硫酸吲哚酚影响人单核细胞源树突状细胞免疫功能的相关研究
发布时间：2018-09-16 19:13
【摘要】：心血管疾病(cardiovascular disease,CVD)是慢性肾脏疾病(chronic kidney disease,CKD)患者最主要的死亡原因[1]。CKD患者动脉粥样硬化(atherosclerosis,AS)的进展极为迅速,极易发生严重的冠状动脉疾病[2,3];虽然接受常规透析治疗可以纠正尿毒症患者的内环境,但仍有超过50%的患者最终因CVD而死亡[4]。硫酸吲哚酚(Indoxyl sulfate,IS)是一种亲蛋白质化合物的尿毒素,不能被目前的透析治疗方式有效清除[5]。IS被认为参与到透析患者AS的发病机制之中,既往研究发现IS可通过破坏血管内皮功能、促使血管平滑肌细胞增生、加速血管钙化等[6,7]来发挥致AS作用,但具体机制尚不明确。近年来,有学说认为AS是一种慢性炎症和自身免疫性疾病[8],而树突状细胞(dendritic cells,DCs)在AS的炎症进展上发挥了重要的作用[9]。我们试图探究IS是否可启动DCs发挥相关免疫作用,促进AS的炎症进展,从而为IS参与AS的病理过程提供更多的理论依据。第一部分人单核细胞源树突状细胞的体外诱导与鉴定目的:应用改良双密度梯度离心法与细胞因子诱导结合的方法分离和培养人单核细胞源树突状细胞(monocyte-derived dendritic cells,mo-DCs),为进一步研究DCs的免疫作用奠定实验基础。方法:利用改良双密度梯度离心法分离人纯化的单核细胞,用rhGM-CSF(1000IU/mL)和rhIL-4(500IU/mL)体外诱导,倒置相差显微镜每天观察细胞状态,培养5天后获得mo-DCs,电子显微镜观察细胞形态,流式细胞仪进行表型鉴定。结果:1、30mL外周血可分离并培养出mo-DCs约(3-6)×106个,台盼蓝染色后细胞存活率平均90%;2、倒置相差显微镜及电子显微镜观察细胞呈典型树突状细胞形态;3、流式细胞仪检测表达CD11c阳性的细胞可达90%以上,获得纯度较高mo-DCs。结论:本实验选用的方法简单、快速,可分离诱导出纯度较高的mo-DCs,可用于后续实验研究。第二部分硫酸吲哚酚对人单核细胞源树突状细胞免疫功能的影响目的:探讨IS对mo-DCs的分化、成熟及免疫功能的影响,为进一步探索IS在AS免疫炎症反应中的机制提供依据。方法:上述方法获得mo-DCs,此时为未成熟树突状细胞(immature dedritic cells,imDCs),用不含血清的RPMI1640继续培养24h后,第6天将imDCs随机分为 5 组:PBS 组、LPS 组(1μg/mL)、IS.1 组(30μmol/L)、IS.2 组(300μmol/L)、IS.3组(600μmol/L)。刺激24h后流式细胞仪检测各组细胞表型和吞噬功能变化,ELISA法检测各组细胞分泌细胞因子IL-12p70的水平变化,电子显微镜观察细胞形态学变化。结果:不同浓度IS作用于imDCs之后,显著上调细胞表面标志物CD80、CD83、CD86、MHCII的表达;降低细胞吞噬功能并促进细胞因子IL-12p70的分泌(P0.05);形态学上看,LPS组和IS.2组细胞呈不规则形,胞体表面有形态不一的突起。认为IS为300μmol/L时为最适宜的刺激浓度,刺激mo-DCs从未成熟到成熟状态。结论:IS可诱导mo-DCs表型及功能成熟,这可能是IS参与AS免疫炎症过程的机制之一。
[Abstract]:Cardiovascular disease (cardiovascular disease,CVD) is the leading cause of death in patients with chronic renal disease (chronic kidney disease,CKD) [1]. The progression of atherosclerosis (atherosclerosis,AS) in patients with chronic renal disease (chronic kidney disease,CKD) is extremely rapid. Although routine dialysis therapy can correct the internal environment of uremic patients, more than 50% of patients end up dying from CVD [4]. Indole sulfate (Indoxyl sulfate,IS) is a proteophile urinary toxin, which can not be effectively cleared by the current dialysis therapy [5] .is considered to be involved in the pathogenesis of AS in dialysis patients. Previous studies have found that IS can destroy vascular endothelial function. Promoting vascular smooth muscle cell proliferation and accelerating vascular calcification [6] to play a role in AS, but the specific mechanism is not clear. In recent years, there have been theories that AS is a chronic inflammation and autoimmune disease [8], and dendritic cells (dendritic cells,DCs) play an important role in the progression of AS inflammation [9]. We try to explore whether IS can activate DCs to play a related immune role and promote the progression of AS inflammation, thus providing more theoretical basis for IS to participate in the pathological process of AS. The first part of the in vitro induction and identification of human monocyte derived dendritic cells objective: to isolate and culture human monocyte derived dendritic cells (monocyte-derived dendritic cells,mo-DCs) by modified double density gradient centrifugation and cytokine induction. Further study of the immune function of DCs laid the experimental foundation. Methods: human monocytes were isolated by modified double density gradient centrifugation. The cells were induced by rhGM-CSF (1000IU/mL) and rhIL-4 (500IU/mL) in vitro. The state of the cells was observed by inverted phase contrast microscope every day. After 5 days of culture, the morphology of the cells was observed by mo-DCs, electron microscope. Phenotypes were identified by flow cytometry. Results mo-DCs could be isolated and cultured from 30 mL of peripheral blood of 1: 1 mil, and mo-DCs was about (3-6) 脳 106. The average cell survival rate of Trypan blue staining was 90 / 2. The morphology of dendritic cells was observed by inverted phase contrast microscope and electron microscope. Flow cytometry was used to detect the expression of CD11c in over 90% of the cells, and a high purity of mo-DCs. was obtained. Conclusion: the method used in this study is simple, rapid, and can be used to separate and induce mo-DCs, with high purity. The second part is the effect of indole sulfate on the immune function of human monocyte derived dendritic cells. Objective: to investigate the effects of IS on the differentiation, maturation and immune function of mo-DCs, and to provide a basis for further exploring the mechanism of IS in the immune inflammation of AS. Methods: mo-DCs, was obtained as immature dendritic cells (immature dedritic cells,imDCs). After 24 hours of RPMI1640 culture without serum, imDCs was randomly divided into 5 groups: 1 渭 g/mL (1 渭 g/mL), IS.1 (30 渭 mol/L), IS.2 (300 渭 mol/L) and 600 渭 mol/L. The changes of phenotype and phagocytic function in each group were detected by flow cytometry 24 hours after stimulation. The level of cytokine IL-12p70 was detected by Elisa and the morphological changes of cells were observed by electron microscope. Results: after imDCs was treated with different concentrations of IS, the expression of CD80,CD83,CD86,MHCII on cell surface was significantly up-regulated, the phagocytic function of cells was decreased and the secretion of cytokine IL-12p70 was promoted (P0.05). There are different protuberances on the surface of the cell body. The optimal concentration of IS was 300 渭 mol/L, and the stimulation of mo-DCs never matured to mature state. Conclusion mo-DCs phenotypic and functional maturation can be induced by 1: is, which may be one of the mechanisms by which IS participates in the process of AS immune inflammation.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
本文编号：2244525
[Abstract]:Cardiovascular disease (cardiovascular disease,CVD) is the leading cause of death in patients with chronic renal disease (chronic kidney disease,CKD) [1]. The progression of atherosclerosis (atherosclerosis,AS) in patients with chronic renal disease (chronic kidney disease,CKD) is extremely rapid. Although routine dialysis therapy can correct the internal environment of uremic patients, more than 50% of patients end up dying from CVD [4]. Indole sulfate (Indoxyl sulfate,IS) is a proteophile urinary toxin, which can not be effectively cleared by the current dialysis therapy [5] .is considered to be involved in the pathogenesis of AS in dialysis patients. Previous studies have found that IS can destroy vascular endothelial function. Promoting vascular smooth muscle cell proliferation and accelerating vascular calcification [6] to play a role in AS, but the specific mechanism is not clear. In recent years, there have been theories that AS is a chronic inflammation and autoimmune disease [8], and dendritic cells (dendritic cells,DCs) play an important role in the progression of AS inflammation [9]. We try to explore whether IS can activate DCs to play a related immune role and promote the progression of AS inflammation, thus providing more theoretical basis for IS to participate in the pathological process of AS. The first part of the in vitro induction and identification of human monocyte derived dendritic cells objective: to isolate and culture human monocyte derived dendritic cells (monocyte-derived dendritic cells,mo-DCs) by modified double density gradient centrifugation and cytokine induction. Further study of the immune function of DCs laid the experimental foundation. Methods: human monocytes were isolated by modified double density gradient centrifugation. The cells were induced by rhGM-CSF (1000IU/mL) and rhIL-4 (500IU/mL) in vitro. The state of the cells was observed by inverted phase contrast microscope every day. After 5 days of culture, the morphology of the cells was observed by mo-DCs, electron microscope. Phenotypes were identified by flow cytometry. Results mo-DCs could be isolated and cultured from 30 mL of peripheral blood of 1: 1 mil, and mo-DCs was about (3-6) 脳 106. The average cell survival rate of Trypan blue staining was 90 / 2. The morphology of dendritic cells was observed by inverted phase contrast microscope and electron microscope. Flow cytometry was used to detect the expression of CD11c in over 90% of the cells, and a high purity of mo-DCs. was obtained. Conclusion: the method used in this study is simple, rapid, and can be used to separate and induce mo-DCs, with high purity. The second part is the effect of indole sulfate on the immune function of human monocyte derived dendritic cells. Objective: to investigate the effects of IS on the differentiation, maturation and immune function of mo-DCs, and to provide a basis for further exploring the mechanism of IS in the immune inflammation of AS. Methods: mo-DCs, was obtained as immature dendritic cells (immature dedritic cells,imDCs). After 24 hours of RPMI1640 culture without serum, imDCs was randomly divided into 5 groups: 1 渭 g/mL (1 渭 g/mL), IS.1 (30 渭 mol/L), IS.2 (300 渭 mol/L) and 600 渭 mol/L. The changes of phenotype and phagocytic function in each group were detected by flow cytometry 24 hours after stimulation. The level of cytokine IL-12p70 was detected by Elisa and the morphological changes of cells were observed by electron microscope. Results: after imDCs was treated with different concentrations of IS, the expression of CD80,CD83,CD86,MHCII on cell surface was significantly up-regulated, the phagocytic function of cells was decreased and the secretion of cytokine IL-12p70 was promoted (P0.05). There are different protuberances on the surface of the cell body. The optimal concentration of IS was 300 渭 mol/L, and the stimulation of mo-DCs never matured to mature state. Conclusion mo-DCs phenotypic and functional maturation can be induced by 1: is, which may be one of the mechanisms by which IS participates in the process of AS immune inflammation.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
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