【摘要】：結(jié)腸癌是常見的惡性腫瘤,具有很高的發(fā)病率和致死率,雖然早期診斷可顯著提高五年生存率,但由于缺乏有效的早期診斷方法,大部分病人仍錯失最佳治療時機,所以尋求有效的血清標志物在結(jié)腸癌的診斷與治療中具有極其重要的作用。由于血清中大量高豐度蛋白的存在,嚴重干擾低豐度蛋白的鑒定,而腫瘤組織間隙液因富集疾病相關(guān)蛋白質(zhì)以及無高豐度蛋白干擾等優(yōu)勢,越來越多的被應(yīng)用于腫瘤標志物的研究中。此外,為了找到能夠準確反映腫瘤發(fā)展進程的血清標志物,只研究腫瘤進程的某一階段顯然是不全面的,而在腫瘤發(fā)展進程中選擇多個時間點進行動態(tài)研究可以為候選標志物的選擇提供更多信息。因此,為了得到動態(tài)研究的樣本,我們首先選擇ApcMin/+結(jié)腸癌小鼠模型進行候選腫瘤標志物的發(fā)現(xiàn)與篩選,然后在結(jié)腸癌病人的臨床樣本中進行驗證,最終確定結(jié)腸癌血清標志物。根據(jù)ApcMin/+小鼠結(jié)腸腫瘤的發(fā)展特點,我們將其分成8、13、18和22周四個時間點,為了排除小鼠周齡對于蛋白質(zhì)表達及分泌的影響,同時設(shè)置了與ApcMin/+小鼠同基因背景并且相同處理的WT小鼠作為對照。通過對ApcMin/+和WT小鼠結(jié)腸組織間隙液蛋白的iTRAQ定量蛋白質(zhì)組學分析,篩選出120個與結(jié)腸癌相關(guān)的蛋白質(zhì),對它們進行變化趨勢分析,46個蛋白質(zhì)隨著腫瘤的發(fā)展進程呈現(xiàn)逐漸變化的趨勢,其中六個蛋白,CELA1, CEL2A, chymopasin, CTRB1, TRY2和TRY4,同屬于絲氨酸蛋白酶家族,且呈現(xiàn)完全一致的上調(diào)變化。多反應(yīng)監(jiān)測(MRM)技術(shù)的驗證結(jié)果表明,六個蛋白酶的逐漸上調(diào)變化趨勢在ApcMin/+小鼠結(jié)腸組織間隙液個體樣本中是普遍存在的,與此同時,結(jié)腸組織的免疫組化檢測以及ApcMin/+小鼠血清MRM檢測也證實它們在結(jié)腸組織中以及血清中的蛋白水平與組織間隙液中呈現(xiàn)完全一致的變化趨勢。以上結(jié)果提示,組織間隙液中某些蛋白質(zhì)的水平可在血清中反映,六個絲氨酸蛋白酶在ApcMin/+小鼠中可用作結(jié)腸癌的血清標志物。四個絲氨酸蛋白酶,CELA1, CEL2A, CTRL (chymopasin的人同源蛋白)和TRY2,可在結(jié)腸癌病人的血清中被成功驗證。與健康對照比較,四個蛋白酶水平均在結(jié)腸癌病人血清中顯著增高,受試者工作特征曲線(ROC)分析表明CELA1和CTRL的聯(lián)合具有最佳診斷效果。隨后,80例結(jié)腸癌病人腫瘤組織和癌旁對照的CELA1和CTRL的免疫組化結(jié)果證實其在腫瘤組織中的高度表達。綜上,通過對ApcMin/+小鼠組織間隙液蛋白質(zhì)的鑒定結(jié)合在人臨床樣本中的驗證,確定CELA1, CEL2A, CTRL和TRY2,四個絲氨酸蛋白酶可作為結(jié)腸癌的血清標志物。
[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.
【學位授予單位】：中國科學院北京基因組研究所
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.35

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