基于AOM-DSS小鼠組織間隙液的結(jié)直腸癌血清蛋白標(biāo)志物的研究
發(fā)布時(shí)間：2019-04-22 17:46
【摘要】：血清蛋白標(biāo)志物的開發(fā)對(duì)結(jié)直腸癌(CRC)的預(yù)防、診斷和預(yù)后等方面具有重要意義。本研究試圖構(gòu)建一種CRC血清蛋白標(biāo)志物開發(fā)的新型路線,以改進(jìn)傳統(tǒng)方法無(wú)法發(fā)現(xiàn)腫瘤發(fā)生發(fā)展早期的提示性標(biāo)志物和候選標(biāo)志物驗(yàn)證效率低下的問(wèn)題。我們以對(duì)CRC發(fā)生發(fā)展過(guò)程中結(jié)腸組織間隙液(TIF)蛋白豐度的動(dòng)態(tài)定量檢測(cè)來(lái)實(shí)現(xiàn)生物標(biāo)志物的尋找,并假設(shè)隨腫瘤發(fā)展呈現(xiàn)持續(xù)上調(diào)變化的TIF蛋白為篩選CRC血清標(biāo)志物的良好選擇,最后通過(guò)在小鼠和人類CRC血清樣本中的一系列實(shí)驗(yàn)來(lái)檢驗(yàn)該假設(shè)并評(píng)估候選標(biāo)志物的應(yīng)用前景。我們成功構(gòu)建了AOM-DSS小鼠結(jié)腸癌模型并建立了使用窄pH范圍(pH 3-6)IPG膠條有效分離TIF肽段的方法。通過(guò)對(duì)該模型腫瘤不同發(fā)展時(shí)期(Cycle Ⅰ、Cycle Ⅱ、Cycle Ⅲ)的結(jié)腸中下段TIF蛋白質(zhì)組的動(dòng)態(tài)定量研究,我們成功定量了584個(gè)TIF蛋白,其中約有66%是理論預(yù)測(cè)的分泌蛋白或已知的血清蛋白。我們從TIF蛋白中篩選得到144個(gè)顯著差異蛋白質(zhì),并根據(jù)其豐度變化趨勢(shì)將其分為持續(xù)上調(diào)(n=45)、持續(xù)下調(diào)(n=17)和非持續(xù)變化(n=82)三類。隨后,我們?cè)?6只小鼠的TIF樣本內(nèi)以多重反應(yīng)監(jiān)測(cè)(MRM)技術(shù)對(duì)24個(gè)持續(xù)性豐度變化蛋白進(jìn)行個(gè)體化定量檢測(cè),確證了其中18個(gè)蛋白質(zhì)(持續(xù)上調(diào)蛋白12個(gè),持續(xù)下調(diào)蛋白6個(gè))的iTRAQ定量結(jié)果。為了檢驗(yàn)CRC相關(guān)TIF蛋白是否具有成為CRC血清標(biāo)志物的潛質(zhì),我們?cè)谠撔∈竽Ｐ偷难鍢颖局虚_展了針對(duì)12個(gè)持續(xù)上調(diào)蛋白的個(gè)體化MRM定量檢測(cè),結(jié)果表明：CycleIII組中LRG1、TUBB5和IGJ的豐度較Control組顯著上調(diào)。隨后,我們同樣以MRM技術(shù)檢測(cè)了16例CRC患者及16例健康人血清樣本中這三個(gè)蛋白質(zhì)的豐度,以初步評(píng)估其作為候選標(biāo)志物在人類血清中應(yīng)用的兼容性。與健康對(duì)照組相比,LRG1和TUBB5在CRC組呈現(xiàn)出了顯著的豐度上調(diào),而IGJ在兩組中無(wú)顯著差異變化。受試者工作特征曲線分析表明,LRG1、TUBB5的曲線下面積分別為0.74和0.70,且這兩個(gè)指標(biāo)的聯(lián)合使用有助于提高檢測(cè)的特異性。為了評(píng)估候選標(biāo)志物的臨床應(yīng)用潛能,我們?cè)跀U(kuò)大的臨床血清樣本中對(duì)LRG1的絕對(duì)濃度進(jìn)行雙抗夾心ELISA檢測(cè),發(fā)現(xiàn)其在CRC組較健康對(duì)照組顯著上調(diào),且當(dāng)閾值為34.56μg/mL時(shí),血清LRG1區(qū)分健康對(duì)照和CRC的靈敏度為59.3%、特異性為79.6%,表明LRG1具有一定的CRC血清標(biāo)志物應(yīng)用前景。隨后,我們以組織芯片免疫組化實(shí)驗(yàn)對(duì)比了結(jié)腸癌及其癌旁組織內(nèi)LRG1的表達(dá)豐度,結(jié)果表明LRG1在結(jié)腸癌上皮細(xì)胞中顯著上調(diào),且其在兩種不同的腫瘤原發(fā)灶情況(T3 vs. T1)、腫瘤遠(yuǎn)端轉(zhuǎn)移與否(M1 vs. M0)及三種不同的腫瘤分期(Stage Ⅳ vs. Stage Ⅰ, Stage Ⅳ vs. Stage Ⅱ)內(nèi)部具有顯著的差異表達(dá)。以上結(jié)果表明,將小鼠模型結(jié)腸TIF樣本的動(dòng)態(tài)定量蛋白質(zhì)組學(xué)研究和在臨床血清樣本中對(duì)候選標(biāo)志物的靶標(biāo)性定量檢驗(yàn)結(jié)合起來(lái),是一種新型的CRC血清蛋白標(biāo)志物開發(fā)的有效技術(shù)路線。我們應(yīng)用這一思路成功地發(fā)現(xiàn)了LRG1這一蛋白質(zhì),并在CRC患者血清及結(jié)腸癌腫瘤組織中驗(yàn)證了其在疾病狀態(tài)下的豐度變化,為CRC的診斷及預(yù)后判斷提供了一個(gè)新的候選血清蛋白標(biāo)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.
【學(xué)位授予單位】：中國(guó)科學(xué)院北京基因組研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.34
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)院北京基因組研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.34
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
1 顧志冬,馬蔚蕓,楊偉宗;運(yùn)用高效毛細(xì)管區(qū)帶電泳法對(duì)人血清蛋白的分析[J];現(xiàn)代檢驗(yàn)醫(yī)學(xué)雜志;2002年02期
2 陸克瓊;“血清蛋白醋酸纖維薄膜電泳”實(shí)驗(yàn)須注意的幾個(gè)環(huán)節(jié)[J];衛(wèi)生職業(yè)教育;2005年17期
3 龍仁玲;何代平;;拉薩地區(qū)藏漢族成人與兒童血清蛋白檢測(cè)分析[J];西南軍醫(yī);2007年01期
4 董矜;田亞平;董振南;劉紅鷹;;應(yīng)激反應(yīng)對(duì)艦艇官兵血清蛋白的影響[J];軍醫(yī)進(jìn)修學(xué)院學(xué)報(bào);2009年04期
5 伍趣京;;“血清蛋白醋酸纖維薄膜電泳”樣板的制備[J];中外醫(yī)療;2010年14期
6 胡洪華;蔣婷;宋海星;胡瀚丹;;四種檢測(cè)法在乙腈沉淀法去除血清蛋白中的比較[J];中國(guó)醫(yī)藥導(dǎo)報(bào);2012年23期
7 ，
本文編號(hào)：2463055
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2463055.html
最近更新
教材專著
