肾移植后Hepcidin的表达及其致慢性移植肾肾病的机制研究
发布时间：2018-09-08 15:44
【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，P0.05；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，P0.05。 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（P0.05），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（P0.05），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（P0.05），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（P0.05）；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。
[Abstract]:In recent years, with the emergence of new immunosuppressive agents and the improvement of clinical immunotherapy, the incidence of acute rejection after renal transplantation has been significantly reduced, but the long-term survival rate has not been significantly improved. Chronic renal allograft nephropathy (CAN) is the main cause of loss of function in the late stage of kidney transplantation. The recipient will develop into a loss of graft function, namely chronic renal failure (CRF).
It is of great significance to study the occurrence and development of chronic allograft nephropathy. We will study the relationship between serum Hepcidin and ischemia-reperfusion and chronic allograft nephropathy.
Part one expression and clinical significance of serum Hepcidin after renal transplantation
Objective:To observe the clinical significance of serum Hepcidin in renal transplantation patients with ischemia-reperfusion injury before and after renal transplantation.
Methods: Peripheral blood samples were collected from renal allograft recipients at different time points before and after transplantation. The levels of IL-6, Hepcidin, sTfR and C (r serum creatinine) in plasma were measured by enzyme-linked immunosorbent assay (ELISA), and the correlation between Hepcidin and inflammatory reaction and reperfusion injury was analyzed.
Results: The levels of serum Hepcidin after renal allograft transplantation were significantly higher than those before transplantation (P 0.05), and correlated with the expressions of IL-6, sTfR and Hb. With the recovery of renal function after renal transplantation, the levels of serum Hepcidin and the ratio of serum creatinine (Cr) to serum Hepcidin were also decreased (P 0.05).
CONCLUSION: Ischemia-reperfusion injury can affect the expression of Hepcidin, and its changes are positively correlated with the inflammatory state in vivo and the degree of renal function injury.
The second part is the expression and significance of Hepcidin in renal ischemia-reperfusion injury in rats.
Objective To observe the changes of liver, Hepcidin, IL-6 and serum creatinine (Cr) in rats with renal ischemia-reperfusion injury (IRI) and analyze its significance.
Methods Forty adult male SD rats were randomly divided into ischemia-reperfusion group (IR) and control group (Control). The rats in the IR group were divided into three groups according to different reperfusion phases: 6 h, 24 h and 48 h, respectively, with 10 rats in each group. Serum levels of Hepcidin and IL-6 were detected by semi-quantitative RT-PCR, serum levels of Hepcidin and IL-6 were detected by ELISA, serum creatinine (Cr) levels were detected, and renal pathological examination was performed in rats.
Results The levels of hepatic and serum hepcidin in renal ischemia-reperfusion group were significantly higher than those in control group (P The expression of Cr in the injection group was higher than that in the control group (P
Conclusion Renal ischemia-reperfusion injury can affect the expression of Hepcidin, which is positively correlated with the inflammatory state in vivo and the degree of renal function injury.
The third part is the expression and significance of Hepcidin in chronic allograft nephropathy in rats.
Objective to investigate the expression and clinical significance of Hepcidin in animal models of chronic allograft nephropathy (CAN) in rats.
Methods Twenty rats with chronic allograft nephropathy were established by orthotopic kidney transplantation with F344 inbred rats as donors and Lewis inbred rats as recipients. Blood and urine samples were collected from the veins of the rats before, 1 month, 2 months and 4 months after transplantation to detect renal function, and the expressions of serum, urinary Hepcidin, serum IL-6 and EPO were detected by ELISA. Ten rats were sacrificed in April and April respectively. The histopathological changes of transplanted kidney in each group were observed and statistically analyzed.
Results The expression of serum Hepcidin and IL-6 increased gradually with the time after transplantation, the excretion of urinary Hepcidin decreased gradually, and the expression of EPO decreased gradually, which was statistically significant before and after transplantation (P 0.05). Positive correlation was negatively correlated with EPO. Renal pathology and HE staining were consistent with pathological changes of chronic allograft nephropathy.
Conclusion With the decrease of renal allograft function, the microinflammatory reaction in rats increases. The expression of Hepcidin in chronic renal allograft nephropathy model is related to renal function and can be used as a marker of renal function damage.
The fourth part is the abnormal expression and molecular mechanism of Hepcidin in inflammatory stimulated renal tubular epithelial cells.
Objective To observe the transdifferentiation and the expression of Hepcidin in HK-2 cells stimulated by IL-6 and explore the molecular mechanism of IL-6 regulating Hepcidin.
Methods: Human HK-2 cells were isolated and cultured and divided into two groups: (1) control group; (2) The final concentration of IL-6 was 10,25,50 ng/ml, respectively. The morphological changes of HK-2 cells were observed after 48 hours. The expression of E-cadherin and aSMA was detected by RT-PCR, and the expression of Hepcidin in supernatant was detected by ELISA and Western blot. The expression of STAT3 in HK-2 cells was determined and the effect of IL-6 on the expression of Hepcidin and STAT3 was analyzed.
Results: After 48 hours of treatment with different concentrations of IL-6, the expression of E-cadherin decreased, the expression of aSMA increased, and the transdifferentiation of HK-2 cells was promoted. The concentration of Hepcidin and the expression of p-STAT3 protein increased significantly in the supernatant of culture medium. IL-6 affected the expression of Hepcidin and p-STAT3 protein.
Conclusion: (1) IL-6 can stimulate the transdifferentiation of HK-2 cells into myofibroblasts. (2) IL-6 may induce the increase of Hepcidin expression by activating JAK/STAT signaling pathway and up-regulating p-STAT3. (3) The change of Hepcidin expression is related to the transdifferentiation ability of HK-2 cells, indicating that Hepcidin plays an important role in renal interstitial fibrosis. Mark.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R699.2
本文编号：2230962
[Abstract]:In recent years, with the emergence of new immunosuppressive agents and the improvement of clinical immunotherapy, the incidence of acute rejection after renal transplantation has been significantly reduced, but the long-term survival rate has not been significantly improved. Chronic renal allograft nephropathy (CAN) is the main cause of loss of function in the late stage of kidney transplantation. The recipient will develop into a loss of graft function, namely chronic renal failure (CRF).
It is of great significance to study the occurrence and development of chronic allograft nephropathy. We will study the relationship between serum Hepcidin and ischemia-reperfusion and chronic allograft nephropathy.
Part one expression and clinical significance of serum Hepcidin after renal transplantation
Objective:To observe the clinical significance of serum Hepcidin in renal transplantation patients with ischemia-reperfusion injury before and after renal transplantation.
Methods: Peripheral blood samples were collected from renal allograft recipients at different time points before and after transplantation. The levels of IL-6, Hepcidin, sTfR and C (r serum creatinine) in plasma were measured by enzyme-linked immunosorbent assay (ELISA), and the correlation between Hepcidin and inflammatory reaction and reperfusion injury was analyzed.
Results: The levels of serum Hepcidin after renal allograft transplantation were significantly higher than those before transplantation (P 0.05), and correlated with the expressions of IL-6, sTfR and Hb. With the recovery of renal function after renal transplantation, the levels of serum Hepcidin and the ratio of serum creatinine (Cr) to serum Hepcidin were also decreased (P 0.05).
CONCLUSION: Ischemia-reperfusion injury can affect the expression of Hepcidin, and its changes are positively correlated with the inflammatory state in vivo and the degree of renal function injury.
The second part is the expression and significance of Hepcidin in renal ischemia-reperfusion injury in rats.
Objective To observe the changes of liver, Hepcidin, IL-6 and serum creatinine (Cr) in rats with renal ischemia-reperfusion injury (IRI) and analyze its significance.
Methods Forty adult male SD rats were randomly divided into ischemia-reperfusion group (IR) and control group (Control). The rats in the IR group were divided into three groups according to different reperfusion phases: 6 h, 24 h and 48 h, respectively, with 10 rats in each group. Serum levels of Hepcidin and IL-6 were detected by semi-quantitative RT-PCR, serum levels of Hepcidin and IL-6 were detected by ELISA, serum creatinine (Cr) levels were detected, and renal pathological examination was performed in rats.
Results The levels of hepatic and serum hepcidin in renal ischemia-reperfusion group were significantly higher than those in control group (P The expression of Cr in the injection group was higher than that in the control group (P
Conclusion Renal ischemia-reperfusion injury can affect the expression of Hepcidin, which is positively correlated with the inflammatory state in vivo and the degree of renal function injury.
The third part is the expression and significance of Hepcidin in chronic allograft nephropathy in rats.
Objective to investigate the expression and clinical significance of Hepcidin in animal models of chronic allograft nephropathy (CAN) in rats.
Methods Twenty rats with chronic allograft nephropathy were established by orthotopic kidney transplantation with F344 inbred rats as donors and Lewis inbred rats as recipients. Blood and urine samples were collected from the veins of the rats before, 1 month, 2 months and 4 months after transplantation to detect renal function, and the expressions of serum, urinary Hepcidin, serum IL-6 and EPO were detected by ELISA. Ten rats were sacrificed in April and April respectively. The histopathological changes of transplanted kidney in each group were observed and statistically analyzed.
Results The expression of serum Hepcidin and IL-6 increased gradually with the time after transplantation, the excretion of urinary Hepcidin decreased gradually, and the expression of EPO decreased gradually, which was statistically significant before and after transplantation (P 0.05). Positive correlation was negatively correlated with EPO. Renal pathology and HE staining were consistent with pathological changes of chronic allograft nephropathy.
Conclusion With the decrease of renal allograft function, the microinflammatory reaction in rats increases. The expression of Hepcidin in chronic renal allograft nephropathy model is related to renal function and can be used as a marker of renal function damage.
The fourth part is the abnormal expression and molecular mechanism of Hepcidin in inflammatory stimulated renal tubular epithelial cells.
Objective To observe the transdifferentiation and the expression of Hepcidin in HK-2 cells stimulated by IL-6 and explore the molecular mechanism of IL-6 regulating Hepcidin.
Methods: Human HK-2 cells were isolated and cultured and divided into two groups: (1) control group; (2) The final concentration of IL-6 was 10,25,50 ng/ml, respectively. The morphological changes of HK-2 cells were observed after 48 hours. The expression of E-cadherin and aSMA was detected by RT-PCR, and the expression of Hepcidin in supernatant was detected by ELISA and Western blot. The expression of STAT3 in HK-2 cells was determined and the effect of IL-6 on the expression of Hepcidin and STAT3 was analyzed.
Results: After 48 hours of treatment with different concentrations of IL-6, the expression of E-cadherin decreased, the expression of aSMA increased, and the transdifferentiation of HK-2 cells was promoted. The concentration of Hepcidin and the expression of p-STAT3 protein increased significantly in the supernatant of culture medium. IL-6 affected the expression of Hepcidin and p-STAT3 protein.
Conclusion: (1) IL-6 can stimulate the transdifferentiation of HK-2 cells into myofibroblasts. (2) IL-6 may induce the increase of Hepcidin expression by activating JAK/STAT signaling pathway and up-regulating p-STAT3. (3) The change of Hepcidin expression is related to the transdifferentiation ability of HK-2 cells, indicating that Hepcidin plays an important role in renal interstitial fibrosis. Mark.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R699.2
【参考文献】
相关期刊论文 前2条
1 刘树欣;卢红梅;刘玉倩;王海涛;康红哲;李文惠;陈军;;铁调素的表达与调节机制[J];中国组织工程研究与临床康复;2010年41期
2 唐亚雄;唐伟;梁思敏;李家兵;李杰;;大鼠慢性移植肾肾病模型的建立[J];重庆医科大学学报;2009年02期
本文编号：2230962
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