雷帕霉素對自身免疫性肝炎的作用及機制
發(fā)布時間：2019-07-05 18:59
【摘要】：一.雷帕霉素對刀豆蛋白A誘導(dǎo)的急性肝損傷的作用及機制目的:觀察尾靜脈注射不同濃度刀豆蛋白A(ConcanavalinA,ConA)后不同時間點小鼠的肝臟損傷、血清學(xué)變化,從而確定造模的最佳劑量與時間。研究Con A對脾臟樹突狀細胞、巨噬細胞、T、B淋巴細胞表型的影響,從而探討Con A模擬自身免疫性肝炎的發(fā)病機制。進一步探討雷帕霉素對刀豆蛋白A(Con A)誘導(dǎo)的急性肝損傷的治療作用,并分析其發(fā)揮作用的機制。方法:1.建立急性肝損傷小鼠模型:8周齡雌性C57BL/6小鼠,平均體重20g。按Con A濃度隨機分為10 mg/kg、15mg/kg、20 mg/kg三組,分別于注射Con A后的6h、24h、48h隨機取6只小鼠,檢測小鼠血清生化水平(AST、ALT);肝組織進行HE染色并作出評分。2.雷帕霉素對急性肝損傷的防治作用:隨機分為對照組、Con A造模組和Con A造模~+雷帕霉素(RAPA)治療組。雷帕霉素處理2h后尾靜脈注射Con A,24h后檢測各組小鼠血清ALT和AST水平;肝組織行HE染色并病理評分。3.流式細胞分析術(shù)檢測脾臟樹突狀細胞(DC,MHC-Ⅱ~+CD11C~+)的比例及表面共刺激分子CD40、CD80、CD86的表達情況,同時檢測CD4~+T、CD8~+T及CD4~+CD25~+調(diào)節(jié)性T細胞(Treg)的比例變化及巨噬細胞和B細胞的變化情況。結(jié)果:1.隨著Con A劑量的增大對肝臟的損傷逐漸加重,10mg/kg劑量對小鼠的肝損傷不顯著,可見局部點狀壞死。15mg/kg劑量可引起點狀或片狀壞死。20mg/kg的劑量注射后24小時內(nèi)小鼠會死亡。Con A組小鼠血清ALT、AST的水平較對照組(PBS組)明顯升高,且Con A注射劑量與小鼠血清ALT、AST的水平呈正相關(guān)。2.ConA對脾臟免疫細胞的影響主要體現(xiàn)在CD4~+T細胞比例顯著上調(diào),巨噬細胞、B細胞、樹突狀細胞的比例呈升高趨勢,且樹突狀細胞共刺激分子(CD80、CD86)顯著上調(diào)。3.RAPA治療組小鼠的血清AST和ALT的水平較Con A組降低,肝臟組織壞死及炎細胞浸潤程度較輕;可以下調(diào)DC的CD80、CD86共刺激分子的表達,同時降低CD4~+T和CD8~+T細胞占脾臟細胞的比例,并且能夠促進CD4~+CD25~+Treg的表達;PAPA對樹突狀細胞共刺激分子CD40的表達無統(tǒng)計學(xué)意義。結(jié)論:1.ConA使小鼠脾臟細胞以CD4T細胞浸潤為主,B細胞、巨噬細胞、樹突狀細胞等抗原提呈細胞的表達有升高的趨勢,且使樹突狀細胞的共刺激分子(CD80、CD86)表達上調(diào)。2.雷帕霉素可以減緩Con A誘導(dǎo)的急性肝損傷,主要通過影響T細胞和DC來發(fā)揮免疫抑制的作用,為自身免疫性肝炎的治療提供了理論依據(jù)。二.雷帕霉素對自身免疫性肝炎肝纖維化的防治作用目的:通過定量多次尾靜脈注射ConA建立小鼠自身免疫性肝炎肝纖維化模型,檢測肝臟淋巴細胞表型及CD4~+T細胞的亞型Th1,Th2,Th3,Tr1細胞的變化情況,從而探討雷帕霉素對其防治作用。方法:1.建立自身免疫性肝炎肝纖維化模型:8周齡雌性C57BL/6小鼠隨機分為對照組、Con A造模組、Con A造模~+雷帕霉素(rapamycin,RAPA)治療組。正常對照組每周一次尾靜脈注射磷酸鹽緩沖液(PBS),模型組和治療組每周一次尾靜脈注射Con A,三組均連續(xù)注射5周。2.評價雷帕霉素的治療效果:檢測各組小鼠血清生化水平;肝臟組織行HE和Masson染色,并行評價肝臟組織炎癥及纖維化程度。3.梯度離心法分離肝臟單個核細胞,流式細胞術(shù)檢測CD4~+T、CD8~+T細胞的比例,并檢測免疫細胞內(nèi)干擾素γ(IFN-γ)、白細胞介素4(IL-4)、白細胞介素10(IL-10)、轉(zhuǎn)化生長因子β(TGF-β)的表達情況。結(jié)果:1.Con A連續(xù)注射第5周時開始出現(xiàn)肝纖維化。雷帕霉素治療組與Con A造模組小鼠相比,血清ALT水平顯著降低(p0.05);肝臟組織炎癥損傷明顯減輕,并無明顯纖維組織增生;2.雷帕霉素治療組與Con A造模組小鼠相比肝臟單個核細胞內(nèi)TGF-β的表達顯著降低(p0.05);肝臟CD4~+T和CD8~+T細胞的比例均下調(diào)(p0.05);Th1細胞的比例降低(p0.05),Th3、Tr1調(diào)節(jié)性T細胞的比例顯著上調(diào)(p0.05),但Th2細胞的差異無統(tǒng)計學(xué)意義(p0.05)。結(jié)論:雷帕霉素可以減緩Con A誘導(dǎo)的慢性肝炎肝纖維化的炎癥程度,與促進肝臟Th3/Tr1細胞的分化,減少肝臟單個核細胞TGF-β的表達有關(guān)。三.雷帕霉素通過誘導(dǎo)免疫耐受性樹突狀細胞的形成減緩自身免疫性肝炎目的:觀察雷帕霉素對樹突狀細胞(dendritic cells,DCs)的共刺激分子及趨化受體的表達、細胞因子、運動速度、特異性免疫反應(yīng)及誘導(dǎo)Treg的能力的影響,探討耐受性樹突狀細胞在自身免疫性肝炎中的作用機制,同時也為治療及預(yù)防自身免疫性肝病提供新的思路。方法:1.免疫磁珠分選CD11C陽性的樹突狀細胞,雷帕霉素刺激后流式檢測共刺激分子及趨化受體表達況,并利用活細胞工作站觀察其動態(tài)運動情況。利用Elisa檢測上清中細胞因子IL-12,IL-6的表達情況。2.體外誘導(dǎo)實驗:將分選的DC與CFSE標記的OT-I,OT-Ⅱ,CD4~+CD25-的T細胞分別共培養(yǎng),流式細胞儀檢測其誘導(dǎo)分化及增殖能力。結(jié)果:1.雷帕霉素刺激后的樹突狀細胞低表達共刺激分子(CD80、CD86)、抑制促炎因子的表達(IL-6、IL-12),符合耐受性樹突狀細胞的特征。促進DC趨化受體CCR7的表達,并促進其運動速度。2.雷帕霉素抑制特異性反應(yīng)中CD4~+T和CD8~+T細胞的增殖,并可以誘導(dǎo)CD4~+CD25-的T細胞分化為CD4~+CD25~+的Treg。結(jié)論:1.雷帕霉素刺激后的樹突狀細胞表現(xiàn)為耐受性樹突狀細胞的特征,同時促進DC趨化受體的表達,并促進其運動速度。2.雷帕霉素降低自身免疫性肝炎的病理損害與抑制CD4~+T、CD8~+T細胞的增殖和誘導(dǎo)CD4~+CD25-的T細胞分化為Treg有著緊密聯(lián)系。
文內(nèi)圖片：
圖片說明：不同劑量ConA在不同時間點對小鼠AST、ALT的影響
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R575.1
，
本文編號：2510740
文內(nèi)圖片：
圖片說明：不同劑量ConA在不同時間點對小鼠AST、ALT的影響
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R575.1
，
本文編號：2510740
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