雷帕霉素对自身免疫性肝炎的作用及机制
发布时间：2019-07-05 18:59
【摘要】：一.雷帕霉素对刀豆蛋白A诱导的急性肝损伤的作用及机制目的:观察尾静脉注射不同浓度刀豆蛋白A(ConcanavalinA,ConA)后不同时间点小鼠的肝脏损伤、血清学变化,从而确定造模的最佳剂量与时间。研究Con A对脾脏树突状细胞、巨噬细胞、T、B淋巴细胞表型的影响,从而探讨Con A模拟自身免疫性肝炎的发病机制。进一步探讨雷帕霉素对刀豆蛋白A(Con A)诱导的急性肝损伤的治疗作用,并分析其发挥作用的机制。方法:1.建立急性肝损伤小鼠模型:8周龄雌性C57BL/6小鼠,平均体重20g。按Con A浓度随机分为10 mg/kg、15mg/kg、20 mg/kg三组,分别于注射Con A后的6h、24h、48h随机取6只小鼠,检测小鼠血清生化水平(AST、ALT);肝组织进行HE染色并作出评分。2.雷帕霉素对急性肝损伤的防治作用:随机分为对照组、Con A造模组和Con A造模~+雷帕霉素(RAPA)治疗组。雷帕霉素处理2h后尾静脉注射Con A,24h后检测各组小鼠血清ALT和AST水平;肝组织行HE染色并病理评分。3.流式细胞分析术检测脾脏树突状细胞(DC,MHC-Ⅱ~+CD11C~+)的比例及表面共刺激分子CD40、CD80、CD86的表达情况,同时检测CD4~+T、CD8~+T及CD4~+CD25~+调节性T细胞(Treg)的比例变化及巨噬细胞和B细胞的变化情况。结果:1.随着Con A剂量的增大对肝脏的损伤逐渐加重,10mg/kg剂量对小鼠的肝损伤不显著,可见局部点状坏死。15mg/kg剂量可引起点状或片状坏死。20mg/kg的剂量注射后24小时内小鼠会死亡。Con A组小鼠血清ALT、AST的水平较对照组(PBS组)明显升高,且Con A注射剂量与小鼠血清ALT、AST的水平呈正相关。2.ConA对脾脏免疫细胞的影响主要体现在CD4~+T细胞比例显著上调,巨噬细胞、B细胞、树突状细胞的比例呈升高趋势,且树突状细胞共刺激分子(CD80、CD86)显著上调。3.RAPA治疗组小鼠的血清AST和ALT的水平较Con A组降低,肝脏组织坏死及炎细胞浸润程度较轻;可以下调DC的CD80、CD86共刺激分子的表达,同时降低CD4~+T和CD8~+T细胞占脾脏细胞的比例,并且能够促进CD4~+CD25~+Treg的表达;PAPA对树突状细胞共刺激分子CD40的表达无统计学意义。结论:1.ConA使小鼠脾脏细胞以CD4T细胞浸润为主,B细胞、巨噬细胞、树突状细胞等抗原提呈细胞的表达有升高的趋势,且使树突状细胞的共刺激分子(CD80、CD86)表达上调。2.雷帕霉素可以减缓Con A诱导的急性肝损伤,主要通过影响T细胞和DC来发挥免疫抑制的作用,为自身免疫性肝炎的治疗提供了理论依据。二.雷帕霉素对自身免疫性肝炎肝纤维化的防治作用目的:通过定量多次尾静脉注射ConA建立小鼠自身免疫性肝炎肝纤维化模型,检测肝脏淋巴细胞表型及CD4~+T细胞的亚型Th1,Th2,Th3,Tr1细胞的变化情况,从而探讨雷帕霉素对其防治作用。方法:1.建立自身免疫性肝炎肝纤维化模型:8周龄雌性C57BL/6小鼠随机分为对照组、Con A造模组、Con A造模~+雷帕霉素(rapamycin,RAPA)治疗组。正常对照组每周一次尾静脉注射磷酸盐缓冲液(PBS),模型组和治疗组每周一次尾静脉注射Con A,三组均连续注射5周。2.评价雷帕霉素的治疗效果:检测各组小鼠血清生化水平;肝脏组织行HE和Masson染色,并行评价肝脏组织炎症及纤维化程度。3.梯度离心法分离肝脏单个核细胞,流式细胞术检测CD4~+T、CD8~+T细胞的比例,并检测免疫细胞内干扰素γ(IFN-γ)、白细胞介素4(IL-4)、白细胞介素10(IL-10)、转化生长因子β(TGF-β)的表达情况。结果:1.Con A连续注射第5周时开始出现肝纤维化。雷帕霉素治疗组与Con A造模组小鼠相比,血清ALT水平显著降低(p0.05);肝脏组织炎症损伤明显减轻,并无明显纤维组织增生;2.雷帕霉素治疗组与Con A造模组小鼠相比肝脏单个核细胞内TGF-β的表达显著降低(p0.05);肝脏CD4~+T和CD8~+T细胞的比例均下调(p0.05);Th1细胞的比例降低(p0.05),Th3、Tr1调节性T细胞的比例显著上调(p0.05),但Th2细胞的差异无统计学意义(p0.05)。结论:雷帕霉素可以减缓Con A诱导的慢性肝炎肝纤维化的炎症程度,与促进肝脏Th3/Tr1细胞的分化,减少肝脏单个核细胞TGF-β的表达有关。三.雷帕霉素通过诱导免疫耐受性树突状细胞的形成减缓自身免疫性肝炎目的:观察雷帕霉素对树突状细胞(dendritic cells,DCs)的共刺激分子及趋化受体的表达、细胞因子、运动速度、特异性免疫反应及诱导Treg的能力的影响,探讨耐受性树突状细胞在自身免疫性肝炎中的作用机制,同时也为治疗及预防自身免疫性肝病提供新的思路。方法:1.免疫磁珠分选CD11C阳性的树突状细胞,雷帕霉素刺激后流式检测共刺激分子及趋化受体表达况,并利用活细胞工作站观察其动态运动情况。利用Elisa检测上清中细胞因子IL-12,IL-6的表达情况。2.体外诱导实验:将分选的DC与CFSE标记的OT-I,OT-Ⅱ,CD4~+CD25-的T细胞分别共培养,流式细胞仪检测其诱导分化及增殖能力。结果:1.雷帕霉素刺激后的树突状细胞低表达共刺激分子(CD80、CD86)、抑制促炎因子的表达(IL-6、IL-12),符合耐受性树突状细胞的特征。促进DC趋化受体CCR7的表达,并促进其运动速度。2.雷帕霉素抑制特异性反应中CD4~+T和CD8~+T细胞的增殖,并可以诱导CD4~+CD25-的T细胞分化为CD4~+CD25~+的Treg。结论:1.雷帕霉素刺激后的树突状细胞表现为耐受性树突状细胞的特征,同时促进DC趋化受体的表达,并促进其运动速度。2.雷帕霉素降低自身免疫性肝炎的病理损害与抑制CD4~+T、CD8~+T细胞的增殖和诱导CD4~+CD25-的T细胞分化为Treg有着紧密联系。
文内图片：
图片说明：不同剂量ConA在不同时间点对小鼠AST、ALT的影响
[Abstract]:I. Objective: To observe the effect and mechanism of rapamycin on acute liver injury induced by bean protein A: to observe the liver injury and serologic change of mice at different time points after different concentration of concanavaline A (ConA) in the tail vein, so as to determine the optimal dosage and time of the model. The effects of Con A on the phenotype of spleen-derived dendritic cells, macrophages, T and B lymphocytes were studied, and the pathogenesis of Con A was discussed. To study the therapeutic effect of rapamycin on acute liver injury induced by Con A (Con A), and to analyze the mechanism of its function. Method:1. An acute liver injury mouse model was established:8-week-old female C57BL/6 mice with an average body weight of 20 g. 6 mice were randomly divided into 10 mg/ kg,15 mg/ kg and 20 mg/ kg according to Con A concentration,6 mice were randomly divided into 6 h,24 h and 48 h after injection of Con A, and the serum biochemical level (AST, ALT) in mice was detected; and the liver tissues were stained with HE and scored. The control effects of rapamycin on acute liver injury were randomly divided into control group, Con A group and Con A model ~ + rapamycin (RAPA) treatment group. The serum levels of ALT and AST in the mice were detected after 2 h of rapamycin treatment, and the levels of serum ALT and AST were detected after 24 h, and the liver tissue was stained with HE and the pathological score was 3. The expression of CD4 ~ + T, CD8 ~ + T and CD4 ~ + CD25 ~ + regulatory T cells (Treg) and the changes of macrophages and B cells were detected by flow cytometry. Results:1. With the increase of Con A dose, the damage of the liver was gradually increased, and the 10 mg/ kg dose was not significant to the liver injury of the mice, and local point necrosis was observed. The 15 mg/ kg dose could cause point-like or sheet-like necrosis. The mice died within 24 hours after a dose of 20 mg/ kg. The level of ALT and AST in the mice of Con A group was significantly higher than that in the control group (PBS group), and the amount of Con A was positively correlated with the level of ALT and AST in the serum of the mice. 3. The level of AST and ALT in the mice in the RAPA group was lower than that of the Con A group, the necrosis of the liver and the degree of inflammatory cell infiltration were light, and the CD80 of the DC could be downregulated. The expression of CD86 co-stimulatory molecules decreased the proportion of CD4 ~ + T and CD8 ~ + T cells in the spleen cells, and can promote the expression of CD4 ~ + CD25 ~ + Treg. Conclusion:1. ConA increases the expression of CD4T cell in the spleen cells of the mouse, and the expression of the antigen-like cells, such as B cells, macrophages and dendritic cells, has a tendency to increase, and the expression of the co-stimulatory molecules (CD80, CD86) of the dendritic cells is up-regulated. Rapamycin can slow the acute liver injury induced by Con A, and can play an immunosuppression effect mainly by influencing the T cell and the DC, and provides a theoretical basis for the treatment of the autoimmune hepatitis. II. The effect of rapamycin on the hepatic fibrosis of autoimmune hepatitis was to establish a model of the liver fibrosis of the mice's autoimmune hepatitis by quantitative multiple-dose intravenous ConA, to detect the changes of Th1, Th2, Th3, Tr1 cells of the phenotype of the liver and the subtypes of CD4 ~ + T cells. So as to explore the prevention and treatment effect of rapamycin. Method:1. The model of hepatic fibrosis of autoimmune hepatitis was established. The 8-week-old female C57BL/6 mice were randomly divided into the control group, the Con A-making module and the Con A-model-+ rapamycin (RAPA) treatment group. In the normal control group, the weekly tail vein phosphate buffer (PBS), the model group and the treatment group were injected intravenously once a week, and the three groups were continuously injected for 5 weeks. The treatment effect of rapamycin was evaluated: the serum biochemical level of each group of mice was detected; the liver tissue was stained with HE and Masson, and the inflammation and fibrosis degree of the liver were evaluated in parallel. The expression of interferon (IFN-1), interleukin-4 (IL-4), interleukin-10 (IL-10) and transformation growth factor (TGF-1) were detected by flow cytometry. Results:1. The hepatic fibrosis started at the fifth week of Con A continuous injection. The serum ALT level was significantly lower in the rapamycin treatment group compared with that of the Con A group (p0.05); the inflammatory injury of the liver tissue was significantly reduced, and there was no significant fibrous tissue proliferation;2. Compared with Con A group, the expression of TGF-1 in the individual nuclear cells decreased significantly (p0.05), and the ratio of CD4 + T and CD8 + T cells in the liver was reduced (p0.05), and the ratio of Th1 cells was decreased (p0.05), and the ratio of Th3 and Tr1 regulatory T cells was significantly increased (p0.05). However, the difference of Th2 cells was not significant (p0.05). Conclusion: Rapamycin can reduce the degree of inflammation of the liver fibrosis induced by Con A and promote the differentiation of the liver Th3/ Tr1 cells and reduce the expression of TGF-1 in the individual nuclear cells of the liver. III. The purpose of the invention is to observe the expression of the co-stimulatory molecules and the chemotactic receptors of the rapamycin on the dendritic cells (DCs) and the expression of the chemotactic receptors, the cytokines, the speed of the movement, The effect of the specific immune response and the ability to induce Treg is to explore the mechanism of tolerance of dendritic cells in autoimmune hepatitis, and to provide a new way for the treatment and prevention of autoimmune liver diseases. Method:1. Immunomagnetic beads were used to sort the CD11C-positive dendritic cells, and the expression of the co-stimulatory molecules and the chemotactic receptors was detected by the flow cytometry after the rapamycin was stimulated, and the dynamic motion of the CD11c-positive dendritic cells was observed by using the living cell workstation. The expression of IL-12 and IL-6 in supernatant was detected by Elisa. In vitro induction experiment: The isolated DC and CFSE-labeled OT-I, OT-II, CD4 ~ + CD25-T cells were co-cultured, and the induced differentiation and proliferation ability were detected by flow cytometry. Results:1. The low-expression co-stimulatory molecules (CD80, CD86) of the dendritic cells after the rapamycin stimulation, and the expression of the pro-inflammatory factor (IL-6, IL-12), met the characteristics of the resistant dendritic cells. To promote the expression of the DC chemotactic receptor CCR7 and to promote the speed of its movement. Rapamycin inhibits the proliferation of CD4 ~ + T and CD8 ~ + T cells in the specific reaction, and can induce the differentiation of CD4 ~ + CD25 ~ + T cells into the Treg of CD4 ~ + CD25 ~ +. Conclusion:1. The dendritic cells stimulated by rapamycin appear to be resistant to the characteristics of dendritic cells, while promoting the expression of the DC chemotactic receptor and promoting its speed of movement. Rapamycin has a close relationship with the inhibition of the proliferation of CD4 ~ + T, CD8 ~ + T cells and the differentiation of CD4 ~ + CD25-T cells into Treg.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.1
，
本文编号：2510740
文内图片：
图片说明：不同剂量ConA在不同时间点对小鼠AST、ALT的影响
[Abstract]:I. Objective: To observe the effect and mechanism of rapamycin on acute liver injury induced by bean protein A: to observe the liver injury and serologic change of mice at different time points after different concentration of concanavaline A (ConA) in the tail vein, so as to determine the optimal dosage and time of the model. The effects of Con A on the phenotype of spleen-derived dendritic cells, macrophages, T and B lymphocytes were studied, and the pathogenesis of Con A was discussed. To study the therapeutic effect of rapamycin on acute liver injury induced by Con A (Con A), and to analyze the mechanism of its function. Method:1. An acute liver injury mouse model was established:8-week-old female C57BL/6 mice with an average body weight of 20 g. 6 mice were randomly divided into 10 mg/ kg,15 mg/ kg and 20 mg/ kg according to Con A concentration,6 mice were randomly divided into 6 h,24 h and 48 h after injection of Con A, and the serum biochemical level (AST, ALT) in mice was detected; and the liver tissues were stained with HE and scored. The control effects of rapamycin on acute liver injury were randomly divided into control group, Con A group and Con A model ~ + rapamycin (RAPA) treatment group. The serum levels of ALT and AST in the mice were detected after 2 h of rapamycin treatment, and the levels of serum ALT and AST were detected after 24 h, and the liver tissue was stained with HE and the pathological score was 3. The expression of CD4 ~ + T, CD8 ~ + T and CD4 ~ + CD25 ~ + regulatory T cells (Treg) and the changes of macrophages and B cells were detected by flow cytometry. Results:1. With the increase of Con A dose, the damage of the liver was gradually increased, and the 10 mg/ kg dose was not significant to the liver injury of the mice, and local point necrosis was observed. The 15 mg/ kg dose could cause point-like or sheet-like necrosis. The mice died within 24 hours after a dose of 20 mg/ kg. The level of ALT and AST in the mice of Con A group was significantly higher than that in the control group (PBS group), and the amount of Con A was positively correlated with the level of ALT and AST in the serum of the mice. 3. The level of AST and ALT in the mice in the RAPA group was lower than that of the Con A group, the necrosis of the liver and the degree of inflammatory cell infiltration were light, and the CD80 of the DC could be downregulated. The expression of CD86 co-stimulatory molecules decreased the proportion of CD4 ~ + T and CD8 ~ + T cells in the spleen cells, and can promote the expression of CD4 ~ + CD25 ~ + Treg. Conclusion:1. ConA increases the expression of CD4T cell in the spleen cells of the mouse, and the expression of the antigen-like cells, such as B cells, macrophages and dendritic cells, has a tendency to increase, and the expression of the co-stimulatory molecules (CD80, CD86) of the dendritic cells is up-regulated. Rapamycin can slow the acute liver injury induced by Con A, and can play an immunosuppression effect mainly by influencing the T cell and the DC, and provides a theoretical basis for the treatment of the autoimmune hepatitis. II. The effect of rapamycin on the hepatic fibrosis of autoimmune hepatitis was to establish a model of the liver fibrosis of the mice's autoimmune hepatitis by quantitative multiple-dose intravenous ConA, to detect the changes of Th1, Th2, Th3, Tr1 cells of the phenotype of the liver and the subtypes of CD4 ~ + T cells. So as to explore the prevention and treatment effect of rapamycin. Method:1. The model of hepatic fibrosis of autoimmune hepatitis was established. The 8-week-old female C57BL/6 mice were randomly divided into the control group, the Con A-making module and the Con A-model-+ rapamycin (RAPA) treatment group. In the normal control group, the weekly tail vein phosphate buffer (PBS), the model group and the treatment group were injected intravenously once a week, and the three groups were continuously injected for 5 weeks. The treatment effect of rapamycin was evaluated: the serum biochemical level of each group of mice was detected; the liver tissue was stained with HE and Masson, and the inflammation and fibrosis degree of the liver were evaluated in parallel. The expression of interferon (IFN-1), interleukin-4 (IL-4), interleukin-10 (IL-10) and transformation growth factor (TGF-1) were detected by flow cytometry. Results:1. The hepatic fibrosis started at the fifth week of Con A continuous injection. The serum ALT level was significantly lower in the rapamycin treatment group compared with that of the Con A group (p0.05); the inflammatory injury of the liver tissue was significantly reduced, and there was no significant fibrous tissue proliferation;2. Compared with Con A group, the expression of TGF-1 in the individual nuclear cells decreased significantly (p0.05), and the ratio of CD4 + T and CD8 + T cells in the liver was reduced (p0.05), and the ratio of Th1 cells was decreased (p0.05), and the ratio of Th3 and Tr1 regulatory T cells was significantly increased (p0.05). However, the difference of Th2 cells was not significant (p0.05). Conclusion: Rapamycin can reduce the degree of inflammation of the liver fibrosis induced by Con A and promote the differentiation of the liver Th3/ Tr1 cells and reduce the expression of TGF-1 in the individual nuclear cells of the liver. III. The purpose of the invention is to observe the expression of the co-stimulatory molecules and the chemotactic receptors of the rapamycin on the dendritic cells (DCs) and the expression of the chemotactic receptors, the cytokines, the speed of the movement, The effect of the specific immune response and the ability to induce Treg is to explore the mechanism of tolerance of dendritic cells in autoimmune hepatitis, and to provide a new way for the treatment and prevention of autoimmune liver diseases. Method:1. Immunomagnetic beads were used to sort the CD11C-positive dendritic cells, and the expression of the co-stimulatory molecules and the chemotactic receptors was detected by the flow cytometry after the rapamycin was stimulated, and the dynamic motion of the CD11c-positive dendritic cells was observed by using the living cell workstation. The expression of IL-12 and IL-6 in supernatant was detected by Elisa. In vitro induction experiment: The isolated DC and CFSE-labeled OT-I, OT-II, CD4 ~ + CD25-T cells were co-cultured, and the induced differentiation and proliferation ability were detected by flow cytometry. Results:1. The low-expression co-stimulatory molecules (CD80, CD86) of the dendritic cells after the rapamycin stimulation, and the expression of the pro-inflammatory factor (IL-6, IL-12), met the characteristics of the resistant dendritic cells. To promote the expression of the DC chemotactic receptor CCR7 and to promote the speed of its movement. Rapamycin inhibits the proliferation of CD4 ~ + T and CD8 ~ + T cells in the specific reaction, and can induce the differentiation of CD4 ~ + CD25 ~ + T cells into the Treg of CD4 ~ + CD25 ~ +. Conclusion:1. The dendritic cells stimulated by rapamycin appear to be resistant to the characteristics of dendritic cells, while promoting the expression of the DC chemotactic receptor and promoting its speed of movement. Rapamycin has a close relationship with the inhibition of the proliferation of CD4 ~ + T, CD8 ~ + T cells and the differentiation of CD4 ~ + CD25-T cells into Treg.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.1
，
本文编号：2510740
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