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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: To observe the differentiation and proliferation of mouse mesenchymal stem cells (mMSCs) under the microenvironment of simulated acute kidney injury (AKI) in vitro. Methods: Rat models of I / R AKI were established by occluding the bilateral renal pedicles of 30 min and then 30 min in male C57BL / 6 mice, and the bilateral renal cortex was immediately harvested for I / R kidney homogenate clear. Bone marrow of C57BL / 6 mice was collected and purified by Percoll density gradient centrifugation combined with adherent culture. The mMSCs were identified by flow cytometry. (1) control group: low glucose DMEM medium containing 10% fetal bovine serum; (2) intervention group: low glucose DMEM medium containing 10% fetal bovine serum + I / R kidney Homogenate the supernatant. The morphological changes of the cells were observed under an inverted microscope at 1 day, 3 days, 5 days and 7 days. The ultrastructure of the cells was observed by transmission electron microscopy. Cytokeratin 18 (CK18) was detected by flow cytometry. The proliferation of mMSCs was detected by CCK- TUNEL method was used to detect apoptosis of mMSCs. Results: The P3-mMSCs isolated highly expressed CD29 and CD44, low expression of CD34 and CD45. Compared with the spindle cells in the control group, some cells in the intervention group showed oval and short fushers on the third day, and most of the cells were round, oval and short fat spindle on the 7th day. Transmission electron microscope also observed The cytoplasm began to appear more rough endoplasmic reticulum, lysosomes, mitochondria. Flow cytometry showed that only a very small amount of CK18 was expressed in the control group, while CK18 in the intervention group was significantly increased. After I / R renal homogenate supernatant was treated, the proliferation of mMSCs was significantly weakened at different time points, while the percentage of cells stained positive by nuclear TUNEL assay was significantly increased (P <0.01). CONCLUSION: In vitro simulated AKI microenvironment induces the differentiation of mMSCs into tubular epithelial-like cells, but at the same time leads to apoptosis and proliferation of cultured mMSCs, which in turn reduces the number of adrenocortical mMSCs that may be One of the reasons for the limited ability of MSCs to promote renal repair in vivo.