本文選題：腎損傷分子-1 + 腎移植　； 參考：《吉林大學(xué)》2014年博士論文

【摘要】：腎損傷分子（KIM-1）是新穎、特異性高的判斷腎小管損傷的生化指標(biāo)，尿液和組織標(biāo)本中均可以檢測(cè)出其表達(dá)；當(dāng)腎近曲小管發(fā)生急慢性損傷時(shí)，KIM-1在腎小管細(xì)胞中表達(dá)，成為凋亡細(xì)胞被吞噬與被清除的受體；KIM-1可能在腎小管損傷和修復(fù)過程中發(fā)揮重要病理生理作用。在移植腎臟中，腎小管損傷的相關(guān)因素有缺血、免疫抑制劑毒性、急性排斥反應(yīng)等，，各種因素聯(lián)合作用或單獨(dú)作用均可以導(dǎo)致慢性移植腎腎病，最終導(dǎo)致移植腎臟丟失。腎組織穿刺活檢，是判斷和識(shí)別移植腎臟損傷的重要檢測(cè)手段。腎穿標(biāo)本病理形態(tài)學(xué)觀察可見到：腎小管上皮刷狀緣的改變、上皮細(xì)胞的凋亡/壞死、小管纖維化/萎縮、上皮脫落等，但因形態(tài)診斷是存在主觀因素的影響，需要尋求一個(gè)早期敏感度高的客觀判斷腎小管輕微損傷的指標(biāo)。 本選題以KIM-1為研究的核心，探索其在移植腎損傷中的變化和意義。 我們選取2009年-2011年69例移植腎穿刺活檢的組織標(biāo)本，應(yīng)用免疫組織化學(xué)方法標(biāo)記移植腎組織中KIM-1的表達(dá)情況，分析移植腎組織中KIM-1表達(dá)水平和其他血液生化指標(biāo)的關(guān)系、移植腎組織中KIM-1表達(dá)與移植腎功能的相關(guān)性。結(jié)果顯示：（1）在不同病理診斷分組中可見KIM-1在近端小管上皮出現(xiàn)不同程度的表達(dá)；（2）在急性T細(xì)胞性排斥反應(yīng)中，KIM-1表達(dá)量隨Banff07分級(jí)升高而呈現(xiàn)增高趨勢(shì)，同時(shí)KIM-1表達(dá)水平與小管炎嚴(yán)重程度相一致。在小管炎出現(xiàn)可見的形態(tài)改變之前，KIM-1蛋白已出現(xiàn)可見的陽(yáng)性表達(dá)。（3）慢性活動(dòng)性抗體介導(dǎo)排異反應(yīng)中，KIM-1呈強(qiáng)陽(yáng)性表達(dá)，強(qiáng)烈提示活動(dòng)性排異反應(yīng)所致腎損傷的存在；（4）在交界性病變時(shí)，KIM-1出現(xiàn)不同程度陽(yáng)性表達(dá)，提示腎小管上皮處于損傷狀態(tài)；（5）未見排斥反應(yīng)發(fā)生和免疫抑制劑中毒組，27.27%（3/11）病例血肌酐在正常范圍內(nèi)，但腎組織中KIM-1呈弱陽(yáng)性表達(dá)。本研究表明：（1）在移植腎組織中KIM-1表達(dá)水平與移植腎損傷程度呈正相關(guān)，提示腎小管細(xì)胞損傷存在。（2）在未見血生化指標(biāo)升高提示腎損傷時(shí)、未見形態(tài)學(xué)可見腎損傷改變時(shí)、未見腎小管炎癥反應(yīng)時(shí)，均可見KIM-1蛋白在腎小管上皮細(xì)胞表達(dá)，提示KIM-1蛋白在移植腎活檢組織中表達(dá)可以成為早期、敏感、特異判定腎小管上皮細(xì)胞損傷的生化指標(biāo)。 本研究通過采用ELISA方法檢測(cè)腎移植患者血清中KIM-1的表達(dá)，研究和探索早期血清中與組織內(nèi)KIM-1的表達(dá)能否成為早期判斷移植腎功能異常的應(yīng)用指標(biāo)。結(jié)果表明：（1）在移植腎患者血清中KIM-1表達(dá)的增強(qiáng)可以提示移植腎損傷程度的強(qiáng)弱。（2）慢性活動(dòng)性排斥造成的損傷可以出現(xiàn)血清KIM-1的高表達(dá)，提示移植腎臟功能狀態(tài)惡化。（3）早期血清中腎損傷分子-1水平升高可能成為提示移植腎功能狀態(tài)的生化指標(biāo)。 在本次實(shí)驗(yàn)研究中，采用體外實(shí)驗(yàn)方法研究環(huán)孢素?fù)p傷腎小管細(xì)胞后導(dǎo)致其產(chǎn)生KIM-1的表達(dá)，并初步探索其中機(jī)制，尋找保護(hù)和延緩環(huán)孢素對(duì)移植腎損傷的藥物。 在實(shí)驗(yàn)中首先建立環(huán)孢素?fù)p傷腎近曲小管上皮細(xì)胞的體外研究模型，采用MTT實(shí)驗(yàn)方法篩選環(huán)孢素的損傷作用的適宜濃度；采用MTT實(shí)驗(yàn)法篩選可能的藥物干預(yù)改善環(huán)孢素對(duì)腎小管上皮細(xì)胞的損傷；篩選出促紅細(xì)胞生成素（EPO）可以保護(hù)腎小管細(xì)胞減少環(huán)孢素對(duì)細(xì)胞的損傷。采用絲裂原活化蛋白激酶系統(tǒng)阻斷劑(p38通路阻斷劑SB202190、ERK1/2通路阻斷劑PD98059)，阻斷相應(yīng)酶的表達(dá)，以期推測(cè)生化過程發(fā)生的機(jī)制。采用ELISA方法鑒定KIM-1在細(xì)胞上清液中的表達(dá)，統(tǒng)計(jì)KIM-1表達(dá)量的變化。采用流式細(xì)胞儀測(cè)定在環(huán)孢素?fù)p傷腎小管中細(xì)胞凋亡發(fā)生特點(diǎn)。 環(huán)孢素使腎近曲小管細(xì)胞損傷時(shí)KIM-1表達(dá)升高并導(dǎo)致細(xì)胞凋亡，在這一過程中絲裂原活化蛋白激酶中的p38通路、ERK1/2通路在其中發(fā)揮作用。阻斷以上2條通路可以減少CsA所致HKC細(xì)胞的凋亡比率（P0.05），增加活細(xì)胞比率（P0.05），減少細(xì)胞的壞死比率（P0.05），同時(shí)減少KIM-1蛋白的表達(dá)量（P0.05）。加入EPO可以減少HKC細(xì)胞在CsA作用下產(chǎn)生KIM-1蛋白（P0.05）；選用EPO15U/ml和30U/ml進(jìn)行實(shí)驗(yàn)，隨EPO作用量的增加，可以減少HKC細(xì)胞在CsA損傷中產(chǎn)生KIM-1的量值，提示EPO可以拮抗CsA對(duì)腎小管上皮的損傷，減少CsA所致腎小管上皮細(xì)胞凋亡和壞死的發(fā)生。 腎損傷分子-1在移植腎中的表達(dá)和變化具有重要的研究?jī)r(jià)值，可以成為判定移植腎損傷早期、敏感、特異性判定腎小管上皮細(xì)胞損傷的生化指標(biāo)；早期血清中腎損傷分子-1水平升高可能成為提示移植腎功能狀態(tài)的生化指標(biāo)。環(huán)孢素使腎近曲小管細(xì)胞損傷時(shí)KIM-1表達(dá)升高，在這一過程中絲裂原活化蛋白激酶中的p38通路、ERK1/2通路在其中起到作用。阻斷p38通路、阻斷ERK1/2通路、加入EPO三種因素均可以減少腎小管細(xì)胞在CsA作用下產(chǎn)生KIM-1蛋白，有效保護(hù)和延緩環(huán)孢素對(duì)移植腎小管上皮損傷的過程。本次選題的實(shí)驗(yàn)結(jié)果與結(jié)論，為腎損傷分子-1在移植腎領(lǐng)域中的應(yīng)用提供有利的基礎(chǔ)實(shí)驗(yàn)依據(jù)，同時(shí)為減少環(huán)孢素對(duì)移植腎損傷方面繼續(xù)探索提供可參考的實(shí)驗(yàn)?zāi)Ｐ团c有效的實(shí)驗(yàn)藥物。
[Abstract]:Renal injury molecule (KIM-1) is a novel and highly specific biochemical indicator of renal tubular injury, which can be detected in urine and tissue specimens. When acute and chronic renal tubule injury occurs, KIM-1 is expressed in renal tubular cells and becomes the receptor for phagocytosis and removal of apoptotic cells; KIM-1 may be damaged in renal tubules and in renal tubules. During the repair process, it plays an important role in pathophysiology. In the transplanted kidney, the related factors of renal tubular injury are ischemia, immunosuppressant toxicity, acute rejection and so on. Various factors combined or alone can lead to chronic renal transplant kidney disease and eventually lead to the loss of kidney transplantation. Renal tissue biopsy is a judgment and recognition shift. The pathological changes of the brush shaped edge of the renal tubular epithelium, the apoptosis / necrosis of the epithelial cells, the tubule fibrosis / atrophy, the epithelia and so on are observed, but the morphological diagnosis is influenced by the subjective factors, and it is necessary to seek an objective judgement of the renal tubules with high early sensitivity. The index of damage.
This topic takes KIM-1 as the core of the study to explore its changes and significance in renal allograft injury.
69 cases of renal transplant biopsy in 2009 -2011 were selected. The expression of KIM-1 in transplanted renal tissue was marked by immunohistochemistry. The relationship between the expression of KIM-1 and other blood biochemical indexes in the transplanted kidney tissue was analyzed. The correlation between the KIM-1 table and the function of the transplanted kidney in the transplanted kidney tissue was analyzed. The results showed: (1 Different levels of expression of KIM-1 in proximal tubule epithelium were found in different pathological diagnosis groups; (2) in acute T cell rejection, the expression of KIM-1 increased with the increase of Banff07 grading, and the expression level of KIM-1 was consistent with the severity of tubulitis. Before the appearance of visible morphological changes in canaliculitis, KIM-1 The positive expression of the protein has been seen. (3) in the reaction of chronic active antibody mediated rejection, KIM-1 is strongly positive and strongly suggests the existence of renal injury caused by active rejection; (4) in borderline lesions, the positive expression of KIM-1 appears in different degrees, suggesting that the renal tubular epithelium is in a state of injury; (5) no rejection occurs. The serum creatinine in 27.27% (3/11) cases was in the normal range, but the expression of KIM-1 in the renal tissue was weakly positive. The study showed that (1) the expression of KIM-1 in the transplanted kidney was positively correlated with the degree of renal transplant injury, suggesting that the renal tubular cells were damaged. (2) when no blood biochemical index was raised, the renal injury was not seen. The expression of KIM-1 protein in renal tubular epithelial cells can be seen in the renal tubular epithelial cells when the renal tubule inflammation is not seen in the morphological changes. It is suggested that the expression of KIM-1 protein in the transplanted renal biopsy tissue can be an early, sensitive and specific biochemical index to determine the injury of renal tubular epithelial cells.
In this study, the expression of KIM-1 in the serum of renal transplant patients was detected by ELISA method. The study and exploration of the early serum and intra tissue KIM-1 expression could be used as an indicator of early renal allograft dysfunction. The results showed that: (1) the enhancement of KIM-1 in the serum of patients with renal allograft could indicate the strong degree of renal transplant injury. (2) the high expression of serum KIM-1 can be found in the injury caused by chronic active rejection, which suggests the deterioration of the function of the transplanted kidney. (3) the increase in the level of renal injury molecules in the early sera may be a biochemical indicator of the function of the transplanted kidney.
In this experimental study, the expression of cyclosporin damaged renal tubule cells in vitro was studied in vitro, and the mechanism of KIM-1 was preliminarily explored to find the protection and delay of cyclosporin in the treatment of renal transplant injury.
In the experiment, we first set up an in vitro model of cyclosporin injury in renal proximal convoluted tubule epithelial cells. The suitable concentration of cyclosporin was screened by MTT method. The possible drug intervention was used to screen the damage of cyclosporin on renal tubular epithelial cells by MTT test, and the screening of erythropoietin (EPO) could protect the epithelial cells of renal tubules. Renal tubule cells reduce cyclosporine damage to cells. The mitogen activated protein kinase blocker (p38 pathway blocker SB202190, ERK1/2 pathway blocker PD98059) was used to block the expression of the corresponding enzyme, in order to speculate on the mechanism of biochemical process. ELISA method was used to determine the expression of KIM-1 in the cell supernatant, and the expression of KIM-1 was statistically expressed. The characteristics of apoptosis in renal tubules damaged by cyclosporine were determined by flow cytometry.
Cyclosporin increases the expression of KIM-1 in renal proximal tubular cells and causes apoptosis. In this process, the p38 pathway in mitogen activated protein kinase and the ERK1/2 pathway play a role in this process. Blocking the above 2 pathways can reduce the apoptosis ratio (P0.05) of HKC cells caused by CsA, increase the living cell ratio (P0.05), and reduce cell damage. The death rate (P0.05) and the decrease of the expression of KIM-1 protein (P0.05). The addition of EPO can reduce the production of KIM-1 protein (P0.05) in HKC cells under the action of CsA, and use EPO15U/ml and 30U/ml to carry out the experiment. Injury can reduce the occurrence of apoptosis and necrosis of renal tubular epithelial cells induced by CsA.
The expression and changes of renal injury molecule -1 in the transplanted kidney have important research value, which can be used to determine the early, sensitive and specific biochemical markers of renal tubular epithelial cell damage in the early stage of renal transplantation. The increase of the level of -1 in the early sera may be a biochemical indicator of the function of the transplanted kidney. Cyclosporin makes the kidney. The expression of KIM-1 increased during the injury of proximal tubule cells. In this process, the p38 pathway in mitogen activated protein kinase and the ERK1/2 pathway play a role in this process. Blocking the p38 pathway, blocking the ERK1/2 pathway, and adding EPO three factors can reduce the formation of KIM-1 protein in the renal tubule cells under the action of CsA and effectively protect and delay the cyclosporin transplantation. The experimental results and conclusions of this topic provide a favorable basis for the application of renal injury molecular -1 in the field of renal transplantation, and provide a reference experimental model and effective experimental drugs for the reduction of the continued exploration of cyclosporin.

【學(xué)位授予單位】：吉林大學(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1條

1 張玉軍;郝軍;劉淑霞;左連富;劉俊茹;吳海江;郭建文;;p38MAPK在戊地昔布誘導(dǎo)Eca109細(xì)胞凋亡中的調(diào)控作用[J];中國(guó)藥理學(xué)通報(bào);2009年04期

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