本文選題：人β-防御素2 + 非小細(xì)胞肺癌��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景及意義原發(fā)性肺癌是世界范圍內(nèi)發(fā)病率和死亡率最高的惡性腫瘤之一,近幾十年來(lái),在所有惡性腫瘤中,男性肺癌的發(fā)病率和死亡率均列第一位,女性肺癌發(fā)病率和死亡率均列第二位,已成為危害人類生命健康的一種主要疾病。根據(jù)相關(guān)統(tǒng)計(jì)學(xué)資料及模型估計(jì),2012年全世界大約有1400萬(wàn)新發(fā)癌癥病例,其中肺癌病例180萬(wàn),占癌癥總發(fā)病率的13%,是癌癥中診斷率最高的病種,也是全球男性以及發(fā)達(dá)國(guó)家女性死亡率最高的癌癥病種。肺癌的確切發(fā)病機(jī)制,目前尚未完全明確,但大量資料表明,長(zhǎng)期大量吸煙與肺癌的發(fā)生有極為密切的關(guān)系,即使是非吸煙者,長(zhǎng)期暴露于二手煙下也是重要的危險(xiǎn)因素。在我國(guó),隨著近年來(lái)經(jīng)濟(jì)發(fā)展和工業(yè)革新所帶來(lái)的嚴(yán)重環(huán)境污染,使得城市居民的肺癌發(fā)病率明顯高于農(nóng)村,而且呈明顯年輕化趨勢(shì)�，F(xiàn)在隨著醫(yī)學(xué)技術(shù)的發(fā)展和健康理念的更新,人們對(duì)肺癌的防范意識(shí)也逐步增強(qiáng),對(duì)于肺癌的早期診斷日趨完善,治療也逐步形成以手術(shù)為主,輔以放療、化療的多學(xué)科綜合治療。近年來(lái)分子靶向治療的出現(xiàn),為許多肺癌晚期患者開(kāi)辟了一條新的道路。盡管各種治療方法不斷完善,全世界范圍內(nèi)肺癌患者的5年生存率還是僅有20%左右,肺癌患者的生存率仍有待提高。局灶浸潤(rùn)擴(kuò)散和遠(yuǎn)處組織器官轉(zhuǎn)移是目前肺癌治療中的難點(diǎn),也是導(dǎo)致肺癌患者死亡的最直接原因。肺癌的轉(zhuǎn)移擴(kuò)散是非常復(fù)雜的過(guò)程,其基本過(guò)程是細(xì)胞外基質(zhì)中腫瘤細(xì)胞脫落,侵襲周圍組織和基底膜并滲入血液,通過(guò)血液流動(dòng)在遠(yuǎn)處位點(diǎn)外滲、遷移,最后形成腫瘤的遠(yuǎn)處轉(zhuǎn)移。上述進(jìn)程涉及多個(gè)基本環(huán)節(jié),如腫瘤細(xì)胞的擴(kuò)散、凋亡逃避、血管和淋巴管生成、遠(yuǎn)處克隆等。理論上講,上述多個(gè)環(huán)節(jié)當(dāng)中的任何一環(huán)受到干擾,都有可能阻斷腫瘤細(xì)胞的擴(kuò)散轉(zhuǎn)移。大量的分子生物學(xué)研究已經(jīng)證實(shí),很多分子及基因的特異性改變均在上述腫瘤發(fā)展進(jìn)程中發(fā)揮了重要作用。因此,尋找能夠影響肺癌轉(zhuǎn)移擴(kuò)散的分子腫瘤標(biāo)志物成為現(xiàn)階段我們亟需解決的問(wèn)題。防御素是一類含有6個(gè)半胱氨酸殘基及3對(duì)二硫鍵的陽(yáng)離子內(nèi)源性抗菌肽,根據(jù)半胱氨酸氨基酸的分子分布和由此產(chǎn)生的二硫鍵,可將防御素分為兩大類,α-防御素(human neutrophil peptide,HNP)和β-防御素(Human β-defensin,HBD),它們廣泛存在于動(dòng)植物體內(nèi)。a-防御素主要由小腸潘氏細(xì)胞和中性粒細(xì)胞合成并儲(chǔ)存于胞漿顆粒中,β-防御素主要由上皮細(xì)胞合成和分泌,其分布廣泛,可被誘導(dǎo)表達(dá),在很多腫瘤中都存在表達(dá)失控的情況。迄今為止,β-防御素已經(jīng)發(fā)現(xiàn)了4種亞型,其中β-防御素2(HBD2)是人體中第一個(gè)被發(fā)現(xiàn)的可誘導(dǎo)性防御素,主要由上皮細(xì)胞,特別是呼吸道上皮細(xì)胞產(chǎn)生,參與機(jī)體抵抗微生物侵入的防御反應(yīng),具有很強(qiáng)的抗細(xì)菌活性、抗病毒活性、免疫學(xué)活性及神經(jīng)損傷修復(fù)活性。有研究發(fā)現(xiàn),HBD2在肺部多種疾病,如社區(qū)獲得性肺炎、肺結(jié)核、COPD、哮喘及肺間質(zhì)性疾病等發(fā)揮著重要的作用。肺癌是呼吸系統(tǒng)極為重要的疾病,是最常見(jiàn)的呼吸道疾病死亡原因,其發(fā)病可能與HBD2也有一定關(guān)聯(lián)。在人體口腔鱗狀上皮細(xì)胞癌、胃癌、子宮頸癌、基底細(xì)胞癌等多種腫瘤的病理組織、細(xì)胞中都存在HBD-2基因和(或)蛋白的異常表達(dá)。已有研究發(fā)現(xiàn),肺癌患者的病變肺組織中HBD2的表達(dá)增高,血清中HBD2的水平也明顯增加,表明其可能參與肺癌的發(fā)病進(jìn)程,其表達(dá)水平的變化可能會(huì)影響肺癌的病情進(jìn)展。細(xì)胞是生物體的基本組成單位,細(xì)胞外基質(zhì)(extracellular matrix, ECM)是分布在細(xì)胞表面或細(xì)胞之間的大分子,其組成復(fù)雜的網(wǎng)架結(jié)構(gòu),支持并連接組織結(jié)構(gòu),調(diào)節(jié)細(xì)胞的生理活動(dòng)和組織發(fā)生。細(xì)胞外基質(zhì)能決定結(jié)締組織的特性,它是動(dòng)物組織的一部分,能通過(guò)信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)影響細(xì)胞的生長(zhǎng)、凋亡、遷移、增殖和分化。目前很多學(xué)者認(rèn)為細(xì)胞外基質(zhì)的降解在惡性腫瘤的發(fā)生、發(fā)展、侵襲和轉(zhuǎn)移過(guò)程中起非常關(guān)鍵的作用。細(xì)胞外基質(zhì)降解酶類主要有3大類：絲氨酸蛋白酶類、半胱氨酸蛋白酶類和基質(zhì)金屬蛋白酶(matrixmetalloproteinases, MMPs)類,其中MMPs是降解細(xì)胞外基質(zhì)的最重要的酶系,占細(xì)胞外基質(zhì)降解酶總活性的70%。MMPs是一種活性依賴于鋅離子和鈣離子的蛋白水解酶,可以降解參與信號(hào)傳導(dǎo)的細(xì)胞外基質(zhì)中的多種成分,破壞抵御腫瘤細(xì)胞侵襲的組織學(xué)屏障,在細(xì)胞遷移、血管生成、惡性腫瘤的浸潤(rùn)轉(zhuǎn)移等病理生理過(guò)程中發(fā)揮非常重要的作用。人體內(nèi)存在它們的天然抑制劑即組織型基質(zhì)金屬蛋白酶抑制物(tissue inhibitors of metalloproteinases, TIMPs), MMPs/TIMPs之間的平衡在調(diào)節(jié)細(xì)胞外基質(zhì)的穩(wěn)態(tài)中起重要作用。MMPs一般在正常組織中表達(dá)量很少,但在某些病理性重建的過(guò)程中高表達(dá)。MMPs的產(chǎn)生和激活一般受以下四個(gè)水平調(diào)節(jié)：(1)基因轉(zhuǎn)錄水平,(2)蛋白合成水平,(3)無(wú)活性酶前體經(jīng)蛋白水解酶的作用而激活,(4)特異性抑制因子作用。目前MMPs家族已分離出26個(gè)成員,分別為MMP1 ～ 26。根據(jù)作用底物及片斷同源性,可將MMPs分為6大類,分別為膠原酶、明膠酶、基質(zhì)降解素、基質(zhì)溶解素、furin活化的MMPs和其他分泌型MMPs。Ⅳ型膠原酶是其中重要的一類,主要有兩種表現(xiàn)形式,一種非糖基化,分子量為72kD,稱為MMP-2,另一種糖基化,分子量為92kD,稱為MMP-9。由于Ⅳ型膠原纖維是組成ECM的主要結(jié)構(gòu)蛋白,而Ⅳ型膠原酶(MMP-2和MMP-9)是降解Ⅳ型膠原纖維的唯一的基質(zhì)蛋白水解酶,所以MMP-2和MMP-9在惡性腫瘤的侵襲和轉(zhuǎn)移過(guò)程中可能起著非常重要的作用。綜上所述,HBD2、MMP2及MMP9可能在肺癌的發(fā)生發(fā)展中發(fā)揮了重要作用,但它們與肺癌之間的關(guān)系尚未完全闡明,基于此,本課題擬進(jìn)行以下兩部分研究：第一部分,探討人β-防御素2(HBD2)在非小細(xì)胞肺癌細(xì)胞增殖以及腫瘤的進(jìn)展和轉(zhuǎn)移中的作用；第二部分,檢測(cè)MMP2、MMP9在非小細(xì)胞肺癌患者病變肺組織中的表達(dá),探討MMP2、MMP9在非小細(xì)胞肺癌中的表達(dá)以及與肺癌轉(zhuǎn)移的關(guān)系。第一部分人p-防御素2通過(guò)ABCG2促進(jìn)肺癌細(xì)胞增殖目的： 探討人p-防御素2(HBD2)與ATP結(jié)合轉(zhuǎn)運(yùn)蛋白G超家族成員2(ATP-binding cassette transporter G2, ABCG2)對(duì)非小細(xì)胞肺癌細(xì)胞增殖的作用。方法：收集130例非小細(xì)胞肺癌(NSCLC)患者的手術(shù)切除腫瘤組織標(biāo)本,同時(shí)取該130例患者的腫瘤旁肺組織標(biāo)本作為對(duì)照組,免疫組織化學(xué)染色法檢測(cè)HBD2與ABCG2的表達(dá)；體外培養(yǎng)人肺癌A549細(xì)胞,以HBD2刺激細(xì)胞后MTT法檢測(cè)細(xì)胞增殖程度,Western blot法檢測(cè)ABCG2表達(dá),再以選擇性ABCG2抑制劑Fumitremorgin C預(yù)處理A549細(xì)胞,觀察HBD2對(duì)ABCG2表達(dá)和細(xì)胞增殖的影響。結(jié)果：NSCLC組HBD2和ABCG2表達(dá)均較對(duì)照組升高(均P0.05); HBD2誘導(dǎo)A549細(xì)胞ABCG2的表達(dá)和細(xì)胞增殖,且均呈劑量和濃度依賴性(均P0.05),Fumitremorgin C可抑制HBD2誘導(dǎo)的ABCG2表達(dá)和細(xì)胞增殖(均P0.05)。結(jié)論：HBD2在非小細(xì)胞肺癌組織中表達(dá)增高,HBD2可通過(guò)誘導(dǎo)ABCG2促進(jìn)肺癌細(xì)胞的增殖,在非小細(xì)胞肺癌的進(jìn)展中發(fā)揮一定作用。第二部分MMP-2、MMP-9在肺癌中的表達(dá)及對(duì)肺癌轉(zhuǎn)移的影響目的：探討MMP2、MMP9在非小細(xì)胞肺癌中的表達(dá)以及對(duì)非小細(xì)胞肺癌侵襲轉(zhuǎn)移的影響。方法：收集1800例非小細(xì)胞肺癌患者的病變肺組織(NSCLC組)及1200例肺部良性病變患者的肺組織(對(duì)照組),采用免疫組織化學(xué)法檢測(cè)肺組織病理切片中MMP-2、MMP-9的表達(dá)水平,分析其表達(dá)以及與非小細(xì)胞肺癌各臨床指標(biāo)之間的關(guān)系。結(jié)果：NSCLC患者中MMP-2和MMP-9相關(guān)的各項(xiàng)指標(biāo)均明顯高于對(duì)照組(均P0.05)。如果肺癌癥狀同時(shí)伴隨淋巴結(jié)轉(zhuǎn)移或遠(yuǎn)處轉(zhuǎn)移,這兩個(gè)指標(biāo)均高于不伴轉(zhuǎn)移的肺癌患者。另外MMP-2和MMP-9在肺鱗狀細(xì)胞癌患者中的表達(dá)水平顯著高于肺腺癌、肺腺鱗癌患者(均P0.05)。結(jié)論：MMP-2、MMP-9的表達(dá)水平與肺癌的發(fā)生發(fā)展和病理類型之間有密切關(guān)聯(lián),確定這種關(guān)系可以為肺癌患者的臨床治療提供科學(xué)的指導(dǎo)數(shù)據(jù),提高治療方案的有效性,有助于提高肺癌患者的生活質(zhì)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R734.2

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