结肠癌血清标志物—丝氨酸蛋白酶的发现和确证

发布时间：2018-08-26 17:26

【摘要】：结肠癌是常见的恶性肿瘤,具有很高的发病率和致死率,虽然早期诊断可显著提高五年生存率,但由于缺乏有效的早期诊断方法,大部分病人仍错失最佳治疗时机,所以寻求有效的血清标志物在结肠癌的诊断与治疗中具有极其重要的作用。由于血清中大量高丰度蛋白的存在,严重干扰低丰度蛋白的鉴定,而肿瘤组织间隙液因富集疾病相关蛋白质以及无高丰度蛋白干扰等优势,越来越多的被应用于肿瘤标志物的研究中。此外,为了找到能够准确反映肿瘤发展进程的血清标志物,只研究肿瘤进程的某一阶段显然是不全面的,而在肿瘤发展进程中选择多个时间点进行动态研究可以为候选标志物的选择提供更多信息。因此,为了得到动态研究的样本,我们首先选择ApcMin/+结肠癌小鼠模型进行候选肿瘤标志物的发现与筛选,然后在结肠癌病人的临床样本中进行验证,最终确定结肠癌血清标志物。根据ApcMin/+小鼠结肠肿瘤的发展特点,我们将其分成8、13、18和22周四个时间点,为了排除小鼠周龄对于蛋白质表达及分泌的影响,同时设置了与ApcMin/+小鼠同基因背景并且相同处理的WT小鼠作为对照。通过对ApcMin/+和WT小鼠结肠组织间隙液蛋白的iTRAQ定量蛋白质组学分析,筛选出120个与结肠癌相关的蛋白质,对它们进行变化趋势分析,46个蛋白质随着肿瘤的发展进程呈现逐渐变化的趋势,其中六个蛋白,CELA1, CEL2A, chymopasin, CTRB1, TRY2和TRY4,同属于丝氨酸蛋白酶家族,且呈现完全一致的上调变化。多反应监测(MRM)技术的验证结果表明,六个蛋白酶的逐渐上调变化趋势在ApcMin/+小鼠结肠组织间隙液个体样本中是普遍存在的,与此同时,结肠组织的免疫组化检测以及ApcMin/+小鼠血清MRM检测也证实它们在结肠组织中以及血清中的蛋白水平与组织间隙液中呈现完全一致的变化趋势。以上结果提示,组织间隙液中某些蛋白质的水平可在血清中反映,六个丝氨酸蛋白酶在ApcMin/+小鼠中可用作结肠癌的血清标志物。四个丝氨酸蛋白酶,CELA1, CEL2A, CTRL (chymopasin的人同源蛋白)和TRY2,可在结肠癌病人的血清中被成功验证。与健康对照比较,四个蛋白酶水平均在结肠癌病人血清中显著增高,受试者工作特征曲线(ROC)分析表明CELA1和CTRL的联合具有最佳诊断效果。随后,80例结肠癌病人肿瘤组织和癌旁对照的CELA1和CTRL的免疫组化结果证实其在肿瘤组织中的高度表达。综上,通过对ApcMin/+小鼠组织间隙液蛋白质的鉴定结合在人临床样本中的验证,确定CELA1, CEL2A, CTRL和TRY2,四个丝氨酸蛋白酶可作为结肠癌的血清标志物。
[Abstract]:Colon cancer is a common malignant tumor with high morbidity and mortality. Although early diagnosis can significantly improve the 5-year survival rate, most patients still miss the best treatment time due to the lack of effective early diagnosis methods. Therefore, seeking effective serum markers plays an important role in the diagnosis and treatment of colon cancer. Due to the presence of a large number of high abundance proteins in serum, the identification of low abundance proteins is seriously interfered with, while the tumor tissue interstitial fluid has the advantages of enriching disease-related proteins and not interfering with high abundance proteins. More and more are used in the study of tumor markers. Moreover, in order to find serum markers that accurately reflect the progression of cancer, it is clearly not comprehensive to study only one stage of the tumor process. Selecting multiple time points for dynamic research in tumor development can provide more information for the selection of candidate markers. Therefore, in order to obtain the dynamic study samples, we first selected the mouse model of ApcMin/ colon cancer for the discovery and screening of candidate tumor markers, and then confirmed the clinical samples of colon cancer patients to determine the serum markers of colon cancer. According to the development characteristics of colon tumors in ApcMin/ mice, we divided them into 8: 1313 ~ 18 and 22 ~ (th) Thursday time points, in order to exclude the effect of weeks of age on protein expression and secretion in mice. WT mice with the same genetic background and same treatment as ApcMin/ mice were also set as controls. By iTRAQ quantitative proteomics analysis of interstitial fluid proteins in colon tissue of ApcMin/ and WT mice, 120 proteins associated with colon cancer were screened. It was found that 46 proteins showed a gradual change with the development of the tumor. Six of them, CELA1, CEL2A, chymopasin, CTRB1, TRY2 and TRY4, belonged to the serine protease family, and showed a completely up-regulation change. The results of multi-reaction monitoring (MRM) showed that the gradual up-regulation of six proteases was common in individual samples of interstitial fluid in colon tissue of ApcMin/ mice, and at the same time, The immunohistochemical detection of colon tissue and the detection of MRM in serum of ApcMin/ mice also confirmed that the protein level in colon tissue and serum was completely consistent with that in interstitial fluid. These results suggest that the level of some proteins in tissue interstitial fluid can be reflected in serum and that six serine proteases can be used as serum markers of colon cancer in ApcMin/ mice. Four serine proteases CELA1, human homologous protein of CEL2A, CTRL (chymopasin) and TRY2, can be successfully validated in serum of colon cancer patients. Compared with the healthy control, the levels of four proteases were significantly higher in the serum of colon cancer patients. The (ROC) analysis of the operating characteristic curve showed that the combination of CELA1 and CTRL had the best diagnostic effect. The high expression of CELA1 and CTRL in tumor tissues of 80 patients with colon cancer was confirmed by immunohistochemistry. In conclusion, CELA1, CEL2A, CTRL and TRY2, four serine proteases can be used as serum markers of colon cancer through the identification of interstitial fluid proteins in ApcMin/ mice combined with human clinical samples.
【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】：R735.35

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