【摘要】：胰腺癌是消化系統(tǒng)常見的惡性腫瘤之一,其惡性程度高、早期診斷率低、進(jìn)展迅速、預(yù)后極差。胰腺癌已經(jīng)成為國內(nèi)外醫(yī)療界需要共同面對的一個(gè)重大的診斷和治療難題,因此加強(qiáng)胰腺癌發(fā)生機(jī)制研究,以發(fā)現(xiàn)新的診斷標(biāo)記物與探索新的治療策略,具有十分重要的意義。生物體內(nèi)細(xì)胞正常的運(yùn)轉(zhuǎn),需要讓合適的分子在合適的時(shí)間到達(dá)合理的靶位置,這一重要作用主要依賴囊泡運(yùn)輸完成。其功能障礙會導(dǎo)致多種細(xì)胞器發(fā)生缺陷,細(xì)胞功能紊亂。近年來的研究顯示囊泡運(yùn)輸異常與腫瘤的發(fā)生發(fā)展相關(guān)。在與蛋白質(zhì)囊泡運(yùn)輸有關(guān)的效應(yīng)分子中,Rab蛋白是Ras相關(guān)的小GTP酶超家族的成員,這些蛋白有著特定的細(xì)胞亞定位,并在細(xì)胞的分泌、胞吞、信號轉(zhuǎn)導(dǎo)和發(fā)育等過程發(fā)揮著重要的作用。有研究表明,Rab27家族中Rab27A及Rab27B與腫瘤的發(fā)生及轉(zhuǎn)移有密切的關(guān)系,有學(xué)者發(fā)現(xiàn)Rab27A與人乳腺癌細(xì)胞的侵襲和轉(zhuǎn)移密切相關(guān);Rab27B可調(diào)節(jié)雌激素受體陽性的乳腺癌細(xì)胞的侵襲性生長和轉(zhuǎn)移;Rab27B和Rab27A與肝癌的進(jìn)展相關(guān),可能是肝癌患者一個(gè)有意義的預(yù)后指標(biāo)。檢索文獻(xiàn)關(guān)于Rab27B和Rab27A與胰腺癌之間的關(guān)系尚未見諸報(bào)道。我們通過將186例胰腺癌組織,54例癌旁周圍組織和25例胰腺良性疾病組織制成組織芯片,以免疫組化技術(shù)分析Rab27A和TP53在胰腺組織中的表達(dá)及臨床意義。我們發(fā)現(xiàn)Rab27A與TP53在胰腺癌組織中較癌旁組織及胰腺良性疾病組織明顯增高。Rab27A和TP53的高表達(dá)均與胰腺癌的腫瘤分期及遠(yuǎn)處轉(zhuǎn)移密切相關(guān)。Rab27A的高表達(dá)還與血管浸潤有關(guān)。在Cox單因素和多因素分析中,我們發(fā)現(xiàn)Rab27A與TP53的陽性表達(dá)與胰腺癌患者不良預(yù)后有密切關(guān)系,差異具有統(tǒng)計(jì)學(xué)意義。雖然研究數(shù)據(jù)不支持TP53可通過Rab27A調(diào)控胰腺癌的胞外分泌功能,但不能排除二者之間存在較微弱的相關(guān)作用的可能性。隨后的研究中,采用免疫組織化學(xué)方法,組織芯片技術(shù),對186例胰腺癌組織,51例對應(yīng)的癌旁周圍組織和23例胰腺良性病變組織標(biāo)本中Rab27B及TP53的表達(dá)進(jìn)行檢測,結(jié)果表明,Rab27B的高表達(dá)可作為胰腺癌的獨(dú)立的預(yù)后因子。通過Cox單因素和多因素分析,我們發(fā)現(xiàn)Rab27B的表達(dá)水平增高是胰腺癌不良預(yù)后的重要的危險(xiǎn)因素,胰腺癌中Rab27B和TP53表達(dá)水平的增高還與血管浸潤、遠(yuǎn)處轉(zhuǎn)移有密切關(guān)系,同時(shí)檢測到Rab27B與TP53蛋白的表達(dá)之間有著相關(guān)性,而TP53與Rab27B之間有著怎樣的作用關(guān)系還有待于進(jìn)一步研究。我們還分別構(gòu)建了針對Rab27A及Rab27B基因的siRNA,通過脂質(zhì)體的方法對胰腺癌細(xì)胞進(jìn)行轉(zhuǎn)染,在細(xì)胞水平上,分別用CCK-8、Transwell小室及Western Blot等方法檢測轉(zhuǎn)染前后對細(xì)胞生物學(xué)特性的影響。結(jié)果顯示,干擾Rab27A及Rab27B基因的表達(dá)可抑制胰腺癌的增殖,減弱其侵襲能力,并可誘導(dǎo)細(xì)胞凋亡,對胰腺癌的增殖轉(zhuǎn)移行為起到反向調(diào)控的作用。綜上所述,本研究首次報(bào)道了Rab27A及TP53在胰腺癌組織中表達(dá)較癌旁組織及胰腺良性疾病組織高,且高表達(dá)的Rab27A與胰腺癌患者的不良預(yù)后之間有著密切關(guān)系。Rab27B在胰腺癌中的表達(dá)雖與癌旁組織及胰腺良性疾病組織中的表達(dá)無明顯統(tǒng)計(jì)學(xué)差異,但在Rab27B高表達(dá)的胰腺癌患者,其五年生存率有明顯的降低。我們沒有檢測到Rab27A與TP53在表達(dá)上明顯的相關(guān),但Rab27B和TP53的表達(dá)有顯著的相關(guān)性,具體的作用方式還有待進(jìn)一步研究。以siRNA技術(shù)干擾Rab27A及Rab27B均可明顯降低胰腺癌細(xì)胞的增殖、侵襲、遷移能力,誘導(dǎo)凋亡,由此為下一步構(gòu)建新型靶向治療藥物提供了理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Pancreatic cancer is one of the most common malignant tumors in the digestive system. Its malignant degree is high, the early diagnosis rate is low, the progress is rapid, and the prognosis is very poor. Pancreatic cancer has become a major diagnosis and treatment problem to be faced by the medical community at home and abroad, so it is of great significance to study the mechanism of pancreatic cancer in order to find new diagnostic markers and explore new therapeutic strategies. The normal operation of the cells in the organism requires the appropriate molecules to reach a reasonable target position at the right time, which is mainly dependent on the completion of the vesicle transport. The dysfunction can lead to the defects of various organelles and the disorder of cell function. In recent years, the study of vesicle transport is related to the development of the tumor. In effector molecules associated with the transport of protein vesicles, the Rab protein is a member of the Ras-associated small GTP-enzyme superfamily, which has specific subcellular localization and plays an important role in the process of cell secretion, endocytosis, signal transduction and development. It is shown that Rab27A and Rab27B in the Rab27 family have a close relationship with the occurrence and metastasis of the tumor, and some scholars have found that Rab27A is closely related to the invasion and metastasis of human breast cancer cells. Rab27B can regulate the invasive growth and metastasis of breast cancer cells that are positive for estrogen receptor. Rab27B and Rab27A are related to the progression of liver cancer, and may be a meaningful prognostic indicator for patients with liver cancer. The literature on the relationship between Rab27B and Rab27A and pancreatic cancer has not been reported. The expression of Rab27A and TP53 in pancreatic tissue and its clinical significance were analyzed by immunohistochemistry. We found that Rab27A and TP53 increased significantly in the tissues of the pancreatic cancer and the tissues of the benign pancreatic diseases in the pancreas. The high expression of Rab27A and TP53 is closely related to the staging and distant metastasis of pancreatic cancer. The high expression of Rab27A is also associated with vascular invasion. In Cox single and multi-factor analysis, we found that the positive expression of Rab27A and TP53 is closely related to the poor prognosis of patients with pancreatic cancer, and the difference is of statistical significance. Although the study data does not support the ability of TP53 to regulate the extracellular secretion of pancreatic cancer by Rab27A, it is not possible to exclude the possibility of a weak correlation between the two. In the following study, the expression of Rab27B and TP53 in the specimens of 186 pancreatic cancer tissues, 51 corresponding adjacent tissues and 23 cases of benign lesions of the pancreas was detected by immunohistochemical method and tissue chip technique. The results showed that, The high expression of Rab27B can be used as an independent prognostic factor for pancreatic cancer. Through the Cox and multi-factor analysis, we found that the increase of the expression level of Rab27B is an important risk factor for the poor prognosis of the pancreatic cancer. The level of the expression of Rab27B and TP53 in the pancreatic cancer is also closely related to the vascular invasion and distant metastasis. The relationship between the expression of Rab27B and TP53 protein is also detected, and the relationship between TP53 and Rab27B remains to be further studied. In addition, the siRNA of Rab27A and Rab27B gene was constructed, and the cells of pancreatic cancer were transfected with the method of liposome, and the effects of the transfection and before and after transfection on the biological characteristics of the cells were detected by the methods of CCK-8, Transwell and Western Blot respectively. The results showed that the expression of Rab27A and Rab27B gene could inhibit the proliferation of pancreatic cancer, reduce its invasion ability and induce cell apoptosis. To sum up, this study first reported the high expression of Rab27A and TP53 in the tissues of pancreatic cancer and the high tissue of the pancreatic benign diseases, and the high expression of Rab27A was closely related to the poor prognosis of the patients with pancreatic cancer. The expression of Rab27B in pancreatic cancer has no significant difference with the expression of the adjacent tissue and the tissue of the pancreatic benign disease, but the five-year survival rate of Rab27B is significantly lower in patients with pancreatic cancer with high expression of Rab27B. We did not detect that Rab27A and TP53 are significantly related to the expression, but the expression of Rab27B and TP53 has a significant correlation, and the specific mode of action is to be further studied. The interference of Rab27A and Rab27B with siRNA technology can significantly reduce the proliferation, invasion, migration and apoptosis of pancreatic cancer cells, thus providing a theoretical basis and experimental basis for the next step to construct a novel targeted therapeutic agent.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.9

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1 王慶慶;Rab27A、Rab27B與TP53在胰腺癌中的作用及部分機(jī)制研究[D];蘇州大學(xué);2015年

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