【作者】 薛冬；

【导师】 何小舟；

【作者基本信息】 苏州大学 ， 泌尿外科， 2014， 博士

【摘要】 近年来，新型免疫抑制剂的不断出现以及临床免疫治疗方案的进一步完善，肾移植术后急性排斥反应的发生率明显降低，但移植肾长期存活率未明显提高。肾移植过程中的肾缺血再灌注损伤是发生最早也是必不可少的环节,缺血再灌注损伤会刺激炎症反应的发生，从而导致慢性移植肾肾病。移植肾后期功能丧失的最主要原因是慢性移植肾肾病(CAN)。慢性移植肾肾病主要表现为肾小管上皮细胞萎缩，肾间质纤维化，其病理变化过程与炎症刺激密不可分。随着病情进展，受者将发展为移植物功能丧失即慢性肾衰竭(chronic renal failure，CRF)。研究慢性移植肾肾病的发生发展，意义重大，，我们将分别研究血清Hepcidin与缺血再灌注以及慢性移植物肾病相关性。第一部分肾移植后血清Hepcidin的表达及临床意义目的：观察血清Hepcidin在肾移植术前、术后患者缺血再灌注的临床意义及损伤中的作用。方法：收集同种异体肾移植患者术前和术后不同时间点外周血，酶联免疫法（ELISA）检测血浆中IL-6、Hepcidin、sTfR（转铁蛋白受体）、C（r血肌酐）的水平，分析Hepcidin与炎症反应和再灌注损伤的相关性。结果：同种异体肾移植术后的血清Hepcidin较术前增高，P<0.05；并且与IL-6、sTfR、Hb的表达有相关性，有统计学差异；随着肾移植术后肾功能的恢复，患者血清Hepcidin降低，血肌酐（Cr）与血清Hepcidin的比值也降低，P<0.05。结论：缺血再灌注损伤可影响Hepcidin的表达，其变化与体内炎症状态及肾功能损伤程度呈正相关。第二部分Hepcidin在大鼠肾缺血再灌注损伤中的表达及意义目的观察肾缺血再灌注损伤（ischemia-reperfusion injury,IRI）对大鼠肝脏及Hepcidin、IL-6、血肌酐（Cr）等指标的变化，并分析其意义。方法将40只成年SD雄性大鼠随机分为缺血再灌注组（IR）和对照组（Control），缺血再灌注组根据再灌注的不同时相分为3组，分别为6h、24h、48h，每组10只。缺血再灌注组切除右肾，完全阻断左肾动脉45min后恢复血流，对照组在暴露双肾45min后关闭腹腔，两组分别在手术6h、24h、48h后取肝脏、肾脏组织标本，抽取静脉血液。采用半定量RT-PCR法检测每组大鼠肝脏Hepcidin的表达水平，ELISA检测每组大鼠血清Hepcidin、IL-6的表达，检测血肌酐（Cr）水平，对大鼠肾脏行病理学检查，并进行统计分析。结果肾缺血再灌注组肝脏和血清Hepcidin水平较对照组明显增高（P<0.05），24小时达到最高，此后随着再灌注时间的延长含量呈下降趋势；肾缺血再灌注组血清IL-6也较对照组增高（P<0.05），而且再灌注组Hepcidin的表达与血清IL-6呈正相关；再灌注组Cr的表达较对照组增高（P<0.05），和Hepcidin亦呈正相关，肾脏病理学检查显示24小时炎性细胞最多。结论肾缺血再灌注损伤可影响Hepcidin的表达，表达的变化与体内炎症状态和肾功能损伤程度呈正相关。因此Hepcidin可作为反映肾缺血再灌注损伤程度的标记物，且对于肾缺血再灌注损伤导致机体铁代谢的改变有临床提示意义。第三部分Hepcidin在大鼠慢性移植肾肾病中的表达及意义目的探讨Hepcidin在大鼠慢性移植肾肾病（CAN）动物模型中的表达及临床意义。方法以F344近交系大鼠为供体、Lewis近交系大鼠为受体，原位肾移植，建立20只慢性移植肾肾病大鼠模型；于建模前、术后1月、2月及4月分别收集大鼠的静脉血和尿样，检测肾功能；ELISA检测血清、尿Hepcidin，血清IL-6、EPO的表达；并于移植后2月及4月分别处死大鼠10只，观察各组大鼠移植肾组织病理学变化，进行统计学分析，探讨其临床意义及相关性。结果血清Hepcidin、IL-6表达随着移植后的时间的推移逐渐增高，尿Hepcidin排出逐渐减少，EPO表达逐渐减少，术前与术后比较有统计学意义（P<0.05）；移植术后大鼠Cr、BUN逐渐升高，并且Cr与血清Hepcidin呈正相关；移植术后血清Hepcidin的表达与IL-6正相关，与EPO负相关；肾脏病理、HE染色符合慢性移植肾肾病的病理改变。结论随着移植肾功能的减退，大鼠体内微炎症反应增加，Hepcidin在大鼠慢性移植肾肾病模型表达变化与肾功能有关，可作为反映肾功能损伤的标记物。第四部分Hepcidin在炎症刺激的肾小管上皮细胞中异常表达及分子机制研究目的采用IL-6刺激肾小管上皮（HK-2）细胞，观察HK-2细胞转分化以及细胞中Hepcidin表达变化，探讨IL-6对Hepcidin调控的分子机制。方法：分离培养人HK-2细胞，将其分成2组，（1）对照组；（2）将用IL-6加入培养液，终浓度分别为10、25、50ng/ml，培养HK-2细胞，48小时后观察细胞形态学变化，RT-PCR检测细胞中E-cadherin，aSMA的表达；ELISA法测定培养液上清Hepcidin的表达，Western blot法测定细胞内STAT3的表达，并分析IL-6对Hepcidin、STAT3表达的影响，以及与HK-2细胞转分化相关性。结果：不同浓度IL-6作用于HK-2细胞48小时后，E-cadherin表达下降，aSMA表达升高，促进HK-2细胞转分化，培养液上清中Hepcidin浓度显著增加，p-STAT3蛋白表达增高；IL-6影响Hepcidin和p-STAT3的表达。讨论：（1）IL-6能刺激HK-2细胞向肌成纤维细胞转分化。（2）IL-6可能通过激活JAK/STAT信号通路上调p-STAT3，诱导Hepcidin表达增加。（3）Hepcidin的表达变化与HK-2细胞转分化能力有关，说明Hepcidin在肾间质纤维化中起到重要作用，可以作为肾功能损伤的标记。更多还原

【Abstract】 In recent years,with the continuous improvement of new immunosuppressive agentsand clinical immunosuppressive scheme, the incidence rate of acute rejection after renaltransplantation has decreased significantly, but the long-term survival rate hasn’tsignificantly improved. The main reason for the late loss of graft function is chronicallograft nephropathy (CAN), accounting for about40%of renal function loss.Thedevelopment of chronic allograft nephropathy, progressive and irreversible, will eventuallydevelop into graft function loss(chronic renal failure, CRF)(end stage renal disease,ESRD). Being unavoidable in the process of renal transplantation, ischemia reperfusioninjury stimulates the inflammation, resulting in chronic allograft nephropathy. Chronicallograft nephropathy mainly leads to for renal tubular epithelial cell atrophy, renalinterstitial fibrosis.Its pathological changes and inflammatory stimuli are inseparable.The first chapter Serum Hepcidin expression and its clinical significance in patientsafter renal transplantationObjective: To observe the clinical significance of serum Hepcidin in kidneytransplantation patients after ischemia reperfusion, to observe the role of Hepcidin in acuteischemia reperfusion injury after renal transplantation.Methods: We collected the peripheral blood of the patients at different time points beforeand after renal transplantation. ELISA was used to detect plasma IL-6, Hepcidin, sTfR(transferrin receptor), Cr (serum creatinine) level, then we analysed the correlation ofHepcidin and acute inflammation and reperfusion injury.Results: After allograft renal transplantation, the serum Hepcidin increased with statistically significant difference; and the serum Hepcidin had correlation with theexpression of IL-6, sTfR, Hb, there is significant difference; with the recovery of the renalfunction after renal transplantation, serum Hepcidin decreased, the ratio of serumcreatinine (Cr) and serum Hepcidin decreased as well, there was statistically significantdifference.Conclusion: Ischemia reperfusion injury can affect the expression of Hepcidin. Theexpression of Hepcidin positively correlates with the inflammatory state and the degree ofrenal function injury.The second chapter Hepcidin expression in rat renal ischemia reperfusion injuryand its significanceObjective: To observe the effect of renal ischemia reperfusion injury (IRI) on rat liver andHepcidin, IL-6, serum creatinine (Cr) and other indexes, and analyze its clinicalsignificance.Methods:40adult male SD rats were randomly divided into ischemia reperfusion group(IR) and control group (Control). According to the different time of reperfusion, ischemiareperfusion group were divided into3groups, respectively6h,24h,48h,10rats in eachgroup, and control group had10rats. Ischemia reperfusion group had resection of the rightkidney, blocked the left renal artery blood flow completely, and recovered it after45min.Liver, kidney specimens, venous blood were taken in6h,24h,48h respectively afteroperation. After45min of left and right kidney exposure, the control group closed theabdominal cavity. Liver, kidney specimens, venous blood were taken in6h,24h,48hrespectively after operation. In each group, Hepcidin levels in the liver of rats weredetected by Semi quantitative RT-PCR method, enzyme linked immunosorbent assay(ELISA) was adopted to detect the expression of hepcidin, IL-6in serum of rats, and serumcreatinine (Cr) was detected in all specimens. Pathological examination was performed onthe kidney of rats, and statistical analysis was adopted to research its correlation andclinical significance. Results: Hepcidin expression level in the liver and serum of renal ischemia reperfusiongroup was significantly higher than the control group (P<0.05),24hours to reach thehighest, then with the prolongation of reperfusion time expression content decreased; IL-6of renal ischemia reperfusion group was higher than control group (P<0.05). In reperfusiongroup, Hepcidin expression positively correlated with serum IL-6; reperfusion group,serum creatinine (Cr) expression was higher than that in control group (P<0.05), and alsopositively correlated with Hepcidin, renal Pathological examination found inflammatorycells reached the highest in24hours.Conclusion: Renal ischemia reperfusion injury can affect the expression of Hepcidin,which positively correlates with the inflammatory state and the degree of renal functioninjury. Therefore, Hepcidin can be used as a marker to reflect the renal ischemiareperfusion injury degree, and has clinical research value in changes of iron metabolisminduced by the renal ischemia reperfusion injury.The third part Hepcidin expression in chronic allograft nephropathy of rats andits significanceObjective: To investigate the Hepcidin expression changes in chronic allograftnephropathy rats (CAN) animal models and clinical significance.Methods: F344inbred rats as donors, inbred Lewis rats as receptor, renal transplantation,20CAN rats were established in the model; before the establishment of model, venousblood and urine were collected respectively1month,2months and4months afteroperation to test renal function. Enzyme linked immunosorbent assay (ELISA) wasused to detect serum, urine hepcidin, serum IL-6, EPO expression, and10rats werekilled respectively in2months and4months after transplantation, then pathologicalchanges of transplanted renal tissue in rats of each group were observed. Statisticalanalysis was adopted to explore its clinical significance and correlation.Results: the serum Hepcidin, IL-6expression gradually increased with the grafting time,urinary hepcidin excretion decreased gradually, EPO expression gradually reduced. The indicators before and after surgery had statistically significant difference (P<0.05). Aftertransplantation, in rats serum creatinine (Cr), blood urea nitrogen (BUN) increasedgradually, and serum creatinine (Cr) correlation with serum Hepcidin was positive. Aftertransplantation, serum Hepcidin had positive correlation with IL-6expression, hadnegative correlation with EPO; renal pathology, HE staining were in accordance withCAN pathologic change.Conclusion:1. With impaired renal allograft function, the micro inflammation in ratsincreases. The changes of Hepcidin expression in rat models of CAN correlate with renalfunction.2. With the changes of time and the inflammatory state, Hepcidin can be usedas markers to reflect renal injury.The fourth chapter The molecular mechanisms of IL-6regulating hepcidin in renaltubular epithelial (HK-2) cellsObjective: Through the stimulation of IL-6on HK-2cells, to observe the effect on thetransdifferentiation of HK-2cells and the regulation of expression of Hepcidin in HK-2cells, and to discuss the molecular mechanism of IL-6in HK-2cells regulating Hepcidin.Methods: HK-2cells were isolated, cultured, and divided into2groups,(1)control group;(2) medium added with IL-6, with final concentration of10,25,50ng/ml concentration ofIL-6added into HK-2cells, we observed the changes of HK-2cell morphology after48H,immunohistochemistry was used to detect the expression of E-cadherin, aSMA expression.ELISA method was used to determine the expression of Hepcidin of culture supernatant;Westblot method was used to determine the intracellular STAT3expression, and weanalysed the correlation of IL-6on Hepcidin, STAT3expression and HK-2celltransdifferentiation.Results: After48hours of different concentration of IL-6acting on HK-2cells, E-cadherindecreased, aSMA increased, precipitated the transdifferentiation of HK-2cells, the culturesupernatant Hepcidin concentration significantly increased, the expression of p-STAT3increased; with the correlation between the expression of IL-6on Hepcidin and p-STAT3. Conclusion：(1) IL-6can stimulate HK-2cell fibroblasts to differentiate into myocytes.(2)IL-6in HK-2cells may be through the activation of JAK/STAT signal pathway inducedupregulation of p-STAT3, increased expression of Hepcidin.(3)The Hepcidin expressionchanges correlate with HK-2Cells Transdifferentiation ability, indicating that hepcidinplays an important role in renal interstitial fibrosis, affects chronic allograft nephropathy,and can be used as markers of renal function damage.更多还原