【作者】 朱辉；

【导师】 吴明拜； 伊力亚尔·夏合丁；

【作者基本信息】 新疆医科大学 ， 内科学， 2011， 博士

【摘要】 食管癌是消化系统常见的恶性肿瘤之一。中国是食管癌高发国家,鳞癌约占我国食管癌总数的80%。食管癌流行病学特征除了显著的地域分布差异之外,民族分布差异亦是其主要的特征之一。食管癌的发生在新疆有着显著的地域性和民族聚集性,提示新疆食管癌的发生受环境和遗传因素共同影响。维吾尔族是新疆的主体民族,但是已有的研究对新疆维吾尔族食管癌的相关研究较少。目前仍然缺乏食管癌的早期诊断方法,大部分食管癌患者确诊时已属中晚期,治疗的效果欠佳。手术治疗是食管癌治疗的主要方法,食管癌患者手术后死亡的主要原因是肿瘤复发和转移,肿瘤淋巴结转移是影响食管癌预后的重要因素之一。目前对手术前有无淋巴结转移判断存在不准确的问题,给手术和治疗方式选择带来了一定的盲目性。提高患者手术前TNM分期的准确率,特别是提高对淋巴结转移判断的准确性,对于提高食管癌患者的治疗水准和改善预后具有重要意义。目前还没有找到可以有效应用于食管癌术后复发和转移早期检测的血清标志物。如何提高食管癌早期诊断率、手术前淋巴结转移诊断准确性和有效监测治疗后复发及转移,尤其是寻找方便有效的血清诊断标志物,已经成为目前临床和基础研究的重要要方向。蛋白质是生命活动的具体执行者和体现者,肿瘤的发展过程中往往有蛋白质的动态变化。蛋白质组学能够识别鉴定细胞、组织或机体的全部蛋白质,提供一组蛋白质的功能及其模式的信息,能够同时反映细胞内部的遗传特性和外界因素的影响的结果。因此,可以在蛋白质组的水平进行进一步探索,寻找用于肿瘤的术前诊断、分期和术后随访监测等方面特异、灵敏的标志物。利用差异蛋白质组学的研究方法对肿瘤和正常人群的差异表达蛋白进行定量、定性、表征分析并且筛选出肿瘤相关的蛋白标记物已成为目前研究的热点。随着蛋白质组学相关技术的迅速发展,临床血清蛋白质组学成为当今生命科学领域中极其活跃的学科,临床血清蛋白质组学从新的角度研究肿瘤,寻找肿瘤标志物,为肿瘤的早期诊断、预后和动态监测提供了重要依据。表面增强激光解吸离子化飞行时间质谱(surface-enhanced laser desorption/ ionization time of flight mass spectrometry, SELDI-TOF-MS或SELDI)技术是一种将生物样品(如细胞液或体液)中的各种蛋白质通过特定的表面基团吸附于蛋白质芯片上,用激光脉冲辐射使芯片上的蛋白质解析形成荷电离子,这些不同质荷比(M/Z)的离子在真空电场中飞行的时间长短不同,据此绘制出质谱图,以获得各种蛋白质的分子量、丰度等信息。将正常人样本的图谱信息同某种疾病患者比较,就有望发现新的疾病相关蛋白质。SELDI-TOF-MS质谱技术已在许多肿瘤标志物研究中应用并取得成功。应用蛋白质组学SELDI-TOF-MS技术对新疆不同民族血清蛋白质指纹图谱的检测结果提示汉族、哈萨克族、维吾尔族食管癌患者间的血清蛋白质指纹图谱存在着明显的差异。本研究采用SELDI-TOF-MS技术对新疆维吾尔族的血清蛋白质指纹图谱进行检测,结合临床以及病理学资料探讨血清目标蛋白质在新疆维吾尔族食管癌患者诊断和术前淋巴结转移判断当中的价值。分析SELDI-TOF-MS技术对食管癌患者手术治疗前后血清蛋白质表达差异检测结果,探讨可能的临床意义。目的：本研究的目的是通过SELDI-TOF-MS质谱技术分析食管癌鳞癌患者和无癌健康者血清蛋白表达谱的改变以及手术前后的血清蛋白表达谱变化,筛选并建立维吾尔族食管鳞癌诊断和患者淋巴结转移诊断的血清标志物模型。寻找食管癌手术前后变化的血清标志物,进一步完善食管癌诊断、淋巴结转移及预后判断意义的食管癌蛋白表达谱系。这将有助于更深层次的理解食管癌基因学、蛋白组学改变以及加深对肿瘤发生机制的认识,为食管癌疾病早期诊断和和检测术后复发提供血清学的依据。本课题主要进行了三个方面的研究：1)新疆地区维吾尔族食管鳞癌患者与健康者血清差异表达蛋白的研究；2)新疆维吾尔族食管鳞癌淋巴结转移血清蛋白质指纹图谱研究；3)食管癌手术前后血清蛋白质组学变化研究。方法：采用弱阳离子交换蛋白芯片(CM10)和表面增强激光解吸离子化飞行时间质谱(SELDI)技术检测血清样本,应用ZUCI-Protein Chip Data Analyze System软件包分析所得到的数据。用支持向量机方法建立蛋白质指纹图诊断模型,用留—法交叉验证作为评估模型判别效果的方法。两组之间的蛋白质峰的比较采用Wilconxon秩和检验,P<0.05有统计学意义。1)检测分析了43例维吾尔族食管鳞癌患者和22例性别、年龄匹配的正常对照血清蛋白表达谱,建立新疆维吾尔族食管鳞癌诊断模型；2)检测分析了21例维吾尔族食管鳞癌有淋巴结转移、22例维吾尔族食管鳞癌无淋巴结转移的患者和22例维吾尔族正常对照的血清,筛选维吾尔族食管鳞癌淋巴结转移的血清标记物,建立维吾尔族食管癌淋巴结转移诊断模型。3)检测分析45例食管鳞癌患者的手术前和手术后血清蛋白表达谱,筛选手术前后发生变化的蛋白质标志物。结果：1)维吾尔族食管鳞癌组与正常对照血清蛋白M/Z峰值图谱明显不同。所得到差异性最大的10个蛋白中(P<0.05),有5个峰在食管癌组呈高表达,M/Z为4487.3973、11693.8038、9160.7745、5494.4509、15963.7368。5个峰呈低表达,M/Z为2763.4477、6648.88、8712.8596、6681.2584、8789.2461。建立了由7个蛋白(M/Z分别为：4487.3973、8712.8596、9160.7745、5494.4509、6681.25843、8789.2461、11693.8038)组成的诊断模型。采用十倍交叉留一法验证,该诊断模型的敏感度为86.05%(37/43),特异度为68.18%(15/22)；2)新疆维吾尔族食管鳞癌淋巴结阳性患者组,淋巴结阴性组和健康人血清的蛋白M/Z峰值图谱明显不同。筛选了6个蛋白质峰(M/Z4487.7591、5494.0576、7992.6041、5874.1964、5899.7251、8584.908)建立了维吾尔族食管癌淋巴结阴性患者诊断模型。用十倍交叉留一法验证,其敏感度为81.82%(18/22),特异度为86.36%(19/22)；建立了由6个蛋白质峰(M/Z 4487.6122、2763.5209、8713.1525、5494.5497、11693.2483、2750.9962)组成的维吾尔族食管鳞癌淋巴结阳性患者诊断模型。用十倍交叉留一法验证,其敏感度为80.95%(17/21),特异度为86.36%(19/22)；建立了由3个蛋白质峰(M/Z 3275.3129、9442.91、2046.0471)组成的维吾尔族食管鳞癌淋巴结转移诊断模型。用十倍交叉留一法验证,模型的敏感度为76.19%,特异度为77.27%；3)食管癌患者手术前后血清蛋白质指纹图谱存在明显差异,表达差异最大的10个蛋白质峰(P<0.05)中M/Z 8712.3909、8788.9114、6450.9811、8585.3391、6649.156、6899.7862手术后表达低于手术前,M/Z 11496.6914、16153.17、7993.1528、11418.0658手术后表达高于手术前。其中3个蛋白质峰(M/Z 8712.3909/8714.1856、8788.9114/8789.6786、6649.156/6649.9783)与此前筛选到的维吾尔族食管鳞癌患者与正常对照之间差异蛋白质峰相同。结论：1)表面增强激光解吸电离飞行时间质谱技术和生物信息学分析软件的联合应用是一种寻找食管癌鳞癌生物标志物的有效方法,构成的诊断模型可以准确灵敏的区分维吾尔族食管鳞癌和健康人；2)维吾尔族食管鳞癌淋巴结阳性患者,淋巴结阴性患者和健康对照组血清蛋白质指纹图谱明显不同。其中5个蛋白峰(M/Z为16081.0634、16152.0842、15962.2598、2019.8519、2044.3842)可能对判定病情预后有重要意义；3)新疆维吾尔族食管癌手术前后血清蛋白质组学变化研究筛选到的3个蛋白质峰(M/Z 8712.3909/8714.1856、8788.9114/8789.6786、6649.156/6649.9783)可能并非是肿瘤分泌的特异性标记物,有可能是与肿瘤患者体内原有的受到抑制免疫相关蛋白或因为进食困难较正常已经降低的营养状况相关指标,其分子特征有待进一步研究。更多还原

【Abstract】 Esophageal carcinoma (EC) is one of the main digestive system malignant tumor. China is a country of high incidence of esophageal cancer and Esophageal squamous cell carcinoma (ESCC) accounts for about 80%of the total number. Epidemiological characteristics of esophageal cancer, the differences in ethnic distribution is one of its main features in addition to significant differences in geographical distribution. Esopha-geal cancer in Xinjiang has a significant regional and national aggregation, which suggest the incidence of esophageal cancer in Xinjiang be affected by the combined effect of environmental and genetic factors. Existing research for Uygur in Xinjiang only few studies on esophageal cancer, and insufficient information available. There is still a lack of early diagnosis of esophageal cancer. Most of the time of diagnosis of esophageal cancer patients are already in the late, their treatment is not so effective. Surgery is the main method of treatment of esophageal cancer. The leading death causes of patients with esophageal cancer after surgery are tumor recurrence and metastasis. Lymphatic metastasis (LM) is an important prognostic factor for esophageal cancer. Current problem is that we can not accurately determine the existence of LM before surgery. So it bring a certain blindness to surgery and treatment options. To improve TNM staging of patients prior to surgery, especially, to improve the accuracy of LM, show great significance for improving the treatment and improving the prognosis of patients with esophageal cancer. So far, there is no effective serum markers application found in diagnosis of esophageal cancer recurrence and metastasis in early stage. How to improve the early diagnosis of esophageal cancer and preoperative diagnostic accuracy of LM, and effective monitoring of recurrence and metastasis after treatment, especially look for convenient and effective diagnostic markers in serum, have become an important direction of clinical and basic research.Protein is the actual implementation and embodiment of those life activities. The development of cancer often have protein dynamics. Proteomics can identify cells, tissues or whole body protein, which provides a set information of protein function and mode and also reflects the genetic characteristics and the results of the impacts of external factors on inside cells. So, looking for specific and sensitive marker for preoperative staging and postoperative follow-up monitoring calls for the further explora-tion for proteome. Using differential proteomics on the expression and quantitative differences between tumor and the normal or benign lesions, and characterization of selected tumor-associated protein markers has become a research hotspot. With the rapid development of proteomics-related technologies, clinical serum proteomics become very active in the field of life sciences disciplines. From a new angle, clinical serum proteomics studies and finds tumor markers, which provides an important basis for early cancer diagnosis, prognosis and dynamic monitoring.A novel technology, surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) evared out such a new path to porteomics research on cancer. As high qunatities of data were obatined from proteomics fields, which traditonal mathematic and statistic methods can not analysis so much data efficiently. Proteins are captured by adsorption, partition, electrostatic interaction, or affinity chromatography on a solid-phase protein chip surface. The protein chip chromatographic surfaces in SELDI are uniquelydesigned to retain proteins from complex mixtures according to their specific properties. After adding a matrix solution, proteins can be ionized with a nitrogen laser and their molecular masses measured by TOF-MS. These different mass to charge ratio (M/Z) of ions in a vacuum electric field in the different lengths of time of flight, thus rendering the mass spectra to obtain a variety of protein molecular weight, abundance and other information. Compared with patients with a disease by their map information, the new disease-related proteins will be found. SELDI-TOF-MS mass spectrometry have been applied to the study biomarkers in many tumor and succeed.Application of SELDI-TOF-MS technique on different nationalities for detection of serum protein fingerprint suggest that information of all health ethnic groups is different from patients with esophageal obviously. In this study, combined clinical and pathological data, SELDI-TOF-MS technology was used for explore the differences in serum proteins between patients with esophageal cancer and the healthy person and estimated the value of diagnosis and preoperative diagnosis of LM in Uygur patient with esophageal cancer in Xinjaing.Objective:The following three aspects analyzed in this study ars to:1) Screen serum tumor biomakers and find diagnostic model of ESCC by SELDI-TOF-MS and ZUCI-Protein Chip Data Analyze System package (ZUCI-PCDAS) in Uygur in Xinjiang; 2) Screen serum tumor biomakers and find diagnostic model of LM of ESCC by SELDI-TOF-MS and PCDAS in Uygur in Xinjiang; 3) Screen serum tumor biomakers and analyze the perioperative dynamic variation of it by SELDI-TOF-MS in patients with ESCC. Methods:1) Serum samples from 42 ESCC Uygur’s patients and 22 Uygur’s healthy controls were analysed on weak cation exchange and hydrophobic surface protein chip (CM 10) using SELDI-TOF-MS technology in screen out the serum differentially expressed markers of ESCC; 2) Serum samples from 42 ESCC Uygur’s patients,21 with and 22 without intrathoracic LM, were screened were detected on weak cation exchange (CM 10) protein chip using SELDI-TOF-MS technology. The obtained protein expression profiles data were devited into two groups named as diagnostic cast, and the results were analyzed using ZUCI-PDAS software in order to screen out serum differentially proteins. 3) Preoperative and postoperative serum samples from 45 patients were detected on weak cation exchangeand hydrophobic surface protein chip (CM10) using SELDI-TOF-MS technology. Also, serum samples of 64 patients and 63 health control the results were were detected. The result of these two groups were comparative analyzed to monitering dynamic variation of biomaker. Results:1) M/Zs were significantly different between ESCC patients and controls in Uygur (P<0.05). Among them, five proteins peaks (4487.3973、15963.7368、9160.7745、5494.4509、11693.8038) were high-expressed and five(2763.4477、8712.8596、6648.88、6681.2584、8789.2461) were low-expressed in ESCC group. According to cross validation, seven proteins composed and established diagnostic model. The sensitivity and specificity of the diagnostic model were 86.05%(37/43) and 68.18%(15/22); 2) M/Zs were significantly different between ESCC patients with and without LM in Uygur (P<0.05). According to cross validation, six proteins composed and established diagnostic model. The sensitivity and specificity of the diagnostic model were 76.19%(16/21) and 77.27%(17/22) respectively; 3) There was significantly difference of serum protein profiling between preoperative and postoperative patients. Three of protein peaks in preoperative sample, which was higher in health control than preoperative patients, was lower postoperatively. Conclusion:1) The combination of Surface-enhanced laser desorption ionization time of flight mass spectrometry and bioinformatics software analysis is a effective method searching for biomarkers of esophageal squamous cell carcinoma, constitutes sensitive diagnostic model can accurately distinguish esophageal squamous cell carcinoma and healthy Uygur. The established sensitive diagnostic model can accurately distinguish esophageal squamous cell carcinoma with healthy Uygur people; 2) The serum protein patterns were significantly different among uygur patients with positive lymph node detection, lymph node-negative patients and healthy controls,5 protein peaks (M/Z%16081.0634, 16152.0842,15962.2598,2019.8519,2044.3842) of which may be important for determining prognosis; 3) The three Screened protein peaks may not be specific markers secreted by tumor, may be suppressed immune related protein in the tumor patients, or relevant indicators of nutritional status because of swallowing difficulties compared with normal. Further study of its molecular characteristics is needed.更多还原