發(fā)布時間：2018-09-08 15:44

【摘要】：近年來，新型免疫抑制劑的不斷出現以及臨床免疫治療方案的進一步完善，腎移植術后急性排斥反應的發(fā)生率明顯降低，但移植腎長期存活率未明顯提高。腎移植過程中的腎缺血再灌注損傷是發(fā)生最早也是必不可少的環(huán)節(jié),缺血再灌注損傷會刺激炎癥反應的發(fā)生，從而導致慢性移植腎腎病。移植腎后期功能喪失的最主要原因是慢性移植腎腎病(CAN)。慢性移植腎腎病主要表現為腎小管上皮細胞萎縮，腎間質纖維化，其病理變化過程與炎癥刺激密不可分。隨著病情進展，受者將發(fā)展為移植物功能喪失即慢性腎衰竭(chronic renal failure，CRF)。 研究慢性移植腎腎病的發(fā)生發(fā)展，意義重大，，我們將分別研究血清Hepcidin與缺血再灌注以及慢性移植物腎病相關性。 第一部分腎移植后血清Hepcidin的表達及臨床意義 目的：觀察血清Hepcidin在腎移植術前、術后患者缺血再灌注的臨床意義及損傷中的作用。 方法：收集同種異體腎移植患者術前和術后不同時間點外周血，酶聯(lián)免疫法（ELISA）檢測血漿中IL-6、Hepcidin、sTfR（轉鐵蛋白受體）、C（r血肌酐）的水平，分析Hepcidin與炎癥反應和再灌注損傷的相關性。 結果：同種異體腎移植術后的血清Hepcidin較術前增高，P0.05；并且與IL-6、sTfR、Hb的表達有相關性，有統(tǒng)計學差異；隨著腎移植術后腎功能的恢復，患者血清Hepcidin降低，血肌酐（Cr）與血清Hepcidin的比值也降低，P0.05。 結論：缺血再灌注損傷可影響Hepcidin的表達，其變化與體內炎癥狀態(tài)及腎功能損傷程度呈正相關。 第二部分Hepcidin在大鼠腎缺血再灌注損傷中的表達及意義 目的觀察腎缺血再灌注損傷（ischemia-reperfusion injury,IRI）對大鼠肝臟及Hepcidin、IL-6、血肌酐（Cr）等指標的變化，并分析其意義。 方法將40只成年SD雄性大鼠隨機分為缺血再灌注組（IR）和對照組（Control），缺血再灌注組根據再灌注的不同時相分為3組，分別為6h、24h、48h，每組10只。缺血再灌注組切除右腎，完全阻斷左腎動脈45min后恢復血流，對照組在暴露雙腎45min后關閉腹腔，兩組分別在手術6h、24h、48h后取肝臟、腎臟組織標本，抽取靜脈血液。采用半定量RT-PCR法檢測每組大鼠肝臟Hepcidin的表達水平，ELISA檢測每組大鼠血清Hepcidin、IL-6的表達，檢測血肌酐（Cr）水平，對大鼠腎臟行病理學檢查，并進行統(tǒng)計分析。 結果腎缺血再灌注組肝臟和血清Hepcidin水平較對照組明顯增高（P0.05），24小時達到最高，此后隨著再灌注時間的延長含量呈下降趨勢；腎缺血再灌注組血清IL-6也較對照組增高（P0.05），而且再灌注組Hepcidin的表達與血清IL-6呈正相關；再灌注組Cr的表達較對照組增高（P0.05），和Hepcidin亦呈正相關，腎臟病理學檢查顯示24小時炎性細胞最多。 結論腎缺血再灌注損傷可影響Hepcidin的表達，表達的變化與體內炎癥狀態(tài)和腎功能損傷程度呈正相關。因此Hepcidin可作為反映腎缺血再灌注損傷程度的標記物，且對于腎缺血再灌注損傷導致機體鐵代謝的改變有臨床提示意義。 第三部分Hepcidin在大鼠慢性移植腎腎病中的表達及意義 目的探討Hepcidin在大鼠慢性移植腎腎�。–AN）動物模型中的表達及臨床意義。 方法以F344近交系大鼠為供體、Lewis近交系大鼠為受體，原位腎移植，建立20只慢性移植腎腎病大鼠模型；于建模前、術后1月、2月及4月分別收集大鼠的靜脈血和尿樣，檢測腎功能；ELISA檢測血清、尿Hepcidin，血清IL-6、EPO的表達；并于移植后2月及4月分別處死大鼠10只，觀察各組大鼠移植腎組織病理學變化，進行統(tǒng)計學分析，探討其臨床意義及相關性。 結果血清Hepcidin、IL-6表達隨著移植后的時間的推移逐漸增高，尿Hepcidin排出逐漸減少，EPO表達逐漸減少，術前與術后比較有統(tǒng)計學意義（P0.05）；移植術后大鼠Cr、BUN逐漸升高，并且Cr與血清Hepcidin呈正相關；移植術后血清Hepcidin的表達與IL-6正相關，與EPO負相關；腎臟病理、HE染色符合慢性移植腎腎病的病理改變。 結論隨著移植腎功能的減退，大鼠體內微炎癥反應增加，Hepcidin在大鼠慢性移植腎腎病模型表達變化與腎功能有關，可作為反映腎功能損傷的標記物。 第四部分Hepcidin在炎癥刺激的腎小管上皮細胞中異常表達及分子機制研究 目的采用IL-6刺激腎小管上皮（HK-2）細胞，觀察HK-2細胞轉分化以及細胞中Hepcidin表達變化，探討IL-6對Hepcidin調控的分子機制。 方法：分離培養(yǎng)人HK-2細胞，將其分成2組，（1）對照組；（2）將用IL-6加入培養(yǎng)液，終濃度分別為10、25、50ng/ml，培養(yǎng)HK-2細胞，48小時后觀察細胞形態(tài)學變化，，RT-PCR檢測細胞中E-cadherin，aSMA的表達；ELISA法測定培養(yǎng)液上清Hepcidin的表達，Western blot法測定細胞內STAT3的表達，并分析IL-6對Hepcidin、STAT3表達的影響，以及與HK-2細胞轉分化相關性。 結果：不同濃度IL-6作用于HK-2細胞48小時后，E-cadherin表達下降，aSMA表達升高，促進HK-2細胞轉分化，培養(yǎng)液上清中Hepcidin濃度顯著增加，p-STAT3蛋白表達增高；IL-6影響Hepcidin和p-STAT3的表達。 討論：（1）IL-6能刺激HK-2細胞向肌成纖維細胞轉分化。（2）IL-6可能通過激活JAK/STAT信號通路上調p-STAT3，誘導Hepcidin表達增加。（3）Hepcidin的表達變化與HK-2細胞轉分化能力有關，說明Hepcidin在腎間質纖維化中起到重要作用，可以作為腎功能損傷的標記。
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R699.2

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本文編號：2230962