间充质干细胞复制性衰老的无标记检测方法研究

发布时间：2018-01-04 07:35

本文关键词：间充质干细胞复制性衰老的无标记检测方法研究　出处：《天津工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：间充质干细胞具有高度增殖、自我更新和多向分化潜能,目前已广泛应用于生物医学领域,成为世界范围内的研究热点。但在间充质干细胞的临床应用过程中,复制性衰老问题始终伴随左右,对其治疗效果产生了严重的影响,因此准确判断间充质干细胞的衰老状态至关重要。传统的检测方法多集中于生物化学手段,这些方法大多具有侵入性和破坏性,不利于间充质干细胞的使用和研究。本课题基于光学技术的无标记、非侵入等优势,利用荧光检测技术和拉曼检测技术分别对不同衰老程度的间充质干细胞进行检测,探寻能够表征细胞衰老的特征光学指标,利用光学技术实现对间充质干细胞复制性衰老的无标记检测,并为深入研究细胞衰老机制提供了实验依据。课题首先采用荧光光谱法检测了间充质干细胞内主要三种内源性荧光物质,即色氨酸(Trp)、还原型辅酶Ⅰ(NADH)和黄素腺嘌呤二核苷酸(FAD),对应的激发波长分别为280nm、340nm和450nm。通过分析相应谱图上物质荧光强度变化,发现Trp、NADH、FAD的荧光强度均随细胞衰老而增强,并且在年轻(复制5代)和衰老(复制20代)细胞之间具有显著差异。但是该方法在细胞衰老状态变化较小时,表现出来的灵敏度和特异性不强,不适于用作表征间充质干细胞衰老的光谱指标。在此基础上采用拉曼显微光谱技术研究了间充质干细胞的衰老变化。研究发现,采用单一拉曼峰表征细胞衰老缺乏准确性和灵敏度,但位于1157cm~(-1)和1174cm~(-1)处的拉曼峰的强度比值I1157/I1174随细胞衰老呈现单调下降的趋势,并且在衰老程度变化较小的细胞之间仍具有统计学差异,具有较高的灵敏度。研究结果表明,拉曼光谱法可作为一种无标记、非侵入的检测技术用于间充质干细胞衰老评估,I1157/I1174可作为表征间充质干细胞衰老的特征光谱指标。基于拉曼光谱检测结果,通过Matlab平台自行设计了一款针对间充质干细胞衰老检测的拉曼数据自动分析软件,实现了自动平滑降噪、去除基线和寻峰等功能,为拉曼数据处理提供了便捷、快速、准确的分析工具。
[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.

【学位授予单位】：天津工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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