發(fā)布時(shí)間：2018-02-01 10:15

  本文關(guān)鍵詞： EBP50 胰腺癌 SW1990 過表達(dá) 機(jī)制　出處：《武漢大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：胰腺癌是常見的消化系統(tǒng)惡性腫瘤,其死亡率高,預(yù)后極差,確診時(shí)多為晚期且伴有淋巴結(jié)或遠(yuǎn)處轉(zhuǎn)移而失去了手術(shù)機(jī)會(huì),原因是其臨床表現(xiàn)隱匿,缺乏有效的早期診斷方法。胰腺癌的發(fā)病機(jī)制尚未完全明確,原癌基因的激活、抑癌基因的功能失調(diào)及信號(hào)轉(zhuǎn)導(dǎo)途徑的異常都被認(rèn)為參與了癌癥的發(fā)生發(fā)展。因此探究胰腺癌的發(fā)病機(jī)制,尋找早期有效的腫瘤標(biāo)志物對(duì)胰腺癌的診斷與預(yù)后意義重大。磷酸化蛋白50(EBP50)又被稱為鈉氫交換子條子因子1(NHERF1)是近年來發(fā)現(xiàn)的一個(gè)與多種腫瘤相關(guān)的抑癌基因,如乳腺癌、肝癌、結(jié)直腸癌、胃癌等。本研究通過檢測(cè)EBP50在胰腺癌組織中的表達(dá)并上調(diào)EBP50對(duì)胰腺癌細(xì)胞系SW1990的影響和具體機(jī)制,旨在為胰腺癌的發(fā)生機(jī)制提供新思路,為尋找早期診斷及靶向治療胰腺癌提供新的依據(jù)。第一部分免疫組化及RT-qPCR檢測(cè)EBP50基因表達(dá)目的：檢測(cè)并比較EBP50基因在胰腺癌組織和癌旁組織的表達(dá)情況方法：120例胰腺癌組織標(biāo)本,采用免疫組化法,檢測(cè)EBP50基因的表達(dá)情況；其中20例胰腺癌組織和20例癌旁組織采用RT-qPCR檢測(cè)EBP50 mRNA的表達(dá)水平。結(jié)果：免疫組化結(jié)果顯示：120例胰腺癌組織標(biāo)本中有45例(37.5%)無EBP50蛋白的表達(dá),另75例(62.5%)表達(dá)EBP50,其中38例(31.67%)染色陽性強(qiáng)度為1+,22例(18.33%)陽性強(qiáng)度2+,僅有15例(12.5%)陽性強(qiáng)度3+,而正常組織標(biāo)本免疫組化結(jié)果陽性強(qiáng)度均為3+。RT-qPCR結(jié)果顯示20例胰腺癌組織標(biāo)本的mRNA平均水平為0.75+心.11,而對(duì)應(yīng)的癌旁組織的EBP50 mRNA平均水平為1.63土0.19。結(jié)論：胰腺癌組織中EBP50表達(dá)較正常組織EBP50的表達(dá)有所降低；胰腺癌組織EBP50 mRNA水平較癌旁組織EBP50 mRNA水平明顯降低；提示EBP50可能參與胰腺癌發(fā)生發(fā)展。第二部分I-調(diào)EBP50基因?qū)�SW1990細(xì)胞生物學(xué)行為影響目的：應(yīng)用質(zhì)粒轉(zhuǎn)染技術(shù)對(duì)EBP50基因進(jìn)行過表達(dá),研究EBP50基因上調(diào)后對(duì)人胰腺癌細(xì)胞系SW1990的增殖,侵襲和克隆形成能力的影響。方法：人胰腺癌細(xì)胞系SW1990細(xì)胞接種培養(yǎng)于含10%胎牛血清的DMEM-F12培養(yǎng)基中培養(yǎng)；脂質(zhì)體介導(dǎo)的質(zhì)粒轉(zhuǎn)染,G418篩選細(xì)胞,Western blot法進(jìn)行鑒定。CCK-8法、軟質(zhì)瓊膠克隆形成實(shí)驗(yàn)及Transwell小室法檢測(cè)細(xì)胞增殖、非錨定依賴性生長(zhǎng)能力及侵襲能力。結(jié)果：成功構(gòu)建穩(wěn)定表達(dá)的EBP50-SW1990細(xì)胞和HA-SW1990細(xì)胞；CCK8顯示EBP50-SW1990細(xì)胞增殖能力明顯低于HA-SW1990細(xì)胞、SW1990細(xì)胞,具有統(tǒng)計(jì)學(xué)差異(P0.05)；克隆形成實(shí)驗(yàn)提示EBP50-SW1990細(xì)胞的非錨定依賴性生長(zhǎng)能力明顯低于兩對(duì)照組,具有統(tǒng)計(jì)學(xué)差異(P0.05); Transwell結(jié)果表明EBP50-SW1990細(xì)胞的侵襲能力較兩對(duì)照組細(xì)胞明顯減弱(P0.05)。結(jié)論：EBP50基因過表達(dá)能抑制胰腺癌SW1990細(xì)胞的增殖、侵襲及非錨定依賴性生長(zhǎng)能力。第三部分上調(diào)EBP50基因?qū)�SW1990細(xì)胞作用的機(jī)制研究目的：探究EBP50基因過表達(dá)后對(duì)人胰腺癌細(xì)胞系SW1990周期和凋亡的影響及其具體機(jī)制。方法：細(xì)胞周期法檢測(cè).EBP50-SW1990細(xì)胞、HA-SW1990細(xì)胞及SW1990細(xì)胞的細(xì)胞周期變化；Hochest 33258檢測(cè)法觀察凋亡影響；Western blot法檢測(cè)三種細(xì)胞細(xì)胞中Bcl-2、β-catenin及E-cadherin的表達(dá)變化。結(jié)果：與SW1990和HA-SW1990兩種細(xì)胞比較,EBP50-SW1990細(xì)胞的G1/G0期細(xì)胞比例增加明顯(62.7%±1.03%),S期細(xì)胞比例明顯減少(15.3%±1.33%),具有統(tǒng)計(jì)學(xué)差異(P,0.05); Hochest 33258檢測(cè)細(xì)胞凋亡結(jié)果表明EBP50-SW1990凋亡的細(xì)胞明顯多于兩組對(duì)照組細(xì)胞(P0.05); EBP50-SW1990細(xì)胞的Bcl-2蛋白表達(dá)明顯較SW1990細(xì)胞低；EBP50-SW1990細(xì)胞的E-cadherin蛋白表達(dá)水平較SW1990細(xì)胞及HA-SW1990細(xì)胞顯著增高,而β-catenin蛋白水平較兩者顯著增高(P0.01)。結(jié)論：BP50基因過表達(dá)后,阻滯了G1-S期進(jìn)程,誘導(dǎo)其凋亡；上調(diào)EBP50基因?qū)σ认侔㏒W1990細(xì)胞的影響是通過抑制Bcl-2的表達(dá)、降低β-catenin和增加E-cadherin的水平來介導(dǎo)的,EBP50發(fā)揮一個(gè)抑癌基因作用。
[Abstract]:Pancreatic cancer is a common malignant tumor of digestive system, its high mortality and poor prognosis, when diagnosed with advanced lymph node or distant metastasis and lost the chance of operation, because of its clinical manifestations, lack of effective early diagnostic methods. The pathogenesis of pancreatic cancer is not completely clear, the activation of oncogene, the dysfunction of tumor suppressor genes and signal transduction pathways of anomalies are considered involved in the occurrence and development of cancer pathogenesis. Therefore research of pancreatic cancer, finding effective early tumor marker for pancreatic cancer diagnosis and prognostic significance. The phosphorylation of protein 50 (EBP50) is also known as sodium hydrogen exchange sub factor 1 (NHERF1) is a tumor suppressor gene discovered in recent years, one is associated with many kinds of tumors, such as breast cancer, liver cancer, colorectal cancer, gastric cancer. This study by detecting the expression of EBP50 in pancreatic carcinoma tissues and up-regulated EBP50 Effect on pancreatic carcinoma cell line SW1990 and the specific mechanism, to provide new ideas for the pathogenesis of pancreatic cancer, for early diagnosis and targeted therapy of pancreatic cancer to provide a new basis. The first part of immunohistochemistry and RT-qPCR detection of EBP50 gene expression objective: used to detect the expression and tissue EBP50 gene in pancreatic cancer and cancer: 120 cases of pancreatic cancer tissue samples by immunohistochemistry, detect the expression of EBP50 gene; the expression level of RT-qPCR EBP50 mRNA by detection of 20 cases of pancreatic cancer and 20 cases of adjacent tissues. Results: immunohistochemical results showed that 120 cases of pancreatic cancer tissues were 45 cases (37.5%) expression of EBP50 protein, the other 75 cases (62.5%) the expression of EBP50, of which 38 cases (31.67%) positive staining intensity of 1+, 22 cases (18.33%) positive intensity of 2+, only 15 cases (12.5%) positive intensity 3+, and normal tissue samples The results of immunohistochemistry staining intensity were 3+.RT-qPCR results showed that 20 cases of pancreatic cancer tissues mRNA the average level of 0.75+.11, and paracancerous tissues of EBP50 mRNA average was 1.63 0.19. conclusion: the expression of EBP50 in pancreatic cancer tissues compared with normal tissues, EBP50 is lower; the level of EBP50 mRNA in pancreatic cancer EBP50 mRNA levels than in adjacent tissue decreased obviously; it suggests that EBP50 may participate in the occurrence and development of pancreatic cancer. The I- gene of second EBP50 influence the biological behavior of SW1990 cells Objective: using plasmid transfection of EBP50 gene expression on the proliferation of human pancreatic cancer cell line SW1990 of up-regulated EBP50 gene, invasion and clone formation the effect. Methods: human pancreatic cancer cell line SW1990 cells were cultured in DMEM-F12 containing 10% fetal bovine serum medium; plasmid mediated by liposome G418 cell transfection, screening, Western blot were identified by.CCK-8 method, soft agar colony formation assay and cell proliferation of Transwell cells, anchorage independent growth and invasiveness. Results: the successful construction of the stable expression of EBP50-SW1990 cells and HA-SW1990 cells; CCK8 showed that the proliferation ability of EBP50-SW1990 cells was significantly lower than that of HA-SW1990 cells. SW1990 cells, with statistical difference (P0.05); cloning experiments showed that EBP50-SW1990 cell anchorage dependent growth capacity was less than two of the control group, with statistical difference (P0.05); Transwell results showed that the invasive ability of EBP50-SW1990 cells was two cells in control group decreased significantly (P0.05). Conclusion: the overexpression of EBP50 can inhibit SW1990 pancreatic cancer cell proliferation, invasion and anchorage independent growth. Effect of up regulation of EBP50 gene on cell SW1990 third The purpose of the study: To explore the mechanism of overexpression of EBP50 gene on human pancreatic cancer cell line SW1990 proliferation and apoptosis and its mechanism. Methods:.EBP50-SW1990 cells to detect cell cycle, cell cycle changes of HA-SW1990 cells and SW1990 cells; Hochest 33258 detection method was used to observe the apoptosis effect of Western detection; blot method three kinds of cells in Bcl-2 expression of -catenin, beta and E-cadherin. Results: compared with the two kinds of SW1990 and HA-SW1990 cells, EBP50-SW1990 cell ratio of G1/G0 phase cells increased significantly (62.7% + 1.03%), the proportion of cells in S phase significantly decreased (15.3% + 1.33%), with statistical difference (P, 0.05); the 33258 Hochest showed that the detection of apoptosis the apoptosis of EBP50-SW1990 cells was more than two groups of cells in the control group (P0.05); the expression of EBP50-SW1990 cells Bcl-2 protein were significantly lower than that in SW1990 cells; EBP50-SW1990 cells The expression level of E-cadherin protein than SW1990 cells and HA-SW1990 cells was significantly increased, while the -catenin protein level is both significantly increased (P0.01). Conclusion: over expression of BP50 gene after block phase G1-S process, induce apoptosis; effect of up regulation of EBP50 gene on pancreatic cancer SW1990 cells by inhibiting the expression of Bcl-2 reduced beta -catenin and increase the level of E-cadherin is mediated by EBP50, play a role of tumor suppressor genes.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.9

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