腎臟缺血再灌注后MicroRNA-146的表達變化及其調(diào)控機制
發(fā)布時間：2018-07-24 20:12
【摘要】：背景： 繼1975年研究者發(fā)現(xiàn)缺血器官復氧后原有損傷加重，并提出缺血再灌注(IschemicReperfusion, IR)損傷的概念之后，上述現(xiàn)象已逐步受到國內(nèi)外眾多學者的重視。器官IR損傷常發(fā)生于阻斷血氧供應的外科手術(shù)以及影響全身循環(huán)的多器官病變中。腎臟作為高灌注器官，對IR損傷的耐受性更差。IR損傷往往對腎臟產(chǎn)生不利影響，嚴重者可導致腎功能進行性惡化或恢復延遲，甚至威脅生命。 隨著近年來對非編碼RNA（Non-coding RNAs）研究的逐步深入，越來越多的實驗表明MicroRNA（微小RNA，miRNA）在許多病理生理過程中發(fā)揮著重要作用。而在目前已發(fā)現(xiàn)的眾多功能各異的miRNA中， miR-146是一類具有免疫調(diào)控功能的miRNA，通過與上游靶基因結(jié)合調(diào)節(jié)下游蛋白質(zhì)的翻譯，借此在免疫應答、炎癥反應、腫瘤發(fā)生等許多領(lǐng)域發(fā)揮調(diào)控作用。在前期研究中，本課題組已經(jīng)發(fā)現(xiàn)一系列miRNA在腎臟IR損傷后表達水平發(fā)生變化，其中包括miR-146，我們推測miR-146可能參與調(diào)控IR損傷，但其機制尚未明確。 目的： 本實驗以小鼠腎臟熱IR損傷模型和體外細胞模型為基礎，探討IR損傷對腎臟實質(zhì)細胞中miR-146表達的影響和調(diào)控機制，以期能夠為臨床和科研中有效減輕并及時發(fā)現(xiàn)腎臟IR損傷提供新的思路和研究靶點。 1.建立腎臟熱缺血IR損傷動物模型。 2.利用腎臟熱缺血IR損傷動物模型，在體內(nèi)實驗中觀察腎臟IR后miR-146的表達變化并分析其可能的原因。 3.通過體外細胞模型，進一步研究IR及氧化應激反應后腎臟實質(zhì)細胞內(nèi)miR-146的表達變化并分析其可能的原因。 方法： 1.以近交系小鼠為研究對象，構(gòu)建腎臟熱缺血IR損傷動物模型。檢測IR后小鼠的生存率、體重變化、血肌酐和尿素氮水平，借此評估動物模型。 2.利用上述動物模型進行實驗，將小鼠分為Sham組和IR組。在不同時間點切取受損腎臟，應用實時熒光定量PCR（qRT-PCR）技術(shù)檢測上述組織內(nèi)miR-146的表達情況。之后按是否使用錳卟啉（MnTMPyP）進行抗氧化預處理將小鼠分為Antioxidant組和Saline組，在反向?qū)嶒炛杏^察抗氧化處理后miR-146的表達變化。 3.利用預先培養(yǎng)的原代腎小管上皮細胞（TEC）進行體外試驗，按是否使用H2O2刺激將細胞分為Stress組和Sham組，利用qRT-PCR檢測相同劑量H2O2刺激不同時長，以及用不同劑量H2O2刺激相同時長后原代TEC內(nèi)miR-146的表達情況。之后利用石蠟油構(gòu)建TEC的體外IR模型，模擬細胞IR過程，進一步觀察miR-146的表達情況。 結(jié)果： 1.IR組小鼠術(shù)后3d內(nèi)部分死亡，但總生存率超過80%。術(shù)后出現(xiàn)顯著體重減輕、精神萎靡、活動減少。與Sham組相比，IR組小鼠血肌酐和尿素氮水平顯著升高。經(jīng)HE染色后鏡下可見IR組腎小管上皮細胞明顯水腫，部分壞死，腎間質(zhì)內(nèi)較多炎細胞浸潤。 2.腎臟再灌注6h后miR-146a表達變化具有統(tǒng)計學意義（P 0.05），24h后表達達到峰值，此時約為Sham組的7.4±1.9倍（P 0.001）。截至再灌注72h，miR-146a表達水平仍為Sham組的5.7±1.3倍（P 0.001），期間miR-146b表達無顯著變化（P0.05）。給予抗氧化處理后，腎臟組織損傷明顯減輕。再灌注24h后Antioxidant組小鼠miR-146a表達的升高幅度出現(xiàn)顯著下調(diào)，與Saline組相比差異有統(tǒng)計學意義（P 0.05）。 3.利用H2O2對原代TEC進行刺激后，發(fā)現(xiàn)上述細胞中miR-146a表達升高。刺激6h后miR-146a表達變化具有統(tǒng)計學意義（P 0.05），24h后達到最高值，約為對照組的15.3±3.8倍（P 0.001），其持續(xù)時間超過48h（P 0.001）。期間miR-146b表達無顯著變化（P0.05）。進一步研究發(fā)現(xiàn)，miR-146a表達變化對H2O2刺激呈劑量依賴性。 4.在細胞體外IR模型中，我們發(fā)現(xiàn)更換培養(yǎng)基（模擬再灌注）后24h內(nèi)，離體的原代TEC內(nèi)miR-146a表達顯著上調(diào)（P 0.05）。 結(jié)論: 1.腎臟IR損傷后miR-146a表達水平在再灌注6h后發(fā)生顯著上調(diào)，并于再灌注后24h后達峰，持續(xù)時間超過72h。 2.腎臟IR損傷后miR-146a表達上調(diào)的主要原因可能是是IR過程中的氧化應激反應。 3.鑒于miR-146的研究背景，在腎臟IR過程中表達上調(diào)的miR-146a可能具有減輕炎癥反應和組織損傷的作用，在未來具有重要的臨床應用價值。此外，，由于IR過程中miR-146a表達改變發(fā)生較早，其潛在的生物學標志功能也不容忽視。
[Abstract]:Background:
After the researchers found that the original damage was aggravated after the reoxygenation of the ischemic organs in 1975, and the concept of IschemicReperfusion (IR) injury was proposed, the above phenomenon has gradually been paid attention to by many scholars at home and abroad. Organ IR injury often occurs in external surgery to block oxygen supply and in multiple organ lesions affecting systemic circulation. The kidney is a high perfusion organ, and the tolerance to IR damage is worse,.IR damage often has adverse effects on the kidney. Serious patients can lead to progressive deterioration of renal function or delay of recovery, and even threaten life.
With the gradual deepening of non coded RNA (Non-coding RNAs) research in recent years, more and more experiments have shown that MicroRNA (small RNA, miRNA) plays an important role in many pathophysiological processes. In many miRNA, which has been found in various functions, miR-146 is a class of miRNA with immune regulation function, through the upstream target base. In the previous study, we have found a series of miRNA changes in the expression level after IR injury in the kidney, including miR-146, and we speculate that miR-146 may be involved in the regulation of IR damage, but its mechanism is The system is not yet clear.
Objective:
In this experiment, the effect of IR damage on the expression of miR-146 in renal parenchymal cells and the regulation mechanism were studied on the basis of the mouse kidney heat IR damage model and in vitro cell model, in order to provide new ideas and targets for the effective mitigation of clinical and scientific research and the timely discovery of renal IR damage in the clinical and scientific research.
1. the animal model of renal ischemia IR injury was established.
2. To observe the expression of microRNA-146 after renal IR in vivo and analyze the possible reasons of the changes.
3. In vitro cell model was used to investigate the expression of microRNAs-146 in renal parenchymal cells after IR and oxidative stress and analyze the possible reasons.
Method:
1. the animal model of kidney hot ischemic IR injury was constructed with the near inbred strain mice. The survival rate, weight change, blood creatinine and urea nitrogen level of the mice after IR were measured, and the animal model was evaluated.
2. using the animal model, the mice were divided into Sham group and IR group. The damaged kidneys were cut at different time points, and the expression of miR-146 in the above tissues was detected by real-time fluorescence quantitative PCR (qRT-PCR). Then the mice were divided into Antioxidant group and Saline group according to the anti oxidation pretreatment using manganese porphyrin (MnTMPyP). The expression of microRNA-146 was observed in reverse experiment after antioxidant treatment.
3. the pre cultured primary renal tubular epithelial cells (TEC) were tested in vitro, and the cells were divided into Stress group and Sham group according to whether H2O2 was used. QRT-PCR was used to detect the same dose of H2O2, and the expression of miR-146 in primary TEC at the same time with different doses of H2O2 stimulation. Then the wax oil was used to construct TEC. In vitro IR model was used to simulate the cellular IR process and further observe the expression of miR-146.
Result:
The mice in group 1.IR died in part of 3D after operation, but the total survival rate was more than that of 80%. after the operation. The level of blood creatinine and urea nitrogen in the IR group was significantly higher than that in the group Sham. After HE staining, the renal tubular epithelial cells in the IR group were obviously edema, partial necrosis, and more inflammatory cells infiltrated in the renal interstitium.
2. after 6h reperfusion, the expression of miR-146a was statistically significant (P 0.05), and the expression reached peak value after 24h, which was about 7.4 + 1.9 times (P 0.001) in Sham group. The expression level of miR-146a was 5.7 + 1.3 times as of Sham group (P 0.001). There was no significant change in miR-146b table during the period of reperfusion (P0.05). The renal tissue loss was given after antioxidant treatment. After 24 hours of reperfusion, the increase of the expression of microRNA-146 a in the Antioxidant group was significantly down-regulated compared with that in the Saline group (P 0.05).
3. using H2O2 to stimulate the primary TEC, it was found that the expression of miR-146a in the above cells increased. The miR-146a expression changes after 6h were statistically significant (P 0.05), after 24h, the maximum value was reached, about 15.3 + 3.8 times of the control group (P 0.001), and its duration exceeded 48h (P 0.001). The miR-146b expression was not significantly changed during the period (P0.05). Further study It was found that the change of miR-146a expression was dose dependent on H2O2 stimulation.
4. in the cell in vitro IR model, we found that the miR-146a expression in the primary TEC of 24h was significantly up-regulated (TEC 0.05) after replacement medium (simulated reperfusion).
Conclusion:
1. the expression level of miR-146a in IR after renal injury increased significantly after reperfusion 6h, and reached the peak after 24h, which lasted for more than 72h..
2. the main reason for the increase of miR-146a expression after renal IR injury may be the oxidative stress reaction during IR.
3. in view of the background of miR-146 research, the expression of up regulated miR-146a in the renal IR process may play a role in alleviating inflammatory and tissue damage, and has important clinical value in the future. In addition, because of the early changes of miR-146a expression during the process of IR, the potential biological marker function can not be ignored.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R699.2
本文編號：2142525
[Abstract]:Background:
After the researchers found that the original damage was aggravated after the reoxygenation of the ischemic organs in 1975, and the concept of IschemicReperfusion (IR) injury was proposed, the above phenomenon has gradually been paid attention to by many scholars at home and abroad. Organ IR injury often occurs in external surgery to block oxygen supply and in multiple organ lesions affecting systemic circulation. The kidney is a high perfusion organ, and the tolerance to IR damage is worse,.IR damage often has adverse effects on the kidney. Serious patients can lead to progressive deterioration of renal function or delay of recovery, and even threaten life.
With the gradual deepening of non coded RNA (Non-coding RNAs) research in recent years, more and more experiments have shown that MicroRNA (small RNA, miRNA) plays an important role in many pathophysiological processes. In many miRNA, which has been found in various functions, miR-146 is a class of miRNA with immune regulation function, through the upstream target base. In the previous study, we have found a series of miRNA changes in the expression level after IR injury in the kidney, including miR-146, and we speculate that miR-146 may be involved in the regulation of IR damage, but its mechanism is The system is not yet clear.
Objective:
In this experiment, the effect of IR damage on the expression of miR-146 in renal parenchymal cells and the regulation mechanism were studied on the basis of the mouse kidney heat IR damage model and in vitro cell model, in order to provide new ideas and targets for the effective mitigation of clinical and scientific research and the timely discovery of renal IR damage in the clinical and scientific research.
1. the animal model of renal ischemia IR injury was established.
2. To observe the expression of microRNA-146 after renal IR in vivo and analyze the possible reasons of the changes.
3. In vitro cell model was used to investigate the expression of microRNAs-146 in renal parenchymal cells after IR and oxidative stress and analyze the possible reasons.
Method:
1. the animal model of kidney hot ischemic IR injury was constructed with the near inbred strain mice. The survival rate, weight change, blood creatinine and urea nitrogen level of the mice after IR were measured, and the animal model was evaluated.
2. using the animal model, the mice were divided into Sham group and IR group. The damaged kidneys were cut at different time points, and the expression of miR-146 in the above tissues was detected by real-time fluorescence quantitative PCR (qRT-PCR). Then the mice were divided into Antioxidant group and Saline group according to the anti oxidation pretreatment using manganese porphyrin (MnTMPyP). The expression of microRNA-146 was observed in reverse experiment after antioxidant treatment.
3. the pre cultured primary renal tubular epithelial cells (TEC) were tested in vitro, and the cells were divided into Stress group and Sham group according to whether H2O2 was used. QRT-PCR was used to detect the same dose of H2O2, and the expression of miR-146 in primary TEC at the same time with different doses of H2O2 stimulation. Then the wax oil was used to construct TEC. In vitro IR model was used to simulate the cellular IR process and further observe the expression of miR-146.
Result:
The mice in group 1.IR died in part of 3D after operation, but the total survival rate was more than that of 80%. after the operation. The level of blood creatinine and urea nitrogen in the IR group was significantly higher than that in the group Sham. After HE staining, the renal tubular epithelial cells in the IR group were obviously edema, partial necrosis, and more inflammatory cells infiltrated in the renal interstitium.
2. after 6h reperfusion, the expression of miR-146a was statistically significant (P 0.05), and the expression reached peak value after 24h, which was about 7.4 + 1.9 times (P 0.001) in Sham group. The expression level of miR-146a was 5.7 + 1.3 times as of Sham group (P 0.001). There was no significant change in miR-146b table during the period of reperfusion (P0.05). The renal tissue loss was given after antioxidant treatment. After 24 hours of reperfusion, the increase of the expression of microRNA-146 a in the Antioxidant group was significantly down-regulated compared with that in the Saline group (P 0.05).
3. using H2O2 to stimulate the primary TEC, it was found that the expression of miR-146a in the above cells increased. The miR-146a expression changes after 6h were statistically significant (P 0.05), after 24h, the maximum value was reached, about 15.3 + 3.8 times of the control group (P 0.001), and its duration exceeded 48h (P 0.001). The miR-146b expression was not significantly changed during the period (P0.05). Further study It was found that the change of miR-146a expression was dose dependent on H2O2 stimulation.
4. in the cell in vitro IR model, we found that the miR-146a expression in the primary TEC of 24h was significantly up-regulated (TEC 0.05) after replacement medium (simulated reperfusion).
Conclusion:
1. the expression level of miR-146a in IR after renal injury increased significantly after reperfusion 6h, and reached the peak after 24h, which lasted for more than 72h..
2. the main reason for the increase of miR-146a expression after renal IR injury may be the oxidative stress reaction during IR.
3. in view of the background of miR-146 research, the expression of up regulated miR-146a in the renal IR process may play a role in alleviating inflammatory and tissue damage, and has important clinical value in the future. In addition, because of the early changes of miR-146a expression during the process of IR, the potential biological marker function can not be ignored.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R699.2
【參考文獻】
相關(guān)期刊論文 前2條
1 ;Virus-Encoded microRNAs: Future Therapeutic Targets?[J];Cellular & Molecular Immunology;2006年06期
2 吳玨;劉芬;阮瓊芳;婁遠蕾;郭菲;楊陽;汪泱;;miRNA-92a在缺血再灌注腎損傷中的表達變化[J];實驗與檢驗醫(yī)學;2010年01期
本文編號：2142525
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