CAJ | 학술논문

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、50 U/ml)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(CK18),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-mBM-MSC高表达CD29和CD44,低表达CD34和CD45.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CK18表达,C、D组CK18阳性表达率显著高于A、B组(均P＜0.01),50 U/mlEPO干预5、7d后CK18表达量显著高于同时间点C组(5 d:35.22 ±4.04比8.72±0.38,7 d: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.
목적 관찰경촉홍세포생성소(EPO)간예후,체외모의급성신손상(AKI)미배경하배양골수간충질간세포(BM-MSC)적분화급분비공능변화.방법 Percoll밀도제도리심연합첩벽배양법분리C57BL/6소서BM-MSC(mBM-MSC).구건결혈재관주(I/R)유도AKI소서모형,제작건강소서급I/R소서신장균장상청.취확증3대적mBM-MSC분조배양:A조:함10％태우혈청적저당DMEM배양액배양；B조:건강소서신장균장상청간예；C조:I/R소서신장균장상청간예,체외모의AKI미배경；D조:I/R소서신장균장상청+불동농도EPO(1、5、10、50 U/ml)간예；배양1、3、5、7d.도치현미경관찰세포형태,투사전경관찰세포초미결구,류식세포의검측세포각단백18(CK18),매련면역흡부시험( ELISA)검측배양상청액중골형태발생단백7(BMP-7)、간세포생장인자(HGF)、혈관내피생장인자(VEGF)수평.결과 분리획득적P3-mBM-MSC고표체CD29화CD44,저표체CD34화CD45.여A、B조장사형세포상비,C조부분세포출현타원형、단사형、원형외관,EPO간예가사세포정현아란석양외관,차C、D조세포적포질내세포기봉부,병가견미융모급교립.A、B조mBM-MSC내부유겁미량CK18표체,C、D조CK18양성표체솔현저고우A、B조(균P＜0.01),50 U/mlEPO간예5、7d후CK18표체량현저고우동시간점C조(5 d:35.22 ±4.04비8.72±0.38,7 d:42.00±5.39비13.20±1.14,균P＜0.01).A、B조상청액중균견유BMP-7、HGF、VEGF표체,량조간차이무통계학의의,C조표체량균현저저우A、B조(P ＜0.05혹P＜0.01).결론 EPO간예가증강mBM-MSC적분화공능,병역전기저분비효응.
Objective 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).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),hepatocyte 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 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).Conclusion The intervention of EPO may boost the differentiation of mBM-MSC but reverse its low secretion.