老年骨髓间充质干细胞外泌微泡的变化及其在肾脏老化中的作用

发布时间：2018-07-07 17:09

  本文选题：骨髓间充质干细胞 + 微泡　； 参考：《南京医科大学》2017年硕士论文

【摘要】：目的:骨髓间充质干细胞(Mesenchyma1 Stem Cells,MSCs)可发生衰老变化。MSCs老化是器官老化和老年患者接受自体干细胞移植治疗效果不佳的重要原因之一。微泡(Microvesicles,MVs)由MSCs旁分泌释放,是MSCs发挥损伤修复作用的有效成分。MVs的改变是否是MSCs功能下调的原因之一,目前尚无相关报道。因此,本研究旨在通过体内、体外实验初步探讨老年MSC-MVs的变化及其在肾脏老化中的作用。方法:全骨髓法提取、培养MSCs,运用β-半乳糖苷酶染色、CCK-8、Transwell、成骨成脂诱导等方法鉴定老年MSCs生物学特性。MSCs培养过程中收集MSCs培养液,经超速冰冻离心提取MVs。体外实验:构建人肾近曲小管上皮细胞系(HK2)老化模型,分别加入青年MVs(Y-MVs)、老年MVs(O-MVs)进行干预治疗。蛋白质印迹法(Western blot)及免疫荧光检测HK2细胞的表型改变。体内实验:在自然衰老的19月龄C57老化小鼠模型中,分别经尾静脉注射Y-MVs、O-MVs进行干预治疗,通过检测肾功能生化指标、老化标志蛋白的表达评估各组小鼠肾脏功能。进一步利用Arraystar mouse lncRNAs芯片检测Y-MVs、O-MVs长链非编码RNA(long non-coding RNA,lncRNAs)表达谱,寻找差异表达的lncRNAs,并运用相关软件进行靶基因预测。研究结果:1.老年MSCs增殖、分化、迁移及外泌MVs等能力降低。2.老年MSC-MVs逆转肾小管上皮细胞和小鼠肾脏组织老化改变能力减低。3.Y-MVs、O-MVs内的lncRNAs表达谱对比分析结果显示,O-MVs中共有1843条lncRNAs表达发生变化,其中39.5%的lncRNAs表达上调。这些上调的lncRNAs可能是老化MSCs在肾脏老化中功能下调的原因之一。结论:老年MSCs生物学功能降低、修复功能减弱,其旁分泌MVs内lncRNAs的差异表达可能是其修复肾脏老化损伤功能减弱的原因之一。
[Abstract]:Objective: the aging of bone marrow mesenchymal stem cells (MSCs) is one of the important reasons for organ aging and autologous stem cell transplantation in elderly patients. The release of microbubbles (MVs) from MSCs paracrine is one of the reasons for the down-regulation of MSCs. Therefore, the purpose of this study was to investigate the changes of MSC-MVs and its role in renal aging in vivo and in vitro. Methods: MSCs were extracted and cultured by whole bone marrow method. The biological characteristics of aged MSCs were identified by 尾 -galactosidase staining and osteogenesis induction. MSCs were collected in culture medium and extracted by freezing centrifugation. In vitro experiment: the aging model of human proximal tubule epithelial cell line (HK2) was established and treated with young MVs (Y-MVs) and aged MVs (O-MVs) respectively. The phenotypic changes of HK2 cells were detected by Western blot and immunofluorescence. In vivo experiment: in the natural aging C57 aging mice model of 19 months old, Y-MVsOMVs were injected into the tail vein respectively. The renal function of each group was evaluated by detecting the biochemical indexes of renal function and the expression of aging marker protein. Furthermore, Arraystar mouse lncRNAs microarray was used to detect the expression profiles of Y-MVsS- O-MVs long strand noncoding RNAs, to search for differentially expressed LNRNAs, and to predict the target genes by using related software. The result of the study was: 1. The ability of proliferation, differentiation, migration and secretion of MVs was decreased in elderly MSCs. The ability of MSC-MVs to reverse the aging of renal tubule epithelial cells and renal tissue in mice was decreased. The results of comparative analysis showed that there were 1843 LNRNAs expression changes in OMVs, 39.5% of which were up-regulated. These up-regulated lncRNAs may be one of the reasons for down-regulating the function of aging MSCs during renal aging. Conclusion: the biological function and repair function of old MSCs are decreased, and the differential expression of lncRNAs in paracrine MVs may be one of the reasons for decreasing the function of repairing renal aging injury.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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本文编号：2105649