本文選題：骨髓間充質(zhì)干細(xì)胞 + 微泡��； 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:骨髓間充質(zhì)干細(xì)胞(Mesenchyma1 Stem Cells,MSCs)可發(fā)生衰老變化。MSCs老化是器官老化和老年患者接受自體干細(xì)胞移植治療效果不佳的重要原因之一。微泡(Microvesicles,MVs)由MSCs旁分泌釋放,是MSCs發(fā)揮損傷修復(fù)作用的有效成分。MVs的改變是否是MSCs功能下調(diào)的原因之一,目前尚無相關(guān)報道。因此,本研究旨在通過體內(nèi)、體外實(shí)驗(yàn)初步探討老年MSC-MVs的變化及其在腎臟老化中的作用。方法:全骨髓法提取、培養(yǎng)MSCs,運(yùn)用β-半乳糖苷酶染色、CCK-8、Transwell、成骨成脂誘導(dǎo)等方法鑒定老年MSCs生物學(xué)特性。MSCs培養(yǎng)過程中收集MSCs培養(yǎng)液,經(jīng)超速冰凍離心提取MVs。體外實(shí)驗(yàn):構(gòu)建人腎近曲小管上皮細(xì)胞系(HK2)老化模型,分別加入青年MVs(Y-MVs)、老年MVs(O-MVs)進(jìn)行干預(yù)治療。蛋白質(zhì)印跡法(Western blot)及免疫熒光檢測HK2細(xì)胞的表型改變。體內(nèi)實(shí)驗(yàn):在自然衰老的19月齡C57老化小鼠模型中,分別經(jīng)尾靜脈注射Y-MVs、O-MVs進(jìn)行干預(yù)治療,通過檢測腎功能生化指標(biāo)、老化標(biāo)志蛋白的表達(dá)評估各組小鼠腎臟功能。進(jìn)一步利用Arraystar mouse lncRNAs芯片檢測Y-MVs、O-MVs長鏈非編碼RNA(long non-coding RNA,lncRNAs)表達(dá)譜,尋找差異表達(dá)的lncRNAs,并運(yùn)用相關(guān)軟件進(jìn)行靶基因預(yù)測。研究結(jié)果:1.老年MSCs增殖、分化、遷移及外泌MVs等能力降低。2.老年MSC-MVs逆轉(zhuǎn)腎小管上皮細(xì)胞和小鼠腎臟組織老化改變能力減低。3.Y-MVs、O-MVs內(nèi)的lncRNAs表達(dá)譜對比分析結(jié)果顯示,O-MVs中共有1843條lncRNAs表達(dá)發(fā)生變化,其中39.5%的lncRNAs表達(dá)上調(diào)。這些上調(diào)的lncRNAs可能是老化MSCs在腎臟老化中功能下調(diào)的原因之一。結(jié)論:老年MSCs生物學(xué)功能降低、修復(fù)功能減弱,其旁分泌MVs內(nèi)lncRNAs的差異表達(dá)可能是其修復(fù)腎臟老化損傷功能減弱的原因之一。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R692

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本文編號：2105649