【摘要】：骨髓增生異常綜合癥(Myeloidysplastic Syndrome,MDS)是一組起源于造血髓系定向干細(xì)胞或多能干細(xì)胞的異質(zhì)性克隆性疾患。在我國(guó),MDS發(fā)病率有很明顯上升趨勢(shì),發(fā)病率已達(dá)到十萬分之三,且患者發(fā)病年齡比西方國(guó)家年輕十歲左右。治療MDS的主要手段是輸血或造血干細(xì)胞移植。越來越多的證據(jù)發(fā)現(xiàn)接受造血干細(xì)胞移植后,病人的肝臟出現(xiàn)典型的鐵過載癥狀進(jìn)而引起病人肝損傷。此外,鐵過載還抑制了病患的造血功能及治療后恢復(fù)。盡管當(dāng)前臨床上利用祛鐵胺(Desferrioxamine,DFO)、祛鐵酮(Deferiprone,DFP)和地拉羅司(Deferasirox,DFX或ICL670)等祛鐵藥物能夠降低肝實(shí)質(zhì)細(xì)胞中的鐵水平、保護(hù)臟器器官并改善MDS患者的預(yù)后,但是對(duì)于病患祛鐵藥物治療所使用的合適劑量仍充滿爭(zhēng)議,同時(shí)祛鐵治療過程漫長(zhǎng)且昂貴。因此,深入研究鐵過載損傷的分子機(jī)制對(duì)病患鐵過載的治療具有重要意義。本課題組以人肝細(xì)胞HH4為研究模型,詳細(xì)研究了體外鐵過載培養(yǎng)條件下細(xì)胞凋亡發(fā)生、基因Hepcidin表達(dá)調(diào)控的信號(hào)通路以及蛋白質(zhì)組和磷酸化蛋白質(zhì)組差異表達(dá),主要內(nèi)容如下:(1)鐵過載誘導(dǎo)細(xì)胞凋亡發(fā)生的機(jī)制研究。枸櫞酸鐵銨(FAC)處理HH4細(xì)胞0-72hr后,對(duì)胞內(nèi)鐵離子含量、細(xì)胞活力、活性氧(ROS)水平、氧化應(yīng)激蛋白表達(dá)變化及細(xì)胞凋亡進(jìn)行考察。結(jié)果表明HH4細(xì)胞鐵過載后,胞內(nèi)鐵離子濃度呈時(shí)間依賴性增加,而細(xì)胞活力呈現(xiàn)濃度和時(shí)間依賴性降低。同時(shí),鐵過載處理顯著增加胞內(nèi)ROS水平,而添加抗氧化劑谷胱甘肽(GSH)和N-乙酰半胱氨酸(NAC)可以明顯降低FAC誘導(dǎo)的胞內(nèi)ROS生成。此外,鐵過載導(dǎo)致蛋白IκB-α、p38 MAPK和NF-κB p65磷酸化水平增加,以及促進(jìn)轉(zhuǎn)錄因子NF-κB p65入核。實(shí)驗(yàn)同時(shí)發(fā)現(xiàn)鐵過載處理導(dǎo)致凋亡相關(guān)基因表達(dá)水平增加,且流式檢測(cè)確認(rèn)凋亡比率顯著增高。而對(duì)凋亡相關(guān)蛋白Fas和Bid進(jìn)行siRNA瞬間干擾后,細(xì)胞活力得到恢復(fù)。此外,添加GSH后,細(xì)胞凋亡率明顯降低且氧化應(yīng)激活化信號(hào)途徑和凋亡信號(hào)途徑相關(guān)蛋白如p-IκB-α、p-p38 MAPK、p-NF-κB、Caspase-8、Cytc和Caspase-3表達(dá)水平呈現(xiàn)顯著下調(diào)。本部分實(shí)驗(yàn)結(jié)果表明鐵過載誘導(dǎo)了ROS介導(dǎo)的HH4細(xì)胞死亡受體途徑及線粒體途徑凋亡發(fā)生。(2)NF-κB參與Hepcidin表達(dá)調(diào)控研究。本部分實(shí)驗(yàn)以不同濃度FAC(0.1、1、5和10 mmol/L)處理HH4細(xì)胞48 hr,半定量PCR法檢測(cè)胞內(nèi)Hepcidin表達(dá)水平。同時(shí)利用染色質(zhì)免疫共沉淀(ChIP)、電泳遷移率實(shí)驗(yàn)(EMSA)和雙熒光素酶報(bào)告基因系統(tǒng)實(shí)驗(yàn),并結(jié)合胞內(nèi)NF-κB活性抑制實(shí)驗(yàn),確定NF-κB對(duì)人Hepcidin轉(zhuǎn)錄活性的影響。結(jié)果表明5 mmol/L和10 mmol/L FAC處理HH4細(xì)胞后,Hepcidin表達(dá)水平顯著增強(qiáng)。ChIP和EMSA實(shí)驗(yàn)證實(shí)NF-κB能夠與Hepcidin基因啟動(dòng)子結(jié)合。重組熒光素酶報(bào)告質(zhì)粒TOPFlash-pHepcidin相對(duì)熒光素酶活性明顯高于對(duì)照組;與重組質(zhì)粒TOPFlash-pHepcidin相比,突變重組熒光素酶報(bào)告質(zhì)粒TOPFlash-pHepcidin-mut相對(duì)熒光素酶活性顯著降低。同時(shí)NF-κB抑制劑BAY 11-7082顯著降低了Hepcidin基因的表達(dá)。本部分實(shí)驗(yàn)結(jié)果表明轉(zhuǎn)錄因子NF-κB參與了FAC過載誘導(dǎo)Hepcidin表達(dá)上調(diào)。(3)肝細(xì)胞在鐵過載模型中的蛋白質(zhì)組學(xué)研究。借助蛋白質(zhì)組學(xué)技術(shù),我們發(fā)現(xiàn)與對(duì)照組相比,鐵過載處理導(dǎo)致HH4細(xì)胞58種蛋白質(zhì)表達(dá)水平顯著上調(diào)以及35種蛋白質(zhì)表達(dá)水平顯著下調(diào)。隨后的GO分析表明鐵過載誘導(dǎo)的93種差異表達(dá)蛋白與內(nèi)吞作用、二價(jià)、三價(jià)金屬陽離子穩(wěn)態(tài)平衡、創(chuàng)傷反應(yīng)、炎癥反應(yīng)、細(xì)胞因子分泌的正調(diào)控反應(yīng)、生長(zhǎng)調(diào)控、抗凋亡以及凋亡的線粒體改變等生物學(xué)過程緊密相關(guān)。此外,蛋白質(zhì)組學(xué)實(shí)驗(yàn)結(jié)果顯示FAC過載處理誘發(fā)TLR2蛋白表達(dá)水平上調(diào)7倍和IL6ST蛋白表達(dá)水平上調(diào)2.9倍,而隨后的蛋白質(zhì)免疫印跡實(shí)驗(yàn)和TLR2基因干擾實(shí)驗(yàn)證實(shí)鐵過載處理激活了HH4細(xì)胞TLR2-MyD88-p-NF-κB-IL-6炎癥路徑。而另一方面,磷酸化蛋白質(zhì)組學(xué)實(shí)驗(yàn)表明HH4細(xì)胞鐵過載處理導(dǎo)致335種磷酸化蛋白出現(xiàn)差異變化,其中高達(dá)11%的磷酸化蛋白與細(xì)胞周期進(jìn)程有關(guān)。磷酸化蛋白質(zhì)組學(xué)實(shí)驗(yàn)結(jié)果同時(shí)顯示HH4細(xì)胞鐵過載處理后G2/M期轉(zhuǎn)換關(guān)鍵蛋白CDK1在Thr14和Tyr15位點(diǎn)磷酸化修飾水平增高約10.9倍。進(jìn)一步的流式檢測(cè)證實(shí)鐵過載能夠引發(fā)HH4和小鼠肝細(xì)胞NMH細(xì)胞G2/M期阻滯,同時(shí)RT-PCR和蛋白免疫印跡檢測(cè)則表明FAC過載可能經(jīng)由p53-p21-CDK1、p53-14-3-3 sigma-CDK1或14-3-3 gamma路徑誘導(dǎo)了HH4細(xì)胞G2/M期阻滯發(fā)生。上述實(shí)驗(yàn)結(jié)果表明鐵過載處理導(dǎo)致HH4細(xì)胞TLR2介導(dǎo)的炎癥反應(yīng)發(fā)生及G2/M期阻滯。
[Abstract]:Myelodysplastic Syndromes (MDS) is a group of heterogeneous clonal disorders derived from hematopoietic stem cells or pluripotent stem cells. In China, the incidence of MDS increased significantly, and the incidence of MDS was up to ten thousand three ter, and the incidence of MDS was about ten years younger than that of Western countries. The main means of treating MDS are transfusion or hemopoietic stem cell transplantation. More and more evidence has found that after transplantation of hematopoietic stem cells, a typical iron overload condition in the liver of a patient leads to a patient's liver injury. In addition, iron overload inhibits the hematopoietic function and post-treatment recovery of the patient. In spite of the current clinical use of desferrioxamine (DFO), deferione (DFP) and deersirox (DFX or ICL670), iron-removing agents can reduce iron levels in liver parenchyma cells, protect organ organs and improve the prognosis of MDS patients, but the appropriate dosage used for the treatment of patients with iron therapy is still controversial while removing iron treatment is a long and expensive process. Therefore, in-depth study of the molecular mechanism of iron overload injury is of great significance to the treatment of iron overload in patients. In this study, human hepatocyte HH4 was used as the research model, the cell apoptosis occurred under the condition of iron overload culture in vitro, the signal pathway of the expression and regulation of gene Hepcidin, and the differential expression of protein group and phosphorylated protein group were studied in detail. The main contents were as follows: (1) The mechanism of iron overload induced apoptosis. The intracellular iron ion content, cell viability, reactive oxygen species (ROS) levels, oxidative stress protein expression and apoptosis were investigated after treatment of HH4 cells for 0-72hr with iron disulfide (FAC). The results showed that the concentration of iron ions increased in the cytoplasm of HH4 cells, while the concentration and time dependence of cell viability decreased. At the same time, iron overload treatment significantly increased intracellular ROS level, while addition of antioxidant glutathione (GSH) and N-cysteine (NAC) could significantly reduce the intracellular ROS generation induced by FAC. In addition, iron overload results in increased phosphorylation of protein I, B-, p38 MAPK, and NF-NADB p65, as well as promoting transcription factor NF-NADB p65 into the nucleus. At the same time, it was found that iron overload treatment resulted in an increase in the expression level of apoptosis-related genes, and the rate of apoptosis was significantly increased. The apoptosis-related proteins Fas and Bid were transiently disrupted by siRNA, and the viability of the cells was restored. In addition, after addition of GSH, the cell apoptosis rate was decreased significantly and oxidative stress activated signaling pathway and apoptosis signal pathway related proteins, such as p-I, B-, p-p38 MAPK, p-NF-Sepharose B, Caspase-8, Cytc and Caspase-3, exhibited a significant downregulation. The results of this part suggest that iron overload induces ROS-mediated HH4 cell death receptor pathway and mitochondrial pathway apoptosis. (2) NF-Sepharose B was involved in the study of the expression and regulation of HepcGVHD. HH4 cells were treated with different concentrations of FAC (0. 1, 1, 5 and 10 mmol/ L) for 48 hr, and the expression level of Hepcidin was detected by semi-quantitative PCR. The effect of NF-Sepharose B on the transcription activity of human Hepci2 was determined by means of chromatin immune co-precipitation (ChIP), electrophoretic mobility assay (EMSA) and double luciferase reporter gene system. The results showed that after treatment of HH4 cells with 5 mmol/ L and 10 mmol/ L FAC, the expression level of HepcGVHD was significantly enhanced. The ChIP and EMSA experiments confirmed that NF-Sepharose B was able to bind to the Hepcidin gene promoter. The relative luciferase activity of the recombinant luciferase reporter plasmid was significantly higher than that of the control group. At the same time, NF-Sepharose B inhibitor BAY 11-7082 significantly reduced the expression of Hepcidin gene. The results of this part show that the transcription factor NF-5B is involved in the upregulation of the expression of FAC overload-induced HepcGVHD. (3) proteomic study of hepatocytes in iron overload models. With the aid of proteomics techniques, we found that iron overload treatment resulted in a significant increase in the expression levels of 58 proteins in HH4 cells and a significant down-regulation of 35 protein expression levels in comparison to the control group. Subsequent GO analysis indicated that 93 differentially expressed proteins induced by iron overload were regulated by positive regulation and growth regulation, such as endocytic action, bivalent, trivalent metal cation homeostasis, wound response, inflammatory response, cytokine secretion, Anti-apoptosis and apoptosis are closely related to biological processes such as mitochondrial change. In addition, the protein group learning experiment showed that the expression level of TLR2 protein increased by 2.7 times and IL6ST protein expression level increased by 2.9 times than that of the FAC overload treatment, while the subsequent Western blot experiment and the TLR2 gene interference experiment confirmed that the iron overload treatment activated the HH4 cell TLR2-MyD88-p-NF-Sepharose B-IL-6 inflammatory pathway. On the other hand, the phosphorylation protein group learning experiment indicated that the HH4 cell iron overload treatment resulted in the variation of 335 phosphorylation proteins, among which 11% of the phosphorylated proteins were related to the cell cycle progression. At the same time, the phosphorylation protein CDK1 in the G2/ M phase after the overload treatment of HH4 cells was increased by about 10. 9 times higher than that in Thr14 and Tyr15 sites. Further flow detection confirmed that iron overload could induce the G2/ M phase arrest of HH4 and mouse liver cells, while RT-PCR and Western blot analysis showed that FAC overload could induce the G2/ M block of HH4 cells via p53-p21-CDK1, p53-14-3-3sigma-CDK1 or 14-3-3 pathway. The results of the above experiment indicated that the iron overload treatment resulted in the inflammation response mediated by TLR2 and G2/ M arrest in HH4 cells.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R551.3

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