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  本文選題：脂肪間質(zhì)干細胞 + 骨髓間質(zhì)干細胞��； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:骨髓來源的間質(zhì)干細胞(Bone marrow mesenchymal stem cells,BM-MSCs)和脂肪來源的間質(zhì)干細胞(Adipose derived mesenchymal stem cells,AD-MSCs)均在心肌損傷修復(fù)中發(fā)揮重要作用,并且這種作用主要通過分泌exosomes來實現(xiàn)。但是,AD-MSCs和BM-MSCs修復(fù)作用的優(yōu)劣尚不明確。本實驗將探究C57BL/6小鼠來源的AD-MSCs和BM-MSCs在細胞特性、exosomes釋放量和對缺血缺氧心肌的保護作用的區(qū)別及機制。方法:6-10周雄性C57BL/6小鼠用于分離培養(yǎng)AD-MSCs和BM-MSCs。MTS實驗和細胞計數(shù)檢測AD-MSCs和BM-MSCs的增殖能力和倍增時間。流式細胞術(shù)檢測AD-MSCs和BM-MSCs的表面標志(CD29,Sca-1和CD34)和autophagy的表達。使用無血清培養(yǎng)基培養(yǎng)48h分別收集第四代AD-MSCs和BM-MSCs上清,利用超濾離心和Exo Quick-TC試劑盒從上清中分離純化出AD-MSCs來源的exosomes(Exo-AD)和BM-MSCs來源的exosomes(Exo-BM),通過Nanosight儀器分析鑒定exosomes的大小和分布。DC蛋白定量檢測exosomes的濃度及其他蛋白濃度。Western blot檢測exosomes的表面標志CD63和細胞自噬相關(guān)分子Beclin-1。根據(jù)心肌細胞提取試劑盒的操作方法體外分離培養(yǎng)新生大鼠心肌細胞,根據(jù)MTS和LDH實驗的檢測指標進行體外構(gòu)建心肌缺血缺氧模型。使用帶紅色熒光的PKH26標記Exo-AD和Exo-BM后與心肌細胞孵育12小時,熒光顯微鏡下檢測exosomes的被攝取情況。MTS實驗檢測Exo-AD和Exo-BM處理心肌細胞缺血缺氧72小時后細胞的存活情況,JC-1實驗檢測Exo-AD和Exo-BM處理后心肌細胞線粒體膜電位(ΔΨm)的變化。miRNAs芯片分析檢測Exo-AD和Exo-BM內(nèi)各miRNAs的含量。結(jié)果:實驗結(jié)果表明AD-MSCs較BM-MSCs的倍增時間短,增殖能力更強,并且分泌exosomes的能力更強。相反地,BM-MSCs內(nèi)自噬體的表達量則更高。本實驗成功構(gòu)建了體外心肌缺血缺氧模型,Exo-AD和Exo-BM均能被新生大鼠心肌細胞攝取。與對照組相比,兩者均具有心肌細胞缺血缺氧過程的保護作用,但是當(dāng)同量exosomes處理時,Exo-BM對心肌細胞的保護作用則更強。miRNAs芯片分析檢測,Exo-AD和Exo-BM內(nèi)miRNAs的含量不盡相同,有243種miRNAs的表達在Exo-AD和Exo-BM中有差異,其中包括與心肌保護作用相關(guān)的miR-21、miR-30、miR-221和miR-223。結(jié)論:本研究發(fā)現(xiàn)AD-MSCs和BM-MSCs在細胞特性、exosomes釋放量及對缺血缺氧的保護作用均有區(qū)別,其主要原因可能與Exo-AD和Exo-BM中miRNAs含量的差異有關(guān)。這些為臨床MSCs的選擇提供了理論依據(jù)。
[Abstract]:Objective: bone marrow mesenchymal stem cells (BM-MSCs) and adipose derived mesenchymal stem cells (AD-MSCs) play an important role in the repair of myocardial injury, and this effect is mainly achieved by secreting exosomes. However, the effect of AD-MSCs and BM-MSCs repair is not clear. The purpose of this study was to investigate the difference and mechanism of AD-MSCs and BM-MSCs derived from C57BL/6 mice in the release of exosomes and their protective effects on ischemic and hypoxic myocardium. Methods male C57BL/6 mice were isolated and cultured for 6 to 10 weeks. The proliferation and doubling time of AD-MSCs and BM-MSCs were measured by cell count and AD-MSCs assay. The expression of CD29 Sca-1 and CD34) and autophagy on the surface of AD-MSCs and BM-MSCs were detected by flow cytometry. The supernatants of the fourth generation AD-MSCs and BM-MSCs were collected in serum-free medium for 48 h. Exosomesus exo-ADfrom AD-MSCs and Exo-BMN from BM-MSCs were isolated and purified from supernatant by ultrafiltration centrifugation and Exo Quick-TC kit. The size and distribution of exosomes were identified by Nanosight instrument. The concentration of exosomes and other protein concentrations were detected by Nanosight. The surface marker of exosomes is CD63 and the cell autophagy related molecule Beclin-1. According to the method of cardiomyocyte extraction kit, neonatal rat cardiomyocytes were isolated and cultured in vitro. Myocardial ischemia and hypoxia model was established in vitro according to MTS and LDH test. Exo-AD and Exo-BM were labeled with red fluorescent PKH26 and incubated with cardiomyocytes for 12 hours. Detection of uptake of exosomes under fluorescence microscope. Detection of survival of cardiomyocytes treated with Exo-AD and Exo-BM for 72 hours after ischemia and hypoxia. JC-1 experiment to detect changes of mitochondrial membrane potential (螖 蠄 m) of cardiomyocytes treated with Exo-AD and Exo-BM. The contents of miRNAs in Exo-AD and Exo-BM were detected by chip analysis. Results: the results showed that the multiplication time of AD-MSCs was shorter than that of BM-MSCs, and the ability of exosomes secretion was stronger. On the contrary, the expression of autophagy in BM-MSCs was higher than that in BM-MSCs. Both Exo-AD and Exo-BM can be absorbed by neonatal rat cardiomyocytes successfully. Compared with the control group, both of them had protective effects on myocardial cells during ischemia and hypoxia, but the protective effect of Exo-BM on cardiomyocytes was stronger when treated with the same amount of exosomes. The contents of miRNAs in Exo-AD and Exo-BM were different by microarray analysis. There were differences in the expression of miRNAs between Exo-AD and Exo-BM, including miR-21 miR-30 miR-221 and miR-223 related to myocardial protection. Conclusion: in this study, we found that there were differences between AD-MSCs and BM-MSCs in the release of exosomes and their protective effects on ischemia and hypoxia. The main reasons may be related to the difference of miRNAs content in Exo-AD and Exo-BM. These provide a theoretical basis for the selection of clinical MSCs.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R542.22

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