本文選題：β3GnT8 + 胃癌��； 參考：《蘇州大學》2016年博士論文

【摘要】：目的胃癌和結腸癌是常見的消化道系統(tǒng)惡性腫瘤,發(fā)病率和死亡率較高。盡管臨床上對胃腸道腫瘤采取了手術、放療、化療、生物治療等綜合治療措施,但治療效果差,仍有相當數(shù)量的患者最終死于癌灶的復發(fā)和轉移,給社會和家庭帶來了沉重的經(jīng)濟及精神負擔。β1,3-N乙酰氨基葡萄糖基轉移酶Ⅷ(β3Gn T8)以UDP-Glc NA為供體,將Glc NAc添加到三天線N-聚糖Galβ1-4Glc NAc的非還原性末端形成多聚乳糖胺型糖鏈。本課題從基因到糖鏈、從細胞內(nèi)到細胞外等多個層次和水平上,通過對糖基轉移酶β3Gn T8、多聚乳糖胺型N-糖鏈展開系統(tǒng)研究,以闡釋β3Gn T8調(diào)控胃癌侵襲轉移和結腸癌化療耐藥的分子機制。方法(1)研究β3Gn T8調(diào)控胃癌細胞侵襲轉移的分子機制(1)運用胃癌組織芯片采用免疫組化染色觀察β3Gn T8、多聚乳糖胺的表達,并將結果與患者臨床病理參數(shù)進行統(tǒng)計分析,進而闡明β3Gn T8、多聚乳糖胺的表達水平與胃癌患者臨床病理特征的關系。(2)培養(yǎng)不同來源的三種胃癌細胞株(AGS、SGC-7901、NCI-N87),通過Tanswell侵襲實驗和劃痕修復實驗,比較各實驗細胞的侵襲性和遷移性;采用熒光定量PCR和Western blot技術,檢測β3Gn T8在各胃癌細胞中的表達高低;采用流式細胞術、Lectin blot、凝集素芯片技術,分析多聚乳糖胺在各胃癌細胞中的表達、結構和功能特點,進而闡明β3Gn T8、多聚乳糖胺的表達與胃癌細胞侵襲轉移潛能的關系。(3)通過基因轉染和RNA干擾技術靶向調(diào)控相應胃癌細胞中β3Gn T8的表達,采用流式細胞術和Lectin blot技術,檢測多聚乳糖胺的表達;通過Transwell實驗比較各實驗細胞的侵襲能力;采用Western blot技術,檢測糖蛋白CD147糖基化修飾改變;采用熒光定量PCR、Western blot,分析基質(zhì)金屬蛋白酶MMPs的表達變化,進而闡明β3Gn T8對胃癌細胞侵襲轉移的調(diào)控機制。(4)采用免疫沉淀技術富集胃癌細胞中β3Gn T8的相互結合蛋白,通過SDSPAGE對免疫沉淀復合物進行分離。然后從膠中切取染色明顯的蛋白條帶,經(jīng)過脫色、還原、烷基化、酶解等處理,進行質(zhì)譜分析。搜索SWISS-PROT數(shù)據(jù)庫,進而篩選和鑒定胃癌細胞中β3Gn T8的靶蛋白。(5)采用PNGase F酶解釋放胃癌細胞中總糖蛋白N-糖鏈,對所得N-糖鏈用Sep-Pak C18柱純化后進行完全甲基化衍生,質(zhì)譜解析N-糖鏈的結構輪廓,進而闡明胃癌細胞中N-糖鏈變化規(guī)律。(2)研究β3Gn T8調(diào)控結腸癌細胞化療耐藥的分子機制(1)采用熒光定量PCR技術檢測β3Gn T8在結腸癌5-氟尿嘧啶(5-FU)耐藥細胞SW620/5-FU及其母細胞SW620中的表達水平;采用流式細胞術、Lectin blot檢測多聚乳糖胺在SW620/5-FU及SW620中的表達特征,進而闡明β3Gn T8、多聚乳糖胺的表達與結腸癌細胞化療耐藥的關系。(2)通過RNA干擾技術抑制SW620/5-FU細胞中β3Gn T8的表達,采用流式細胞術、Lectin blot檢測多聚乳糖胺的表達改變;采用MTT藥物敏感性實驗檢測細胞耐藥性狀的改變;采用Annexin V-FITC/PI雙染法檢測細胞凋亡;采用PI單染檢測細胞周期,進而闡明β3Gn T8對結腸癌細胞化療耐藥的調(diào)控機制。(3)通過多聚乳糖胺合成抑制劑3'-疊氮胸苷(AZT)干預SW620/5-FU細胞中多聚乳糖胺的合成,采用MTT藥物敏感性實驗檢測細胞耐藥性狀的改變,進而闡明多聚乳糖胺對結腸癌細胞化療耐藥的調(diào)控機制。結果(1)β3Gn T8高表達通過誘導多聚乳糖胺的合成進而促進胃癌細胞侵襲轉移(1)β3Gn T8陽性產(chǎn)物主要定位于胃癌組織細胞的胞核和胞漿中,呈棕黃色顆粒狀分布。多聚乳糖胺陽性表達主要集中在胃癌組織細胞的胞漿,表現(xiàn)為胞漿內(nèi)呈現(xiàn)棕黃色或者棕褐色顆粒,也有少量為胞膜中呈現(xiàn)棕黃色。胃癌患者中β3Gn T8的表達與年齡、性別、分化程度無明顯相關性,但是與腫瘤的病理分級和臨床分期正相關。胃癌患者中多聚乳糖胺的表達與腫瘤病理分級和臨床分期存在正相關,而與年齡、性別、分化程度不具有相關性。β3Gn T8、多聚乳糖胺在胃癌組織中的表達強度呈正相關關系。(2)胃癌AGS細胞的侵襲轉移能力最強,其次是SGC-7901細胞,而NCI-N87細胞的侵襲轉移能力最弱。AGS細胞中β3Gn T8基因和蛋白表達最高,其次是SGC-7901細胞,而NCI-N87細胞中β3Gn T8基因和蛋白表達最低。AGS細胞中多聚乳糖胺含量最多,其次是SGC-7901細胞,而NCI-N87細胞中多聚乳糖胺含量最少。β3Gn T8、多聚乳糖胺在胃癌細胞中的表達水平存在一致性。(3)上調(diào)胃癌NCI-N87細胞中β3Gn T8的基因表達,多聚乳糖胺的含量上升,細胞侵襲能力增強,高糖基化(HG)-CD147表達升高,MMP-14的基因和蛋白表達增多。下調(diào)胃癌AGS細胞中β3Gn T8的基因表達,多聚乳糖胺的含量下降,細胞侵襲能力減弱,高糖基化(HG)-CD147表達降低,MMP-14的基因和蛋白表達受到抑制。(4)在胃癌AGS細胞中,共分離鑒定出45種可與β3Gn T8相互結合的蛋白;在NCI-N87細胞中,共分離鑒定出32種可與β3Gn T8相互結合的蛋白。通過對比分析,發(fā)現(xiàn)AGS和NCI-N87細胞中存在23種可與β3Gn T8結合的共有蛋白。針對這些蛋白進行功能研究,并結合免疫共沉淀技術,發(fā)現(xiàn)膜聯(lián)蛋白A2(annexin A2,ANXA2)是β3Gn T8潛在靶蛋白。(5)胃癌AGS、SGC-7901、NCI-N87細胞中所含的N-糖鏈種類沒有明顯差別,但存在量上的差異。AGS細胞中巖藻糖和唾液酸的N-糖鏈含量均很少;SGC-7901細胞中巖藻糖的N-糖鏈所占比例明顯高于其他兩種細胞;NCI-N87細胞中唾液酸的N-糖鏈所占比例明顯高于其他兩種細胞。(2)β3Gn T8高表達通過誘導多聚乳糖胺的合成進而促進結腸癌細胞耐藥(1)與結腸癌SW620細胞相比,耐藥細胞SW620/5-FU中β3Gn T8的表達和多聚乳糖胺的含量均明顯增加。(2)抑制SW620/5-FU細胞中β3Gn T8的基因表達,多聚乳糖胺的含量下降,對化療藥物5-FU的敏感性上升,細胞凋亡增多,G1期細胞減少,而S期和G2/M期細胞增加。(3)AZT可有效抑制SW620/5-FU細胞中多聚乳糖胺的合成,提高其對化療藥物5-FU的敏感性。結論(1)β3Gn T8、多聚乳糖胺在胃癌組織中高表達,且均與胃癌患者的病理分級和臨床分期有關聯(lián),二者在胃癌組織的表達具有正相關性,提示其與胃癌的惡性進展有密切關系。隨著胃癌細胞侵襲轉移能力的增強,β3Gn T8的表達和多聚乳糖胺的含量也隨之升高。β3Gn-T8通過催化CD147分子中多聚乳糖的合成調(diào)控MMP-14的表達,從而改變胃癌細胞侵襲轉移能力,這可能需要ANXA2的協(xié)同作用。此外,N-糖鏈表達異常也與其胃癌細胞惡性表型有關。(2)β3Gn T8和多聚乳糖胺在結腸癌5-FU耐藥細胞中高表達。抑制β3Gn T8的表達和多聚乳糖胺的生物合成,可增強結腸癌耐藥細胞對5-FU的敏感性,實現(xiàn)其耐藥性的逆轉�？傊�,β3Gn T8及多聚乳糖胺在調(diào)控胃腸道腫瘤侵襲轉移和化療耐藥過程中起著關鍵作用。這一研究為針對胃腸道腫瘤治療相關分子藥物設計、篩選提供新的靶點,為臨床個體化治療提供基礎,具有重要的科學意義和實用價值。
[Abstract]:Objective gastric cancer and colon cancer are common malignant tumors of the digestive tract, with high morbidity and mortality. Despite the clinical treatment of gastrointestinal tumors, such as surgery, radiotherapy, chemotherapy and biological treatment, the curative effect is poor, and a considerable number of patients eventually die from the recurrence and metastasis of the cancer and bring to the society and family. The heavy economic and spiritual burden. The beta 1,3-N acetylglucosaminotransferase VIII (beta 3Gn T8), with UDP-Glc NA as the donor, added Glc NAc to the non reductive terminal of the three antenna N- Gal beta 1-4Glc NAc to form a polygalactamine chain. This subject has passed from genes to sugar chains, from intracellular to extracellular and other levels and levels. A systematic study of glycosyltransferase beta 3Gn T8 and poly lactamamine type N- chain expansion to explain the molecular mechanism of beta 3Gn T8 regulating invasion and metastasis of gastric cancer and chemotherapeutic resistance of colon cancer. Method (1) the molecular mechanism of the invasion and metastasis of gastric cancer cells by beta 3Gn T8 was studied (1) using immunohistochemical staining of gastric carcinoma tissue to observe the beta 3Gn T8 and polygalamines. The expression of the results and the clinicopathological parameters of the patients were analyzed, and the relationship between the expression level of beta 3Gn T8 and polygalamines and the clinicopathological features of gastric cancer patients. (2) to cultivate three kinds of gastric cancer cell lines (AGS, SGC-7901, NCI-N87) from different sources, through the Tanswell invasion experiment and the scratch repair experiment, to compare the experimental cells. The expression of beta 3Gn T8 in gastric cancer cells was detected by fluorescence quantitative PCR and Western blot. Flow cytometry, Lectin blot, and agglutinin chip technology were used to analyze the expression, structure and energy characteristics of polygalamine in gastric cancer cells, and then clarify the expression of beta 3Gn T8 and the expression of polygalamine. The relationship between the invasion and metastasis potential of gastric cancer cells. (3) the expression of beta 3Gn T8 in the corresponding gastric cancer cells was regulated by gene transfection and RNA interference technique. Flow cytometry and Lectin blot were used to detect the expression of polyamines; the invasion ability of the experimental cells was compared by Transwell experiment; and the Western blot technology was used to detect the sugar eggs. White CD147 glycosylation modification, fluorescence quantitative PCR, Western blot, analysis of the expression changes of matrix metalloproteinase MMPs, and further elucidate the regulation mechanism of the invasion and metastasis of gastric cancer cells by beta 3Gn T8. (4) enrichment of the mutual binding protein of beta 3Gn T8 in gastric cancer cells by immunoprecipitation technique, and the immunoprecipitation complex by SDSPAGE. Then, the obvious protein bands were cut from the glue, and the SWISS-PROT database was used to search and identify the target protein of the beta 3Gn T8 in gastric cancer cells. (5) the PNGase F enzyme was used to explain the sugar chain of the total glycoprotein N- in the gastric cancer cells and the Sep-Pak C18 in the N- chain. After purification, complete methylation was carried out, the structural profile of N- sugar chain was analyzed by mass spectrometry, and then the changes of N- sugar chain in gastric cancer cells were elucidated. (2) the molecular mechanism of chemotherapy resistance of colon cancer cells by beta 3Gn T8 was studied (1) the fluorescence quantitative PCR technique was used to detect the 3Gn T8 in the colon cancer 5- fluorouracil (5-FU) resistant cells SW620/5-FU and its mother The expression level in cell SW620; using flow cytometry, Lectin blot to detect the expression of polygalamines in SW620/5-FU and SW620, and further elucidate the relationship between the expression of beta 3Gn T8, the expression of polygalamines and chemotherapeutic resistance of colon cancer cells. (2) the expression of beta 3Gn T8 in SW620/ 5-FU cells was suppressed by RNA interference technique, and flow cytometry was used. In blot detected the changes in the expression of polygalactamine; detected the change of cell resistance by MTT drug sensitivity test; Annexin V-FITC/PI double staining was used to detect cell apoptosis; PI single staining was used to detect cell cycle, and then the regulation mechanism of beta 3Gn T8 to chemotherapy resistance of colon cancer cells was elucidated. (3) 3'synthesis of inhibitor 3' by polygalamines. - azidoside (AZT) interfered with the synthesis of polygalactamine in SW620/5-FU cells. The sensitivity test of MTT was used to detect the change of cell resistance characteristics and the regulation mechanism of polygalactamine on chemotherapy resistance of colon cancer cells. Results (1) the high expression of beta 3Gn T8 was induced by the synthesis of polygalamine to promote the invasion of gastric cancer cells. The positive products of transfer (1) beta 3Gn T8 were mainly located in the nucleus and cytoplasm of gastric carcinoma tissue cells, with brown and yellow granular distribution. The positive expression of polygalamines mainly concentrated in the cytoplasm of gastric cancer tissue cells, showing brown or brown brown granules in the cytoplasm, and a small amount of brown yellow in the membrane. The beta 3Gn T in gastric cancer patients. The expression of 8 was not associated with age, sex and differentiation, but it was positively correlated with the pathological grade and clinical stage of the tumor. There was a positive correlation between the expression of polygalactamine and the pathological grade and clinical stage of tumor, but not related to age, sex and differentiation. Beta 3Gn T8, polyamines were in gastric cancer tissue. The expression intensity was positive correlation. (2) the invasion and metastasis of gastric cancer AGS cells was the strongest, followed by SGC-7901 cells, while the weakest.AGS cells in the NCI-N87 cells were the weakest.AGS cells with the highest expression of the 3Gn T8 gene and protein, followed by the SGC-7901 cells, while the beta 3Gn T8 gene and protein expression in NCI-N87 cells expressed the lowest concentration of polygalactose in the.AGS cells. The content of amines is the most, followed by SGC-7901 cells, and the content of polygalamines in NCI-N87 cells is the least. The expression level of beta 3Gn T8 and polyamines in gastric cancer cells is consistent. (3) the gene expression of beta 3Gn T8 in gastric cancer NCI-N87 cells, the rise of polygalamines, the enhancement of cell invasiveness, and the -CD147 expression of high glycosylation (HG) in gastric cancer cells are up. The gene and protein expression of MMP-14 increased. The gene expression of beta 3Gn T8 in gastric cancer AGS cells decreased, the content of polygalamine decreased, the cell invasiveness decreased, the expression of high glycosylated (HG) -CD147 decreased, and the expression of MMP-14 gene and protein was inhibited. (4) 45 kinds of beta 3Gn T8 were identified in the gastric cancer AGS cells. In NCI-N87 cells, 32 proteins that could be combined with beta 3Gn T8 were isolated and identified. Through comparative analysis, 23 common proteins associated with beta 3Gn T8 were found in AGS and NCI-N87 cells. The functional study of these proteins and the combination of immunoprecipitation techniques showed that the membrane associated protein A2 (annexin A2, ANXA2) was beta 3G. N T8 potential target protein. (5) there is no significant difference in the N- sugar chain in AGS, SGC-7901, NCI-N87 cells of gastric cancer, but there is a significant difference in the N- sugar chain content of fucose and sialic acid in.AGS cells; the N- sugar chain of fucose in SGC-7901 cells is higher than the other two cells; the N- sugar of sialic acid in NCI-N87 cells The proportion of the chain was significantly higher than that of the other two cells. (2) the high expression of beta 3Gn T8 was induced by the synthesis of polyamines to promote the drug resistance of colon cancer cells (1), compared with the colon cancer SW620 cells, the expression of beta 3Gn T8 and the content of polylactiamines in the drug resistant cell SW620/5-FU increased significantly. (2) the inhibition of the gene of T8 in SW620/5-FU cells. It was expressed that the content of lactamines decreased, the sensitivity of the chemotherapeutic drug 5-FU increased, cell apoptosis increased, G1 cells decreased, and S and G2/M cells increased. (3) AZT could effectively inhibit the synthesis of polygalactamine in SW620/5-FU cells and improve its sensitivity to chemotherapy drug 5-FU. Conclusion (1) beta 3Gn T8 and polygalamines in gastric cancer tissue The expression of middle and high levels is associated with the pathological grade and clinical stage of gastric cancer patients. The expression of the two is positively related to the expression of gastric cancer, suggesting that it is closely related to the malignant progression of gastric cancer. With the enhancement of the invasion and metastasis of gastric cancer cells, the expression of beta 3Gn T8 and the content of polyamines also increase. Beta 3Gn-T8 through the catalytic C The synthesis of polygalactose in D147 molecules regulates the expression of MMP-14 and changes the invasion and metastasis of gastric cancer cells, which may require a synergistic effect of ANXA2. In addition, the abnormal expression of N- sugar chain is related to the malignant phenotype of gastric cancer cells. (2) the expression of beta 3Gn T8 and Polyamines in colon cancer 5-FU resistant cells is highly expressed and the expression of T8 in beta 3Gn is inhibited and the expression of beta 3Gn T8 is inhibited. The biosynthesis of polygalamines can enhance the sensitivity of colon cancer resistant cells to 5-FU and achieve a reversal of their resistance. In a word, beta 3Gn T8 and polyamines play a key role in regulating the invasion and metastasis of gastrointestinal tumors and chemotherapeutic resistance. This study is to screen for the design of molecular drugs for the treatment of gastrointestinal tumors. It is of great scientific significance and practical value to provide new targets for clinical individualized treatment.

【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735

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相關期刊論文 前1條

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本文編號：1780434