水通道蛋白-3在食管鳞癌区域癌化中的表达及临床相关研究
发布时间：2018-08-05 19:53
【摘要】：食管癌(Esophageal carcinoma,EC)是一种最常见的消化道恶性肿瘤,位居全世界癌症发病率第四位。在中国食管癌已经成为主要的消化道肿瘤死因之一。现阶段尚不清楚食管癌的确切病因,临床上大多数食管癌患者就诊时已处于中晚期,近年来随着外科诊疗水平的提高其术后5年生存率仅仅为25%左右。然而,早期食管癌术后5年生存率可达90%以上。为此探讨针对食管癌的早期致病机制,已经成为临床食管癌主要的研究方向之一。目前为止,食管癌致病机制的临床和基础研究仍无突破。随着分子生物学技术研究的提高,已经证实在头颈部鳞癌、肺癌、口腔癌、结肠癌中包括食管鳞癌在内的多种上皮来源性的肿瘤中都有可能存在“区域癌化”等早期癌变现象。水通道蛋白-3(AQP-3,aquaporin-3)作为水通道蛋白家族中一重要亚型,具有该蛋白家族基因的共同特性,研究发现AQP3曾作为致癌基因在食管癌的分化增殖和侵袭转移中起着重要的作用。目前对于食管鳞癌的研究国内很少见从“区域癌化”理论角度来探讨食管癌早期发病机制,尤其缺乏对食管鳞癌区域癌化内水通道蛋白-3(AQP3)表达异常的系统性认识及相关临床研究。近年研究结果显示,AQP3在多种不同恶性肿瘤内表达水平与肿瘤的增殖分化、远处侵袭转移及生存预后存在相关性。依据现有文献报道,在食管鳞癌不同组织区域中存在AQP3表达差异,其中AQP3表达含量与食管鳞癌的临床预后具有相关性。本研究采用Lugol's染色及HE化学染色法初步筛选出食管鳞癌中具有“区域癌化组织”的标本,通过Real-Time PCR实时荧光定量和Western-blot蛋白质免疫印迹技术从基因和蛋白质水平验证AQP3基因在食管正常组织、区域癌化及配对鳞癌组织中的表达差异,并分析其与相关影响因素之间的关系;结合术后随访系统评估食管鳞癌患者AQP3表达水平及相关临床预后因素对生存时间的影响;最后从基因、蛋白质组学方面为食管鳞癌的早期发病机制提供新的思路。目的:依据食管鳞癌“区域癌化”理论基础,用Lugol's染色及HE化学染色法初步筛选出食管鳞癌中“区域癌化组织”即Lugol's液染色阳性病理表型正常的食管粘膜组织。利用Real-Time PCR实时荧光定量和Western-blot蛋白质免疫印迹技术验证水通道蛋白-3(AQP3)在食管正常上皮、区域癌化及食管鳞癌三种不同组织中的表达,并结合临床相关预后因素探讨AQP3表达与食管鳞癌生存时间的关系,初步了解AQP3基因在食管鳞癌癌变过程中的作用。方法:(1)回顾性分析2005年1月至2015年12月间甘肃省人民医院及甘肃省肿瘤医院胸外科食管鳞癌手术标本共102例。所有收集的标本均行食管粘膜Lugos’s液染色,初步筛选出65例患者存在“区域癌化”组织;(2)将65例符合纳入标准的标本分别采用Real-Time PCR和Western-blot从基因及蛋白层面检测水通道蛋白-3(AQP3)在65例食管鳞癌区域癌化组织中的表达情况。(3)依据病历查询系统及生存期回访系统回顾性收集65例患者临床相关信息。采用Kaplan-Meier生存曲线分析比较各相关独立因素生存期;采用Log-Rank检验比较各组间生存时间差异;Cox回归模型分析判断影响食管鳞癌预后的独立因素,以α=0.05为差异有显著统计学意义。结果1.Lugol's染色及HE化学染色法初步筛选出食管鳞癌田野癌化区域。证实了碘染色异常但病理表型正常的食管上皮符合区域癌化的条件,是食管鳞癌的早期病变。2.AQP3mRNA、蛋白表达水平与食管鳞癌的关系。AQP3mRNA、蛋白表达在三种不同食管组织中存在显著的统计学差异,其表达含量依次升高(P0.05)。男性、家族史、淋巴结转移、高中分化、浸润深度、肿瘤大小,与AQP3mRNA阳高性表达率相关,且AQP3表达蛋白的变化趋势与AQP3基因表达具有一致性。3.生存分析Kaplan-Meier生存分析显示:食管鳞癌生存期与性别、家族史、淋巴结转移、浸润深度、分化程度、肿瘤病理形态、AQP3mRNA及蛋白表达等单因素相关。Cox风险回归模型显示:性别、淋巴结转移、分化程度、浸润深度及AQP3mRNA、蛋白阳性表达情况为食管鳞癌的独立预后因素。结论总之本课题首次筛选出食管鳞癌病灶外区域癌化组织,然后从基因组、蛋白组等不同层面验证了食管鳞癌区域癌化中差异基因AQP3的表达情况,并结合临床病理指标分析AQP3表达与食管鳞癌生存期之间的相关性,从而使人们对区域癌化的本质有更深入的了解,为寻找食管鳞癌致病的上游靶点提供了新的研究方向。
[Abstract]:Esophageal carcinoma (EC) is one of the most common malignant tumors in the digestive tract, ranking fourth in the world. In China, esophageal cancer has become one of the main causes of death in the digestive tract. The exact cause of esophageal cancer is not clear at this stage. Most of the patients with esophageal cancer have been in the middle and late stages of clinic, and in recent years, the most of the patients are in the middle and late stages. With the improvement of surgical diagnosis, the survival rate of 5 years after surgery is only about 25%. However, the 5 year survival rate of early esophageal carcinoma can reach 90%. Therefore, the early pathogenesis of esophageal cancer has become one of the main research directions in the clinical esophagus cancer. So far, the clinical and basic Research of the pathogenesis of esophageal cancer is still still in the clinical and basic research. No breakthrough. With the improvement of molecular biological technology, it has been proved that early cancerous phenomena such as "regional carcinogenesis" are possible in the head and neck squamous cell carcinoma, lung cancer, oral cancer, and carcinoma of the esophagus including squamous carcinoma of the esophagus. The water channel protein -3 (AQP-3, aquaporin-3) is used as the aquaporin family. An important subtype, with the common characteristics of the protein family, has been found that AQP3 has played an important role in the proliferation and invasion and metastasis of esophageal cancer as a oncogene. At present, it is rare to explore the early pathogenesis of esophageal cancer from the perspective of "regional carcinogenicity", especially in the study of esophageal squamous cell carcinoma. Systematic understanding of abnormal expression of -3 (AQP3) in carcinogenesis of squamous cell carcinoma and related clinical studies. Recent results show that the expression level of AQP3 in a variety of different malignant tumors is related to tumor proliferation and differentiation, distant invasion and metastasis and survival prognosis. According to the existing literature, different tissues of esophageal squamous cell carcinoma are reported. There is a difference in the expression of AQP3 in the domain, in which the expression of AQP3 is correlated with the clinical prognosis of esophageal squamous cell carcinoma. In this study, the specimens with "regional cancerous tissue" in esophageal squamous cell carcinoma were screened by Lugol's staining and HE chemical staining, and the Real-Time PCR real-time fluorescence determination and Western-blot protein immunoblotting technique were used. Gene and protein levels were used to verify the difference in the expression of AQP3 gene in normal esophageal tissue, regional carcinogenesis and paired squamous cell carcinoma, and to analyze the relationship between the gene and related factors, and to evaluate the expression level of AQP3 in the patients with esophageal squamous cell carcinoma and the effect of related clinical prognostic factors on the survival time. The white matter group provides a new idea for the early pathogenesis of esophageal squamous cell carcinoma. Objective: Based on the theory of "regional carcinogenicity" of esophageal squamous cell carcinoma, Lugol's staining and HE chemical staining were used to preliminarily screen the esophageal mucosa tissues of "regional cancerous tissue" in esophageal squamous cell carcinoma, that is, the positive pathological phenotype of Lugol's liquid staining, and the use of Real-Time. PCR real-time fluorescence quantification and Western-blot protein immunoblotting were used to verify the expression of aquaporin -3 (AQP3) in three different tissues of normal esophageal epithelium, regional carcinogenesis and esophageal squamous cell carcinoma, and the relationship between the expression of AQP3 and the survival time of esophageal squamous cell carcinoma combined with clinical prognostic factors, and a preliminary understanding of the AQP3 gene in squamous carcinoma of the esophagus Methods: (1) retrospective analysis of 102 cases of esophageal squamous cell carcinoma in Gansu people's Hospital and Gansu cancer hospital from January 2005 to December 2015. All the collected specimens were stained with Lugos' s solution of esophageal mucosa, and 65 cases were preliminarily screened for "regional cancerous" tissue; (2) 65 cases were conformed to Nana. The standard specimens were used to detect the expression of aquaporin -3 (AQP3) in the cancerous tissues of 65 cases of esophageal squamous cell carcinoma using Real-Time PCR and Western-blot, respectively. (3) the clinical information of 65 patients was collected according to the medical record query system and the survival time return system. The Kaplan-Meier survival curve was used. The survival time of each related independent factor was compared and compared. Log-Rank test was used to compare the difference of survival time between each group; Cox regression model was used to determine the independent factors affecting the prognosis of esophageal squamous cell carcinoma, and the difference of alpha =0.05 was significant. Results 1.Lugol's staining and HE chemical staining were used to screen out the carcinogenesis area of esophageal squamous cell carcinoma. The esophageal epithelium with abnormal iodine staining but normal pathological phenotype accords with the condition of regional carcinogenesis. It is an early pathological change of esophageal squamous cell carcinoma (.2.AQP3mRNA). The relationship between protein expression level and esophageal squamous cell carcinoma is.AQP3mRNA. The protein expression in three different esophageal tissues has significant statistical difference, and the expression level of the esophageal squamous cell carcinoma is increased in turn (P0.05). History, lymph node metastasis, high school differentiation, depth of invasion, tumor size, correlation with AQP3mRNA Yang high expression rate, and the variation trend of AQP3 expression protein with AQP3 gene expression,.3. survival analysis Kaplan-Meier survival analysis showed that the survival period of esophageal squamous cell carcinoma and sex, family history, lymph node metastasis, depth, degree of differentiation, tumor A single factor related.Cox risk regression model, such as pathological morphology, AQP3mRNA and protein expression, showed that sex, lymph node metastasis, degree of differentiation, depth of infiltration and AQP3mRNA, and positive expression of protein were independent prognostic factors of esophageal squamous cell carcinoma. Conclusion in conclusion, this topic first screened out cancerous tissue in the outer region of the lesions of the squamous cell carcinoma of the tube, and then from the genome, The expression of differential gene AQP3 in the carcinogenesis of esophageal squamous cell carcinoma was verified at different levels, and the correlation between the expression of AQP3 and the survival period of esophageal squamous cell carcinoma was analyzed in combination with the clinicopathological indexes, thus making people more understanding of the nature of regional carcinogenesis, and providing a new research for the search of the upstream target of the esophageal squamous cell carcinoma. Look at the direction.
【学位授予单位】：甘肃中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1
，
本文编号：2166847
[Abstract]:Esophageal carcinoma (EC) is one of the most common malignant tumors in the digestive tract, ranking fourth in the world. In China, esophageal cancer has become one of the main causes of death in the digestive tract. The exact cause of esophageal cancer is not clear at this stage. Most of the patients with esophageal cancer have been in the middle and late stages of clinic, and in recent years, the most of the patients are in the middle and late stages. With the improvement of surgical diagnosis, the survival rate of 5 years after surgery is only about 25%. However, the 5 year survival rate of early esophageal carcinoma can reach 90%. Therefore, the early pathogenesis of esophageal cancer has become one of the main research directions in the clinical esophagus cancer. So far, the clinical and basic Research of the pathogenesis of esophageal cancer is still still in the clinical and basic research. No breakthrough. With the improvement of molecular biological technology, it has been proved that early cancerous phenomena such as "regional carcinogenesis" are possible in the head and neck squamous cell carcinoma, lung cancer, oral cancer, and carcinoma of the esophagus including squamous carcinoma of the esophagus. The water channel protein -3 (AQP-3, aquaporin-3) is used as the aquaporin family. An important subtype, with the common characteristics of the protein family, has been found that AQP3 has played an important role in the proliferation and invasion and metastasis of esophageal cancer as a oncogene. At present, it is rare to explore the early pathogenesis of esophageal cancer from the perspective of "regional carcinogenicity", especially in the study of esophageal squamous cell carcinoma. Systematic understanding of abnormal expression of -3 (AQP3) in carcinogenesis of squamous cell carcinoma and related clinical studies. Recent results show that the expression level of AQP3 in a variety of different malignant tumors is related to tumor proliferation and differentiation, distant invasion and metastasis and survival prognosis. According to the existing literature, different tissues of esophageal squamous cell carcinoma are reported. There is a difference in the expression of AQP3 in the domain, in which the expression of AQP3 is correlated with the clinical prognosis of esophageal squamous cell carcinoma. In this study, the specimens with "regional cancerous tissue" in esophageal squamous cell carcinoma were screened by Lugol's staining and HE chemical staining, and the Real-Time PCR real-time fluorescence determination and Western-blot protein immunoblotting technique were used. Gene and protein levels were used to verify the difference in the expression of AQP3 gene in normal esophageal tissue, regional carcinogenesis and paired squamous cell carcinoma, and to analyze the relationship between the gene and related factors, and to evaluate the expression level of AQP3 in the patients with esophageal squamous cell carcinoma and the effect of related clinical prognostic factors on the survival time. The white matter group provides a new idea for the early pathogenesis of esophageal squamous cell carcinoma. Objective: Based on the theory of "regional carcinogenicity" of esophageal squamous cell carcinoma, Lugol's staining and HE chemical staining were used to preliminarily screen the esophageal mucosa tissues of "regional cancerous tissue" in esophageal squamous cell carcinoma, that is, the positive pathological phenotype of Lugol's liquid staining, and the use of Real-Time. PCR real-time fluorescence quantification and Western-blot protein immunoblotting were used to verify the expression of aquaporin -3 (AQP3) in three different tissues of normal esophageal epithelium, regional carcinogenesis and esophageal squamous cell carcinoma, and the relationship between the expression of AQP3 and the survival time of esophageal squamous cell carcinoma combined with clinical prognostic factors, and a preliminary understanding of the AQP3 gene in squamous carcinoma of the esophagus Methods: (1) retrospective analysis of 102 cases of esophageal squamous cell carcinoma in Gansu people's Hospital and Gansu cancer hospital from January 2005 to December 2015. All the collected specimens were stained with Lugos' s solution of esophageal mucosa, and 65 cases were preliminarily screened for "regional cancerous" tissue; (2) 65 cases were conformed to Nana. The standard specimens were used to detect the expression of aquaporin -3 (AQP3) in the cancerous tissues of 65 cases of esophageal squamous cell carcinoma using Real-Time PCR and Western-blot, respectively. (3) the clinical information of 65 patients was collected according to the medical record query system and the survival time return system. The Kaplan-Meier survival curve was used. The survival time of each related independent factor was compared and compared. Log-Rank test was used to compare the difference of survival time between each group; Cox regression model was used to determine the independent factors affecting the prognosis of esophageal squamous cell carcinoma, and the difference of alpha =0.05 was significant. Results 1.Lugol's staining and HE chemical staining were used to screen out the carcinogenesis area of esophageal squamous cell carcinoma. The esophageal epithelium with abnormal iodine staining but normal pathological phenotype accords with the condition of regional carcinogenesis. It is an early pathological change of esophageal squamous cell carcinoma (.2.AQP3mRNA). The relationship between protein expression level and esophageal squamous cell carcinoma is.AQP3mRNA. The protein expression in three different esophageal tissues has significant statistical difference, and the expression level of the esophageal squamous cell carcinoma is increased in turn (P0.05). History, lymph node metastasis, high school differentiation, depth of invasion, tumor size, correlation with AQP3mRNA Yang high expression rate, and the variation trend of AQP3 expression protein with AQP3 gene expression,.3. survival analysis Kaplan-Meier survival analysis showed that the survival period of esophageal squamous cell carcinoma and sex, family history, lymph node metastasis, depth, degree of differentiation, tumor A single factor related.Cox risk regression model, such as pathological morphology, AQP3mRNA and protein expression, showed that sex, lymph node metastasis, degree of differentiation, depth of infiltration and AQP3mRNA, and positive expression of protein were independent prognostic factors of esophageal squamous cell carcinoma. Conclusion in conclusion, this topic first screened out cancerous tissue in the outer region of the lesions of the squamous cell carcinoma of the tube, and then from the genome, The expression of differential gene AQP3 in the carcinogenesis of esophageal squamous cell carcinoma was verified at different levels, and the correlation between the expression of AQP3 and the survival period of esophageal squamous cell carcinoma was analyzed in combination with the clinicopathological indexes, thus making people more understanding of the nature of regional carcinogenesis, and providing a new research for the search of the upstream target of the esophageal squamous cell carcinoma. Look at the direction.
【学位授予单位】：甘肃中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1
，
本文编号：2166847
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