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AGGF1-EPCs对小鼠急性肾损伤保护作用的研究
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Abstract:
目的：探究AGGF1预处理的EPCs对肾IRI的治疗作用。方法：用淋巴细胞分离液(histopaque-1083)离心分离出单个核细胞层，分离的单个核细胞(Bone marrow mononuclear cells, MNCs)进行鉴定(Dil-ac-LDL和FITC-UEA-1染色)后用分离纯化的AGGF1蛋白预处理(0.5 μg/mL，洗涤后尾静脉注射) 12 h，之后进行小鼠细胞治疗。小鼠分Sham组、生理盐水组(Saline组，I/R小鼠)、EPCs组(移植EPCs，I/R小鼠)、AGGF1-EPCs组(移植AGGF1蛋白预处理的EPCs，I/R小鼠)共4组，为了评估AGGF1预处理的EPCs植入疗法对肾IRI的影响，我们实施了免疫荧光实验、免疫组化和ELISA等实验。结果：实验结果显示，MNCs来源的EPCs呈现Dil-ac-LDL和FITC-UEA-1阳性。ELISA实验结果显示，AGGF1-EPCs组小鼠的尿素氮水平(BUN)，肌酐(Creatine)与急性肾损伤标志物NGAL水平均较Saline组、EPCs组显著下降，同时，免疫组化分析也显示AGGF1-EPCs组小鼠肾脏的F4/80较Saline组、EPCs组显著下降。此外，血清IL-1β，TNF-α的检测结果也显示，炎症因子(IL-1β, TNF-α)在AGGF1-EPCs组小鼠的中表达最低。结论：AGGF1预处理的EPCs植入疗法可以显著降低缺血再灌注引起的急性肾损伤与炎症反应，对IRI具有良好的改善保护作用，这为未来肾IR的临床干预提供了一种新的潜在治疗方案。
Objective: To explore the therapeutic effect of AGGF1 primed-EPCs on renal ischemia-reperfusion injury (IRI). Methods: We centrifuge and separate the bone marrow mononuclear cells (MNCs) using histopaque-1083, and identify the MNCs with Dil-ac-LDL and FITC-UEA-1 staining. After pretreatment with purified AGGF1 protein (0.5 μg/mL), EPCs were washed and injected into the C57BL/6J mice via tail vein. The mice were divided into four groups: Sham group, Saline group (I/R mice), EPCs group (transplanted EPCs, I/R mice), and AGGF1-EPCs group (EPCs pretreated with AGGF1 protein, I/R mice). To evaluate the effect of EPCs implantation on renal IRI, we carried out western blot, ELISA, and immunohistochemistry experiments. Results: Our results showed that EPCs derived from MNCs exhibited Dil-ac-LDL and FITC-UEA-1 positive. The ELISA results showed that the levels of urea nitrogen (BUN), creatinine and acute kidney injury marker NGAL in the AGGF1-EPCs group mice were significantly lower than those in the Saline group and EPCs group. At the same time, immunohistochemical analysis also showed that the NGAL levels and F4/80 in the kidneys of AGGF1-EPCs group mice were significantly lower than those in the Saline group and EPCs group. In addition, we also observed that the inflammatory factors IL-1β, TNF-α were significantly reduced in the AGGF1-EPCs group of mice. Conclusion: The implantation therapy of AGGF1 primed-EPCs significantly reduce acute renal injury and inflammatory response caused by ischemia-reperfusion. This provides a new potential treatment for clinical intervention of renal IR.
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