided into 6 groups with 20 in each group. The normal control group was treated with 10 mg / kg FTY720 in EAE group and 3 mg / kg FTY720 in EAE group and 1 mg / kg FTY720 in EAE group. The model of EAE mice was established. The mice were immunized with different doses of FTY720 on the 14th day, and normal saline was given to the normal control group. All the mice were killed on the 30th day after FTY720 intervention. Immunohistochemical technique was used to detect the expression of MMP-9 protein in brain tissue. The mRNA expression of MMP-9 was detected by real-time fluorescence quantitative PCR. Results: 1. Observation on the clinical manifestations of the mice in the control group: compared with the EAE group, the 1 mg / kg intervention group could relieve the clinical symptoms and reduce the weight loss, compared with the control group. Prolonging incubation period (p0.05) had no effect on the incidence (P0.05). The intervention group of 10 mg / kg or 3 mg / kg could alleviate clinical symptoms, reduce weight loss, prolong incubation period, and reduce incidence rate (p0.05) .3 compared with 1mg/kg intervention group, 10 mg / kg intervention group and 3 mg / kg intervention group could alleviate clinical symptoms. Loss of weight, prolonged incubation period, the difference was statistically significant (p0.05). 2. The pathological results of brain tissue in each group of mice showed that the number of inflammatory lesions decreased in 10 mg / kg intervention group and 1 mg / kg intervention group compared with that in EAE group. The difference was statistically significant (p0.05) .3 compared with the 1mg/kg intervention group, the inflammatory lesions in the 10 mg / kg intervention group decreased (p0.05) .4LFB staining showed that there were more typical demyelinating lesions in the white matter around the lateral ventricle of the normal group. There were only a few demyelinating lesions in 1 mg / kg group in 10 mg / kg intervention group. The expression of MMP-9 protein in the white matter of each group was significantly higher than that in the control group, and the expression of MMP-9 protein in the white matter of each group was significantly higher than that in the control group. There was a significant difference in the expression of MMP-9 protein between 10 mg / kg group and 10 mg / kg group compared with EAE group (p0.05). The expression of MMP-9 protein in 10 mg / kg group was significantly lower than that in 10 mg / kg group, and the expression level of MMP-9 protein in 10 mg / kg group was significantly lower than that in 1mg/kg group (p0.05). The expression of MMP-9 protein in the 10 mg / kg group was significantly lower than that in the control group (p0.05), and the expression level of MMP-9 protein in the 10 mg / kg group was significantly lower than that in the 1mg/kg group. The difference was statistically significant (p0.05). PCR quantitative detection of MMP-9 in brain tissue of mice in each group compared with control group, the mRNA expression of MMP-9 in MMP-9 group was significantly higher than that in control group (p0.05). 2 the mRNA expression of MMP-9 in 10 mg / kg group was significantly lower than that in 10 mg / kg group compared with EAE group, and the mRNA expression level in 1 mg / kg group was significantly lower than that in 10 mg / kg group. The difference was statistically significant (p0.05) .3 compared with the 1mg/kg intervention group, the mRNA expression level of MMP-9 in the 10 mg / kg intervention group was lower than that in the 3 mg / kg intervention group. The difference was significant (p0. 05). There was a negative correlation between the intervention dose of 5.FTY720 and the neurological function score (r-0.779, p0. 000), the expression of MMP-9 protein (r-0. 759, p0. 000) and the expression of MMP-9mRNA (r-0. 801, p0. 000). Conclusion: the therapeutic effect of 1.FTY720 on EAE mice may be dose-dependent to some extent. One of the mechanisms of 2.FTY720 on EAE mice may be through down-regulating the expression of MMP-9 mRNA and protein in brain tissue. Then the blood-brain barrier was protected and the inflammatory response of brain tissue in EAE mice was alleviated.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.3;R744.51
本文编号：2218692
[Abstract]:Aim: to observe the effect of FTY720 (fingolimod, Fengomod) on the expression of mRNA and protein of matrix metalloproteinase-9 (matrix metalloproteinase-9,MMP-9) in brain tissue of mice with experimental autoimmune encephalomyelitis (experimental autoimmune eneephalomyelitis,EAE), and to compare the effects of different doses of FTY720 on the expression of MMP-9. Methods: 120 female C57BL/6 mice were randomly divided into 6 groups with 20 in each group. The normal control group was treated with 10 mg / kg FTY720 in EAE group and 3 mg / kg FTY720 in EAE group and 1 mg / kg FTY720 in EAE group. The model of EAE mice was established. The mice were immunized with different doses of FTY720 on the 14th day, and normal saline was given to the normal control group. All the mice were killed on the 30th day after FTY720 intervention. Immunohistochemical technique was used to detect the expression of MMP-9 protein in brain tissue. The mRNA expression of MMP-9 was detected by real-time fluorescence quantitative PCR. Results: 1. Observation on the clinical manifestations of the mice in the control group: compared with the EAE group, the 1 mg / kg intervention group could relieve the clinical symptoms and reduce the weight loss, compared with the control group. Prolonging incubation period (p0.05) had no effect on the incidence (P0.05). The intervention group of 10 mg / kg or 3 mg / kg could alleviate clinical symptoms, reduce weight loss, prolong incubation period, and reduce incidence rate (p0.05) .3 compared with 1mg/kg intervention group, 10 mg / kg intervention group and 3 mg / kg intervention group could alleviate clinical symptoms. Loss of weight, prolonged incubation period, the difference was statistically significant (p0.05). 2. The pathological results of brain tissue in each group of mice showed that the number of inflammatory lesions decreased in 10 mg / kg intervention group and 1 mg / kg intervention group compared with that in EAE group. The difference was statistically significant (p0.05) .3 compared with the 1mg/kg intervention group, the inflammatory lesions in the 10 mg / kg intervention group decreased (p0.05) .4LFB staining showed that there were more typical demyelinating lesions in the white matter around the lateral ventricle of the normal group. There were only a few demyelinating lesions in 1 mg / kg group in 10 mg / kg intervention group. The expression of MMP-9 protein in the white matter of each group was significantly higher than that in the control group, and the expression of MMP-9 protein in the white matter of each group was significantly higher than that in the control group. There was a significant difference in the expression of MMP-9 protein between 10 mg / kg group and 10 mg / kg group compared with EAE group (p0.05). The expression of MMP-9 protein in 10 mg / kg group was significantly lower than that in 10 mg / kg group, and the expression level of MMP-9 protein in 10 mg / kg group was significantly lower than that in 1mg/kg group (p0.05). The expression of MMP-9 protein in the 10 mg / kg group was significantly lower than that in the control group (p0.05), and the expression level of MMP-9 protein in the 10 mg / kg group was significantly lower than that in the 1mg/kg group. The difference was statistically significant (p0.05). PCR quantitative detection of MMP-9 in brain tissue of mice in each group compared with control group, the mRNA expression of MMP-9 in MMP-9 group was significantly higher than that in control group (p0.05). 2 the mRNA expression of MMP-9 in 10 mg / kg group was significantly lower than that in 10 mg / kg group compared with EAE group, and the mRNA expression level in 1 mg / kg group was significantly lower than that in 10 mg / kg group. The difference was statistically significant (p0.05) .3 compared with the 1mg/kg intervention group, the mRNA expression level of MMP-9 in the 10 mg / kg intervention group was lower than that in the 3 mg / kg intervention group. The difference was significant (p0. 05). There was a negative correlation between the intervention dose of 5.FTY720 and the neurological function score (r-0.779, p0. 000), the expression of MMP-9 protein (r-0. 759, p0. 000) and the expression of MMP-9mRNA (r-0. 801, p0. 000). Conclusion: the therapeutic effect of 1.FTY720 on EAE mice may be dose-dependent to some extent. One of the mechanisms of 2.FTY720 on EAE mice may be through down-regulating the expression of MMP-9 mRNA and protein in brain tissue. Then the blood-brain barrier was protected and the inflammatory response of brain tissue in EAE mice was alleviated.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.3;R744.51
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