發(fā)布時(shí)間：2018-09-08 15:44

【摘要】：近年來，新型免疫抑制劑的不斷出現(xiàn)以及臨床免疫治療方案的進(jìn)一步完善，腎移植術(shù)后急性排斥反應(yīng)的發(fā)生率明顯降低，但移植腎長(zhǎng)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(huì)刺激炎癥反應(yīng)的發(fā)生，從而導(dǎo)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(xiàn)為腎小管上皮細(xì)胞萎縮，腎間質(zhì)纖維化，其病理變化過程與炎癥刺激密不可分。隨著病情進(jìn)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(guān)性。 第一部分腎移植后血清Hepcidin的表達(dá)及臨床意義 目的：觀察血清Hepcidin在腎移植術(shù)前、術(shù)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(shù)前和術(shù)后不同時(shí)間點(diǎn)外周血，酶聯(lián)免疫法（ELISA）檢測(cè)血漿中IL-6、Hepcidin、sTfR（轉(zhuǎn)鐵蛋白受體）、C（r血肌酐）的水平，分析Hepcidin與炎癥反應(yīng)和再灌注損傷的相關(guān)性。 結(jié)果：同種異體腎移植術(shù)后的血清Hepcidin較術(shù)前增高，P0.05；并且與IL-6、sTfR、Hb的表達(dá)有相關(guān)性，有統(tǒng)計(jì)學(xué)差異；隨著腎移植術(shù)后腎功能的恢復(fù)，患者血清Hepcidin降低，血肌酐（Cr）與血清Hepcidin的比值也降低，P0.05。 結(jié)論：缺血再灌注損傷可影響Hepcidin的表達(dá)，其變化與體內(nèi)炎癥狀態(tài)及腎功能損傷程度呈正相關(guān)。 第二部分Hepcidin在大鼠腎缺血再灌注損傷中的表達(dá)及意義 目的觀察腎缺血再灌注損傷（ischemia-reperfusion injury,IRI）對(duì)大鼠肝臟及Hepcidin、IL-6、血肌酐（Cr）等指標(biāo)的變化，并分析其意義。 方法將40只成年SD雄性大鼠隨機(jī)分為缺血再灌注組（IR）和對(duì)照組（Control），缺血再灌注組根據(jù)再灌注的不同時(shí)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(dòng)脈45min后恢復(fù)血流，對(duì)照組在暴露雙腎45min后關(guān)閉腹腔，兩組分別在手術(shù)6h、24h、48h后取肝臟、腎臟組織標(biāo)本，抽取靜脈血液。采用半定量RT-PCR法檢測(cè)每組大鼠肝臟Hepcidin的表達(dá)水平，ELISA檢測(cè)每組大鼠血清Hepcidin、IL-6的表達(dá)，檢測(cè)血肌酐（Cr）水平，對(duì)大鼠腎臟行病理學(xué)檢查，并進(jìn)行統(tǒng)計(jì)分析。 結(jié)果腎缺血再灌注組肝臟和血清Hepcidin水平較對(duì)照組明顯增高（P0.05），24小時(shí)達(dá)到最高，此后隨著再灌注時(shí)間的延長(zhǎng)含量呈下降趨勢(shì)；腎缺血再灌注組血清IL-6也較對(duì)照組增高（P0.05），而且再灌注組Hepcidin的表達(dá)與血清IL-6呈正相關(guān)；再灌注組Cr的表達(dá)較對(duì)照組增高（P0.05），和Hepcidin亦呈正相關(guān)，腎臟病理學(xué)檢查顯示24小時(shí)炎性細(xì)胞最多。 結(jié)論腎缺血再灌注損傷可影響Hepcidin的表達(dá)，表達(dá)的變化與體內(nèi)炎癥狀態(tài)和腎功能損傷程度呈正相關(guān)。因此Hepcidin可作為反映腎缺血再灌注損傷程度的標(biāo)記物，且對(duì)于腎缺血再灌注損傷導(dǎo)致機(jī)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(dá)及意義 目的探討Hepcidin在大鼠慢性移植腎腎�。–AN）動(dòng)物模型中的表達(dá)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(shù)后1月、2月及4月分別收集大鼠的靜脈血和尿樣，檢測(cè)腎功能；ELISA檢測(cè)血清、尿Hepcidin，血清IL-6、EPO的表達(dá)；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(xué)變化，進(jìn)行統(tǒng)計(jì)學(xué)分析，探討其臨床意義及相關(guān)性。 結(jié)果血清Hepcidin、IL-6表達(dá)隨著移植后的時(shí)間的推移逐漸增高，尿Hepcidin排出逐漸減少，EPO表達(dá)逐漸減少，術(shù)前與術(shù)后比較有統(tǒng)計(jì)學(xué)意義（P0.05）；移植術(shù)后大鼠Cr、BUN逐漸升高，并且Cr與血清Hepcidin呈正相關(guān)；移植術(shù)后血清Hepcidin的表達(dá)與IL-6正相關(guān)，與EPO負(fù)相關(guān)；腎臟病理、HE染色符合慢性移植腎腎病的病理改變。 結(jié)論隨著移植腎功能的減退，大鼠體內(nèi)微炎癥反應(yīng)增加，Hepcidin在大鼠慢性移植腎腎病模型表達(dá)變化與腎功能有關(guān)，可作為反映腎功能損傷的標(biāo)記物。 第四部分Hepcidin在炎癥刺激的腎小管上皮細(xì)胞中異常表達(dá)及分子機(jī)制研究 目的采用IL-6刺激腎小管上皮（HK-2）細(xì)胞，觀察HK-2細(xì)胞轉(zhuǎn)分化以及細(xì)胞中Hepcidin表達(dá)變化，探討IL-6對(duì)Hepcidin調(diào)控的分子機(jī)制。 方法：分離培養(yǎng)人HK-2細(xì)胞，將其分成2組，（1）對(duì)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(xì)胞，48小時(shí)后觀察細(xì)胞形態(tài)學(xué)變化，，RT-PCR檢測(cè)細(xì)胞中E-cadherin，aSMA的表達(dá)；ELISA法測(cè)定培養(yǎng)液上清Hepcidin的表達(dá)，Western blot法測(cè)定細(xì)胞內(nèi)STAT3的表達(dá)，并分析IL-6對(duì)Hepcidin、STAT3表達(dá)的影響，以及與HK-2細(xì)胞轉(zhuǎn)分化相關(guān)性。 結(jié)果：不同濃度IL-6作用于HK-2細(xì)胞48小時(shí)后，E-cadherin表達(dá)下降，aSMA表達(dá)升高，促進(jìn)HK-2細(xì)胞轉(zhuǎn)分化，培養(yǎng)液上清中Hepcidin濃度顯著增加，p-STAT3蛋白表達(dá)增高；IL-6影響Hepcidin和p-STAT3的表達(dá)。 討論：（1）IL-6能刺激HK-2細(xì)胞向肌成纖維細(xì)胞轉(zhuǎn)分化。（2）IL-6可能通過激活JAK/STAT信號(hào)通路上調(diào)p-STAT3，誘導(dǎo)Hepcidin表達(dá)增加。（3）Hepcidin的表達(dá)變化與HK-2細(xì)胞轉(zhuǎn)分化能力有關(guān)，說明Hepcidin在腎間質(zhì)纖維化中起到重要作用，可以作為腎功能損傷的標(biāo)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(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R699.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 劉樹欣;盧紅梅;劉玉倩;王海濤;康紅哲;李文惠;陳軍;;鐵調(diào)素的表達(dá)與調(diào)節(jié)機(jī)制[J];中國組織工程研究與臨床康復(fù);2010年41期

2 唐亞雄;唐偉;梁思敏;李家兵;李杰;;大鼠慢性移植腎腎病模型的建立[J];重慶醫(yī)科大學(xué)學(xué)報(bào);2009年02期

本文編號(hào)：2230962