【作者】 唐琢；

【导师】 邓艳春；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 硕士

【摘要】 研究背景脑胶质瘤是神经系统最常见的肿瘤,可以来源于多种神经外胚层间质细胞,甚至实质细胞,发病率高,且以恶性者多见,传统手术难以根除,常随复发其恶性度不断增高,导致大量患者致残、丧生,危害极大。单纯放射疗法、化学疗法的疗效也远不能令人满意。随着肿瘤是基因疾病概念的深化,新发展起来的基因治疗肿瘤的技术给克服这类疾病带来了希望。NDRG2 (N-myc downstream regulated gene 2)基因是我的导师邓艳春教授于1999年首先发现的新的在正常脑组织和脑胶质瘤中差异表达的基因,在后者低表达或者无表达,而在前者高表达。系列研究表明,该基因还在多种组织的恶性肿瘤中低表达,而在相应的正常组织中高表达。应用重组NDRG2蛋白对肿瘤细胞系进行干预或者在恶性肿瘤细胞系内转染该基因,发现这些细胞系恶性表型明显下降。因此将该基因列为抑癌候选基因,并对其抑癌作用机制进行系列研究,以期开发其临床应用价值。NDRG2功能的发挥可能是通过其蛋白表达产物在不同的信号转导通路中扮演不同的角色而实现的。PI3K-AKT通路在脑胶质瘤的发生和发展中常常异常激活,促进其进一步向恶性演变。国外在研究胰岛素信号通路时,发现NDRG2是AKT的底物,受其磷酸化作用的调节。由于NDRG2在各种组织内广泛表达,提示其可能发挥多重和重要的功能,这些功能的发挥也可能是由不同或者相同的信号转导通路参与的。因此,为了初步探索在脑胶质瘤中NDRG2与蛋白激酶AKT的关系,我们设计了如下实验。实验方法:1.收取临床脑胶质瘤手术切除组织标本24例,按WHO病理分级为Ⅱ、Ⅲ、Ⅳ级各8例,低温速冻保存。分别提取总蛋白,BCA法测定其浓度。培养胶质瘤细胞系U87和U251,应用浓度为10uM的PI3K抑制剂LY294002处理细胞1小时,抑制内源PI3K活性,蛋白提取方法同前。2.采用蛋白半定量的Western-blotting方法测定相同含量总蛋白中的NDRG2蛋白、AKT2蛋白、332位丝氨酸磷酸化的NDRG2蛋白(P-NDRG2-SER332)、348位苏氨酸磷酸化的NDRG2蛋白(P-NDRG2-THR348)、474位丝氨酸磷酸化的AKT2蛋白(P-AKT2-SER474)的含量,统计分析不同病理级别的标本中各种蛋白含量变化及之间的关系。3.在胶质瘤细胞系U87和U251中,仍采用Western-blotting方法测定上述蛋白在应用LY294002抑制PI3K-AKT通路的磷酸化前后的变化情况并进行分析。实验结果1. Western-blot检测到在Ⅱ、Ⅲ、Ⅳ级胶质瘤标本中,随着胶质瘤病理分级的升高,NDRG2蛋白的表达量下降,级别之间的差异具有统计学意义,P<0.05。AKT2蛋白在各个胶质瘤标本中均有表达, 474位丝氨酸磷酸化的AKT2蛋白(P-AKT2-SER474)在58%的胶质瘤标本中表达;332位丝氨酸磷酸化的NDRG2蛋白(P-NDRG2-SER332)和348位苏氨酸磷酸化的NDRG2蛋白(P-NDRG2-THR348)在Ⅱ、Ⅲ、Ⅳ级胶质瘤标本中含量不等,均是在Ⅲ级胶质瘤中表达量最高。2.在多数高表达P-AKT2-SER474的标本中,P-NDRG2-SER332和P-NDRG2-THR348低表达或无表达;反之,在低表达或无表达P-AKT2-SER474的标本中,P-NDRG2-SER332和P-NDRG2-THR348均高表达。3.在U87和U251细胞系中,NDRG2蛋白的表达量在PI3K磷酸化抑制后均有所减低。P-NDRG2-SER332蛋白和P-NDRG2-THR348蛋白的表达量在两个细胞系PI3K磷酸化抑制后也均有所减低。在U87和U251细胞系中,AKT2蛋白均有表达,P-AKT2-SER474蛋白在U87细胞系中表达,抑制后有所降低;而在U251细胞系中无论抑制前后均无表达。结论1. NDRG2蛋白在不同病理级别的胶质瘤中表达有差异,随着病理级别的升高,其含量明显下降(P<0.05),支持其为抑癌基因。2.在胶质瘤中,NDRG2的磷酸化不依赖于AKT2的磷酸化,并且和AKT2蛋白的磷酸化可能存在竞争关系。3. NDRG2是PI3K下游信号分子之一,除了AKT2外,尚有其他PI3K下游激酶分子可以磷酸化NDRG2。更多还原

【Abstract】 Background Human gliomas are the most common primary central nervous system neoplasm. The vast majority of these tumors developing from glial cells and the minority from neuron cells are collectively called gliomas. The patient’s prognosis was still not satisfactory with poor effect of traditional treats,such as surgical operation, radiotherapy and chemotherapy. Gene therapy has been thought of as a promising method to treat gliomas nowadays for higher understanding that tumors are gene related diseases .NDRG2 (N-myc downstream regulated gene 2)is a novel gene found firstly by my teacher professor Deng Yanchun in 1999 and its expression level is decreased apparently in gliomas than in normal tissues. A series of researches have showed that the level of NDRG2 was lower in all kinds of tumors than in their counterpart normal tissues. Interfering some tumor cell lines with recombinant protein of NDRG2 or transfection of tumor cell lines with NDRG2 could inhibited proliferation and progression of these cells. So we regard NDRG2 as a candidate tumor suppressor gene,and are going to study its mechanisem of being downregulated and inhibiting tumors,wishing that it may be a tool to help curing tumors.Extensive expression of NDRG2 in all kinds of tissues implies its important meaning.NDRG2 may function by acting a role in some signal transduction path. PI3K-AKT signaling pathway is often activated in gliomas and encourages tumors’malignant progression.It has reported that NDRG2 is a substrate of AKT and is phosphorylated by AKT in rats’skeletal muscle cells when investigating insulin signaling pathway, so we design this experiment to investigate the expression level of NDRG2 and AKT2 protein and their phosphoralated forms in different pathology grades gliomas and to study the quantitive changes of these proteins through PI3K activity inhibition in both U87 and U251 cell lines in order to get more information available to more research of action mechanism of NDRG2.Methods1.Every 8 samples belonging toⅡ,Ⅲ,Ⅳpathology grade gliomas were collected and total protein was extracted from them respectively.BCA method was applied to test concentration of total protein.At the same time,glioma cell lines U87 and U251 were cultured,and 10uM LY294002, a PI3K signaling pathway inhibitor, was used to inhibit the activity of PI3K for 1 hour.Total protein was extracted from two cell lines before and after the inhibition with the same ways.2. Western blot analysis was applied to detect the expression level of NDRG2 and AKT2 protein and their phosphoralated forms in all of tissue samples. At last,all data was analyzed statistically.3. The level of five kinds of proteins was still detected in U87 and U251 cell lines before and after the inhibition and analyzed.Result1.The level of NDRG2 protein decreased apparently in gliomas with the increasing of grades and the variance was significant statistically ( P<0.05 ) .The AKT2 protein is expressed in all samples ,while the AKT2 phosphorylated at Ser474 site (P-AKT2-SER474) is only expressed in 58% gliomas samples.The levels of the NDRG2 phosphorylated at Ser332 (P-NDRG2-SER332) and the NDRG2 phosphorylated at thr348 (P-NDRG2-THR348) were different in gliomas from every grade and the levels of two kinds of protein in gradeⅢwere both the highest of the three grades.2. Both P-NDRG2-SER332 protein and P-NDRG2-THR348 protein highly expressed in the glioma samples in which P-AKT2-SER474 lowly or didn’t express,while both proteins lowly or didn’t express in the glioma samples in which P-AKT2-SER474 highly expressed.3. After phosphorylation inhibition of PI3K,the level of NDRG2 protein, P-NDRG2-SER332 protein and P-NDRG2-THR348 protein decreased in both U87 cell line and U251 cell line.AKT2 protein expressed in both cell lines whenever before or after PI3K activity inhibition,but P-AKT2-SER474 expressed only in U87 cell line,and its level decreased after inhibion of PI3K activity.Conclusion1.The expression level of NDRG2 in gliomas correlates with the pathology grades of gliomas,which supports NDRG2 may a tumor suppressor gene.2.In gliomas,the phosphorylation of NDRG2 doesn’t depend on the phosphorylation of the AKT2,and the competitive relation between the phosphorylation of NDRG2 and that of AKT2 maybe exist.3.NDRG2 protein is a probable molecule in PI3K signaling pathway,but other molecules in downstream of PI3K must phosphorylate it except for AKT2.更多还原