荧光差示凝胶电泳技术的建立及其在角质细胞特异性Smad4剔除小鼠表皮差异蛋白质组学研究中的应用

发布时间：2018-08-20 19:23

【摘要】：Smad4是转化生长因子-β(TGF-β)超家族信号转导途径中一个共同的中介因子，在哺乳动物的生长发育过程中有重要作用。为了研究Smad4在皮肤发育中的作用，杨晓教授(军事医学科学院生物工程研究所发育和疾病遗传学研究室)实验室利用Cre-loxP介导的重组系统成功建立了角质细胞特异性Smad4基因剔除小鼠(Smad4~(co/co)；K5-Cre)。角质细胞Smad4剔除的小鼠毛囊不能进入退化期，出现进行性脱毛。70％的Smad4~(co/co)；K5-Cre小鼠出生12个月内发生包括皮肤肿瘤在内的自发性肿瘤。细胞的生长发育依靠复杂的代谢和调节通路，蛋白质则是这些通路中发挥功能的执行体。蛋白质组学是规模化研究蛋白质表达组成及其调控变化的新兴学科，近年来其成长速度令人刮目相看。差异蛋白质组学通过比较样本间蛋白质表达水平的变化，寻找差异分子，，用以解决各种各样的生物学问题。目前已涌现出许多新颖的差异蛋白质组分析方法，如差示荧光凝胶电泳(difference gel electrophoresis，2D DIGE)、可裂解同位素编码亲和标签技术(cleavable isotope-coded affinity tags，cICAT)和同位素标签相对和绝对定量(isobaric tags for relmive and absolute quantification，iTRAQ)等技术。由于继承了二维凝胶电泳的高分辨率，同时又具备高重现性、高通量和高动态范围等优势，使得2D DIGE日益受到关注，成为最受欢迎的定量蛋白质组研究手段之一。为了高通量、大规模地筛选与SMAD4信号通路中有相互关系的蛋白质，进一步揭示脱发和皮肤肿瘤形成的分子机制，我们考察建立了2D DIGE技术并应用该技术对角质细胞特异性Smad4条件基因剔除小鼠的表皮进行了差异蛋白质组学分析。文中优化了小鼠表皮全蛋白的提取方案，考查了2D DIGE的灵敏度、重现性，评估了该样本的差异域值，建立了角质细胞特异性Smad4条件基因剔除小鼠的表皮差异蛋白质表达谱。采用液相色谱—电喷雾串联质谱技术，对241个差异表达的蛋白质点进行了鉴定，其中167个点得到可靠鉴定结果。对鉴定的差异蛋白进行了详细的生物信息学分析，这些差异表达的蛋白包括14-3-3δ、Galectin-7、FABP5、cathepsin D和Enolase 1等。对部分差异蛋白做了Western验证。
[Abstract]:Smad4 is a common mediator in the signal transduction pathway of transforming growth factor-beta (TGF-beta) superfamily and plays an important role in the growth and development of mammals. In order to study the role of Smad4 in skin development, Professor Yang Xiao (Laboratory of Development and Disease Genetics, Institute of Bioengineering, Academy of Military Medical Sciences) used it. Cre-loxP-mediated recombination system was successfully established in keratinocyte-specific Smad4 knockout mice (Smad4~ (co/co); K5-Cre). The hair follicles of the mice with keratinocyte-specific Smad4 knockout could not enter the degenerative stage, and progressive hair loss occurred in 70% of the Smad4~ (co/co); spontaneous tumors including skin tumors occurred in K5-Cre mice within 12 months of birth.
The growth and development of cells depend on complex metabolic and regulatory pathways, and proteins are the executors of these pathways. Proteomics is a new subject that studies the composition and regulation of protein expression on a large scale. In recent years, its growth rate is remarkable. Differential proteomics compares the protein tables between samples. Many novel differential proteomic analysis methods have emerged, such as differential gel electrophoresis (2D DIGE), cleavable isotope-coded affinity tags (cICAT). (iTRAQ) and isobaric tags for relmive and absolute quantification (isobaric tags for relmive and absolute quantification, iTRAQ) have attracted increasing attention and become the most popular quantitative proteomic research due to their inheritance of high resolution, high reproducibility, high throughput and high dynamic range of two-dimensional gel electrophoresis. One of the means.
In order to screen proteins associated with SMAD4 signaling pathway in a high throughput and large scale, and to further reveal the molecular mechanisms of hair loss and skin tumorigenesis, we investigated the establishment of a two-dimensional DIGE technique and applied it to the differential proteomic analysis of the epidermis of keratinocyte-specific Smad4 conditioned gene knockout mice. The sensitivity and reproducibility of 2D DIGE were examined, and the difference domain values of the samples were evaluated. The differential protein expression profiles of the cuticle-specific Smad4 conditioned gene knockout mice were established. 241 differentially expressed proteins were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS). The identified differentially expressed proteins, including 14-3-3 delta, Galectin-7, FABP5, cathepsin D and Enolase 1, were analyzed in detail by bioinformatics.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R341

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