促红细胞生成素对急性肾损伤微环境下骨髓间充质干细胞分化及分泌功能的影响

Effects of erythropoietin on mesenchymal stem cells＇ function of differentiation and secretion cultured under acute kidney injury microenvironment

目的观察经促红细胞生成素（EPO）干预后，体外模拟急性肾损伤（AKI）微环境下培养骨髓间充质干细胞（BM-MSC）的分化及分泌功能变化。方法Percoll密度梯度离心联合贴壁培养法分离C57BL／6小鼠BM-MSC（mBM-MSC）。构建缺血再灌注（I／R）诱导AKI小鼠模型，制作健康小鼠及I／R小鼠肾脏匀浆上清。取扩增3代的mBM-MSC分组培养：A组：含10％胎牛血清的低糖DMEM培养液培养；B组：健康小鼠肾脏匀浆上清干预；C组：I／R小鼠肾脏匀浆上清干预，体外模拟AKI微环境；D组：I／R小鼠肾脏匀浆上清＋不同浓度EPO（1、5、10、50U／m1）干预；培养1、3、5、7d。倒置显微镜观察细胞形态，透射电镜观察细胞超微结构，流式细胞仪检测细胞角蛋白18（CKl8），酶联免疫吸附试验（ELISA）检测培养上清液中骨形态发生蛋白7（BMP-7）、肝细胞生长因子（HGF）、血管内皮生长因子（VEGF）水平。结果分离获得的P3一mBM-MSC高表达CD29和CIM4，低表达CD34和CIM5。与A、B组长梭形细胞相比，C组部分细胞出现椭圆形、短梭形、圆形外观，EPO干预可使细胞呈现鹅卵石样外观，且C、D组细胞的胞质内细胞器丰富，并可见微绒毛及桥粒。A、B组mBM-MSC内仅有极微量CKl8表达，C、D组CKl8阳性表达率显著高于A、B组（均P〈0．01），50U／mlEPO干预5、7d后CKl8表达量显著高于同时间点C组（5d：35．22±4．04比8．72±0．38，7d：42．00±5．39比13．20±1．14，均P〈0．01）。A、B组上清液中均见有BMP-7、HGF、VEGF表达，两组间差异无统计学意义，C组表达量均显著低于A、B组（P〈0．05或P〈0．01）。结论EPO干预可增强mBM-MSC的分化功能，并逆转其低分泌效应。

Objective To explore the effects of erythropoietin （EPO） on the differentiation and secretion of euhured bone marrow-derived mesenchymal stem cells （ BM-MSC ） in the microenvironment of acute kidney injury （AKI）. Methods C57BL/6 murine BM-MSC （mBM-MSC） were successfully isolated by the methods of Percoll density gradient centrifugation and adherence cultivation. The AKI murine model was induced by ischemia/reperfusion （I/R）. The homogenate supernatants were prepared for normal and I/R murine kidney. P3-mBM-MSC were treated differently： Group A： low glucose DMEM medium with 10% fetal bovine serum, Group B： normal murine kidney homogenate supernatant intervention, Group C： I/R kidney homogenate supernatant intervention, Group D： I/R kidney homogenate supernatant plus different concentrations of EPO （ 1, 5, 10, 50 U/ml）. Each group was incubated for 1, 3, 5 and 7 days. Inverted microscope was used to observe the morphological changes of these cells and their ultrastructural changes were observed by transmission electron microscope. Cytokeratin-18 was detected by flow cytometry. The levels of bone morphogenetic protein-7 （ BMP-7）, hepatoeyte growth factor （HGF） and vascular endothelial growth factor （VEGF） were detected by ELISA in culture medium. Results The cells yielded a high expression of CD29 and CD44 and a low expression of CD34 and CIM5. Compared with Groups A and B, thecells of Group C presented oval and short fusiform shapes. After the intervention of EPO, Group D showed a cobble appearance. More organelles were also found. A trace expression of CK18 was found in Groups A and B. A positive expression of CK18 was significantly higher in Groups C and D than Groups A and B （P 〈 0. 01 ）. The expression of EPO 50 U/ml at Day 5 and 7 was higher than Group C of the same time （5 d ： 35.22 ± 4.04 vs 8.72 ±O. 38, 7 d ：42.00±5.39 vs 13.20 ± 1.14, both P 〈 0.01 ）. The results of ELISA showed that the levels of BMP-7, HGF and VEGF in Group C decreased significantly （ P 〈 0. O1 or P 〈 0.05 ）. Conclusion The intervention of EPO may boost the differentiation of mBM-MSC but reverse its low secretion.