NDRG2基因对胃癌细胞恶性表型的影响

发布时间：2018-05-18 11:36

本文选题：胃癌 + NDRG2　；参考：《郑州大学》2015年硕士论文

【摘要】：胃癌是世界范围内最常见的恶性肿瘤之一,其发病率和死亡率在我国均居第二位,远远高于世界水平。大多数患者在有症状就诊时已进入中晚期,失去了外科手术机会,这主要是由于胃癌的发生发展机制尚不清楚,临床上缺乏有效的早期诊断指标及早期有效的治疗。恶性肿瘤的局部浸润、远处转移、再次复发和多药耐药是胃癌患者晚期死亡的主要原因,因此研究其恶性表型的机制十分有必要,同时对于胃癌的早期诊断及治疗也有一定的指导意义。人类NDRG家族由NDRG1、NDRG2、NDRG3、NDRG4共同组成,各成员之间约57%~65%的氨基酸具有同源性,其中NDRG2是由我国第四军医大学生物化学与分子生物学教研室首先克隆并报道的。众多研究表明,NDRG2在大多数肿瘤中表达下调,在胃癌中也有类似报道,但其对胃癌的细胞学水平影响及其相关分子机制尚不完全清楚,有待进一步深入的研究。目的:NDRG2是一个潜在的肿瘤抑制基因,在胃癌组织中低表达。本课题旨在探讨NDRG2基因对胃癌细胞恶性表型的影响。方法:1.通过Western Blot、Real-Time PCR验证NDRG2在永生化的正常胃上皮细胞GES及胃癌细胞系(MKN-28、MKN-45、AGS、SGC-7901、BGC-823)中的表达水平。2.通过瞬时转染使pc DNA3.1-NDRG2、pc DNA3.1、si RNA-NDRG2、si RNA-negetive进入AGS细胞内,使用RT-PCR及Western Blot验证转染效率;将实验设置为pc DNA3.1-NDRG2(上调表达载体组)、pc DNA3.1(对照组)、si RNA-NDRG2(下调表达载体组)、si RNA-negetive(对照组)四组,并采用MTT及平板克隆实验检测细胞的体外增殖能力、采用流式细胞仪分析细胞的凋亡。3.转染pc DNA3.1-NDRG2、pc DNA3.1、si RNA-NDRG2、si RNA-negetive后通过Western Blot检测NDRG2与Bcl-2之间的关系;通过购买组织芯片运用免疫组织化学技术初步检测NDRG2和Bcl-2在胃癌组织中的表达模式,再次通过收集临床标本及相关病理资料,扩大样本量运用免疫组织化学技术检测NDRG2和Bcl-2在胃癌组织中的表达模式,并利用统计学软件分析NDRG2和Bcl-2与临床病理资料间的关系。结果:1.通过Western Blot、Real-Time PCR发现NDRG2在胃癌细胞中低表达,在AGS细胞中表达量居中,可用于后续构建过表达载体及低表达载体的研究。2.通过Western Blot、RT-PCR发现AGS细胞转染pc DNA3.1-NDRG2、si RNA-NDRG2后与对照组相比NDRG2 m RNA及蛋白水平会分别增高与降低;MTT发现pc DNA3.1-NDRG2组与pc DNA3.1组相比细胞增殖能力明显减弱,si RNA-NDRG2组与si RNA-negetive组相比细胞增殖能力明显增强;平板克隆实验发现pc DNA3.1-NDRG2组与pc DNA3.1组相比细胞克隆形成能力明显减弱,si RNA-NDRG2组与si RNA-negetive组相比细胞克隆形成能力明显增强;流式细胞仪检测细胞凋亡发现pc DNA3.1-NDRG2组与pc DNA3.1组相比细胞凋亡明显增加,si RNA-NDRG2组与si RNA-negetive组相比细胞凋亡明显减少。3.通过Western Blot发现转染pc DNA3.1-NDRG2后Bcl-2明显减少,而转染si RNA-NDRG2后Bcl-2明显增加;利用免疫组织化学技术发现NDRG2在胃癌组织中与胃正常组织相比低表达,而Bcl-2在胃癌组织中与胃正常组织相比高表达,二者并呈负相关,通过统计相关临床病理资料发现NDRG2和Bcl-2在胃癌组织中的表达阳性率与年龄、性别无关,而与肿瘤分化程度、临床分期、淋巴结有无转移相关。结论:NDRG2能够抑制胃癌细胞的增殖、促进胃癌细胞的凋亡,初步发现NDRG2在胃癌中发挥抑癌作用可能与Bcl-2有关,但具体两者之间的机制还有待进一步深入的研究。
[Abstract]:Gastric cancer is one of the most common malignant tumors in the world. Its incidence and mortality rate are second in China, which is far higher than the world level. Most patients have entered the middle and late stages of symptomatic treatment and lost the chance of surgical operation. This is mainly due to the unclear mechanism of the occurrence and development of gastric cancer and the lack of effective early clinical practice. The main cause of late death of gastric cancer patients is local invasion, distant metastasis, recurrence and multidrug resistance, so it is necessary to study the mechanism of malignant phenotype, and it also has certain guiding significance for the early diagnosis and treatment of gastric cancer. The human NDRG family is NDRG1, N DRG2, NDRG3, and NDRG4 are common, and the amino acids of approximately 57%~65% are homologous among members. NDRG2 is first cloned and reported by the Department of Biochemistry and molecular biology at the The Fourth Military Medical University. Many studies have shown that NDRG2 has been downregulated in most tumors and has similar reports in gastric cancer, but it is fine for gastric cancer. NDRG2 is a potential tumor suppressor gene and low expression in gastric cancer tissue. The purpose of this study is to explore the effect of NDRG2 gene on the malignant phenotype of gastric cancer cells. Methods: 1. through Western Blot, Real-Time PCR, NDRG2 is proved to be immortalized. The expression level of the normal gastric epithelial cells GES and gastric cancer cell lines (MKN-28, MKN-45, AGS, SGC-7901, BGC-823).2. through transient transfection makes PC DNA3.1-NDRG2, PC DNA3.1, Si, into the cells. PC DNA3.1 (control group), Si RNA-NDRG2 (down regulated expression vector group) and Si RNA-negetive (control group) were used to detect the proliferation of cells in vitro by MTT and flat clones. The apoptotic.3. was transfected to PC DNA3.1-NDRG2, PC DNA3.1. The relationship between NDRG2 and Bcl-2; using immunohistochemical technique to detect the expression pattern of Bcl-2 in gastric cancer tissue by using immunohistochemical technique, and by collecting the clinical specimens and related pathological data, expanding the sample size and using immunohistochemical technique to detect the expression pattern of NDRG2 and Bcl-2 in gastric cancer tissue, and use the system. Analysis of the relationship between NDRG2 and Bcl-2 and clinicopathological data. Results: 1. through Western Blot and Real-Time PCR, the expression of NDRG2 in gastric cancer cells is low, and the expression of NDRG2 in AGS cells is middle, and the study of.2. through Western Blot can be used for subsequent construction of overexpression vector and low expression vector. Compared with the control group, the levels of NDRG2 m RNA and protein were increased and decreased respectively after DRG2 and Si RNA-NDRG2, and MTT found that the proliferation ability of PC DNA3.1-NDRG2 group was significantly lower than that of PC DNA3.1 group. Compared with the cell clone formation ability, the cell clone formation ability of Si RNA-NDRG2 group was obviously enhanced compared with that of Si RNA-negetive group. The apoptosis of PC DNA3.1-NDRG2 group was obviously increased compared with that of PC DNA3.1 group, and the apoptosis of Si RNA-NDRG2 group was significantly reduced than that of Si RNA-negetive group. Western Blot found that Bcl-2 decreased significantly after transfection of PC DNA3.1-NDRG2, and Bcl-2 increased obviously after transfection of Si RNA-NDRG2, and the expression of NDRG2 in gastric cancer tissues was lower than that of normal gastric tissue, while Bcl-2 was highly expressed in gastric carcinoma tissue compared with normal gastric tissue, and the two were negatively correlated, by statistical correlation. It is found that the positive rate of NDRG2 and Bcl-2 in gastric cancer tissues is not related to age and sex, but is related to the degree of differentiation, clinical stage, and lymph node metastasis. Conclusion: NDRG2 can inhibit the proliferation of gastric cancer cells and promote the apoptosis of gastric cancer cells. It is found that NDRG2 may play a role in inhibiting cancer in gastric cancer and Bcl-2 in gastric cancer. But the mechanism between them should be further studied.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

【共引文献】