Liu Nan-mei, Tian Jun, Wang Wei-wei, Han Guo-feng, Cheng Jin, Huang Jian, Zhang Jin-yuan
Department of Nephrology, No. 455 Hospital of PLA, Shanghai, China.
Zhonghua Yi Xue Za Zhi. 2012 Feb 14;92(6):417-21.
To explore the effects of erythropoietin (EPO) on the differentiation and secretion of cultured bone marrow-derived mesenchymal stem cells (BM-MSC) in the microenvironment of acute kidney injury (AKI).
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), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) were detected by ELISA in culture medium.
The cells yielded a high expression of CD29 and CD44 and a low expression of CD34 and CD45. Compared with Groups A and B, the cells 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 ± 0.38, 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 < 0.01 or P < 0.05).
The intervention of EPO may boost the differentiation of mBM-MSC but reverse its low secretion.
探讨促红细胞生成素（EPO）对急性肾损伤（AKI）微环境中培养的骨髓间充质干细胞（BM-MSC）分化及分泌的影响。
采用Percoll密度梯度离心法和贴壁培养法成功分离C57BL/6小鼠骨髓间充质干细胞（mBM-MSC）。通过缺血/再灌注（I/R）诱导建立AKI小鼠模型。制备正常小鼠和I/R小鼠肾脏的匀浆上清液。对P3-mBM-MSC进行不同处理：A组：含10%胎牛血清的低糖DMEM培养基；B组：正常小鼠肾脏匀浆上清液干预；C组：I/R肾脏匀浆上清液干预；D组：I/R肾脏匀浆上清液加不同浓度EPO（1、5、10、50 U/ml）。每组孵育1、3、5和7天。用倒置显微镜观察细胞形态变化，用透射电子显微镜观察其超微结构变化。通过流式细胞术检测细胞角蛋白-18。用酶联免疫吸附测定法（ELISA）检测培养基中骨形态发生蛋白-7（BMP-7）、肝细胞生长因子（HGF）和血管内皮生长因子（VEGF）的水平。
细胞高表达CD29和CD44，低表达CD34和CD45。与A组和B组相比，C组细胞呈椭圆形和短梭形。EPO干预后，D组呈现鹅卵石样外观，且细胞器增多。A组和B组中发现CK18微量表达。C组和D组中CK18阳性表达显著高于A组和B组（P<0.01）。第5天和第7天50 U/ml EPO组的表达高于同期C组（5天：35.22±4.04对8.72±0.38，7天：42.00±5.39对13.20±1.14，均P<0.01）。ELISA结果显示，C组中BMP-7、HGF和VEGF水平显著降低（P<0.01或P<0.05）。
EPO干预可能促进mBM-MSC的分化，但逆转其低分泌状态。
