【摘要】：據(jù)統(tǒng)計(jì),胃癌是世界上第五大惡性腫瘤,致死率占世界第三位[1]。胃癌在不同國(guó)家和地區(qū)之間的發(fā)病率存在很大差異,亞洲地區(qū)胃癌病例數(shù)占世界總病例數(shù)的73.6%,其中中國(guó)的胃癌患者數(shù)占世界的42.7%。在2015年中國(guó)確診的癌癥病例中,男性的胃癌發(fā)病率占全部癌癥發(fā)病率的第二位,女性的為第三位。針對(duì)胃癌的主要治療方法包括手術(shù)切除、化療、免疫療法和定向治療。雖然近年來(lái)診斷和治療技術(shù)都有所提高,但是晚期胃癌病人的生存期仍舊低于1年,臨床上缺乏診斷早期胃癌的有效方法,晚期胃癌病人預(yù)后很差。CUL4B(Cullin 4B)作為支架蛋白參與構(gòu)成E3泛素連接酶復(fù)合物Cullin 4B-Ring E3 Ligase(CRL4B),參與調(diào)節(jié)包括細(xì)胞周期、細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、基因轉(zhuǎn)錄和生物體個(gè)體生長(zhǎng)發(fā)育等多項(xiàng)重要生命活動(dòng)。CRL4B不僅可以通過(guò)泛素化降解p53和cyclin E參與調(diào)控細(xì)胞增殖和衰老過(guò)程,還能通過(guò)單泛素化H2A蛋白,調(diào)節(jié)靶基因啟動(dòng)子組蛋白H3的乙酰化以及甲基化等過(guò)程,從而在表觀遺傳水平上調(diào)控p16、p21、PTGDS、miRNA-371/372等基因的表達(dá)。越來(lái)越多的證據(jù)顯示CRL4B在腫瘤發(fā)生發(fā)展中發(fā)揮重要作用。CRL4B可以通過(guò)單泛素化H2AK119,抑制Wnt拮抗因子、PTEN、IGFBP3和miR-194的表達(dá)。在宮頸癌、肝癌和非小細(xì)胞肺癌等癌癥中,CRL4B通過(guò)抑制腫瘤抑癌基因的表達(dá)發(fā)揮癌基因的功能。有趣的是,本實(shí)驗(yàn)室還有一項(xiàng)研究結(jié)果提示CUL4B可通過(guò)調(diào)節(jié)微環(huán)境中MDSC細(xì)胞的分化抑制腫瘤的生長(zhǎng)。因此,CUL4B在不同環(huán)境下在腫瘤的發(fā)生和發(fā)展發(fā)揮不同的功能。截止到目前,CUL4B在胃癌中的作用還未見(jiàn)報(bào)道,為了探究CUL4B在胃癌中發(fā)揮的功能,我們首先檢測(cè)了 CUL4B在胃癌組織中的表達(dá)水平,并分析了 CUL4B表達(dá)對(duì)胃癌細(xì)胞增殖、遷移侵襲以及對(duì)化療藥物敏感性的影響。首先,我們檢測(cè)了CUL4B在胃癌和癌旁組織中的表達(dá),結(jié)果顯示,相對(duì)于癌旁組織,CUL4B在胃癌組織中表達(dá)水平明顯上調(diào),并且CUL4B的表達(dá)與胃癌的病理分級(jí)呈正相關(guān)。隨后我們選用了三種胃癌細(xì)胞—MKN-45、BGC-823和SGC-7901,利用病毒感染,分別構(gòu)建了三種CUL4B低表達(dá)的胃癌細(xì)胞系和對(duì)應(yīng)的的空載體對(duì)照細(xì)胞系,并且構(gòu)建了MKN-45 CUL4B高表達(dá)細(xì)胞系和對(duì)應(yīng)的空載體對(duì)照細(xì)胞系。我們利用MTT實(shí)驗(yàn)檢測(cè)了 CUL4B表達(dá)降低后胃癌細(xì)胞增殖活性的改變,結(jié)果發(fā)現(xiàn)CUL4B表達(dá)降低抑制胃癌細(xì)胞增殖活性。利用流式法檢測(cè)細(xì)胞周期時(shí)我們并未發(fā)現(xiàn)CUL4B表達(dá)降低對(duì)胃癌細(xì)胞周期存在顯著影響,利用細(xì)胞動(dòng)力學(xué)分析顯示CUL4B不影響胃癌細(xì)胞G1/S其以及M/G1期進(jìn)程,EdU摻入實(shí)驗(yàn)也顯示CUL4B表達(dá)降低對(duì)胃癌細(xì)胞DNA合成無(wú)明顯影響,提示CUL4B調(diào)控胃癌細(xì)胞增殖活性并不是通過(guò)對(duì)以上過(guò)程的調(diào)控而實(shí)現(xiàn)的。接下來(lái)我們利用transwell實(shí)驗(yàn)和劃痕實(shí)驗(yàn)分析CUL4B表達(dá)對(duì)胃癌細(xì)胞遷移侵襲能力的影響,劃痕實(shí)驗(yàn)結(jié)果顯示,相對(duì)于對(duì)照細(xì)胞,CUL4B表達(dá)降低的細(xì)胞遷移能力明顯下降。隨后,我們采用MTT實(shí)驗(yàn)分析發(fā)現(xiàn),相對(duì)于對(duì)照細(xì)胞,低表達(dá)CUL4B的胃癌細(xì)胞對(duì)于同等濃度化療藥物順鉑和吡柔比星的敏感性增加。TUNEL實(shí)驗(yàn)分析顯示CUL4B低表達(dá)胃癌細(xì)胞在順鉑處理后凋亡比例更高。這些結(jié)果表明CUL4B參與調(diào)控胃癌細(xì)胞對(duì)化療藥物的敏感性。總之,通過(guò)本研究我們證實(shí)了 CUL4B在胃癌中表達(dá)上調(diào),并且CUL4B表達(dá)水平與胃癌的病理分級(jí)呈正相關(guān);細(xì)胞學(xué)實(shí)驗(yàn)結(jié)果顯示抑制CUL4B表達(dá)會(huì)抑制胃癌細(xì)胞的增殖活性、侵襲遷移能力并增加胃癌細(xì)胞對(duì)化療藥物的敏感性,這些結(jié)果提示我們CUL4B在胃癌中發(fā)揮原癌基因的作用。該結(jié)果為進(jìn)一步深入探究CUL4B功能提供了一定的實(shí)驗(yàn)數(shù)據(jù),為闡明胃癌的發(fā)生機(jī)制、篩選新的腫瘤標(biāo)志基因和研究新的療法奠定了一定的理論基礎(chǔ)。
[Abstract]:According to statistics, gastric cancer is the fifth largest malignant tumor in the world. The mortality rate of the third [1]. gastric cancer in the world is very different in different countries and regions. In Asia, the number of cases of gastric cancer accounts for 73.6% of the total number of cases in the world, of which the number of gastric cancer patients in China accounts for the world's 42.7%. in the 2015 Chinese confirmed cancer cases. The incidence of sexual gastric cancer accounts for second of the total cancer incidence and third in women. The main treatment methods for gastric cancer include surgical resection, chemotherapy, immunotherapy, and directional therapy. Although diagnosis and treatment techniques have improved in recent years, the survival period of patients with advanced gastric cancer is still less than 1 years, and the early diagnosis is lack of early diagnosis. Effective methods of gastric cancer, the prognosis of patients with advanced gastric cancer is poor.CUL4B (Cullin 4B) as a scaffold protein involved in the formation of E3 ubiquitin ligase complex Cullin 4B-Ring E3 Ligase (CRL4B), involved in the regulation of cell cycle, cell signal transduction, gene transcription and individual growth and development of organisms, such as many important activities of.CRL4B can not only pass through the life of.CRL4B. Excessive ubiquitination of p53 and cyclin E participates in the regulation of cell proliferation and senescence, and can also regulate the expression of p16, p21, PTGDS, and miRNA-371/372 by regulating the expression of p16, p21, PTGDS, and miRNA-371/372 through a single ubiquitin H2A protein, regulating the acetylation and methylation of the target gene promoter H3. More and more evidence shows that CRL4B is swollen. .CRL4B can play an important role in the development of tumor. H2AK119 can be used to inhibit the expression of Wnt antagonist, PTEN, IGFBP3 and miR-194. In cancer of cervical cancer, liver cancer and non-small cell lung cancer, CRL4B plays oncogene function by inhibiting the expression of tumor suppressor genes. Interestingly, there is also a research result in this laboratory. It is suggested that CUL4B can inhibit the growth of tumor by regulating the differentiation of MDSC cells in microenvironment. Therefore, CUL4B plays different functions in the occurrence and development of tumor in different environments. To the present, the role of CUL4B in gastric cancer has not been reported. In order to explore the function of CUL4B in gastric cancer, we first detected CUL4B in gastric cancer. The expression level in the tissue, and the effect of CUL4B expression on the proliferation, migration and invasion of gastric cancer cells and the sensitivity to chemotherapeutic drugs. First, we detected the expression of CUL4B in gastric and paracancerous tissues. The results showed that the expression level of CUL4B in gastric cancer tissues was significantly up-regulated compared with the Para cancerous tissues, and the expression of CUL4B and gastric cancer was associated with gastric cancer. Three kinds of gastric cancer cells, MKN-45, BGC-823 and SGC-7901, were then used to construct three kinds of CUL4B low expression gastric cancer cell lines and corresponding unloaded body control cell lines, and the MKN-45 CUL4B high expression cell line and the corresponding empty body control cell line were constructed. MTT test was used to detect the proliferation activity of gastric cancer cells after the decrease of CUL4B expression. The results showed that the expression of CUL4B decreased to inhibit the proliferation activity of gastric cancer cells. We did not find that the decrease of CUL4B expression had a significant effect on the cell cycle of gastric cancer by flow method. The results of cell dynamics analysis showed that CUL4B did not affect the stomach. The cancer cell G1/S and the M/G1 phase process, EdU incorporation test also showed that the decrease of CUL4B expression had no obvious effect on the DNA synthesis of gastric cancer cells, suggesting that CUL4B regulation of the proliferation activity of gastric cancer cells was not realized by the regulation of the above process. Then we used Transwell experiment and scratch test to analyze the migration of CUL4B expression to gastric cancer cell migration. The effect of invasive ability, the results of scratch test showed that the cell migration ability of CUL4B expression decreased significantly compared with the control cells. Then, we used MTT test to find that the sensitivity of gastric cancer cells with low expression of CUL4B to cisplatin and pirirubicin with the same concentration was increased by.TUNEL experimental analysis relative to the control cells. The results showed that the CUL4B low expression of gastric cancer cells had a higher percentage of apoptosis after cisplatin treatment. These results showed that CUL4B was involved in regulating the sensitivity of gastric cancer cells to chemotherapeutic drugs. In conclusion, we confirmed that the expression of CUL4B in gastric cancer was up regulated by this study and that the level of CUL4B expression was positively correlated with the classification of gastric cancer; cytological experimental results showed that Inhibition of CUL4B expression can inhibit the proliferation of gastric cancer cells, invasion and migration and increase the sensitivity of gastric cancer cells to chemotherapeutic drugs. These results suggest that CUL4B plays the role of proto oncogene in gastric cancer. The results provide some experimental data for further exploring the function of CUL4B, in order to elucidate the mechanism of gastric cancer. The selection of new tumor marker genes and the study of new therapies have laid a theoretical foundation.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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2 胡偉國(guó);腫瘤相關(guān)成纖維細(xì)胞中Thy-1激活誘導(dǎo)上皮間質(zhì)轉(zhuǎn)化促進(jìn)胃癌細(xì)胞侵襲轉(zhuǎn)移的研究[D];上海交通大學(xué);2014年

3 周韻斕;應(yīng)激條件下胃癌細(xì)胞高表達(dá)線粒體活性氧和前梯度蛋白2促進(jìn)腫瘤進(jìn)展的研究[D];上海交通大學(xué);2014年

4 徐同鵬;SP1誘導(dǎo)的長(zhǎng)非編碼RNA TINCR通過(guò)調(diào)控KLF2 mRNA穩(wěn)定性影響胃癌細(xì)胞的增殖與凋亡[D];南京醫(yī)科大學(xué);2015年

5 王暢;miR-34a通過(guò)miR-34a-c-myc/Bmi-1負(fù)反饋通路負(fù)調(diào)控胃癌干細(xì)胞樣特性[D];復(fù)旦大學(xué);2014年

6 羅貫虹;MGr1-Ag/37LRP參與PrP~C介導(dǎo)的胃癌細(xì)胞多藥耐藥[D];第四軍醫(yī)大學(xué);2014年

7 默董亮;人類解旋酶RecQL4調(diào)控胃癌細(xì)胞耐藥性分子機(jī)制研究[D];中國(guó)科學(xué)院北京基因組研究所;2016年

8 劉皎婧;PI3K/Akt/GSK3β/p190ARhoGAP/RhoA信號(hào)通路在Wnt5a誘導(dǎo)胃癌細(xì)胞遷移中作用的研究[D];南京醫(yī)科大學(xué);2014年

9 Muhammad Kamran（卡姆拉）;[D];大連醫(yī)科大學(xué);2015年

10 王瑛;PKGⅡ?qū)PA誘導(dǎo)的胃癌細(xì)胞徖移及RhoA和Rac1激活的影響[D];江蘇大學(xué);2015年

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1 林光錟;CyclinA/CDK2介導(dǎo)BAG3 Ser291位點(diǎn)的磷酸化并影響胃癌細(xì)胞的增殖和凋亡[D];福建醫(yī)科大學(xué);2015年

2 蔡洙哲;p12-LOX對(duì)胃癌細(xì)胞MKN-28的增殖和遷移的影響[D];延邊大學(xué);2015年

3 李龍;Her-2高表達(dá)對(duì)胃癌細(xì)胞侵襲、遷移的影響[D];長(zhǎng)江大學(xué);2015年

4 趙婷婷;GPIbα對(duì)胃癌細(xì)胞粘附、遷移和侵襲能力的影響及其機(jī)制的初步探討[D];蘭州大學(xué);2015年

5 謝瑞霞;盤狀結(jié)構(gòu)域受體1調(diào)控上皮—間質(zhì)轉(zhuǎn)化影響胃癌侵襲轉(zhuǎn)移分子機(jī)制的研究[D];蘭州大學(xué);2015年

6 王東倉(cāng);NM23-H2通過(guò)抑制Wnt/β-catenin信號(hào)通路抑制胃癌細(xì)胞的侵襲遷移[D];蘭州大學(xué);2015年

7 朱瑞雪;NDRG2基因?qū)ξ赴┘?xì)胞惡性表型的影響[D];鄭州大學(xué);2015年

8 董凱楠;內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)胃癌細(xì)胞侵襲遷移能力的影響[D];鄭州大學(xué);2015年

9 單爭(zhēng)爭(zhēng);二甲雙胍在IL-6誘導(dǎo)胃癌細(xì)胞EMT中的作用[D];鄭州大學(xué);2015年

10 田莉;NM23-H2通過(guò)誘導(dǎo)自噬抑制胃癌細(xì)胞EMT的發(fā)生[D];蘭州大學(xué);2015年

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