發(fā)布時間：2018-01-04 07:35

  本文關(guān)鍵詞：間充質(zhì)干細胞復制性衰老的無標記檢測方法研究　出處：《天津工業(yè)大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 間充質(zhì)干細胞 復制性衰老 無標記檢測 熒光光譜 拉曼光譜

【摘要】：間充質(zhì)干細胞具有高度增殖、自我更新和多向分化潛能,目前已廣泛應用于生物醫(yī)學領(lǐng)域,成為世界范圍內(nèi)的研究熱點。但在間充質(zhì)干細胞的臨床應用過程中,復制性衰老問題始終伴隨左右,對其治療效果產(chǎn)生了嚴重的影響,因此準確判斷間充質(zhì)干細胞的衰老狀態(tài)至關(guān)重要。傳統(tǒng)的檢測方法多集中于生物化學手段,這些方法大多具有侵入性和破壞性,不利于間充質(zhì)干細胞的使用和研究。本課題基于光學技術(shù)的無標記、非侵入等優(yōu)勢,利用熒光檢測技術(shù)和拉曼檢測技術(shù)分別對不同衰老程度的間充質(zhì)干細胞進行檢測,探尋能夠表征細胞衰老的特征光學指標,利用光學技術(shù)實現(xiàn)對間充質(zhì)干細胞復制性衰老的無標記檢測,并為深入研究細胞衰老機制提供了實驗依據(jù)。課題首先采用熒光光譜法檢測了間充質(zhì)干細胞內(nèi)主要三種內(nèi)源性熒光物質(zhì),即色氨酸(Trp)、還原型輔酶Ⅰ(NADH)和黃素腺嘌呤二核苷酸(FAD),對應的激發(fā)波長分別為280nm、340nm和450nm。通過分析相應譜圖上物質(zhì)熒光強度變化,發(fā)現(xiàn)Trp、NADH、FAD的熒光強度均隨細胞衰老而增強,并且在年輕(復制5代)和衰老(復制20代)細胞之間具有顯著差異。但是該方法在細胞衰老狀態(tài)變化較小時,表現(xiàn)出來的靈敏度和特異性不強,不適于用作表征間充質(zhì)干細胞衰老的光譜指標。在此基礎(chǔ)上采用拉曼顯微光譜技術(shù)研究了間充質(zhì)干細胞的衰老變化。研究發(fā)現(xiàn),采用單一拉曼峰表征細胞衰老缺乏準確性和靈敏度,但位于1157cm~(-1)和1174cm~(-1)處的拉曼峰的強度比值I1157/I1174隨細胞衰老呈現(xiàn)單調(diào)下降的趨勢,并且在衰老程度變化較小的細胞之間仍具有統(tǒng)計學差異,具有較高的靈敏度。研究結(jié)果表明,拉曼光譜法可作為一種無標記、非侵入的檢測技術(shù)用于間充質(zhì)干細胞衰老評估,I1157/I1174可作為表征間充質(zhì)干細胞衰老的特征光譜指標�；�于拉曼光譜檢測結(jié)果,通過Matlab平臺自行設(shè)計了一款針對間充質(zhì)干細胞衰老檢測的拉曼數(shù)據(jù)自動分析軟件,實現(xiàn)了自動平滑降噪、去除基線和尋峰等功能,為拉曼數(shù)據(jù)處理提供了便捷、快速、準確的分析工具。
[Abstract]:Mesenchymal stem cells have high proliferation, self-renewal and differentiation potential, has been widely used in the biomedical field, has become a hotspot in the world. But in the process of clinical application of mesenchymal stem cells, the problem is always accompanied by the replicative senescence, resulting in a serious impact on the treatment effect, therefore accurate judgment of mesenchymal stem cells. The aging state is one of the most important traditional detection methods focus on biochemical methods, most of these methods are invasive and destructive, is not conducive to the use of mesenchymal stem cells and research. This paper based on the technology of optical label free, non-invasive and other advantages, the use of fluorescence detection technology and Raman detection techniques were used to detect different senescent mesenchymal stem cells, to explore the characteristics of optical characterization indexes of cell senescence of mesenchymal stem using optical technology Label free detection of cell replicative senescence, and provide an experimental basis for the study of cell senescence mechanism deeply. Firstly, using fluorescence spectroscopy to detect the stem cells of three main endogenous fluorophores mesenchymal, namely tryptophan (Trp), NADH (NADH) and flavin adenine dinucleotide (FAD) the corresponding excitation wavelength, respectively 280nm, 340nm and 450nm. through the analysis of the corresponding spectrum fluorescence intensity changes, Trp, NADH, FAD fluorescence intensity increased with cell senescence and enhancement, and in the young (copy 5 generation) and aging (copy 20 generation) have significant differences between cells. But the method in cell senescence state change is small, the sensitivity and specificity of the show is not strong, is not suitable for spectral index characterization of mesenchymal stem cells of aging. The research of mesenchymal stem cells by Raman micro spectroscopy technology based on the Aging changes. The study found that using single cell Raman characterization of senescence of the lack of accuracy and sensitivity, but is located in 1157cm~ (-1) and 1174cm~ (-1) I1157/I1174 Raman peak intensity ratio with cell aging decreases, and still has a statistically significant difference between the degree of aging in changes of small cells, with high sensitivity. The results show that Raman spectroscopy can be used as a label free, non-invasive detection technology for mesenchymal stem cell aging evaluation, I1157/I1174 can be used as indicators of spectral characterization of mesenchymal stem cell aging. Raman spectrum detection based on the results, through the Matlab platform to design a automatic analysis software to do the Raman data of mesenchymal cell aging detection, automatic smooth noise reduction, removal of baseline and peak searching function for Raman data processing provides a convenient, fast and accurate A definite analytical tool.

【學位授予單位】：天津工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R329.2

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