發(fā)布時間：2018-06-04 09:40

  本文選題：肝癌 + 轉(zhuǎn)移�。� 參考：《安徽醫(yī)科大學(xué)》2015年碩士論文

【摘要】：肝細胞癌(hepatocellular carcinoma, HCC)是世界上發(fā)病率排名第七,死亡率排名第三的惡性腫瘤。在我國每年約有383,000人死于肝癌,大約占全世界肝癌死亡人數(shù)的50%,且逐年增加。肝癌的轉(zhuǎn)移是臨床治療失敗、致死率居高不下的主要因素。目前肝癌轉(zhuǎn)移的分子機制尚不完全明確,仍沒有可用于肝癌轉(zhuǎn)移復(fù)發(fā)臨床檢測的生物標(biāo)志物和有效的治療靶標(biāo),發(fā)掘肝癌轉(zhuǎn)移相關(guān)的重要蛋白具有重要的意義。腫瘤的轉(zhuǎn)移涉及腫瘤細胞之間、腫瘤細胞和胞外基質(zhì)環(huán)境之間的相互作用,包括胞外基質(zhì)(extracellular matrix, ECM)重塑、免疫抑制、靶器官組織的損害和血管生成等過程。腫瘤分泌蛋白既可以自分泌、旁分泌的形式調(diào)控腫瘤發(fā)生發(fā)展的過程,也可直接進入血液、尿液、組織間隙液等體液中作為腫瘤轉(zhuǎn)移標(biāo)志物。蛋白質(zhì)組學(xué)作為一種全局性、高通量的蛋白質(zhì)分析策略,被廣泛應(yīng)用于以腫瘤為代表的多基因、多因素復(fù)雜疾病的發(fā)病機制研究。本課題將細胞培養(yǎng)條件下穩(wěn)定同位素標(biāo)記(stable isotope labeling with amino acids in cell culture,SILAC)定量蛋白質(zhì)組學(xué)技術(shù)應(yīng)用到肝癌分泌蛋白的分析之中,以期發(fā)現(xiàn)肝癌轉(zhuǎn)移相關(guān)蛋白。研究選擇背景相同、轉(zhuǎn)移能力逐漸增高的三株肝癌細胞MHCC97L、 MHCC97H和HCCLM6作為分析材料,利用SILAC技術(shù)對三株細胞無血清培養(yǎng)上清中提取的分泌蛋白進行分析,共鑒定到1287個蛋白,其中分泌蛋白、膜蛋白和細胞膜連接蛋白共占總鑒定蛋白的18%。MHCC97H/MHCC97L中共鑒定到890個蛋白,有174個是差異蛋白(ratio±1.5倍),其中40個蛋白在轉(zhuǎn)移能力較高的MHCC97H中上調(diào),134個蛋白下調(diào)；在HCCLM6/MHCC97L中共鑒定到983個蛋白,有182個差異蛋白(ratio≥±1.5倍),其中67個蛋白在轉(zhuǎn)移能力較高的HCCLM6中上調(diào),115個蛋白下調(diào)。對上調(diào)蛋白進行生物過程分析,發(fā)現(xiàn)顯著富集的前十位生物過程為細胞黏附、基因調(diào)控,核酸代謝,蛋白代謝,糖代謝、細胞分化、DNA代謝、細胞識別、生物聚合物修飾和脂類代謝。對上調(diào)蛋白進行通路分析,發(fā)現(xiàn)上調(diào)蛋白顯著富集到溶酶體、聚糖降解和ECM-受體相互作用等通路。差異蛋白中有14個蛋白質(zhì)的表達量隨肝癌細胞轉(zhuǎn)移能力增強而逐步上調(diào),包括6種已知與腫瘤轉(zhuǎn)移正相關(guān)的蛋白質(zhì)如基質(zhì)金屬蛋白酶-1 (Matrix metalloproteinase-1, MMP1)、膜聯(lián)蛋白A1 (Annexin A1, ANXA1)和角蛋白18(Keratin, type I cytoskeletal 18, KRT18)等,這也反映了我們數(shù)據(jù)的可靠性。另外還有4種蛋白質(zhì)膠原蛋白六α-1鏈(Collagen alpha-1 (VI) chain, COL6A1)、光蛋白聚糖(Lumican, LUM)、N-乙酰葡糖胺-6-硫酸酯酶(N-acetylglucosamine-6-sulfatase, GNS)和前蛋白轉(zhuǎn)化酶枯草溶菌素9(Proprotein convertase subtilisin/kexin type 9, PCSK9)未曾有與肝癌轉(zhuǎn)移相關(guān)的報道,可作為候選蛋白進一步關(guān)注。對上調(diào)倍數(shù)較大的分泌蛋白COL6A1 (MHCC97H/MHCC97L=2.77、 HCCLM6/MHCC97L=4.23)進行進一步驗證。通過Western Blot的方法,在正常肝細胞LO2、無轉(zhuǎn)移能力的兩株肝癌細胞系SK-Hep1和HepG2、以及三株轉(zhuǎn)移能力依次升高的肝癌細胞系MHCC97L、MHCC97H、HCCLM6的分泌蛋白中檢測COL6A1的蛋白含量。結(jié)果表明,COL6A1在LO2、SK-Hep1和HepG2分泌蛋白中沒有被檢測到,而該蛋白在轉(zhuǎn)移能力依次升高的細胞系MHCC97L、MHCC97H和HCCLM6分泌蛋白中表達量依次升高。同時,COL6A1在LO2、SK-Hep1和HepG2的全細胞蛋白中也沒有被檢測到,而在MHCC97L、MHCC97H和HCCLM6的全細胞蛋白中表達量依次降低。上述結(jié)果表明COL6A在分泌蛋白中的含量與肝癌細胞的轉(zhuǎn)移能力正相關(guān),同時提示轉(zhuǎn)移能力強的肝癌細胞可能具有更強的分泌COL6A1的能力。COL6A1廣泛存在于胞外基質(zhì),涉及纖連蛋白和細胞的連接過程,并可與其它膠原蛋白(如膠原蛋白4,膠原蛋白1)連接,介導(dǎo)微纖維網(wǎng)絡(luò)的形成。它與轉(zhuǎn)移的相關(guān)性可能與改變ECM特性、促進細胞與ECM黏附和支持細胞運動有關(guān)。本研究發(fā)現(xiàn)并初步驗證了COL6A1是與肝癌轉(zhuǎn)移正相關(guān)的分泌蛋白,但其具體機制尚待進一步研究。為了進一步驗證上述體外實驗的結(jié)果,我們對轉(zhuǎn)移能力不同的肝癌病人血清進行了Western Blot檢測。遺憾的是由于目標(biāo)蛋白在血中的豐度太低,Western Blot未能檢測出COL6A1.我們下一步將積極尋找配對抗體用于臨床血清樣本的ELISA檢測。除此之外,我們還對不同轉(zhuǎn)移能力的人肝癌細胞系(HCCLM6和MHCC97)全細胞蛋白進行了iTRAQ (sobaric tags for relative and absolute quantitation)差異蛋白質(zhì)組分析。共鑒定4804種蛋白質(zhì),4790種蛋白質(zhì)有定量信息,發(fā)現(xiàn)103種差異蛋白(ratio≥±1.5),其中46種蛋白在高轉(zhuǎn)移細胞HCCLM6中上調(diào),57種下調(diào)。差異蛋白的細胞定位主要為細胞質(zhì)和細胞膜,GO分析顯著富集到細胞黏附等生物過程和蛋白結(jié)合等生物功能。發(fā)現(xiàn)了4種新的與肝癌轉(zhuǎn)移相關(guān)的候選蛋白層粘連蛋白亞基a 5 (Laminin subunit alpha-5, LAMA5)、微管蛋白鏈β-2B(Tubulin beta-2B chain, TUBB2B)、著絲粒蛋白F (Centromere protein F, CENPF)和囊泡相關(guān)膜蛋白5 (Vesicle-associated membrane protein 5, VAMP5),為進一步肝癌轉(zhuǎn)移機制研究提供了重要的數(shù)據(jù)參考。
[Abstract]:Hepatocellular carcinoma (HCC) is the seventh most malignant tumor in the world, and the mortality rate is third. In China, about 383000 people die of liver cancer every year, accounting for 50% of the death toll in the world, and the metastasis of liver cancer is the main factor of clinical treatment failure and high mortality. The molecular mechanism of the metastasis of liver cancer is still not completely clear. There is still no biomarker and effective target for the clinical detection of the metastasis of liver cancer. It is of great significance to explore the important proteins related to the metastasis of liver cancer. The metastasis of tumor involves the interaction between tumor cells, the tumor cell and the extracellular matrix environment, including the interaction between tumor cells and the extracellular matrix environment. Extracellular matrix (extracellular matrix, ECM) remodeling, immunosuppression, damage to target organ tissue and angiogenesis. Tumor secreting proteins can be autocrine, paracrine in the form of regulation of tumor development, and can also directly enter the blood, urine, interstitial fluid and other body fluids as tumor metastasis markers. As a global, high throughput protein analysis strategy, it is widely used in the study of the pathogenesis of multifactorial complex diseases represented by tumors. This topic uses stable isotope labeling (stable isotope labeling with amino acids in cell culture, SILAC) quantitative proteomics technology under cell culture. In the analysis of the secretory protein of liver cancer, the metastasis related protein of liver cancer was found. Three hepatoma cells, MHCC97L, MHCC97H and HCCLM6, which had the same background and increasing metastasis ability, were selected as the analytical materials. The secretory proteins extracted from the serum free culture supernatant of three cells were analyzed by SILAC technology, and 1287 of them were identified. Protein, in which the secretory protein, membrane protein and membrane connexin accounted for 890 proteins, 174 were differential proteins (ratio 1.5 times), of which 40 proteins were up regulated in MHCC97H with higher transfer ability and 134 proteins down; 983 proteins were identified in HCCLM6/MHCC97L and 18 in HCCLM6/MHCC97L. 2 differential proteins (ratio > 1.5 times), of which 67 proteins were up-regulated in HCCLM6 with higher transfer ability, and 115 proteins were downregulated. The biological process analysis of up regulated proteins showed that the first ten biological processes that were significantly enriched were cell adhesion, gene regulation, nucleic acid metabolism, egg white metabolism, sugar metabolism, cell differentiation, DNA metabolism, cell recognition and birth. The pathway analysis of up-regulated proteins showed that the up-regulated proteins were significantly enriched in lysosomes, chitosan degradation and ECM- receptor interaction. The expression of 14 proteins in the differential proteins increased gradually with the enhancement of the metastasis ability of hepatoma cells, including 6 eggs known to be positively related to tumor metastasis. White matter such as matrix metalloproteinase -1 (Matrix metalloproteinase-1, MMP1), membrane protein A1 (Annexin A1, ANXA1) and keratin 18 (Keratin, type I cytoskeletal 18, etc.), which also reflect the reliability of our data. There are also 4 kinds of protein collagen protein six alpha chain. Lumican, LUM, N- acetyl glucosamine -6- sulfate (N-acetylglucosamine-6-sulfatase, GNS) and preprotein invertase, wit lysozyme 9 (Proprotein convertase subtilisin/kexin type 9, PCSK9) have not been reported to be associated with metastasis of liver cancer. It can be used as a candidate protein for further attention. (MHCC97H/MHCC97L=2.77, HCCLM6/MHCC97L=4.23) for further validation. Through Western Blot, the content of COL6A1 protein was detected in the normal hepatocyte LO2, two liver cancer cell lines without metastasis, SK-Hep1 and HepG2, and the secretory proteins of the liver cancer cell line, which had the three metastatic ability in turn, in the secretory proteins of MHCC97H and HCCLM6. COL6A1 was not detected in the secretory proteins of LO2, SK-Hep1 and HepG2, and the protein expression in the cell lines, MHCC97L, MHCC97H and HCCLM6 secreting protein, in turn, increased in turn, and COL6A1 was not detected in LO2, SK-Hep1 and HepG2 whole cell proteins. The above results indicate that the content of COL6A in the secretory protein is positively related to the metastasis ability of the liver cancer cells, and suggests that the hepatoma cells with strong metastatic ability may have a stronger ability to secrete COL6A1,.COL6A1 is widely existed in the extracellular matrix, involving the connection between fibronectin and cells, and can be related to it. Its collagen (such as collagen 4, collagen 1) is connected to mediate the formation of a microfiber network. Its correlation with metastasis may be related to changes in ECM characteristics, promoting cell adhesion to ECM and supporting cell movement. This study found and preliminarily verified that COL6A1 is a positive secretory protein associated with metastasis of liver cancer, but its specific mechanism remains to be advanced. Step study. In order to further verify the results of the above in vitro experiments, we detected Western Blot in the serum of patients with different metastatic liver cancer. Unfortunately, the abundance of the target protein in the blood was too low, and Western Blot failed to detect COL6A1. we would be actively looking for the paired antibody for the clinical serum samples of ELISA next. In addition, we also carried out iTRAQ (sobaric tags for relative and absolute quantitation) differential proteomic analysis of human hepatoma cell lines (HCCLM6 and MHCC97) of human hepatocellular carcinoma cell lines (sobaric tags for relative and). 4804 proteins were identified, 4790 proteins had quantitative information, and 103 different proteins (ratio > 1.5) were found. The 46 proteins were up-regulated and 57 down regulated in the high transfer cell HCCLM6. The cell location of the differential proteins was mainly cytoplasm and cell membrane, GO analysis significantly enriched the biological processes of cell adhesion and other biological processes, and found 4 new candidate egg white laminin a 5 (Laminin subunit ALP) associated with the metastasis of liver cancer. Ha-5, LAMA5), microtubule protein chain beta -2B (Tubulin beta-2B chain, TUBB2B), centromere protein F (Centromere protein F, CENPF) and vesicular related membrane protein 5, provide important data reference for further study of the mechanism of liver cancer metastasis.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.7

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相關(guān)期刊論文 前1條

1 Toshiyuki Ishiwata;Yoko Matsuda;Zenya Naito;;Nestin in gastrointestinal and other cancers: Effects on cells and tumor angiogenesis[J];World Journal of Gastroenterology;2011年04期

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本文編號：1976901