EBP50基因过表达对胰腺癌SW1990细胞作用的机制研究

发布时间：2018-02-01 10:15

本文关键词： EBP50 胰腺癌 SW1990 过表达机制　出处：《武汉大学》2016年博士论文　论文类型：学位论文

【摘要】：胰腺癌是常见的消化系统恶性肿瘤,其死亡率高,预后极差,确诊时多为晚期且伴有淋巴结或远处转移而失去了手术机会,原因是其临床表现隐匿,缺乏有效的早期诊断方法。胰腺癌的发病机制尚未完全明确,原癌基因的激活、抑癌基因的功能失调及信号转导途径的异常都被认为参与了癌症的发生发展。因此探究胰腺癌的发病机制,寻找早期有效的肿瘤标志物对胰腺癌的诊断与预后意义重大。磷酸化蛋白50(EBP50)又被称为钠氢交换子条子因子1(NHERF1)是近年来发现的一个与多种肿瘤相关的抑癌基因,如乳腺癌、肝癌、结直肠癌、胃癌等。本研究通过检测EBP50在胰腺癌组织中的表达并上调EBP50对胰腺癌细胞系SW1990的影响和具体机制,旨在为胰腺癌的发生机制提供新思路,为寻找早期诊断及靶向治疗胰腺癌提供新的依据。第一部分免疫组化及RT-qPCR检测EBP50基因表达目的：检测并比较EBP50基因在胰腺癌组织和癌旁组织的表达情况方法：120例胰腺癌组织标本,采用免疫组化法,检测EBP50基因的表达情况；其中20例胰腺癌组织和20例癌旁组织采用RT-qPCR检测EBP50 mRNA的表达水平。结果：免疫组化结果显示：120例胰腺癌组织标本中有45例(37.5%)无EBP50蛋白的表达,另75例(62.5%)表达EBP50,其中38例(31.67%)染色阳性强度为1+,22例(18.33%)阳性强度2+,仅有15例(12.5%)阳性强度3+,而正常组织标本免疫组化结果阳性强度均为3+。RT-qPCR结果显示20例胰腺癌组织标本的mRNA平均水平为0.75+心.11,而对应的癌旁组织的EBP50 mRNA平均水平为1.63土0.19。结论：胰腺癌组织中EBP50表达较正常组织EBP50的表达有所降低；胰腺癌组织EBP50 mRNA水平较癌旁组织EBP50 mRNA水平明显降低；提示EBP50可能参与胰腺癌发生发展。第二部分I-调EBP50基因对SW1990细胞生物学行为影响目的：应用质粒转染技术对EBP50基因进行过表达,研究EBP50基因上调后对人胰腺癌细胞系SW1990的增殖,侵袭和克隆形成能力的影响。方法：人胰腺癌细胞系SW1990细胞接种培养于含10%胎牛血清的DMEM-F12培养基中培养；脂质体介导的质粒转染,G418筛选细胞,Western blot法进行鉴定。CCK-8法、软质琼胶克隆形成实验及Transwell小室法检测细胞增殖、非锚定依赖性生长能力及侵袭能力。结果：成功构建稳定表达的EBP50-SW1990细胞和HA-SW1990细胞；CCK8显示EBP50-SW1990细胞增殖能力明显低于HA-SW1990细胞、SW1990细胞,具有统计学差异(P0.05)；克隆形成实验提示EBP50-SW1990细胞的非锚定依赖性生长能力明显低于两对照组,具有统计学差异(P0.05); Transwell结果表明EBP50-SW1990细胞的侵袭能力较两对照组细胞明显减弱(P0.05)。结论：EBP50基因过表达能抑制胰腺癌SW1990细胞的增殖、侵袭及非锚定依赖性生长能力。第三部分上调EBP50基因对SW1990细胞作用的机制研究目的：探究EBP50基因过表达后对人胰腺癌细胞系SW1990周期和凋亡的影响及其具体机制。方法：细胞周期法检测.EBP50-SW1990细胞、HA-SW1990细胞及SW1990细胞的细胞周期变化；Hochest 33258检测法观察凋亡影响；Western blot法检测三种细胞细胞中Bcl-2、β-catenin及E-cadherin的表达变化。结果：与SW1990和HA-SW1990两种细胞比较,EBP50-SW1990细胞的G1/G0期细胞比例增加明显(62.7%±1.03%),S期细胞比例明显减少(15.3%±1.33%),具有统计学差异(P,0.05); Hochest 33258检测细胞凋亡结果表明EBP50-SW1990凋亡的细胞明显多于两组对照组细胞(P0.05); EBP50-SW1990细胞的Bcl-2蛋白表达明显较SW1990细胞低；EBP50-SW1990细胞的E-cadherin蛋白表达水平较SW1990细胞及HA-SW1990细胞显著增高,而β-catenin蛋白水平较两者显著增高(P0.01)。结论：BP50基因过表达后,阻滞了G1-S期进程,诱导其凋亡；上调EBP50基因对胰腺癌SW1990细胞的影响是通过抑制Bcl-2的表达、降低β-catenin和增加E-cadherin的水平来介导的,EBP50发挥一个抑癌基因作用。
[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.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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