β防御素和基质金属蛋白酶在非小细胞肺癌的表达及与肺癌转移关系的研究

发布时间：2018-06-26 04:07

本文选题：人β-防御素2 + 非小细胞肺癌　；参考：《山东大学》2016年博士论文

【摘要】：研究背景及意义原发性肺癌是世界范围内发病率和死亡率最高的恶性肿瘤之一,近几十年来,在所有恶性肿瘤中,男性肺癌的发病率和死亡率均列第一位,女性肺癌发病率和死亡率均列第二位,已成为危害人类生命健康的一种主要疾病。根据相关统计学资料及模型估计,2012年全世界大约有1400万新发癌症病例,其中肺癌病例180万,占癌症总发病率的13%,是癌症中诊断率最高的病种,也是全球男性以及发达国家女性死亡率最高的癌症病种。肺癌的确切发病机制,目前尚未完全明确,但大量资料表明,长期大量吸烟与肺癌的发生有极为密切的关系,即使是非吸烟者,长期暴露于二手烟下也是重要的危险因素。在我国,随着近年来经济发展和工业革新所带来的严重环境污染,使得城市居民的肺癌发病率明显高于农村,而且呈明显年轻化趋势。现在随着医学技术的发展和健康理念的更新,人们对肺癌的防范意识也逐步增强,对于肺癌的早期诊断日趋完善,治疗也逐步形成以手术为主,辅以放疗、化疗的多学科综合治疗。近年来分子靶向治疗的出现,为许多肺癌晚期患者开辟了一条新的道路。尽管各种治疗方法不断完善,全世界范围内肺癌患者的5年生存率还是仅有20%左右,肺癌患者的生存率仍有待提高。局灶浸润扩散和远处组织器官转移是目前肺癌治疗中的难点,也是导致肺癌患者死亡的最直接原因。肺癌的转移扩散是非常复杂的过程,其基本过程是细胞外基质中肿瘤细胞脱落,侵袭周围组织和基底膜并渗入血液,通过血液流动在远处位点外渗、迁移,最后形成肿瘤的远处转移。上述进程涉及多个基本环节,如肿瘤细胞的扩散、凋亡逃避、血管和淋巴管生成、远处克隆等。理论上讲,上述多个环节当中的任何一环受到干扰,都有可能阻断肿瘤细胞的扩散转移。大量的分子生物学研究已经证实,很多分子及基因的特异性改变均在上述肿瘤发展进程中发挥了重要作用。因此,寻找能够影响肺癌转移扩散的分子肿瘤标志物成为现阶段我们亟需解决的问题。防御素是一类含有6个半胱氨酸残基及3对二硫键的阳离子内源性抗菌肽,根据半胱氨酸氨基酸的分子分布和由此产生的二硫键,可将防御素分为两大类,α-防御素(human neutrophil peptide,HNP)和β-防御素(Human β-defensin,HBD),它们广泛存在于动植物体内。a-防御素主要由小肠潘氏细胞和中性粒细胞合成并储存于胞浆颗粒中,β-防御素主要由上皮细胞合成和分泌,其分布广泛,可被诱导表达,在很多肿瘤中都存在表达失控的情况。迄今为止,β-防御素已经发现了4种亚型,其中β-防御素2(HBD2)是人体中第一个被发现的可诱导性防御素,主要由上皮细胞,特别是呼吸道上皮细胞产生,参与机体抵抗微生物侵入的防御反应,具有很强的抗细菌活性、抗病毒活性、免疫学活性及神经损伤修复活性。有研究发现,HBD2在肺部多种疾病,如社区获得性肺炎、肺结核、COPD、哮喘及肺间质性疾病等发挥着重要的作用。肺癌是呼吸系统极为重要的疾病,是最常见的呼吸道疾病死亡原因,其发病可能与HBD2也有一定关联。在人体口腔鳞状上皮细胞癌、胃癌、子宫颈癌、基底细胞癌等多种肿瘤的病理组织、细胞中都存在HBD-2基因和(或)蛋白的异常表达。已有研究发现,肺癌患者的病变肺组织中HBD2的表达增高,血清中HBD2的水平也明显增加,表明其可能参与肺癌的发病进程,其表达水平的变化可能会影响肺癌的病情进展。细胞是生物体的基本组成单位,细胞外基质(extracellular matrix, ECM)是分布在细胞表面或细胞之间的大分子,其组成复杂的网架结构,支持并连接组织结构,调节细胞的生理活动和组织发生。细胞外基质能决定结缔组织的特性,它是动物组织的一部分,能通过信号转导系统影响细胞的生长、凋亡、迁移、增殖和分化。目前很多学者认为细胞外基质的降解在恶性肿瘤的发生、发展、侵袭和转移过程中起非常关键的作用。细胞外基质降解酶类主要有3大类：丝氨酸蛋白酶类、半胱氨酸蛋白酶类和基质金属蛋白酶(matrixmetalloproteinases, MMPs)类,其中MMPs是降解细胞外基质的最重要的酶系,占细胞外基质降解酶总活性的70%。MMPs是一种活性依赖于锌离子和钙离子的蛋白水解酶,可以降解参与信号传导的细胞外基质中的多种成分,破坏抵御肿瘤细胞侵袭的组织学屏障,在细胞迁移、血管生成、恶性肿瘤的浸润转移等病理生理过程中发挥非常重要的作用。人体内存在它们的天然抑制剂即组织型基质金属蛋白酶抑制物(tissue inhibitors of metalloproteinases, TIMPs), MMPs/TIMPs之间的平衡在调节细胞外基质的稳态中起重要作用。MMPs一般在正常组织中表达量很少,但在某些病理性重建的过程中高表达。MMPs的产生和激活一般受以下四个水平调节：(1)基因转录水平,(2)蛋白合成水平,(3)无活性酶前体经蛋白水解酶的作用而激活,(4)特异性抑制因子作用。目前MMPs家族已分离出26个成员,分别为MMP1 ～ 26。根据作用底物及片断同源性,可将MMPs分为6大类,分别为胶原酶、明胶酶、基质降解素、基质溶解素、furin活化的MMPs和其他分泌型MMPs。Ⅳ型胶原酶是其中重要的一类,主要有两种表现形式,一种非糖基化,分子量为72kD,称为MMP-2,另一种糖基化,分子量为92kD,称为MMP-9。由于Ⅳ型胶原纤维是组成ECM的主要结构蛋白,而Ⅳ型胶原酶(MMP-2和MMP-9)是降解Ⅳ型胶原纤维的唯一的基质蛋白水解酶,所以MMP-2和MMP-9在恶性肿瘤的侵袭和转移过程中可能起着非常重要的作用。综上所述,HBD2、MMP2及MMP9可能在肺癌的发生发展中发挥了重要作用,但它们与肺癌之间的关系尚未完全阐明,基于此,本课题拟进行以下两部分研究：第一部分,探讨人β-防御素2(HBD2)在非小细胞肺癌细胞增殖以及肿瘤的进展和转移中的作用；第二部分,检测MMP2、MMP9在非小细胞肺癌患者病变肺组织中的表达,探讨MMP2、MMP9在非小细胞肺癌中的表达以及与肺癌转移的关系。第一部分人p-防御素2通过ABCG2促进肺癌细胞增殖目的：探讨人p-防御素2(HBD2)与ATP结合转运蛋白G超家族成员2(ATP-binding cassette transporter G2, ABCG2)对非小细胞肺癌细胞增殖的作用。方法：收集130例非小细胞肺癌(NSCLC)患者的手术切除肿瘤组织标本,同时取该130例患者的肿瘤旁肺组织标本作为对照组,免疫组织化学染色法检测HBD2与ABCG2的表达；体外培养人肺癌A549细胞,以HBD2刺激细胞后MTT法检测细胞增殖程度,Western blot法检测ABCG2表达,再以选择性ABCG2抑制剂Fumitremorgin C预处理A549细胞,观察HBD2对ABCG2表达和细胞增殖的影响。结果：NSCLC组HBD2和ABCG2表达均较对照组升高(均P0.05); HBD2诱导A549细胞ABCG2的表达和细胞增殖,且均呈剂量和浓度依赖性(均P0.05),Fumitremorgin C可抑制HBD2诱导的ABCG2表达和细胞增殖(均P0.05)。结论：HBD2在非小细胞肺癌组织中表达增高,HBD2可通过诱导ABCG2促进肺癌细胞的增殖,在非小细胞肺癌的进展中发挥一定作用。第二部分MMP-2、MMP-9在肺癌中的表达及对肺癌转移的影响目的：探讨MMP2、MMP9在非小细胞肺癌中的表达以及对非小细胞肺癌侵袭转移的影响。方法：收集1800例非小细胞肺癌患者的病变肺组织(NSCLC组)及1200例肺部良性病变患者的肺组织(对照组),采用免疫组织化学法检测肺组织病理切片中MMP-2、MMP-9的表达水平,分析其表达以及与非小细胞肺癌各临床指标之间的关系。结果：NSCLC患者中MMP-2和MMP-9相关的各项指标均明显高于对照组(均P0.05)。如果肺癌症状同时伴随淋巴结转移或远处转移,这两个指标均高于不伴转移的肺癌患者。另外MMP-2和MMP-9在肺鳞状细胞癌患者中的表达水平显著高于肺腺癌、肺腺鳞癌患者(均P0.05)。结论：MMP-2、MMP-9的表达水平与肺癌的发生发展和病理类型之间有密切关联,确定这种关系可以为肺癌患者的临床治疗提供科学的指导数据,提高治疗方案的有效性,有助于提高肺癌患者的生活质量。
[Abstract]:Background and significance primary lung cancer is one of the most malignant tumors in the world with the highest morbidity and mortality. In the last few decades, the incidence and mortality of male lung cancer are the first in all malignant tumors. The incidence and mortality of the female lung cancer are second, which has become a major disease that endangers human life and health. According to the relevant statistical data and model estimates, there are about 1400 million new cases of cancer in the world in 2012, of which 1 million 800 thousand of the cases of lung cancer, accounting for 13% of the total cancer incidence, are the highest diagnosed diseases in cancer, and the highest mortality rate of women in the world as well as in developed countries. The exact pathogenesis of lung cancer is not yet available. It is clear, but a large amount of information shows that long term smoking is closely related to the occurrence of lung cancer. Even non smokers, exposed to second-hand smoke for a long time are also important risk factors. In China, the incidence of lung cancer in urban residents is obvious with the serious environmental pollution caused by economic development and industrial innovation in recent years. With the development of medical technology and the renewal of health concept, the awareness of the prevention of lung cancer is gradually increasing. The early diagnosis of lung cancer is becoming more and more perfect, and the treatment is gradually formed in the multidisciplinary integrated treatment with surgery, radiotherapy and chemotherapy. It opens up a new path for many advanced patients with lung cancer. Despite the continuous improvement of various treatments, the 5 year survival rate of lung cancer patients around the world is only about 20%, and the survival rate of lung cancer patients remains to be improved. The most direct cause of death of lung cancer patients. Metastasis and diffusion of lung cancer is a very complicated process. The basic process is that the tumor cells in the extracellular matrix fall off, invade the surrounding tissue and the basement membrane and infiltrate into the blood, and migrate through the blood flow at the distant loci, and finally form the distant metastasis of the tumor. Segments, such as the proliferation of tumor cells, apoptosis evasion, vascular and lymphatic formation, distant cloning, etc. theoretically, any of the rings in the above-mentioned links are interfered and may block the proliferation and metastasis of tumor cells. A large number of molecular biological studies have confirmed that the specific changes in many molecules and genes have been found in the above tumor. It has played an important role in the development process. Therefore, it is an urgent problem to find the molecular tumor markers that can affect the metastasis and diffusion of lung cancer. The defensin is a class of cationic endogenous antibacterial peptides containing 6 cysteine residues and 3 pairs of two sulfur bonds, based on the molecular distribution of cysteine amino acids and the resulting production Two sulfur bonds can be divided into two major groups, human neutrophil peptide (HNP) and beta defensin (Human beta -defensin, HBD). They are widely found in animals and plants by the synthesis and storage of.A- defensins mainly from small intestine PAM cells and neutrophils in cytoplasm particles. It is widely distributed and can be induced to express, in many tumors there are out of control. So far, beta defensin has found 4 subtypes, of which beta defensin 2 (HBD2) is the first inducible defensin found in the human body, mainly produced by epithelial cells, especially respiratory epithelial cells, and is involved in the body's resistance to microbiology. The defensive reaction of intrusive substances has strong anti bacterial activity, antiviral activity, immunological activity and nerve damage repair activity. Some studies have found that HBD2 plays an important role in a variety of lung diseases such as community-acquired pneumonia, tuberculosis, COPD, asthma and pulmonary interstitial diseases. Lung cancer is the most important disease of the respiratory system. The most common cause of respiratory disease death may be associated with HBD2. In human oral squamous cell carcinoma, gastric cancer, cervical cancer, basal cell carcinoma and other pathological tissues, there is an abnormal expression of HBD-2 gene and (or) protein in the cells. It is found that HBD2 in the lung tissue of the patients with lung cancer has been found. The expression of HBD2 also increased significantly in serum, indicating that it may be involved in the pathogenesis of lung cancer. The changes in the expression level may affect the progression of lung cancer. Cells are the basic unit of the organism, and the extracellular matrix (extracellular matrix, ECM) is a large molecule distributed on the surface of cells or between cells. A complex lattice structure that supports and connects the tissue structure to regulate the physiological activities and tissue of cells. The extracellular matrix can determine the characteristics of connective tissue. It is part of the animal tissue and can affect cell growth, apoptosis, migration, proliferation and differentiation through the signal transduction system. Many scholars believe that the extracellular matrix is reduced. There are 3 major types of extracellular matrix degradation enzymes: serine protease, cysteine protease and matrixmetalloproteinases (MMPs), and MMPs is the most important enzyme system for the degradation of extracellular matrix. 70%.MMPs of the total activity of extracellular matrix degrading enzyme is a kind of protein hydrolase, which is dependent on zinc ions and calcium ions. It can degrade various components of extracellular matrix involved in signal transduction, destroy the tissue barrier against tumor cell invasion, and the pathophysiological processes such as cell migration, angiogenesis, invasion and metastasis of malignant tumor. It plays a very important role. There are natural inhibitors in the human body, the tissue matrix metalloproteinase inhibitor (tissue inhibitors of metalloproteinases, TIMPs), and the balance between MMPs/TIMPs plays an important role in regulating the homeostasis of the extracellular matrix, and the expression of.MMPs is very small in normal tissues, but in some diseases. In the process of rational reconstruction, the production and activation of high expression.MMPs are generally regulated by the following four levels: (1) gene transcription level, (2) protein synthesis level, (3) activation of inactive enzyme precursors through the action of protein hydrolase, (4) specific inhibitory factors. At present, the MMPs family has separated 26 members, which are MMP1 to 26., respectively. The substrate and fragment homology can be divided into 6 categories: collagenase, gelatinase, matrix degrading, matrix lysin, furin activated MMPs and other secretory MMPs. IV collagenase, which are one of the most important types, including two forms, one non glycosylated, a molecular weight of 72kD, a MMP-2, another glycosylation, and a molecular weight of 9 2kD, called MMP-9. because type IV collagen fibers are the main structural proteins that make up ECM, and type IV collagenase (MMP-2 and MMP-9) is the only matrix protein hydrolase for the degradation of type IV collagen fibers, so MMP-2 and MMP-9 may play an unusual role in the invasion and metastasis of malignant tumors. To sum up, HBD2, MMP2 and MMP9 may be possible. It plays an important role in the development of lung cancer, but the relationship between them and lung cancer has not been fully elucidated. Based on this, the following two parts are planned: the first part is to explore the role of human beta defensin 2 (HBD2) in the proliferation of non-small cell lung cancer cells and the progression and metastasis of tumor; the second part, detection of MMP2, MMP 9 expression in the lung tissue of non small cell lung cancer patients and the expression of MMP2, MMP9 in non-small cell lung cancer and the relationship with the metastasis of lung cancer. The first part of human p- defensin 2 promotes the proliferation of lung cancer cells through ABCG2: the exploration of human p- defensin 2 (HBD2) and ATP binding transporter G superfamily member 2 (ATP-binding cassette TR) The effect of ansporter G2, ABCG2) on the proliferation of non small cell lung cancer cells. Methods: 130 cases of non small cell lung cancer (NSCLC) were collected and the tumor tissue specimens were collected, and the para lung tissue specimens of the 130 patients were taken as the control group. The expression of HBD2 and ABCG2 was detected by immunohistochemical staining, and the human lung cancer A549 was cultured in vitro A549. Cell proliferation was detected by MTT method after HBD2 stimulation, ABCG2 expression was detected by Western blot method, and A549 cells were pretreated with selective ABCG2 inhibitor Fumitremorgin C. The effect of HBD2 on ABCG2 expression and cell proliferation was observed. The expression and cell proliferation are both dose and concentration dependent (all P0.05), and Fumitremorgin C can inhibit the ABCG2 expression and cell proliferation induced by HBD2 (all P0.05). Conclusion: HBD2 is expressed in non small cell lung cancer tissues, and HBD2 can promote the proliferation of lung cancer cells by inducing ABCG2, and play a certain role in the progress of non-small cell lung cancer. Use. Second part MMP-2, MMP-9 expression in lung cancer and its effect on metastasis of lung cancer: To explore the expression of MMP2, MMP9 in non-small cell lung cancer and to the invasion and metastasis of non-small cell lung cancer. Methods: to collect 1800 cases of non small cell lung cancer (NSCLC group) and 1200 cases of lung benign lesions The expression level of MMP-2, MMP-9, expression and the relationship with various clinical indexes of non-small cell lung cancer were detected by immunohistochemistry. Results: all the indexes related to MMP-2 and MMP-9 in NSCLC patients were significantly higher than those in the control group (P0.05). If the lung cancer symptoms were accompanied by the same symptoms The two indexes were higher than those of non metastatic lung cancer patients. In addition, the expression level of MMP-2 and MMP-9 in patients with lung squamous cell carcinoma was significantly higher than that of lung adenocarcinoma and adenosscc (all P0.05). Conclusion: MMP-2, the expression level of MMP-9 is closely related to the development and pathological types of lung cancer, and it is determined that there is a close association between the expression level of lung cancer and the pathological type of lung cancer. This relationship can provide scientific guidance data for clinical treatment of lung cancer patients, improve the effectiveness of the treatment plan, and help improve the quality of life of patients with lung cancer.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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