FTY720對EAE小鼠腦組織MMP-9表達影響
發(fā)布時間：2018-09-02 07:34
【摘要】：目的：觀察FTY720(fingolimod,芬戈莫德)對實驗性自身免疫性腦脊髓炎(experimental autoimmune eneephalomyelitis,EAE)小鼠腦組織基質(zhì)金屬蛋白酶-9（matrix metalloproteinase-9,MMP-9）的mRNA及蛋白表達的影響,并比較不同劑量FTY720的干預對MMP-9表達影響差異。 方法：按照隨機的原則將120只雌性C57BL/6小鼠分為6組，每組20只。正常對照組；CFA組；EAE組；10mg/kg FTY720干預EAE組；3mg/kg FTY720干預EAE組；1mg/kg FTY720干預EAE組。構(gòu)建EAE小鼠模型，免疫第14天，給予不同劑量FTY720干預，相應的正常對照組、CFA組、EAE組給予生理鹽水。于FTY720干預后第30天處死所有小鼠。免疫組化技術(shù)對腦組織中的MMP-9蛋白表達進行檢測。實時熒光定量PCR技術(shù)檢測MMP-9的mRNA表達。 結(jié)果： 1.各組小鼠的臨床表現(xiàn)觀察:⑴正常組、CFA組無小鼠發(fā)病。⑵與EAE組比較,1mg/kg干預組能減輕臨床癥狀、減少體重下降、延長潛伏期(p0.05),對發(fā)病率無影響(P0.05);10mg/kg干預組、3mg/kg干預組能減輕臨床癥狀、減少體重下降、延長潛伏期、降低發(fā)病率(p0.05)。⑶與1mg/kg干預組比較,10mg/kg干預組、3mg/kg干預組能減輕臨床癥狀、減少體重下降、延長潛伏期，差異有統(tǒng)計學意義(p0.05)。 2.各組小鼠腦組織的病理結(jié)果觀察:⑴HE染色顯示正常組、CFA組小鼠側(cè)腦室周圍白質(zhì)無炎性病灶。⑵與EAE組比較,10mg/kg干預組、3mg/kg干預組、1mg/kg干預組炎性病灶數(shù)減少，差異有統(tǒng)計學意義(p0.05)。⑶與1mg/kg干預組比較,10mg/kg干預組、3mg/kg干預組炎性病灶數(shù)減少(p0.05)。⑷LFB染色顯示正常組、CFA組小鼠側(cè)腦室周圍白質(zhì)無髓鞘脫失,EAE組有較多典型脫髓鞘病灶,10mg/kg干預組、3mg/kg干預組、1mg/kg干預組中僅少數(shù)脫髓鞘病灶。 3.各組小鼠腦白質(zhì)免疫組化觀察結(jié)果:⑴與正常組、CFA組比較,EAE組MMP-9蛋白的表達水平明顯升高，差異有統(tǒng)計學意義(p0.05)。⑵與EAE組比較,10mg/kg干預組、3mg/kg干預組、1mg/kg干預組MMP-9蛋白的表達水平降低，差異有統(tǒng)計學意義(p0.05)。⑶與1mg/kg干預組比較,10mg/kg干預組、3mg/kg干預組MMP-9蛋白的表達水平降低，差異有統(tǒng)計學意義(p0.05)。 4.各組小鼠腦組織MMP-9的PCR定量檢測：⑴與正常組、CFA組比較,EAE組MMP-9的mRNA表達水平明顯升高，差異有統(tǒng)計學意義(p0.05)。⑵與EAE組比較,10mg/kg干預組、3mg/kg干預組、1mg/kg干預組MMP-9的mRNA表達水平降低，差異有統(tǒng)計學意義(p0.05)。⑶與1mg/kg干預組比較,10mg/kg干預組、3mg/kg干預組MMP-9的mRNA表達水平降低，差異有統(tǒng)計學意義(p0.05)。 5.FTY720干預劑量與神經(jīng)功能評分呈負相關(guān)(r=-0.779, p=0.000)，，與MMP-9蛋白表達呈負相關(guān)(r=-0.759, p=0.000)，與MMP-9mRNA表達呈負相關(guān)(r=-0.801, p=0.000)，均具有統(tǒng)計學意義。 結(jié)論： 1.FTY720對EAE小鼠治療作用可能在一定程度上呈劑量相關(guān)性。 2.FTY720對EAE小鼠的作用機制之一可能是通過下調(diào)腦組織中MMP-9的mRNA及蛋白表達水平,進而保護血腦屏障，從而減輕EAE小鼠腦組織的炎性反應。
[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.
【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R744.3;R744.51
本文編號：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.
【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R744.3;R744.51
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本文編號：2218692
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