【作者】 韩潇；

【导师】 周道斌； 赵永强；

【作者基本信息】 北京协和医学院 ， 血液学， 2010， 博士

【摘要】 [研究背景]目前认为,慢性病贫血(ACD)的发病机制是由原发病启动的免疫反应所介导：异常升高的炎性细胞因子主要是IL-6通过上调肝脏hepcidin,导致铁利用障碍,引发贫血；此外,可直接抑制红系前体细胞增殖分化,缩短红细胞寿命,钝化对EPO的反应,干扰铁代谢相关蛋白的转录等导致慢性病贫血。单核细胞是慢性病贫血中产生炎性细胞因子的主要来源,同时是铁储存细胞,接受hepcidin的调控,在ACD的发病中发挥关键作用,近年来研究显示,单核细胞与肝脏相似,受到炎症刺激后hepcidin mRNA表达明显增加。单核细胞的hepcidin也对铁代谢有调节作用,同时在ACD患者中表达升高,与贫血和升高的IL-6明显相关,显示其可能在患者体内参与ACD的发病。促红细胞生成素(EPO)是目前治疗ACD的主要手段,通过与EPO受体结合刺激造血、抑制肝脏hepcidin以及降低炎症因子等发挥治疗作用。但是EPO降低hepcidin的具体机制尚不清楚,其降低肝脏hepcidin可能与下调C/EBPa,抑制STAT3的磷酸化有关；但是否存在其他的信号途径、对单核细胞hepcidin是否也有类似的降低作用和分子机制并不清楚,此外,EPO降低前炎症因子的机制也并不清楚。多发性骨髓瘤和非霍奇金淋巴瘤是肿瘤相关ACD的代表性疾病,骨髓瘤患者血清和尿hepcidin水平升高,非霍奇金淋巴瘤则缺乏相关研究。对于这两种疾病,单核细胞hepcidin是否升高,是否在ACD中发挥作用,尚不清楚。[研究目的]1观察EPO是否能降低单核细胞hepcidin并探讨其分子学机制。2探讨EPO降低IL-6的分子机制。3探讨单核细胞hepcidin在MM和NHL患者相关ACD中可能发挥的作用。[研究方法]1 IL-6诱导THP-1单核细胞,Real time PCR法检测hepcidin;加入EPO,观察其对hepcidin的影响。Western blot免疫印迹法检测hepcidin蛋白及信号分子：C/EBPa、Smadl/5/8、P-Smad1/5/8及P-STAT3,加入EPOR抗体,观察对EPO的拮抗作用。2 LPS诱导THP-1单核细胞和人原代单核细胞,Real time PCR法检测IL-6及TNF-a表达。Western blot法检测信号分子PARP-1,加入EPOR抗体或PARP-1抑制剂3AB,观察对EPO的拮抗作用及信号蛋白的影响。3收集多发性骨髓瘤、非霍奇金淋巴瘤患者的临床资料及血清,ELISA法检测患者血清IL-6及TNF-a的浓度。4磁珠分选法分离外周血CD14+单核细胞,Real time PCR实时定量法检测单核细胞hepcidin、IL-6、TNF-a及C/EBPa的表达。[结果]1 EPO可降低IL-6诱导的THP-1单核细胞hepcidin mRNA表达及蛋白量：同时降低C/EBPa、P-Smad1/58、P-STAT3, EPOR抗体可拮抗EPO对hepcidin及信号蛋白的影响。2 EPO可降低LPS诱导的THP-1细胞和人原代单核细胞IL-6和TNF-a的表达,同时降低PARP-1的蛋白量,EPOR抗体及3AB均对可拮抗EPO对IL-6及PARP-1的下降作用。3 MM患者单核细胞hepcidin的表达高于正常,初治者与Hb、TSAT%负相关,与SF、体内IL-6水平、CRP及β2微球蛋白正相关,与体内TNF-a水平、LDH无关联。单核细胞CEBPa表达与hepcidin水平正相关。4 NHL患者单核细胞hepcidin表达高于正常,初治组与Hb负相关,与体内IL-6水平、CRP、LDH正相关,与TSAT%、SF及体内TNF-a水平无关联。单核细胞CEBPa表达与hepcidin水平无关。[结论]1 EPO可从基因水平和蛋白水平明显降低IL-6诱导的单核细胞hepcidin;其可能的分子机制：通过与EPOR结合,降低信号分子C/EBPa,减少Smad1/5/8及STAT3磷酸化。2 EPO可明显降低LPS诱导的单核细胞IL-6和TNF-a mRNA的表达,EPO可能是通过与EPOR结合,抑制信号分子PARP-1,实现对IL-6的下调作用。3多发性骨髓瘤和非霍奇金淋巴瘤患者单核细胞hepcidin mRNA表达升高,在初治患者中与血红蛋白存在关联,可能是导致其ACD的原因之一；单核细胞hepcidin mRNA表达升高可能是由患者体内升高的IL-6所诱导。CRP或LDH可能反应初治患者体内hepcidin的水平。更多还原

【Abstract】 [BACKGROUND]The current view is that the pathogenesis of anemia of chronic disease (ACD) is mediated by the immune response initiated by primary disease. Abnormal elevated inflammatory cytokines, mostly IL-6, lead to the iron metabolism disorder and anemia through elevating hepcidin level in liver, and directly inhibit the proliferation and differentiation of erythroid precursor cells, shorten life span of red blood cell, blunt response to EPO, interfere with iron metabolism related proteins in transcription. Monocytes are major source of inflammatory cytokines generated in chronic anemia; they are also iron storage cells, regulated by hepcidin, which play a key role in the pathogenesis of ACD. Recent studies have shown that monocyte is similar to liver in increasing the expression of hepcidin significantly by inflammation stimulation. Hepcidin from monocyte also regulate iron metabolism. The monocyte hepcidin is increased in patients with ACD and related significantly with anemia and elevated IL-6, indicated that it may play a role in the pathogenesis of ACD. Erythropoietin (EPO) is the primary means of treating ACD through combining with the receptors and stimulating hematopoiesis, inhibiting hepatic hepcidin and decreasing inflammatory factors. EPO can reduce hepcidin expression but the specific mechanism is unclear now. It may be associated with decreased C/EBPa, and depressed phosphorylation of STAT3; but whether there are other signaling pathways, whether there is similar role on monocyte is not clear, in addition, the mechanism of EPO decreasing inflammatory cytokines is not clear, either. Multiple myeloma and non-Hodgkin’s lymphoma can lead to tumor-associated ACD. Patients of multiple myeloma have elevated levels of serum and urinary hepcidin, but there is not such research on patients of non-Hodgkin’s lymphoma. In both diseases, whether monocyte hepcidin is increased, and whether it plays a role in ACD is not clear.[OBJECTIVE]1 The effect of EPO on monocytes hepcidin and its molecular mechanism.2 The molecular mechanism of IL-6 reduced by EPO. 3 The role of monocytes hepcidin in MM and NHL patients.[METHODS]1 THP-1 monocytes are stimulated by IL-6, hepcidin is detected by Real time PCR.Observing the impact of EPO on monocytes hepcidin.Hepcidin and signaling molecule including C/EBPa, Smadl/5/8, P-Smadl/5/8 and P-STAT3 are detected by Western blot. To observe the antagonistic effect on EPO by adding EPOR antibody.2 THP-1 monocytes and primary human monocytes stimulated by LPS, the expression of IL-6 and TNF-a mRNA detected by Real time PCR. PARP-1 detected by Western blot. Adding EPOR antibody and/or PARP-1 inhibitor to observe the antagonistic effect on EPO and the impact on the signaling proteins.3 Collecting the clinical information and serum of multiple myeloma and non-Hodgkin’s lymphoma.Serum concentration of IL-6 and TNF-a is detected by ELISA.4 Peripheral blood monocytes is isolated by Magnetic beads.Hepcidin, IL-6, TNF-a and C/EBPa mRNA of monocytes is detected by Real time PCR quantitative assay.[RESULTS]1 EPO can suppress the monocyte hepcidin mRNA expression and protein induced by IL-6 in THP-1 cells. C/EBPa, P-Smadl/5/8 and P-STAT3 reduced at the same time. EPOR antibody can antagonize the effect of EPO on hepcidin and signaling proteins.2 EPO can decrease the levels of IL-6 and TNF-a stimulated by LPS both in THP-1 cells and in primary human monocytes. PARP-1 protein is decreased at the same time. The decline of IL-6 by EPO can be antagonized by both EPOR antibody and 3AB.3 In MM patients, the expression of monocyte hepcidin mRNA is higher than normal and negatively correlated with Hb,positively correlated with SF, IL-6 levels, CRP and 132 microglobulin in newly diagnosed patients,but unrelated with LDH and the TNF-a levels. C/EBPa expression in monocytes is positively correlated with monocyte hepcidin and IL-6 levels,but unrelated with the TNF-a levels.4 In NHL patients, the expression of monocyte hepcidin mRNA is higher than normal.Monocyte hepcidin in patients with newly diagnosed negatively correlated with Hb, IL-6 levels, CRP and LDH, but not associated with TSAT%, SF and TNF-a levels. C/EBPa expression in monocyte is unrelated with monocyte hepcidin levels. [CONCLUSIONS]1 Monocyte hepcidin mRNA and protein levels can be reduced by EPO when stimulated by IL-6 in monoytes. EPO inhibits the monocyte hepcidin, at least in part,via combination with EPOR and suppression of C/EBPa, P-Smadl/5/8 and P-STAT3 signaling in vitrol.2 EPO can block the expression of IL-6 and TNF-a in monocytes stimulated by LPS, EPO may inhibit IL-6 expression via combination of EPOR and suppression of PARP-1.3 The expression of monocyte hepcidin is increased in patients of multiple myeloma and non-Hodgkin’s lymphoma, and has an association with ACD, the increased hepcidin levels play an etiologic role in ACD.CRP or LDH may be able to monitor the level of hepcidin.更多还原