肾脏缺血再灌注后MicroRNA-146的表达变化及其调控机制
发布时间：2018-07-24 20:12
【摘要】：背景： 继1975年研究者发现缺血器官复氧后原有损伤加重，并提出缺血再灌注(IschemicReperfusion, IR)损伤的概念之后，上述现象已逐步受到国内外众多学者的重视。器官IR损伤常发生于阻断血氧供应的外科手术以及影响全身循环的多器官病变中。肾脏作为高灌注器官，对IR损伤的耐受性更差。IR损伤往往对肾脏产生不利影响，严重者可导致肾功能进行性恶化或恢复延迟，甚至威胁生命。 随着近年来对非编码RNA（Non-coding RNAs）研究的逐步深入，越来越多的实验表明MicroRNA（微小RNA，miRNA）在许多病理生理过程中发挥着重要作用。而在目前已发现的众多功能各异的miRNA中， miR-146是一类具有免疫调控功能的miRNA，通过与上游靶基因结合调节下游蛋白质的翻译，借此在免疫应答、炎症反应、肿瘤发生等许多领域发挥调控作用。在前期研究中，本课题组已经发现一系列miRNA在肾脏IR损伤后表达水平发生变化，其中包括miR-146，我们推测miR-146可能参与调控IR损伤，但其机制尚未明确。 目的： 本实验以小鼠肾脏热IR损伤模型和体外细胞模型为基础，探讨IR损伤对肾脏实质细胞中miR-146表达的影响和调控机制，以期能够为临床和科研中有效减轻并及时发现肾脏IR损伤提供新的思路和研究靶点。 1.建立肾脏热缺血IR损伤动物模型。 2.利用肾脏热缺血IR损伤动物模型，在体内实验中观察肾脏IR后miR-146的表达变化并分析其可能的原因。 3.通过体外细胞模型，进一步研究IR及氧化应激反应后肾脏实质细胞内miR-146的表达变化并分析其可能的原因。 方法： 1.以近交系小鼠为研究对象，构建肾脏热缺血IR损伤动物模型。检测IR后小鼠的生存率、体重变化、血肌酐和尿素氮水平，借此评估动物模型。 2.利用上述动物模型进行实验，将小鼠分为Sham组和IR组。在不同时间点切取受损肾脏，应用实时荧光定量PCR（qRT-PCR）技术检测上述组织内miR-146的表达情况。之后按是否使用锰卟啉（MnTMPyP）进行抗氧化预处理将小鼠分为Antioxidant组和Saline组，在反向实验中观察抗氧化处理后miR-146的表达变化。 3.利用预先培养的原代肾小管上皮细胞（TEC）进行体外试验，按是否使用H2O2刺激将细胞分为Stress组和Sham组，利用qRT-PCR检测相同剂量H2O2刺激不同时长，以及用不同剂量H2O2刺激相同时长后原代TEC内miR-146的表达情况。之后利用石蜡油构建TEC的体外IR模型，模拟细胞IR过程，进一步观察miR-146的表达情况。 结果： 1.IR组小鼠术后3d内部分死亡，但总生存率超过80%。术后出现显著体重减轻、精神萎靡、活动减少。与Sham组相比，IR组小鼠血肌酐和尿素氮水平显著升高。经HE染色后镜下可见IR组肾小管上皮细胞明显水肿，部分坏死，肾间质内较多炎细胞浸润。 2.肾脏再灌注6h后miR-146a表达变化具有统计学意义（P 0.05），24h后表达达到峰值，此时约为Sham组的7.4±1.9倍（P 0.001）。截至再灌注72h，miR-146a表达水平仍为Sham组的5.7±1.3倍（P 0.001），期间miR-146b表达无显著变化（P0.05）。给予抗氧化处理后，肾脏组织损伤明显减轻。再灌注24h后Antioxidant组小鼠miR-146a表达的升高幅度出现显著下调，与Saline组相比差异有统计学意义（P 0.05）。 3.利用H2O2对原代TEC进行刺激后，发现上述细胞中miR-146a表达升高。刺激6h后miR-146a表达变化具有统计学意义（P 0.05），24h后达到最高值，约为对照组的15.3±3.8倍（P 0.001），其持续时间超过48h（P 0.001）。期间miR-146b表达无显著变化（P0.05）。进一步研究发现，miR-146a表达变化对H2O2刺激呈剂量依赖性。 4.在细胞体外IR模型中，我们发现更换培养基（模拟再灌注）后24h内，离体的原代TEC内miR-146a表达显著上调（P 0.05）。 结论: 1.肾脏IR损伤后miR-146a表达水平在再灌注6h后发生显著上调，并于再灌注后24h后达峰，持续时间超过72h。 2.肾脏IR损伤后miR-146a表达上调的主要原因可能是是IR过程中的氧化应激反应。 3.鉴于miR-146的研究背景，在肾脏IR过程中表达上调的miR-146a可能具有减轻炎症反应和组织损伤的作用，在未来具有重要的临床应用价值。此外，，由于IR过程中miR-146a表达改变发生较早，其潜在的生物学标志功能也不容忽视。
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R699.2
本文编号：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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R699.2
【参考文献】
相关期刊论文 前2条
1 ;Virus-Encoded microRNAs: Future Therapeutic Targets?[J];Cellular & Molecular Immunology;2006年06期
2 吴珏;刘芬;阮琼芳;娄远蕾;郭菲;杨阳;汪泱;;miRNA-92a在缺血再灌注肾损伤中的表达变化[J];实验与检验医学;2010年01期
本文编号：2142525
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