本文選題：ESCC + 血清標(biāo)志物�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：食管鱗狀細(xì)胞癌(esophageal squamous cell carcinoma, ESCC)是全球常見惡性腫瘤之一,由于缺乏明顯的早期癥狀和可靠的診斷技術(shù),多數(shù)患者就診時已為中晚期,治療效果欠佳,5年平均生存率僅為3040%。如能早期診斷,其5年生存率可達(dá)90%以上。目前尚無ESCC可靠的血清學(xué)標(biāo)志分子。本研究首先應(yīng)用酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay, ELISA)檢測了ESCC患者、食管癌前病變不典型增生和健康對照個體血清中角化素蛋白片段19(Cyfra21-1)及鱗狀細(xì)胞癌抗原(squamous cell carcinoma antigen, SCC)水平。結(jié)果發(fā)現(xiàn),與健康對照組相比,Cyfra21-1和SCC在食管不典型增生和食管癌患者血清中均高表達(dá)(P0.05)。伴有淋巴結(jié)轉(zhuǎn)移的ESCC患者血清Cyfra21-1水平顯著高于無轉(zhuǎn)移組(P=0.005)。而血清Cyfra21-1和SCC水平與患者年齡、性別、腫瘤部位、分化程度、腫瘤大小和TNM分期等特征無關(guān)�；瘜W(xué)發(fā)光法定量檢測Cyfra21-1和SCC表達(dá)水平并計算曲線下面積(area under curve, AUC),結(jié)果提示聯(lián)合檢測Cyfra21-1和SCC血清標(biāo)志診斷食管癌效果(AUC=0.777)優(yōu)于Cyfra21-1(AUC=0.757)或SCC (AUC=0.661)單獨檢測的效果�；瘜W(xué)發(fā)光法與ELISA法檢測血清Cyfra21-1和SCC表達(dá)水平結(jié)果一致。接下來,為了發(fā)現(xiàn)和篩選新的ESCC循環(huán)標(biāo)志物,應(yīng)用弱陽離子交換磁珠與基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜(magnetic bead-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MB-based MALDI-TOF-MS)聯(lián)用,利用477例腫瘤和健康對照樣本,建立了一個由三個多肽差異峰(1,925.5,2,950.6and 5,900.0 Da)組成的ESCC血清診斷模型,該模型在100例ESCC和98例健康對照樣本組成的訓(xùn)練集中的診斷靈敏度和特異度分別達(dá)到97.00%(97/100)和95.92%(94/98),101例ESCC和98例健康對照樣本組成的驗證集的診斷靈敏度和特異度分別達(dá)到97.03% (98/101)和100.00% (98/98)。該模型的診斷效果優(yōu)于SCC和Cyfra 21-1聯(lián)用,且該模型對于早期ESCC樣本的診斷靈敏度達(dá)到42/43(97.67%)。通過軌道阱組合式高分辨液質(zhì)聯(lián)用系統(tǒng)對差異峰進(jìn)行了鑒定,三個差異峰分別來源于α2 HS糖蛋白(Alpha2-HS glycoprotein, AHSG),血小板反應(yīng)素-1(Thrombospondin-1, TSP1)和纖維蛋白原α鏈(Fibrinogen A alpha-chain, FGA)蛋白片段。此外,TSP1在ESCC組織和血清檢測中表達(dá)升高且與ESCC進(jìn)展相關(guān),組織中TSP1的表達(dá)是ESCC不良預(yù)后的獨立風(fēng)險因素。由于TSP1蛋白N端的一個多肽在ESCC患者血清中明顯升高,據(jù)此序列制備了特異性識別該多肽片段的抗體,并初步建立了一種特異性檢測血清中的該多肽片段的競爭性ELISA方法。測試分析發(fā)現(xiàn),食管癌患者血清中該TSP1N端多肽的表達(dá)水平顯著高于健康對照組(P0.05)。新制備的抗TSP1 N端多肽片段的抗體,以及基于此抗體的競爭性ELISA方法,可以作為檢測和診斷ESCC的新工具。這一工作仍在進(jìn)行中,還需更多的樣本來進(jìn)一步驗證其臨床意義。最后,本實驗室前期研究發(fā)現(xiàn)ABCC4基因的種系DNA拷貝數(shù)擴增和蛋白的異常表達(dá)促進(jìn)食管癌的發(fā)生發(fā)展。ABCC4 (ATP-binding cassette transporter family class C4, ABCC4)是跨膜的ABC轉(zhuǎn)運蛋白C家族成員之一,在轉(zhuǎn)運生理性、內(nèi)源性和外源性物質(zhì)中發(fā)揮重要作用,也被稱為多藥抗藥性相關(guān)蛋白4(Multidrug resistance-associated protein 4,MRP4)。本研究進(jìn)一步檢測了ESCC細(xì)胞系中ABCC4表達(dá)情況與化療藥順鉑耐藥的關(guān)系。結(jié)果發(fā)現(xiàn)與ABCC4低表達(dá)細(xì)胞系相比,ABCC4高表達(dá)細(xì)胞系對順鉑更耐藥。用siRNA敲降KYSE140細(xì)胞中ABCC4表達(dá)后,細(xì)胞對順鉑的敏感性顯著增高。此外,基于前期在ABCC4基因內(nèi)含子區(qū)發(fā)現(xiàn)的增強子元件,進(jìn)一步用報告基因?qū)嶒灚@得其核心區(qū)段,并通過染色質(zhì)免疫共沉淀-聚合酶鏈?zhǔn)椒磻?yīng)(Chromatin Immunoprecipitation-Polymerase Chain Reaction, ChIP-PCR)實驗發(fā)現(xiàn)了與該核心區(qū)段結(jié)合的轉(zhuǎn)錄因子。這些結(jié)果提示ABCC4在ESCC化療敏感性調(diào)控中發(fā)揮重要作用,而ESCC中ABCC4基因的表達(dá)調(diào)控還需要進(jìn)一步深入研究。
[Abstract]:Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors in the world. Because of the lack of obvious early symptoms and reliable diagnostic techniques, most patients have been in the middle and late stages of medical treatment. The treatment effect is poor and the average survival rate of 5 years is only 3040%. If early diagnosis, the 5 year survival rate can reach 90%. There is no ESCC reliable serological marker. In this study, enzyme-linked immunosorbent assay (ELISA) was used to detect the serum keratinin fragment 19 (Cyfra21-1) and squamous cell carcinoma antigen (squamous cell carcinoma antigen) in the serum of ESCC patients, atypical hyperplasia of precancerous lesions and healthy controls. The results showed that Cyfra21-1 and SCC were highly expressed in the sera of the atypical hyperplasia of the esophagus and the patients with esophageal cancer compared with the healthy control group (P0.05). The serum Cyfra21-1 level of the ESCC patients with lymph node metastasis was significantly higher than that in the non metastasis group (P=0.005). The serum Cyfra21-1 and SCC levels were associated with the patient's age, sex, tumor location, and differentiation. The extent, tumor size and TNM staging were not related. Chemiluminescence was used to quantify the expression of Cyfra21-1 and SCC and to calculate the area under the curve (area under curve, AUC). The results suggested that the combined detection of Cyfra21-1 and SCC serum markers in the diagnosis of esophageal cancer (AUC=0.777) was better than that of Cyfra21-1 (AUC=0.757) or individual detection. The results of chemiluminescence and ELISA detection were consistent with the levels of serum Cyfra21-1 and SCC. Next, in order to find and screen new ESCC cycle markers, the weak cation exchange magnetic beads and matrix assisted laser desorption ionization time of flight mass spectrometry (magnetic bead-based matrix-assisted laser desorption/ionization time-of-flight MAS) S spectrometry, MB-based MALDI-TOF-MS) combined with 477 cases of tumor and healthy control samples, a ESCC serum diagnostic model consisting of three polypeptide difference peaks (1925.5,2950.6and 5900 Da) was established. The diagnostic sensitivity and specificity of the model were 9 in the training concentration of 100 cases of ESCC and 98 healthy control samples. The diagnostic sensitivity and specificity of 7% (97/100) and 95.92% (94/98), 101 cases of ESCC and 98 healthy control samples are 97.03% (98/101) and 100% (98/98) respectively. The diagnostic effect of this model is better than SCC and Cyfra 21-1, and the diagnostic sensitivity of the model to early ESCC samples is 42/43 (97.67%). Through orbits The well combined high resolution liquid chromatography-mass spectrometry system was used to identify the difference peaks. Three different peaks were derived from alpha 2 HS glycoprotein (Alpha2-HS glycoprotein, AHSG), thrombocytopenin -1 (Thrombospondin-1, TSP1) and fibrinogen alpha chain (Fibrinogen A alpha-chain, FGA) protein fragments. The expression of TSP1 in the tissue is an independent risk factor for the poor prognosis of ESCC. As a polypeptide in the N terminal of the TSP1 protein is obviously elevated in the serum of the patients with the N, the specific antibody to identify the polypeptide fragment is prepared, and a preliminary establishment of a specific detection of the polypeptide fragment in the serum of the ESCC has been established. Contention ELISA method. Test analysis found that the expression level of the TSP1N terminal polypeptide in the sera of the patients with esophageal cancer was significantly higher than that of the healthy control group (P0.05). The new antibody against the TSP1 N peptide fragment and the competitive ELISA method based on this antibody could be used as a new tool for detecting and diagnosing ESCC. This work is still in progress and needs to be done. More samples were used to further verify its clinical significance. Finally, the previous study found that the amplification of the DNA copy number of the ABCC4 gene and the abnormal expression of the protein promoted the development of.ABCC4 (ATP-binding cassette transporter family class C4, ABCC4), one of the member of the ABC transporter C family in the transmembrane. Physiological, endogenous and exogenous substances play an important role, also known as drug resistance related protein 4 (Multidrug resistance-associated protein 4, MRP4). This study further detected the relationship between the expression of ABCC4 in the ESCC cell line and the resistance of chemotherapy to cisplatin. The results showed that the high expression of ABCC4 was compared with the ABCC4 low expression cell line. The cell line was more resistant to cisplatin. The sensitivity of the cells to cisplatin was significantly increased after the expression of ABCC4 in the KYSE140 cells of siRNA knockdown. Furthermore, based on the enhancers found in the intron of the ABCC4 gene, the core segments were obtained by the reporter gene experiment, and the chromatin immunoprecipitation polymerase chain reaction (Chrom) was obtained. Atin Immunoprecipitation-Polymerase Chain Reaction, ChIP-PCR) found the transcription factors associated with the core section. These results suggest that ABCC4 plays an important role in the regulation of chemosensitivity in ESCC, and the regulation of the expression of ABCC4 gene in ESCC needs further study.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.1

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