小型猪肾损伤疾病模型的构建及干预研究

发布时间：2018-04-11 12:04

本文选题：庆大霉素 + 自噬　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：目的：急性肾损伤（acute kidney injury, AKI）是一种常见，具有高致死率的特点的临床综合征，目前尚缺乏有效的预防和治疗措施。同时，AKI也是导致慢性肾脏病和终末期肾病最终进展的重要因素，给社会带来了巨大的经济负担。庆大霉素等氨基糖苷类抗生素是治疗革兰阴性菌感染最常用的抗生素。肾毒性和耳毒性等严重并发症极大限制了氨基糖苷类抗生素的临床应用。目前，研究发现氧化损伤在庆大霉素肾毒性的发生中具有重要作用。自噬（autophagy）是细胞中清除损伤生物大分子和线粒体等细胞器，维持细胞稳态的重要保护机制。同时，自噬具有减轻细胞氧化损伤的作用。目前，自噬在庆大霉素诱导肾毒性中的作用机制以及具有抗氧化作用的N-乙酰半胱氨酸（NAC)和自噬增强剂雷帕霉素对庆大霉素肾损伤中自噬的影响还未明确。本研究拟构建庆大霉素肾损伤小型猪模型，观察在不同庆大霉素给药时间肾脏自噬水平，氧化损伤，炎症和凋亡的变化。此外，观察N-乙酰半胱氨酸和雷帕霉素对庆大霉素肾病理损伤，肾脏氧化损伤，凋亡和自噬的影响。这对于进一步明确庆大霉素肾损伤的发生和发展机制以及探索改善庆大霉素肾毒性的新型治疗方案具有重要意义。方法：第一，我们采用5,6.85,10,15,20,40,60,80和100mg/kg庆大霉素连续肌注10天，观察小型猪血生化和肾脏病理损伤。第二，连续10天肌注80mg/kg庆大霉素，观察0天，1天，3天，5天，7天和10天组肾脏病理，血生化，凋亡，氧化损伤，炎症和自噬的变化。第三，我们观察给予N-乙酰半胱氨酸或雷帕霉素干预后，庆大霉素肾损伤中肾脏病理损伤，血生化，氧化损伤以及自噬的变化。结果：首先，在剂量依赖性研究中，与正常对照组相比，80mg/kg组和100mg/kg组小型猪血肌酐和尿素氮水平显著升高，肾脏病理病理损伤显著加重。第二，在时间依赖性研究中，与0天组相比，10天组血肌酐和尿素氮水平显著升高，10天组肾脏损伤显著加重，此外，与0天组小型猪相比，炎症因子表达显著升高，凋亡加重，线粒体自噬标志物Bnip3以及其上游转录因子HIF-1α显著降低，自噬流标志物p62/SQSTM1和多聚泛素聚集体表达显著升高，凋亡，蛋白质，脂质和DNA氧化损伤加重。第三，，我们观察了庆大霉素注射10天的同时，给予N-乙酰半胱氨酸和雷帕霉素干预。与庆大霉素模型组相比，N-乙酰半胱氨酸组和雷帕霉素组血肌酐和尿素氮水平，肾小管损伤评分均无显著性改变。此外，肾脏自噬流标志物p62/SQSTM1和多聚泛素聚集体表达显著降低，Bnip3以及其上游转录因子HIF-1α显著升高，氧化损伤标志物8-OHdG，4HNE和蛋白质羰基表达下降，凋亡细胞减少。结论：随着庆大霉素给药时间的延长，小型猪肾脏的自噬功能下降，近端肾小管上皮细胞中线粒体肿胀和嵴断裂加剧，凋亡细胞数目增多和氧化损伤显著加重,表明自噬功能受损可能在庆大霉素肾毒性的发生和发展中发挥重要作用。N-乙酰半胱氨酸和雷帕霉素可减轻庆大霉素肾损伤的氧化损伤，凋亡水平，但尚未显著改善庆大霉素肾毒性的血生化和肾脏病理损伤。
[Abstract]:Objective: acute kidney injury (acute kidney, injury, AKI) is a common clinical features with high death rate of the syndrome, there is still a lack of effective prevention and treatment measures. At the same time, AKI is also an important factor leading to chronic kidney disease and end-stage renal disease eventually progress, brought huge economic burden to the society aminoglycoside antibiotics such as gentamicin. Treatment of leather is the most commonly used antibiotics of Gram-negative bacteria infection. Serious complications such as nephrotoxicity and ototoxicity has greatly limited the clinical application of aminoglycoside antibiotics. At present, the study found that oxidative damage plays an important role in the occurrence of gentamicin nephrotoxicity. Autophagy (autophagy) is to remove the biological damage molecular and cell organelles such as cells, an important protective mechanism for maintaining cellular homeostasis. At the same time, autophagy has decreased cell oxidative damage. At present, autophagy in. The mechanism of action of mycin induced nephrotoxicity and the effect of N- acetylcysteine (NAC) and rapamycin, a antioxidant enhancer, on the autophagy in gentamicin induced renal injury are not yet clear.
This study intends to construct gentamicin renal injury models of pigs, were observed at different time of gentamicin administration renal autophagy, oxidative damage, inflammatory changes and apoptosis. In addition, the observation of N- acetylcysteine and rapamycin on renal pathological injury of kidney of gentamicin, oxidative damage, apoptosis and autophagy. It has important significance for new treatment to further clarify the occurrence and development of renal injury and explore the mechanism of gentamicin gentamycin improve renal toxicity.
Methods: first, we use 5,6.85,10,15,20,40,60,80 and 100mg/kg for 10 days to observe the intramuscular injection of gentamicin, small blood biochemical and renal pathological damage. Second, 10 consecutive days of intramuscular injection of 80mg/kg gentamicin, observed for 0 days, 1 days, 3 days, 5 days, 7 days and 10 days were kidney pathology, blood biochemistry, apoptosis, oxidative damage, changes inflammation and autophagy. Third, we observe the N- acetylcysteine or rapamycin intervention, renal damage, blood biochemical gentamicin renal injury, oxidative damage and autophagy changes.
Results: first, in a dose-dependent study, compared with the normal control group, the serum creatinine and urea nitrogen levels of 80mg/kg group and 100mg/kg group increased significantly, and the pathological and pathological lesions of kidney increased significantly.
In second, time dependent study, compared with the 0 day group, 10 day group, serum creatinine and blood urea nitrogen levels increased significantly, 10 days group renal injury significantly increased, in addition, compared with the 0 day mini pigs, the expression of inflammatory factors increased significantly, apoptosis increased, mitochondrial autophagy marker Bnip3 and its upstream transcription factor HIF-1 alpha was significantly reduced, flow autophagy markers p62/SQSTM1 and polyubiquitin aggregates significantly increased expression, apoptosis, protein, lipid and increased oxidative DNA damage.
Third, we observed and gentamicin injection for 10 days, give N- acetylcysteine and rapamycin. Compared with gentamicin group, N- acetylcysteine group and rapamycin group, serum creatinine and blood urea nitrogen levels, there were no significant changes in the renal tubular injury score. In addition, renal flow autophagy markers p62/SQSTM1 and polyubiquitin aggregates expression significantly, Bnip3 and its upstream transcription factor HIF-1 alpha significantly increased oxidative damage markers 8-OHdG, 4HNE and protein carbonyl decreased expression of apoptotic cells decreased.
Conclusion: with the prolonging of gentamicin administration time, the function of autophagy miniature pig kidney decreased proximal increased mitochondrial swelling and cristae in renal tubular epithelial cells, and the number of apoptotic cells increased significantly aggravated oxidative damage, demonstrated the role of.N- acetylcysteine and rapamycin, the occurrence and development of the function of autophagy may be impaired in Gentamycin nephrotoxicity. Can alleviate the oxidative damage, gentamicin renal injury level of apoptosis, but not significantly improve gentamycin nephrotoxicity of blood biochemical and renal pathological damage.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.5;R-332

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