基于AOM-DSS小鼠组织间隙液的结直肠癌血清蛋白标志物的研究

发布时间：2019-04-22 17:46

【摘要】：血清蛋白标志物的开发对结直肠癌(CRC)的预防、诊断和预后等方面具有重要意义。本研究试图构建一种CRC血清蛋白标志物开发的新型路线,以改进传统方法无法发现肿瘤发生发展早期的提示性标志物和候选标志物验证效率低下的问题。我们以对CRC发生发展过程中结肠组织间隙液(TIF)蛋白丰度的动态定量检测来实现生物标志物的寻找,并假设随肿瘤发展呈现持续上调变化的TIF蛋白为筛选CRC血清标志物的良好选择,最后通过在小鼠和人类CRC血清样本中的一系列实验来检验该假设并评估候选标志物的应用前景。我们成功构建了AOM-DSS小鼠结肠癌模型并建立了使用窄pH范围(pH 3-6)IPG胶条有效分离TIF肽段的方法。通过对该模型肿瘤不同发展时期(Cycle Ⅰ、Cycle Ⅱ、Cycle Ⅲ)的结肠中下段TIF蛋白质组的动态定量研究,我们成功定量了584个TIF蛋白,其中约有66%是理论预测的分泌蛋白或已知的血清蛋白。我们从TIF蛋白中筛选得到144个显著差异蛋白质,并根据其丰度变化趋势将其分为持续上调(n=45)、持续下调(n=17)和非持续变化(n=82)三类。随后,我们在16只小鼠的TIF样本内以多重反应监测(MRM)技术对24个持续性丰度变化蛋白进行个体化定量检测,确证了其中18个蛋白质(持续上调蛋白12个,持续下调蛋白6个)的iTRAQ定量结果。为了检验CRC相关TIF蛋白是否具有成为CRC血清标志物的潜质,我们在该小鼠模型的血清样本中开展了针对12个持续上调蛋白的个体化MRM定量检测,结果表明：CycleIII组中LRG1、TUBB5和IGJ的丰度较Control组显著上调。随后,我们同样以MRM技术检测了16例CRC患者及16例健康人血清样本中这三个蛋白质的丰度,以初步评估其作为候选标志物在人类血清中应用的兼容性。与健康对照组相比,LRG1和TUBB5在CRC组呈现出了显著的丰度上调,而IGJ在两组中无显著差异变化。受试者工作特征曲线分析表明,LRG1、TUBB5的曲线下面积分别为0.74和0.70,且这两个指标的联合使用有助于提高检测的特异性。为了评估候选标志物的临床应用潜能,我们在扩大的临床血清样本中对LRG1的绝对浓度进行双抗夹心ELISA检测,发现其在CRC组较健康对照组显著上调,且当阈值为34.56μg/mL时,血清LRG1区分健康对照和CRC的灵敏度为59.3%、特异性为79.6%,表明LRG1具有一定的CRC血清标志物应用前景。随后,我们以组织芯片免疫组化实验对比了结肠癌及其癌旁组织内LRG1的表达丰度,结果表明LRG1在结肠癌上皮细胞中显著上调,且其在两种不同的肿瘤原发灶情况(T3 vs. T1)、肿瘤远端转移与否(M1 vs. M0)及三种不同的肿瘤分期(Stage Ⅳ vs. Stage Ⅰ, Stage Ⅳ vs. Stage Ⅱ)内部具有显著的差异表达。以上结果表明,将小鼠模型结肠TIF样本的动态定量蛋白质组学研究和在临床血清样本中对候选标志物的靶标性定量检验结合起来,是一种新型的CRC血清蛋白标志物开发的有效技术路线。我们应用这一思路成功地发现了LRG1这一蛋白质,并在CRC患者血清及结肠癌肿瘤组织中验证了其在疾病状态下的丰度变化,为CRC的诊断及预后判断提供了一个新的候选血清蛋白标志物。
[Abstract]:The development of serum protein markers is of great significance in the prevention, diagnosis and prognosis of colorectal cancer (CRC). This study was an attempt to construct a new route for the development of a CRC serum protein marker to improve the conventional approach to the inability to identify early warning markers and candidate markers for low efficiency in the development of tumorigenesis. We use the dynamic quantitative detection of the abundance of the colon tissue interstitial fluid (TIF) protein in the development of CRC to realize the search for biomarkers, and assume that the TIF protein, which is continuously up-regulated with the development of the tumor, is a good choice for screening the CRC serum markers, Finally, the hypothesis is tested and the application prospect of the candidate marker is evaluated by a series of experiments in the mouse and human CRC serum samples. We successfully constructed the AOM-DSS mouse colon cancer model and established a method of effectively isolating the TIF peptide segment using a narrow pH range (pH 3-6) IPG strip. By the dynamic quantitative study of the lower TIF protein in the colon of the model with different stages of development (Cycle I, Cycle II, Cycle III),584 TIF proteins were successfully quantified, of which about 66% were the theoretically predicted secreted proteins or known serum proteins. A total of 144 significant difference proteins were selected from TIF protein and divided into three categories: continuous up-regulation (n = 45), continuous down-regulation (n = 17) and non-continuous change (n = 82) according to the change trend of its abundance. Subsequently, we tested 24 persistent abundance change proteins with multiple response monitoring (MRM) techniques in the TIF samples of 16 mice, and confirmed the iTRAQ quantitative results of 18 proteins (12 continuous up-regulation proteins and 6 continuous down-regulation proteins). In order to check whether the CRC-related TIF protein has a potential to be a CRC serum marker, we conducted an individualized MRM quantitative test for 12 continuous up-regulation proteins in the serum samples of the mouse model, and the results showed that the abundance of LRG1, TUBB5 and IGJ in the CycleIII group was significantly increased in the control group. Subsequently, we also examined the abundance of these three proteins in the serum samples of 16 CRC patients and 16 healthy controls with the MRM technique to preliminarily assess the compatibility of these three proteins as candidate markers in human serum. Compared with the healthy control group, LRG1 and TUBB5 showed significant increase in abundance in the CRC group, while IGJ had no significant difference in the two groups. The subject's work characteristic curve analysis showed that the area under the curve of LRG1 and TUBB5 was 0.74 and 0.70, respectively, and the combined use of these two indexes could help to improve the specificity of the test. In order to evaluate the clinical application potential of the candidate marker, we tested the absolute concentration of LRG1 in the expanded clinical serum sample and found that it was significantly up-regulated in the CRC and the healthy control group, and when the threshold was 34.56. m u.g/ mL, The sensitivity of the serum LRG1 to the healthy control and CRC was 59.3%, and the specificity was 79.6%, indicating that the LRG1 has a certain application prospect of CRC serum markers. In that follow, we compare the expression abundance of LRG1 in the colon cancer and its adjacent tissue with the immunohistochemical study of the tissue chip, and the results show that the LRG1 is up-regulated in the colon cancer epithelial cells and it is in the case of two different tumor origin (T3 vs. T1). The distal metastasis of the tumor (M1 vs. M0) and the three different stages of tumor (Stage IV vs. Stage I, Stage IV vs. Stage II) had significant differences in expression. The above results show that the dynamic quantitative proteomics of the mouse model colon TIF sample and the target quantitative test of the candidate marker in the clinical serum sample are combined, which is a new effective technical route for the development of the CRC serum protein marker. We used this idea to successfully find the protein of LRG1, and in the CRC patient serum and colon cancer tumor tissues, the abundance changes of the LRG1 in the disease state are verified, and a new candidate serum protein marker is provided for the diagnosis and the prognosis judgment of CRC.
【学位授予单位】：中国科学院北京基因组研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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