【摘要】：心血管疾病(cardiovascular disease,CVD)是慢性腎臟疾病(chronic kidney disease,CKD)患者最主要的死亡原因[1]。CKD患者動(dòng)脈粥樣硬化(atherosclerosis,AS)的進(jìn)展極為迅速,極易發(fā)生嚴(yán)重的冠狀動(dòng)脈疾病[2,3];雖然接受常規(guī)透析治療可以糾正尿毒癥患者的內(nèi)環(huán)境,但仍有超過(guò)50%的患者最終因CVD而死亡[4]。硫酸吲哚酚(Indoxyl sulfate,IS)是一種親蛋白質(zhì)化合物的尿毒素,不能被目前的透析治療方式有效清除[5]。IS被認(rèn)為參與到透析患者AS的發(fā)病機(jī)制之中,既往研究發(fā)現(xiàn)IS可通過(guò)破壞血管內(nèi)皮功能、促使血管平滑肌細(xì)胞增生、加速血管鈣化等[6,7]來(lái)發(fā)揮致AS作用,但具體機(jī)制尚不明確。近年來(lái),有學(xué)說(shuō)認(rèn)為AS是一種慢性炎癥和自身免疫性疾病[8],而樹(shù)突狀細(xì)胞(dendritic cells,DCs)在AS的炎癥進(jìn)展上發(fā)揮了重要的作用[9]。我們?cè)噲D探究IS是否可啟動(dòng)DCs發(fā)揮相關(guān)免疫作用,促進(jìn)AS的炎癥進(jìn)展,從而為IS參與AS的病理過(guò)程提供更多的理論依據(jù)。第一部分人單核細(xì)胞源樹(shù)突狀細(xì)胞的體外誘導(dǎo)與鑒定目的:應(yīng)用改良雙密度梯度離心法與細(xì)胞因子誘導(dǎo)結(jié)合的方法分離和培養(yǎng)人單核細(xì)胞源樹(shù)突狀細(xì)胞(monocyte-derived dendritic cells,mo-DCs),為進(jìn)一步研究DCs的免疫作用奠定實(shí)驗(yàn)基礎(chǔ)。方法:利用改良雙密度梯度離心法分離人純化的單核細(xì)胞,用rhGM-CSF(1000IU/mL)和rhIL-4(500IU/mL)體外誘導(dǎo),倒置相差顯微鏡每天觀察細(xì)胞狀態(tài),培養(yǎng)5天后獲得mo-DCs,電子顯微鏡觀察細(xì)胞形態(tài),流式細(xì)胞儀進(jìn)行表型鑒定。結(jié)果:1、30mL外周血可分離并培養(yǎng)出mo-DCs約(3-6)×106個(gè),臺(tái)盼藍(lán)染色后細(xì)胞存活率平均90%;2、倒置相差顯微鏡及電子顯微鏡觀察細(xì)胞呈典型樹(shù)突狀細(xì)胞形態(tài);3、流式細(xì)胞儀檢測(cè)表達(dá)CD11c陽(yáng)性的細(xì)胞可達(dá)90%以上,獲得純度較高mo-DCs。結(jié)論:本實(shí)驗(yàn)選用的方法簡(jiǎn)單、快速,可分離誘導(dǎo)出純度較高的mo-DCs,可用于后續(xù)實(shí)驗(yàn)研究。第二部分硫酸吲哚酚對(duì)人單核細(xì)胞源樹(shù)突狀細(xì)胞免疫功能的影響目的:探討IS對(duì)mo-DCs的分化、成熟及免疫功能的影響,為進(jìn)一步探索IS在AS免疫炎癥反應(yīng)中的機(jī)制提供依據(jù)。方法:上述方法獲得mo-DCs,此時(shí)為未成熟樹(shù)突狀細(xì)胞(immature dedritic cells,imDCs),用不含血清的RPMI1640繼續(xù)培養(yǎng)24h后,第6天將imDCs隨機(jī)分為 5 組:PBS 組、LPS 組(1μg/mL)、IS.1 組(30μmol/L)、IS.2 組(300μmol/L)、IS.3組(600μmol/L)。刺激24h后流式細(xì)胞儀檢測(cè)各組細(xì)胞表型和吞噬功能變化,ELISA法檢測(cè)各組細(xì)胞分泌細(xì)胞因子IL-12p70的水平變化,電子顯微鏡觀察細(xì)胞形態(tài)學(xué)變化。結(jié)果:不同濃度IS作用于imDCs之后,顯著上調(diào)細(xì)胞表面標(biāo)志物CD80、CD83、CD86、MHCII的表達(dá);降低細(xì)胞吞噬功能并促進(jìn)細(xì)胞因子IL-12p70的分泌(P0.05);形態(tài)學(xué)上看,LPS組和IS.2組細(xì)胞呈不規(guī)則形,胞體表面有形態(tài)不一的突起。認(rèn)為IS為300μmol/L時(shí)為最適宜的刺激濃度,刺激mo-DCs從未成熟到成熟狀態(tài)。結(jié)論:IS可誘導(dǎo)mo-DCs表型及功能成熟,這可能是IS參與AS免疫炎癥過(guò)程的機(jī)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R392

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