急性肾损伤微环境对培养骨髓间充质干细胞分化及分裂增殖的影响

Differentiation,division and proliferation of cultured mesenchymal stem cells under acute kidney injury microenvironment

目的:观察体外模拟急性肾损伤(acute kidney injury,AKI)的微环境下,小鼠骨髓间充质干细胞(mouse mesenchymal stem cells,mMSCs)分化及分裂增殖情况。方法:采用夹闭雄性C57BL/6小鼠双侧肾蒂30min再开放30min的方法制作缺血再灌注(I/R)性AKI鼠模型,即刻取双侧肾脏皮质制作I/R肾脏匀浆上清。抽取C57BL/6小鼠的骨髓,经Percoll密度梯度离心联合贴壁培养法分离纯化出mMSCs,以流式细胞仪鉴定。取扩增3代的mMSCs分组培养:(1)对照组:含10%胎牛血清的低糖DMEM培养基;(2)干预组:含10%胎牛血清的低糖DMEM培养基+I/R肾脏匀浆上清。诱导1d、3d、5d、7d后倒置显微镜下观察细胞形态学变化;透射电镜观察细胞超微结构;流式细胞仪检测角蛋白18(cytokeratin18,CK18);CCK-8法检测培养mMSCs的增生;TUNEL法检测mMSCs凋亡。结果:分离获得的P3-mMSCs高表达CD29和CD44,低表达CD34和CD45。与对照组长梭形细胞相比,干预组第3天可见部分细胞为椭圆形、短梭形,至第7天大部分细胞呈圆形、椭圆形、短胖梭形;透射电镜也观察到胞质内开始出现较多的粗面内质网、溶酶体、线粒体。流式细胞仪检测发现,对照组mMSCs内仅有极微量CK18表达,而干预组CK18阳性表达率显著增加。经I/R肾脏匀浆上清干预后,不同时间点mMSCs的增殖效应均显著减弱,而TUNEL检测显示胞核染色阳性的细胞百分比有显著升高(P<0.01)。结论:体外模拟的AKI微环境可诱导mMSCs部分分化为肾小管上皮样细胞,但同时也会导致培养的mMSCs凋亡,增殖能力减弱,进而减少了可肾向分化的mMSCs数量,推测这可能是MSCs体内移植促肾修复能力有限的原因之一。

Objective： Establishing mice＇s model with ichemia-reperfusion （l/R） kidney injury, drawing bilateral renal cortex to make kidney homogenate, culturing mouse mesenchymal stem cells （mMSCs） with kidney homogenate to simulate acute kidney injury （AKI） microenvironment, and to investigate mMSCs＇ differentiation and replication under this Methodology： To make AKI mice models by clamping bilateral renal pedicles 30 minutes and reopening 30 minutes. Then immediately drew bilateral renal cortex to make I/R kidney homogenate supernatant. C57BL/6 mice＇s mMSCs had been successfully isolated by percoll density gradient centrifugation and adherence cultivation, their surface markers were identified by flow cytometry. Then V3-mMSCs were treated with different group： control group （low glucose DMEM medium with 10% fetal bovine serum）, and intervention group （low glucose DMEM medium with 10% fetal bovine serum plus I/R kidney homogenate supernatant ）. Each group was incubated for 1 d, 3d, 5d, and 7d, morphological changes of these cells were observed by inverted microscope and uhrastructure changes were examined by transmission electron microscope. Cytokeratin-18, proliferation of mMSCs, and apoptosis were detected by flow cytometry, CCK-8 and TUNEL respectively. Results： The cells （P3-mMSC） were highly expressed CD29 and CD44, lowly expressed CD34 and CIMS. Compared with the control group, the cells of intervention group presented oval shape, and short fusiform on the third day, round shape, oval shape, and short-pang fusiform on the 7th day. At the same time, there were much rough endoplasmic, lysosome, and mitochondria appearing in cytoplasm. There was only trace expression of CK18 in control group, while the expression rate was increased significantly after intervention of the I/R kidney homogenate supernatant. The I/R kidney homogenate supernatant could alleviate the proliferation ability of mMSCs while the apoptosis percentage increased significantly （P 〈 0. 01 ）. Conclusion：AKI microenvironment can not only induce mMSCs partially to transfer to renal tubular epithelial -shaping cells,but also induce mMSCs＇ apoptosis, weaken their proliferation ability, and as a result, decrease the number of mMSCs that can make trans-differentiation. It may be a reason that renal repair ability of MSCs＇transplantation is limited.