β-actin作为抑郁症患者血浆Western blot内参的初步研究

发布时间：2018-10-22 13:46

【摘要】：研究背景抑郁症是常见的精神疾病，以显著而持久的情绪低落为主要临床特征，其终生患病率高达16%，但发病机制尚不明确，缺乏客观有效的诊断标志物。由于血浆是一种取材容易、应用广泛的研究样品，检测血浆特异蛋白的改变越来越广泛应用于临床疾病的诊断。近几年发展起来的差异蛋白质组学技术在研究疾病发病机制、寻找疾病相关标志物、探索并揭示许多未知但具有重要作用的蛋白质方面具有明显的优势。因此，运用血浆差异蛋白组学的方法，寻找抑郁症相关的蛋白分子，有望为抑郁症的早期诊断提供客观标志物。虽然血浆差异蛋白质组学技术在寻找疾病相关标志物方面较传统方法更有优势，但方法本身也有不足和局限性。首要问题是必须对蛋白质组学筛查的差异表达蛋白进行深入验证，以确保结果的可靠性。Western blot是常用的进行差异蛋白验证方法之一。在血浆Western blot实验中，选择合适的内参对Western blot验证结果的准确性尤为重要。β-actin是一种重要的细胞骨架蛋白，广泛分布于细胞浆内，在各组织和细胞中的表达丰富且相对恒定，是组织和细胞样品中常用的内参。然而β-actin是否存在于血浆中？如果存在，其在血浆中的含量如何，能否作为抑郁症患者血浆Western blot的内参？这些问题目前还不清楚。目的探索β-actin是否存在于人的血浆中，并进一步探讨β-actin能否作为抑郁症患者血浆Western blot的内参。方法 1.经肘正中静脉采集6例空腹抑郁症患者与6例空腹健康对照者血液5ml（EDTA抗凝），分离血浆保存以备使用。 2.运用ELISA方法检测抑郁症患者及健康人的血浆中β-actin含量。 3.分析血浆蛋白上样量与Western blot方法检测的β-actin灰度值的线性关系。 4.运用Western blot的方法检测β-actin在抑郁症患者和健康人血浆中的表达情况，分析两组间血浆β-actin表达是否存在差异。结果 1. β-actin存在于抑郁症患者及健康人血浆中，并且占血浆总蛋白的9.4×10~(-8)。 2.上样量与β-actin灰度值的线性关系非常好（R2=0.974±0.012）。 3.抑郁症患者与健康人血浆中β-actin的表达没有统计学差异（P0.05），且两组β-actin灰度值变异系数很小（CV=9.2±1.2%）。结论 1. β-actin存在于人的血浆中，但其含量较组织和细胞中少。 2.初步研究证明β-actin可以作为抑郁症患者血浆Western blot的内参。
[Abstract]:Background Depression is a common mental disorder with significant and persistent depression as the main clinical feature. The lifetime prevalence rate of depression is as high as 16. However, the pathogenesis of depression is still unclear and lacks of objective and effective diagnostic markers. Because plasma is a kind of easy material, it is widely used to detect the change of plasma specific protein in the diagnosis of clinical diseases. The differential proteomics technology developed in recent years has obvious advantages in studying the pathogenesis of diseases, searching for disease related markers, and exploring and revealing many unknown but important proteins. Therefore, using the method of plasma differential proteomics to search for depressive related protein molecules may provide an objective marker for the early diagnosis of depression. Although plasma differential proteomics technique has more advantages than traditional methods in finding disease-related markers, the method itself has its own shortcomings and limitations. The most important problem is that the differential expression proteins of proteomics screening must be thoroughly verified to ensure the reliability of the results. Western blot is one of the commonly used differential protein verification methods. In plasma Western blot assay, it is very important to select suitable internal parameters to verify the accuracy of Western blot results. 尾-actin is an important cytoskeleton protein, widely distributed in the cytoplasm, and its expression is abundant and relatively constant in all tissues and cells. It is a common internal reference in tissue and cell samples. However, does 尾-actin exist in plasma? If so, what is its content in plasma and can it be used as an internal reference for plasma Western blot in patients with depression? These problems are still unclear. Objective to investigate whether 尾-actin exists in human plasma and whether 尾-actin can be used as an internal reference for plasma Western blot in patients with depression. Method 1. Blood 5ml (EDTA anticoagulant) was collected from 6 patients with depression and 6 healthy controls through the median cubital vein. The content of 尾-actin in the plasma of patients with depression and healthy persons was detected by ELISA method. To analyze the linear relationship between the amount of plasma protein sample and the gray value of 尾-actin detected by Western blot method. 4. Western blot was used to detect the expression of 尾-actin in the plasma of depression patients and healthy people, and the difference of 尾-actin expression between the two groups was analyzed. Results 1. 尾-actin existed in the plasma of patients with depression and healthy people, and accounted for 9.4 脳 10 ~ (-8) 路2. 2 of the total plasma protein. 2. The linear relationship between the amount of sample and the gray value of 尾-actin is very good (R20.974 卤0.012). There was no significant difference in the expression of 尾-actin between patients with depression and healthy subjects (P0.05), and the coefficient of variation of 尾-actin in the two groups was very small (CV=9.2 卤1.2%). Conclusion 1. 尾-actin exists in human plasma, but its content is less than that in tissue and cell. 2. Preliminary studies have demonstrated that 尾-actin can be used as an internal reference for plasma Western blot in patients with depression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.4

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