腎臟缺血再灌注后MicroRNA-146的表達(dá)變化及其調(diào)控機(jī)制
發(fā)布時(shí)間：2018-07-24 20:12
【摘要】：背景： 繼1975年研究者發(fā)現(xiàn)缺血器官?gòu)?fù)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(guó)內(nèi)外眾多學(xué)者的重視。器官IR損傷常發(fā)生于阻斷血氧供應(yīng)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(duì)IR損傷的耐受性更差。IR損傷往往對(duì)腎臟產(chǎn)生不利影響，嚴(yán)重者可導(dǎo)致腎功能進(jìn)行性惡化或恢復(fù)延遲，甚至威脅生命。 隨著近年來(lái)對(duì)非編碼RNA（Non-coding RNAs）研究的逐步深入，越來(lái)越多的實(shí)驗(yàn)表明MicroRNA（微小RNA，miRNA）在許多病理生理過(guò)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(guò)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(yīng)答、炎癥反應(yīng)、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(dá)水平發(fā)生變化，其中包括miR-146，我們推測(cè)miR-146可能參與調(diào)控IR損傷，但其機(jī)制尚未明確。 目的： 本實(shí)驗(yàn)以小鼠腎臟熱IR損傷模型和體外細(xì)胞模型為基礎(chǔ)，探討IR損傷對(duì)腎臟實(shí)質(zhì)細(xì)胞中miR-146表達(dá)的影響和調(diào)控機(jī)制，以期能夠?yàn)榕R床和科研中有效減輕并及時(shí)發(fā)現(xiàn)腎臟IR損傷提供新的思路和研究靶點(diǎn)。 1.建立腎臟熱缺血IR損傷動(dòng)物模型。 2.利用腎臟熱缺血IR損傷動(dòng)物模型，在體內(nèi)實(shí)驗(yàn)中觀察腎臟IR后miR-146的表達(dá)變化并分析其可能的原因。 3.通過(guò)體外細(xì)胞模型，進(jìn)一步研究IR及氧化應(yīng)激反應(yīng)后腎臟實(shí)質(zhì)細(xì)胞內(nèi)miR-146的表達(dá)變化并分析其可能的原因。 方法： 1.以近交系小鼠為研究對(duì)象，構(gòu)建腎臟熱缺血IR損傷動(dòng)物模型。檢測(cè)IR后小鼠的生存率、體重變化、血肌酐和尿素氮水平，借此評(píng)估動(dòng)物模型。 2.利用上述動(dòng)物模型進(jìn)行實(shí)驗(yàn)，將小鼠分為Sham組和IR組。在不同時(shí)間點(diǎn)切取受損腎臟，應(yīng)用實(shí)時(shí)熒光定量PCR（qRT-PCR）技術(shù)檢測(cè)上述組織內(nèi)miR-146的表達(dá)情況。之后按是否使用錳卟啉（MnTMPyP）進(jìn)行抗氧化預(yù)處理將小鼠分為Antioxidant組和Saline組，在反向?qū)嶒?yàn)中觀察抗氧化處理后miR-146的表達(dá)變化。 3.利用預(yù)先培養(yǎng)的原代腎小管上皮細(xì)胞（TEC）進(jìn)行體外試驗(yàn)，按是否使用H2O2刺激將細(xì)胞分為Stress組和Sham組，利用qRT-PCR檢測(cè)相同劑量H2O2刺激不同時(shí)長(zhǎng)，以及用不同劑量H2O2刺激相同時(shí)長(zhǎng)后原代TEC內(nèi)miR-146的表達(dá)情況。之后利用石蠟油構(gòu)建TEC的體外IR模型，模擬細(xì)胞IR過(guò)程，進(jìn)一步觀察miR-146的表達(dá)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(guò)80%。術(shù)后出現(xiàn)顯著體重減輕、精神萎靡、活動(dòng)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(jiàn)IR組腎小管上皮細(xì)胞明顯水腫，部分壞死，腎間質(zhì)內(nèi)較多炎細(xì)胞浸潤(rùn)。 2.腎臟再灌注6h后miR-146a表達(dá)變化具有統(tǒng)計(jì)學(xué)意義（P 0.05），24h后表達(dá)達(dá)到峰值，此時(shí)約為Sham組的7.4±1.9倍（P 0.001）。截至再灌注72h，miR-146a表達(dá)水平仍為Sham組的5.7±1.3倍（P 0.001），期間miR-146b表達(dá)無(wú)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(dá)的升高幅度出現(xiàn)顯著下調(diào)，與Saline組相比差異有統(tǒng)計(jì)學(xué)意義（P 0.05）。 3.利用H2O2對(duì)原代TEC進(jìn)行刺激后，發(fā)現(xiàn)上述細(xì)胞中miR-146a表達(dá)升高。刺激6h后miR-146a表達(dá)變化具有統(tǒng)計(jì)學(xué)意義（P 0.05），24h后達(dá)到最高值，約為對(duì)照組的15.3±3.8倍（P 0.001），其持續(xù)時(shí)間超過(guò)48h（P 0.001）。期間miR-146b表達(dá)無(wú)顯著變化（P0.05）。進(jìn)一步研究發(fā)現(xiàn)，miR-146a表達(dá)變化對(duì)H2O2刺激呈劑量依賴性。 4.在細(xì)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(dá)顯著上調(diào)（P 0.05）。 結(jié)論: 1.腎臟IR損傷后miR-146a表達(dá)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(dá)峰，持續(xù)時(shí)間超過(guò)72h。 2.腎臟IR損傷后miR-146a表達(dá)上調(diào)的主要原因可能是是IR過(guò)程中的氧化應(yīng)激反應(yīng)。 3.鑒于miR-146的研究背景，在腎臟IR過(guò)程中表達(dá)上調(diào)的miR-146a可能具有減輕炎癥反應(yīng)和組織損傷的作用，在未來(lái)具有重要的臨床應(yīng)用價(jià)值。此外，，由于IR過(guò)程中miR-146a表達(dá)改變發(fā)生較早，其潛在的生物學(xué)標(biāo)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R699.2
本文編號(hào)：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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【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 ;Virus-Encoded microRNAs: Future Therapeutic Targets?[J];Cellular & Molecular Immunology;2006年06期
2 吳玨;劉芬;阮瓊芳;婁遠(yuǎn)蕾;郭菲;楊陽(yáng);汪泱;;miRNA-92a在缺血再灌注腎損傷中的表達(dá)變化[J];實(shí)驗(yàn)與檢驗(yàn)醫(yī)學(xué);2010年01期
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