本文關(guān)鍵詞： EBP50 胰腺癌 SW1990 過表達 機制　出處：《武漢大學》2016年博士論文　論文類型：學位論文

【摘要】：胰腺癌是常見的消化系統(tǒng)惡性腫瘤,其死亡率高,預后極差,確診時多為晚期且伴有淋巴結(jié)或遠處轉(zhuǎn)移而失去了手術(shù)機會,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)及信號轉(zhuǎn)導途徑的異常都被認為參與了癌癥的發(fā)生發(fā)展。因此探究胰腺癌的發(fā)病機制,尋找早期有效的腫瘤標志物對胰腺癌的診斷與預后意義重大。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EBP50在胰腺癌組織中的表達并上調(diào)EBP50對胰腺癌細胞系SW1990的影響和具體機制,旨在為胰腺癌的發(fā)生機制提供新思路,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EBP50基因表達目的：檢測并比較EBP50基因在胰腺癌組織和癌旁組織的表達情況方法：120例胰腺癌組織標本,采用免疫組化法,檢測EBP50基因的表達情況；其中20例胰腺癌組織和20例癌旁組織采用RT-qPCR檢測EBP50 mRNA的表達水平。結(jié)果：免疫組化結(jié)果顯示：120例胰腺癌組織標本中有45例(37.5%)無EBP50蛋白的表達,另75例(62.5%)表達EBP50,其中38例(31.67%)染色陽性強度為1+,22例(18.33%)陽性強度2+,僅有15例(12.5%)陽性強度3+,而正常組織標本免疫組化結(jié)果陽性強度均為3+。RT-qPCR結(jié)果顯示20例胰腺癌組織標本的mRNA平均水平為0.75+心.11,而對應(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表達較正常組織EBP50的表達有所降低；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)W1990細胞生物學行為影響目的：應(yīng)用質(zhì)粒轉(zhuǎn)染技術(shù)對EBP50基因進行過表達,研究EBP50基因上調(diào)后對人胰腺癌細胞系SW1990的增殖,侵襲和克隆形成能力的影響。方法：人胰腺癌細胞系SW1990細胞接種培養(yǎng)于含10%胎牛血清的DMEM-F12培養(yǎng)基中培養(yǎng)；脂質(zhì)體介導的質(zhì)粒轉(zhuǎn)染,G418篩選細胞,Western blot法進行鑒定。CCK-8法、軟質(zhì)瓊膠克隆形成實驗及Transwell小室法檢測細胞增殖、非錨定依賴性生長能力及侵襲能力。結(jié)果：成功構(gòu)建穩(wěn)定表達的EBP50-SW1990細胞和HA-SW1990細胞；CCK8顯示EBP50-SW1990細胞增殖能力明顯低于HA-SW1990細胞、SW1990細胞,具有統(tǒng)計學差異(P0.05)；克隆形成實驗提示EBP50-SW1990細胞的非錨定依賴性生長能力明顯低于兩對照組,具有統(tǒng)計學差異(P0.05); Transwell結(jié)果表明EBP50-SW1990細胞的侵襲能力較兩對照組細胞明顯減弱(P0.05)。結(jié)論：EBP50基因過表達能抑制胰腺癌SW1990細胞的增殖、侵襲及非錨定依賴性生長能力。第三部分上調(diào)EBP50基因?qū)W1990細胞作用的機制研究目的：探究EBP50基因過表達后對人胰腺癌細胞系SW1990周期和凋亡的影響及其具體機制。方法：細胞周期法檢測.EBP50-SW1990細胞、HA-SW1990細胞及SW1990細胞的細胞周期變化；Hochest 33258檢測法觀察凋亡影響；Western blot法檢測三種細胞細胞中Bcl-2、β-catenin及E-cadherin的表達變化。結(jié)果：與SW1990和HA-SW1990兩種細胞比較,EBP50-SW1990細胞的G1/G0期細胞比例增加明顯(62.7%±1.03%),S期細胞比例明顯減少(15.3%±1.33%),具有統(tǒng)計學差異(P,0.05); Hochest 33258檢測細胞凋亡結(jié)果表明EBP50-SW1990凋亡的細胞明顯多于兩組對照組細胞(P0.05); EBP50-SW1990細胞的Bcl-2蛋白表達明顯較SW1990細胞低；EBP50-SW1990細胞的E-cadherin蛋白表達水平較SW1990細胞及HA-SW1990細胞顯著增高,而β-catenin蛋白水平較兩者顯著增高(P0.01)。結(jié)論：BP50基因過表達后,阻滯了G1-S期進程,誘導其凋亡；上調(diào)EBP50基因?qū)σ认侔㏒W1990細胞的影響是通過抑制Bcl-2的表達、降低β-catenin和增加E-cadherin的水平來介導的,EBP50發(fā)揮一個抑癌基因作用。
[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.

【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.9

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