本文選題：鼻咽癌 + 血清標(biāo)志物��； 參考：《廣西醫(yī)科大學(xué)》2013年碩士論文

【摘要】：鼻咽癌(nasopharyngeal carcinoma, NPC)是源于鼻咽部上皮組織的一種惡性腫瘤,95%以上臨床病例屬低分化癌和未分化癌,惡性程度高。目前確診鼻咽癌主要依靠病理診斷,由于鼻咽癌早期不易察覺,大部分病人確診時(shí)已處于中晚期,遠(yuǎn)處轉(zhuǎn)移機(jī)會(huì)多,治療效果差,患者生存期較短。因此,尋找鼻咽癌相關(guān)標(biāo)志物對(duì)于NPC的篩選、治療靶標(biāo)、探究腫瘤發(fā)生發(fā)展機(jī)理重要意義。本研究旨在運(yùn)用定量蛋白質(zhì)組學(xué)方法篩選能作為篩選與診斷的鼻咽癌血清標(biāo)志物,并對(duì)候選標(biāo)志物進(jìn)行驗(yàn)證和初步的研究。 第一部分iTRAQ結(jié)合2D LC-MS/MS篩選鼻咽癌相關(guān)血清標(biāo)志物 目的：運(yùn)用基于二維液相色譜/質(zhì)譜聯(lián)用的穩(wěn)定同位素標(biāo)記方法(iTRAQ結(jié)合2D LC-MS/MS)對(duì)鼻咽癌患者血清進(jìn)行定量蛋白組學(xué)分析,獲得差異蛋白譜,從中篩選潛在的腫瘤標(biāo)志物。 方法：正常組、鼻咽癌組(Ⅱ期,Ⅲ期,Ⅳ期)共4組血清樣本(每組6例)經(jīng)多重親和免疫去除14種高豐度蛋白后運(yùn)用iTRAQ結(jié)合2D LC-MS/MS技術(shù)進(jìn)行定量蛋白組學(xué)分析,重復(fù)實(shí)驗(yàn)一次,匯總2次實(shí)驗(yàn)結(jié)果,獲得差異蛋白譜并通過生物信息學(xué)方法從中挖掘潛在的生物標(biāo)志物。 結(jié)果：兩次質(zhì)譜實(shí)驗(yàn)共鑒定出274個(gè)置信蛋白,兩次實(shí)驗(yàn)鑒定蛋白重復(fù)率為70.8%,在2次實(shí)驗(yàn)中均被鑒定和定量到的差異蛋白有15個(gè),其中5個(gè)相對(duì)于正常組表達(dá)上調(diào)(1.4倍),10個(gè)表達(dá)下調(diào)(0.7倍)。S100A8和S100A9在2次定量實(shí)驗(yàn)中均穩(wěn)定顯著高表達(dá),而S100A8/A9兩者作為炎癥相關(guān)因子的功能已被廣泛認(rèn)可,近來越來越多的研究表明其不僅可作為腫瘤標(biāo)志物,在腫瘤發(fā)展和轉(zhuǎn)移過程中也發(fā)揮重要作用。 第二部分S100A8/A9,有潛力的鼻咽癌標(biāo)志物 目的：對(duì)質(zhì)譜實(shí)驗(yàn)得到的差異蛋白S100A8/A9進(jìn)行驗(yàn)證,確認(rèn)它們的差異表達(dá)并初步研究其與鼻咽癌的關(guān)系。 方法：①ELISA技術(shù)檢測(cè)S100A8/A9在血清中的表達(dá)水平；②IHC技術(shù)檢測(cè)S100A8/A9在鼻咽癌組織和鼻咽炎組織中的表達(dá)和分布；③ICC和Western Blot技術(shù)檢測(cè)S100A8/A9在永生化正常人鼻咽細(xì)胞系NP69、高分化鼻咽癌細(xì)胞系CNE1、低分化鼻咽癌細(xì)胞系CNE2中表達(dá)與分布；④RT-PCR技術(shù)檢測(cè)S100A8/A9/A12在3個(gè)細(xì)胞系中mRNA的表達(dá)水平；⑤通過不同濃度的Zn2+作用細(xì)胞,CCK8法檢測(cè)3個(gè)細(xì)胞系的活性與增值并細(xì)胞計(jì)數(shù),⑥RT-PCR檢測(cè)隨Zn2+濃度改變S100A8/A9/A12的mRNA表達(dá)水平變化。 結(jié)果：①血清和病理組織中S100A8/A9含量水平變化趨勢(shì)符合質(zhì)譜結(jié)果,癌癥組高于正常組。②S100A8/A9在鼻咽癌細(xì)胞系、其他頭頸部高分化鱗癌細(xì)胞、高分化的表皮細(xì)胞中強(qiáng)表達(dá)而在低分化的腺體細(xì)胞、基底細(xì)胞中不表達(dá)提示S100A8/A9可能與分化有關(guān)。③S100A8/A9/A12的mRNA水平隨培養(yǎng)時(shí)間的延長(zhǎng)在癌細(xì)胞系CNE1、CNE2中表達(dá)降低,正常細(xì)胞NP69中表達(dá)升高。④Zn2+50μM時(shí)可以促進(jìn)3個(gè)細(xì)胞系的增殖但濃度超過70gM后則迅速表現(xiàn)出細(xì)胞毒性,20μM時(shí)絕大部分細(xì)胞已死亡,CNE2表現(xiàn)出更強(qiáng)的耐受性。另外值得一提的是當(dāng)細(xì)胞生長(zhǎng)發(fā)生堆疊后特別是癌細(xì)胞系,對(duì)Zn2+的耐受性明顯增強(qiáng),110μM時(shí)大部分細(xì)胞仍存活。⑤隨著Zn2+濃度增加,當(dāng)S100A8/A9/A12的mRNA表達(dá)降低時(shí)細(xì)胞增殖與活性加強(qiáng),表明S100A8/A9/A12可能參與細(xì)胞的生長(zhǎng)進(jìn)程。 結(jié)論：iTRAQ結(jié)合2DLC-MS/MS是強(qiáng)有力的定量蛋白組學(xué)手段,其高通量、高重復(fù)性與準(zhǔn)確性有助于篩選生物標(biāo)志物。本研究首次采用iTRAQ技術(shù)篩選鼻咽癌血清相關(guān)標(biāo)志物并獲得一系列差異蛋白,從中挖掘分析將能獲得潛在的標(biāo)志物。對(duì)差異蛋白S100A8/A9的驗(yàn)證和研究表明是有潛力的鼻咽癌標(biāo)志物,與鼻咽癌的發(fā)展密切相關(guān)。對(duì)這些潛在標(biāo)志物的進(jìn)一步研究將有助于鼻咽癌的篩選和診斷,發(fā)現(xiàn)新的治療靶標(biāo)和探究癌癥發(fā)生發(fā)展的機(jī)制。
[Abstract]:Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the epithelial tissue of the nasopharynx. More than 95% of the clinical cases are low differentiated and undifferentiated, and the malignant degree is high. At present, the diagnosis of nasopharyngeal carcinoma is mainly based on the pathological diagnosis. Because the early diagnosis of nasopharyngeal carcinoma is not easy to detect, most patients are in the middle and late stage, and the distant metastasis machine has been diagnosed. There are many, poor therapeutic effects and shorter survival time. Therefore, it is important to search for the related markers of nasopharyngeal carcinoma for the screening of NPC, the target of treatment, and the significance of the mechanism of the development of the tumor. A preliminary study.
Part 1 screening of nasopharyngeal carcinoma related serum markers by iTRAQ combined with 2D LC-MS/MS
Objective: to use the stable isotope labeling method based on two-dimensional liquid chromatography / mass spectrometry (iTRAQ combined with 2D LC-MS/MS) to analyze the serum of patients with nasopharyngeal carcinoma by quantitative proteomic analysis, to obtain the differential protein spectrum and to screen the potential tumor markers.
Methods: the normal group and the nasopharyngeal carcinoma group (stage II, stage III, IV) had 4 groups of serum samples (6 cases in each group) after multiple affinity immunization to remove 14 kinds of high abundance proteins, and then used iTRAQ combined with 2D LC-MS/MS to carry out quantitative proteomic analysis. Excavate potential biomarkers.
Results: 274 confidence proteins were identified in the two mass spectrometry, and the rate of protein repetition in two tests was 70.8%. There were 15 differential proteins identified and quantified in the 2 experiments. 5 of them were up to up (1.4 times) relative to the normal group (1.4 times), and 10 expressions down down (0.7 times).S100A8 and S100A9 were all stable and significant in the 2 quantitative experiments. S100A8/A9, as a function of inflammation related factors, has been widely recognized. Recently, more and more studies have shown that it not only can be used as a tumor marker, but also plays an important role in the process of tumor development and metastasis.
The second part is S100A8/A9, a potential marker for nasopharyngeal carcinoma.
Objective: to verify the differential protein S100A8/A9 obtained by mass spectrometry, confirm their differential expression and preliminarily study their relationship with nasopharyngeal carcinoma.
Methods: (1) ELISA technique was used to detect the expression level of S100A8/A9 in serum; (2) IHC technique was used to detect the expression and distribution of S100A8/A9 in nasopharyngeal carcinoma tissues and nasopharyngitis tissues; (3) ICC and Western Blot techniques were used to detect NP69 in nasopharyngeal cell line in immortalized normal human, CNE1 of highly differentiated nasopharyngeal carcinoma cell line, and CN of low differentiated nasopharyngeal carcinoma cell line. Expression and distribution in E2; (4) RT-PCR technique was used to detect the expression level of mRNA in S100A8/A9/A12 in 3 cell lines; (5) the activity and increment of 3 cell lines were detected by CCK8 method by different concentrations of Zn2+ acting cells, and RT-PCR was used to detect the change of mRNA expression level of S100A8/A9/A12 with Zn2+ concentration.
Results: (1) the change trend of S100A8/A9 content in serum and pathological tissue conforms to the results of mass spectrometry, and the cancer group is higher than the normal group. (2) S100A8/A9 is in the nasopharyngeal carcinoma cell line, the other high differentiated squamous cell carcinoma cells in the head and neck, the highly differentiated epidermal cells are strongly expressed in the low differentiated glandular cell, and the non expression of S100A8/A9 in the basal cells may be suggested as possible. The mRNA level of S100A8/A9/A12 was increased with the prolongation of culture time in the cancer cell line CNE1, CNE2, and the expression of normal cells increased in NP69. (4) Zn2+50 mu M could promote the proliferation of 3 cell lines, but the cytotoxicity was quickly displayed after the concentration of more than 70gM, and most of the cells were dead at 20 u M and CNE2 showed stronger. In addition, it is worth mentioning that when the cell growth is stacked, especially the cancer cell lines, the tolerance to Zn2+ is obviously enhanced, and most of the cells still survive at 110 M. 5. As the concentration of Zn2+ increases, the proliferation and activity of the cell is strengthened when the mRNA expression of S100A8/A9/A12 decreases. It shows that S100A8/A9/A12 may be involved in the cell growth process.
Conclusion: iTRAQ combined with 2DLC-MS/MS is a powerful quantitative proteomic method. Its high flux, high repeatability and accuracy are helpful for screening biomarkers. This study is the first to use iTRAQ technology to screen serum related markers of nasopharyngeal carcinoma and obtain a series of differential proteins. The validation and research of white S100A8/A9 indicate that it is a potential marker for nasopharyngeal carcinoma and is closely related to the development of nasopharyngeal carcinoma. Further research on these potential markers will help the screening and diagnosis of nasopharyngeal carcinoma, discover new therapeutic targets and explore the mechanism of cancer development.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R739.63

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