claudin-1与cyclinB1在下咽癌中的表达及其临床意义

发布时间：2018-05-16 04:04

本文选题：claudin-1 + cyclinB1　；参考：《山东大学》2015年博士论文

【摘要】：下咽癌在头颈部恶性肿瘤中相对较少见,90%以上为鳞状细胞癌；虽然发病率较低,但却是恶性程度较高的肿瘤之一,其具体病因尚不明确,绝大多数病人有长期大量的吸烟和饮酒史,考虑可能与长期吸烟饮酒关系密切,临床上主要表现为咽部不适、咽异物感、咽痛、吞咽疼痛、颈部包块等,晚期可出现声音嘶哑、吞咽困难、呼吸困难等,因早期症状不典型,同时下咽部有丰富的淋巴管网,往往患者发现时已经处于晚期,多伴有颈部淋巴结转移和/或邻近部位侵犯；目前多采用以手术治疗为主,辅以放疗、化疗的综合治疗的方法,但患者预后仍然较差,国外报道5年的生存率只有30%。而早发现、早治疗将有效的改善下咽癌患者的预后,因此寻找一些理想的肿瘤标志物会帮助我们了解下咽癌肿瘤的生物学特性,从而有利于制定个性化的治疗方案,改善肿瘤患者的预后。紧密连接是位于上皮细胞及内皮细胞间的一种重要结构,在维持细胞稳态、维系上皮细胞间黏附和保持细胞的极性中发挥重要作用,其主要封闭蛋白为claudin蛋白,其结构及功能的变化直接受到claudin蛋白表达数量的变化所影响；目前研究认为claudin蛋白可在多种恶性肿瘤中出现异常表达,使细胞极性丢失,细胞间的黏附性下降,从而改变了紧密连接的正常结构及功能,促使肿瘤细胞获得侵袭力及转移的潜能。claudin-1属claudins蛋白家族成员之一,在多种肿瘤中出现异常表达,研究表明,claudin-1在肾细胞癌、卵巢癌、胃腺癌和大肠癌中过度表达,促进了肿瘤发生发展的过程,而在对乳腺癌和肺腺癌的研究中,发现claudin-1表达显著降低。这种差异性的表达方式已被证实与肿瘤的发生、发展密切相关。正常细胞的周期是在特定的细胞周期蛋白和细胞周期蛋白依赖性激酶的作用下,忠实而有序的启动和完成的,而不受控制的增殖是恶性肿瘤的标志之一,细胞周期调节失控是细胞过度增殖及癌变的重要原因cyclinB1是一个经典的细胞周期蛋白,通过改变细胞G2/M期的进程,改变了细胞周期进程,从而参与调控细胞周期检查点。目前研究显示,在多种恶性肿瘤细胞中,如在乳腺癌、胃癌、早期非小细胞肺癌、结肠直肠癌、前列腺癌中都发现cyclinBl(?)过表达,cyclinB1失控性的表达与肿瘤细胞的转化和恶性增殖密切相关,而进一步研究显示,cyclinB1的过表达与食管癌、舌癌、肺癌患者的预后有关,所以cyclinB1有可能成为一个潜在的肿瘤诊断标志。恶性肿瘤的重要特性是浸润、扩散和转移,肿瘤的转移是一个复杂的过程,一般可分为黏附、降解、移动3个步骤,受到多种因素的影响,而通过血管及淋巴管转移是多数肿瘤最常见、最主要的转移方式,血管和淋巴管成为肿瘤转移的重要通道,目前认为在恶性肿瘤的过度生长、局部侵袭及转移过程中,新生的血管及淋巴管起到了极其重要的作用。大量的研究使肿瘤通过血管转移得到了很大的发展；而淋巴管的转移在肿瘤的转移中也起着重要的作用,肿瘤细胞在原发灶快速生长,并从原发灶上脱离,浸润、进入附近的微淋巴管,沿微淋巴管运行至目标淋巴结,在目标淋巴管内进行大量增殖,最终形成转移淋巴结。随着特异性的抗淋巴管内皮细胞抗体的发现,微淋巴管作为肿瘤侵袭、转移的通道,在肿瘤的侵袭、转移过程中的作用越来越被人们所重视。微淋巴管密度(microlymphatic vessel density, MLVD)成为评估恶性肿瘤转移与预后的重要因子。综上所述,我们发现claudin-1和cylinBl在下咽鳞状细胞癌组织中的表达水平、与肿瘤临床分期、病理学分级、患者临床预后之间的相关性尚有待研究；claudin-1蛋白与下咽癌微淋巴管生成是否相关尚不清楚。本研究采用免疫组化和实时定量PCR的方法,测定claudin-1和cyclinBl在下咽癌组织及癌旁组织中的表达情况,分析claudin-1口cyclinBl与下咽鳞癌的临床病理特征之间的关系,同时分析它们与患者预后之间的关系,采用免疫组化的方法检测D2-40标记的微淋巴管密度,研究claudin-1蛋白与下咽鳞癌淋巴管生成的关系,以期为下咽癌的早期诊断和预后判断提供分子生物学指标,为寻求肿瘤靶向治疗提供研究方向,同时应用体外实验通过沉默claudin-1观察对FaDu细胞增殖及侵袭的影响,进一步研究claudin-1的生物学特性。第一部分claudin-1与cyclinBl在下咽癌中的表达及其临床意义下咽鳞状细胞癌的发病率虽然较低,但因原发肿瘤位置隐蔽,不易发现,多数下咽癌患者在就诊时已经是晚期,往往已发生临近部位侵犯和颈部淋巴结转移,患者预后较差。claudin-1属于claudins蛋白家族成员之一,在多种肿瘤中出现异常表达,目前研究表明,这种异常表达与肿瘤的发生、发展有关；cyclinBl是一个经典的细胞周期蛋白,目前发现cyclinBl失控性的表达与肿瘤细胞的转化和恶性增殖密切相关。但是claudin-1与cyclinB1在下咽鳞癌中的表达还罕有报道。因此,我们首先研究claudin-1与cyclinB1在下咽鳞癌及癌旁组织中的表达情况,分析claudin-1和cyclinB1与下咽鳞癌的临床病理特征之间的关系,同时分析它们与患者预后之间的关系。目的与方法：1、选取97例具有完整临床病理资料的下咽鳞癌患者的组织学标本为研究对象,并选取90例癌旁正常黏膜组织作为对照,采用免疫组化的方法从蛋白水平比较claudin-1和cyclinB1在下咽鳞癌及正常黏膜组织中的表达的差异,分析两者在下咽鳞癌中的表达与临床病理参数及患者预后之间的关系。2、选取30位下咽癌病人术后的癌组织及癌旁正常黏膜,采用RT-PCR方法,在mRNA水平检测claudin-1口cyclinB1的表达并定量分析,探讨两者的相关性。结果：1.claudin-1与cyclinB1在下咽癌中均为高表达；claudin-1蛋白表达与肿瘤的分化程度和淋巴结转移有关；cyclinB1蛋白表达与肿瘤的分化程度有关；而与其他临床病理资料无明显相关性。Kaplan-Meier分析显示claudin-1与患者的生存率相关(P＝0.003)。claudin-1与cyclinB1呈明显正相关关系。2.claudin-1 mRNA和cyclinB1mRNA在下咽癌组织中的表达量均高于癌旁黏膜组织中的表达量。Spearman等级分析结果显示在肿瘤组织和癌旁黏膜组织中claudin-1mRNA与cyclinB1mRNA之间存在明显正相关关系。结论：(1)claudin-1在下咽鳞状细胞癌组织中呈高表达,且与肿瘤的分化程度和淋巴结转移有关；并与患者的生存率相关,claudin-1低表达者有较高的生存率(2)cyclinB1在下咽鳞状细胞癌组织中亦呈高表达,并与肿瘤的分化程度有关,与患者的生存率无关。(3)claudin-1和cyclinB1在下咽鳞状细胞癌组织中的表达具有显著正相关关系。第二部分claudin-1蛋白与下咽癌微淋巴管密度的关系研究下咽鳞癌发生部位比较隐匿,同时下咽部有丰富的淋巴管网,容易较早出现颈部淋巴结转移,临床分期N1-N。患者颈部淋巴结转移率为65%-80%,目前认为,颈部淋巴结转移成为下咽癌治疗失败的主要原因之一,严重影响了患者的预后。恶性肿瘤的重要特性包括浸润和转移,而多数肿瘤通过淋巴管发生转移,目前认为淋巴管的生成在肿瘤的生长、侵袭及转移过程中发挥了极其重要的作用。微淋巴管密度(microlymphatic vessel density, MLVD)成为评估恶性肿瘤转移与预后的重要因子。D2-40被认为是目前发现的最具有特异性的淋巴管标记物,广泛的应用于肿瘤微淋巴管生成的研究,我们前期研究中证实下咽鳞状细胞癌中claudin-1蛋白表达情况与肿瘤淋巴结转移有关。但claudin-1蛋白与下咽癌微淋巴管生成是否相关尚不清楚,是否通过微淋巴管生成导致了淋巴结的转移尚待研究；本研究通过研究claudin-1蛋白与下咽鳞癌淋巴管生成的关系,探讨下咽鳞癌淋巴转移的机制。目的与方法：选取97例具有完整临床病理资料的下咽鳞癌患者的组织学标本为研究对象,并选取90例癌旁正常黏膜组织作为对照,采用免疫组化的方法检测claudin-1 和微淋巴管密度在下咽鳞癌及正常黏膜组织中的表达,分析微淋巴管密度与下咽鳞癌临床病理资料的关系,分析claudin-1和微淋巴管密度在下咽鳞癌及正常黏膜组织中表达的相关性,从而探讨下咽鳞癌淋巴转移的机制。结果：1、下咽鳞癌中,癌巢周边区的微淋巴管(MLVD)数目显著高于癌巢中心区微淋巴管数目(MLVD)及癌旁正常粘膜组织中MLVD,两者间差异均有统计学意义(P0.05)；而癌巢中心区MLVD与癌旁正常粘膜组织中MLVD相比,差异无统计学意义(P0.05)。2、癌巢中心区的MLVD与患者各临床指标进行相关分析均无统计学意义(P0.05)。癌巢周边区的MLVD与组织病理学分级、临床分期和淋巴结转移等指标显著相关,分析均有统计学意义(P0.05)。3、癌巢中心区的claudin-1表达阳性组的MLVD与阴性组相比差异无统计学意义,癌巢周边区claudin-1表达阳性MLVD与阴性组MLVD相比有统计学意义(P0.05),相关分析表明在下咽鳞癌癌巢周边区中claudin-1表达与MLVD之间呈正相关表达。结论：1、下咽癌癌巢周边区微淋巴管密度和淋巴结转移相关(P0.05),提示癌巢周边区微淋巴管可能促进了局部淋巴结的转移。2、claudin-1阳性表达和阴性表达在癌巢周边区的微淋巴管密度存在差异,提示高表达claudin-1可能会诱导肿瘤淋巴管生成,从而促进淋巴结转移。第三部分沉默claudin-1在体外对细胞增殖及侵袭的影响我们在前期研究中发现,claudin-1在下咽鳞状细胞癌中呈现高表达,与细胞分化及淋巴结转移相关,且claudin-1mRNA在下咽癌细胞中的含量增高,考虑claudin-1在下咽鳞癌中起了促进肿瘤细胞增殖及局部侵袭转移的功能,因此我们通过体外实验,在FaDu细胞系中通过沉默claudin-1观察FaDu细胞增殖及侵袭能力的变化,从而进一步研究claudin-1的生物学特性。目的与方法：通过脂质体2000转染人咽癌细胞系(FaDu)沉默claudin-1,利用细胞培养技术进行增殖实验和侵袭实验。结果：1、培养的FaDu细胞增殖活力良好。2、应用RNA干扰技术,沉默claudin-1 后, FaDu-siRNA瞬时沉默的效果良好,经Western blot实验证实。3、沉默claudin-1后,FaDu-siRNA (沉默组)、FaDu-control(阴性对照组)以及FaDu未处理组,三组细胞均出现了不同程度的增殖现象,三组的吸光值在48小时后,沉默组与阴性对照组及未处理组比较,细胞的增殖明显受影响,沉默组增殖能力降低,差异具有统计学意义(P0.01),而阴性对照组及未处理组之间比较,差异没有统计学意义(P0.05)。4、侵袭实验表明：进入小室的细胞数,沉默组与阴性对照组及空白对照组分别为15士6.45、47士6.36、50士8.73,差异有统计学意义(P0.001)；沉默claudin-1后,细胞的侵袭能力明显下降,FaDu-control以及FaDu未处理组比FaDu-siRNA组具有更强的侵袭能力。结论：体外实验显示claudin-1能促进下咽癌细胞的增殖、黏附及侵袭。
[Abstract]:Hypopharyngeal carcinoma is relatively rare in the malignant tumor of the head and neck, and more than 90% is squamous cell carcinoma. Although the incidence is low, it is one of the most malignant tumors. The specific cause is not clear. Most of the patients have a long history of smoking and drinking, which may be closely related to long term smoking and drinking. For pharynx discomfort, pharynx sensation, pharynx pain, swallowing pain, neck lump and so on, at the late stage can appear hoarseness, dysphagia, dyspnea and so on. The early symptoms are not typical, and the lower pharynx has a rich lymphatic network. With the method of surgical treatment, combined with radiotherapy and chemotherapy, the prognosis of patients is still poor. The survival rate of 5 years in foreign countries is only 30%.. Early treatment will effectively improve the prognosis of hypopharynx cancer patients, so finding some ideal tumor markers will help us understand the biological characteristics of hypopharyngeal cancer. It is beneficial to develop a personalized treatment scheme and improve the prognosis of cancer patients. Close connection is an important structure between epithelial cells and endothelial cells. It plays an important role in maintaining cell homeostasis, maintaining the adhesion of epithelial cells and maintaining the polarity of cells. The main closed protein is claudin protein, its structure and function. The changes in the expression of claudin protein are directly affected by the changes in the number of expression of the protein. The present study suggests that the abnormal expression of claudin protein in a variety of malignant tumors can cause the loss of cell polarity and the decrease of intercellular adhesion, thus changing the normal structure and function of tightly connected cells, and promoting the potential of invasion and metastasis of tumor cells,.C Laudin-1 is one of the members of the claudins protein family. Abnormal expression in a variety of tumors shows that claudin-1 is overexpressed in renal cell carcinoma, ovarian cancer, gastric adenocarcinoma and colorectal cancer, which promotes the process of tumor development. In the study of breast and lung adenocarcinoma, it is found that the expression of claudin-1 is significantly reduced. The expression pattern has been proved to be closely related to the occurrence and development of tumors. The cycle of normal cells is initiated and completed faithfully and orderly under the action of specific cyclin and cyclin dependent kinase, and uncontrolled proliferation is one of the markers of malignant tumor, and the control of cell cycle control is the overgrowth of cells. The important cause of colonization and carcinogenesis cyclinB1 is a classic cycyclin protein that changes the process of cell cycle by changing the process of G2/M phase and participates in the regulation of cell cycle checkpoints. Current studies show that in many malignant tumor cells, such as breast, gastric, early non small cell lung cancer, colorectal cancer, prostate CyclinBl (?) overexpression is found in cancer, and the expression of cyclinB1 out of control is closely related to the transformation and proliferation of tumor cells. Further studies have shown that the overexpression of cyclinB1 is associated with the prognosis of esophageal, tongue cancer, and lung cancer patients, so cyclinB1 may be a potential diagnostic marker. The important characteristics of malignant tumor It is infiltration, diffusion and metastasis, the metastasis of tumor is a complicated process, which can be divided into 3 steps, adhesion, degradation and movement, which are affected by many factors, and the most common and most important way of metastasis is the most common mode of metastasis through blood vessels and lymphatic metastasis, and the blood vessels and drenching tube are the important channels of tumor metastasis. The neovascularization and lymphatic vessels play an important role in the overgrowth of the tumor, local invasion and metastasis. A large number of studies have made the tumor develop greatly through the metastasis of blood vessels, and the metastasis of the lymphatic tube plays an important role in the metastasis of the tumor. The tumor cells grow rapidly in the primary focus and are from the primary focus. Disconnect, infiltrate, enter the nearby microlymphatics, run along the microlymphatics to the target lymph nodes, proliferate in the target lymphatics, and eventually form a metastatic lymph node. With the specific anti lymphatic endothelial cell antibody, the microlymphatic tube acts as a tumor invasion, the transfer channel, the role of the tumor in the invasion and metastasis process. Microlymphatic vessel density (MLVD) is an important factor in the assessment of metastasis and prognosis of malignant tumors. To sum up, we have found that the expression level of claudin-1 and cylinBl in the hypopharyngeal squamous cell carcinoma tissue is associated with the tumor stage, pathological grade, and the clinical prognosis of the tumor. The correlation between claudin-1 protein and the formation of microlymphatic vessels in hypopharyngeal carcinoma is not clear. The present study uses immunohistochemical and real-time quantitative PCR methods to determine the expression of claudin-1 and cyclinBl in hypopharyngeal and paracancerous tissues, and to analyze the clinicopathological features of claudin-1 mouth cyclinBl and hypopharyngeal squamous cell carcinoma. The relationship between them and the prognosis of patients was analyzed. The microlymphatic density of D2-40 markers was detected by immunohistochemical method, and the relationship between claudin-1 protein and the formation of lymphatic vessels in hypopharyngeal squamous cell carcinoma was studied in order to provide molecular biological indicators for the early diagnosis and prognosis of hypopharyngeal carcinoma and provide a study for the search for tumor targeting therapy. At the same time, the effects of claudin-1 observation on proliferation and invasion of FaDu cells were also applied in vitro, and the biological characteristics of claudin-1 were further studied. The first part of the expression of claudin-1 and cyclinBl in hypopharyngeal carcinoma and the incidence of squamous cell carcinoma of the pharynx in the clinical significance were low, but the location of the primary tumor was not easy to occur because of the location of the primary tumor. Now, most of the patients with hypopharyngeal cancer are in the advanced stage, and often have the invasion of adjacent areas and cervical lymph node metastasis. The poor prognosis of the patients is one of the members of the claudins protein family, which is abnormal expression in a variety of tumors. The present study shows that this abnormal expression is related to the development of the tumor and the development of the tumor; cyclinBl is the.Claudin-1 A classic cycyclin protein has been found to be closely related to the expression of cyclinBl out of control with the transformation and proliferation of tumor cells. However, the expression of claudin-1 and cyclinB1 in hypopharyngeal squamous cell carcinoma is rare. Therefore, we first study the expression of claudin-1 and cyclinB1 in hypopharyngeal squamous carcinoma and para cancer tissues, and analyze Claudi The relationship between n-1 and cyclinB1 with the clinicopathological features of hypopharyngeal squamous cell carcinoma and the relationship between them and the prognosis of the patients. Objective and methods: 1, 97 cases of hypopharyngeal squamous cell carcinoma with complete clinicopathological data were selected as the subjects, and 90 cases of normal mucosa adjacent to the cancer were selected as the control, and the immunological group was used. The expression of claudin-1 and cyclinB1 in hypopharyngeal squamous cell carcinoma and normal mucosal tissue was compared from the protein level. The relationship between the expression of the two in the hypopharyngeal squamous cell carcinoma and the clinicopathological parameters and the prognosis of the patients was analyzed.2. The cancer tissues and the normal mucosa adjacent to the cancer in 30 patients with hypopharynx cancer were selected and the RT-PCR method was used in mRNA water. The expression and quantitative analysis of the claudin-1 mouth cyclinB1 were measured and analyzed. Results: both 1.claudin-1 and cyclinB1 were highly expressed in the hypopharyngeal carcinoma; the expression of claudin-1 protein was related to the degree of differentiation and lymph node metastasis; the expression of cyclinB1 protein was related to the degree of differentiation of the tumor; and it was associated with other clinicopathological data. No significant correlation.Kaplan-Meier analysis showed that claudin-1 was associated with the survival rate of patients (P = 0.003).Claudin-1 and cyclinB1 had a significant positive correlation between.2.claudin-1 mRNA and cyclinB1mRNA in hypopharyngeal carcinoma tissues, which were higher than those in the paracancerous mucosa. There is a significant positive correlation between claudin-1mRNA and cyclinB1mRNA in mucous tissue. Conclusion: (1) claudin-1 is highly expressed in the hypopharyngeal squamous cell carcinoma and is related to the degree of differentiation and lymph node metastasis, and is associated with the survival rate of the patients. The low expression of claudin-1 has a higher survival rate (2) cyclinB1 in the hypopharyngeal squamous cell. The expression of claudin-1 and cyclinB1 in the tissues of hypopharyngeal squamous cell carcinoma has a significant positive correlation. (3) the relationship between the expression of the second part of the hypopharyngeal squamous cell carcinoma and the relationship between the density of the microlymphatic tube of the hypopharynx cancer and the location of the hypopharyngeal squamous cell carcinoma is relatively concealed. At the same time, there is a rich lymphatic network in the lower pharynx, which is prone to cervical lymph node metastasis earlier. The cervical lymph node metastasis rate of N1-N. patients is 65%-80%. It is considered that cervical lymph node metastasis is one of the main reasons for the failure of hypopharyngeal cancer treatment, which seriously affects the prognosis of the patients. Microlymphatic vessel density (MLVD) has become the most important factor in the assessment of metastasis and prognosis of malignant tumors..D2-40 is considered to be the most important factor to be found at present. Specific lymphatic markers are widely used in the study of the formation of tumor microlymphatics. Our previous study has confirmed that the expression of claudin-1 protein in hypopharyngeal squamous cell carcinoma is associated with tumor lymph node metastasis. However, whether claudin-1 protein is associated with the formation of the microlymphatics of hypopharyngeal carcinoma is not clear, and whether the microlymphatic vessels are generated by microlymphatic formation In this study, the mechanism of lymphatic metastasis of hypopharyngeal squamous cell carcinoma was investigated by studying the relationship between claudin-1 protein and the formation of lymphatic vessels in hypopharyngeal squamous cell carcinoma. Objective and methods: to select 97 cases of hypopharyngeal squamous cell carcinoma with complete clinicopathological data and 90 cases of normal paracancerous normal. As a control, the expression of claudin-1 and microlymphatic density in hypopharyngeal squamous cell carcinoma and normal mucous tissue were detected by immunohistochemical method. The relationship between the density of microlymphatic tube and the clinicopathological data of hypopharyngeal squamous cell carcinoma was analyzed. The correlation between the expression of claudin-1 and the density of microlymphatic tube in the hypopharyngeal squamous cell carcinoma and normal mucosa was analyzed. The mechanism of lymphatic metastasis of hypopharyngeal squamous cell carcinoma was investigated. Results: 1, the number of microlymphatic tubes (MLVD) in the surrounding area of the nests of the hypopharyngeal squamous cell carcinoma is significantly higher than the number of microlymphatics (MLVD) in the center of the carcinoma nest and the MLVD in the normal mucosa adjacent to the carcinoma (P0.05), while MLVD in the center of the carcinoma nest and the normal mucosa adjacent to the carcinoma are in the mucosa. Compared with MLVD, there was no significant difference (P0.05).2. There was no statistical significance in the correlation analysis between the MLVD of the cancer nest center and the clinical indexes of the patients (P0.05). The MLVD in the surrounding area of the nests was significantly correlated with the histopathological classification, the clinical stage and lymph node metastasis, and the analysis was statistically significant (P0.05).3, and claudin in the center of the cancer nest. There was no significant difference in the MLVD in the -1 positive group compared with the negative group. The claudin-1 expression positive MLVD in the surrounding area of the nests was statistically significant compared with the negative group MLVD (P0.05). The correlation analysis showed that the expression of Claudin-1 in the nests surrounding the hypopharyngeal squamous cell carcinoma was positively correlated with MLVD. Conclusion: 1, the microlymphatic lymph nodes around the nests of the hypopharyngeal carcinoma are microlymphatic. The correlation between tube density and lymph node metastasis (P0.05) suggests that the microlymphatics in the surrounding area of the carcinoma nest may promote the metastasis of local lymph nodes. The density of claudin-1 positive and negative expression in the peripheral region of the carcinoma nest is different, suggesting that high expression of claudin-1 may induce lymphatic angiogenesis and promote lymph node metastasis. Third The effect of partial silencing of claudin-1 on cell proliferation and invasion in vitro, we found that claudin-1 is highly expressed in hypopharyngeal squamous cell carcinoma, related to cell differentiation and lymph node metastasis, and the content of claudin-1mRNA in hypopharyngeal cancer cells is higher, considering that claudin-1 promotes tumor cells in hypopharyngeal squamous cell carcinoma. The function of proliferation and local invasion and metastasis, therefore, we observed the proliferation of FaDu cells in FaDu cell line by silencing Claudin-1 in vitro.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.63

【共引文献】