【作者】 刘洪义；

【导师】 所剑；

【作者基本信息】 吉林大学 ， 外科学， 2009， 博士

【摘要】 胃癌是消化系统最为常见的恶性肿瘤,恶性程度高。在当前缺乏有效的一级预防措施的情况下,早期诊断尤为困难,造成根治机会少,五年生存率低。研究结果显示,Twist基因的高表达与多种肿瘤的发生、发展及侵袭进程密切相关,然而,它与胃癌发生发展的具体相关关系目前尚未阐述清楚。本研究应用免疫组化法检测了Twist基因在胃癌组织中的表达与临床病理特征之间的关系,并进一步以人胃腺癌SGC7901细胞系为对象,利用RNAi技术沉默Twist基因的表达,观察其对人胃腺癌细胞恶性生物学行为的影响并探讨其可能的作用机制。研究提示:针对Twist基因表达产物的检测,将对胃癌的恶性程度及侵袭性提供一个有意义的判断指标。沉默胃癌细胞中Twist蛋白的表达,能抑制肿瘤细胞的生长,Twist可能成为胃癌基因治疗的潜在靶点。更多还原

【Abstract】 Twist is a new transcription factor which can regulate cell apoptosis. Twist can extent cell survival time by inhibiting cell apoptosis through many pathways. Twist shows a high level expression in may kinds of tumors and is related to tumor progression. At present, the study on the relationship between twist and gastric carcinoma is on the initial stage. The roles and mechanisms of twist involved in gastric carcinoma are still unknown. And also, the mechanisms of twist involved in gastric carcinoma need further research and demonstration.These years, RNA interference are used to be a new gene research technique which can decrease specific gene expression. RNAi are applied in many researches such as gene function, gene knock out and gene regulation. In this study, we applied RNAi technique to silence the target Twist gene, and demonstrated its effects on the biological behavior of gastric carcinoma cells. We focused our study on the effects of Twist gene silence on SGc7901 apoptosis and its related mechanism, and the roles in tumor invasion, migration and their related mechanisms.1 Expression of Twist in gastric carcinoma tissue and its relationship between clinical factorsObjective: To investigate the relationship between the Twist expression and clinical factors in gastric carcinoma, and detected Twist gene expression in gastric carcinoma cell line. Methods: Immunohistochemistry was applied to detect the Twist expression in 127 cases of gastric carcinoma and 19 cases of normal gastric tissue. Results: 3 cases of normal gastric tissue showed twist positive staining. Twist showed positive expression on all four stage gastric carcinoma. There was significance increased expression of Twist in gastric carcinoma compared with normal gastric tissue. In gastric carcinoma, Twist expression increased significantly from high differentiation to low differentiation. Twist showed stronger expression in malignant gastric carcinoma. Twist showed positive expression in SGC7901 cells through RT-PCR and Western Blot. Conclusion: Twist showed high expression in gastric carcinoma tissue and was related with clinical factors. SGC7901 cell could be the cell model for Twist gene silence to investigate the function of Twist.2 Construction and identification of Twist RNAi vectorObjective: to construct Twist RNAi vector for twist silence in gastric carcinoma cell SGC7901 and detect its silence effects. Methods: Determination suitable RNAi sequence according to the GeneBank Twist genen sequence and construct pGensil siRNA Twist vector. Gene sequencing was used to identify the vector. RT-PCR and Western Blot were applied to evaluate the silence effects. Results: The Twist siRNA was constructed correctly through gene sequencing. Twist expression at protein level and mRNA level were significantly decreased by Twist siRNA in SGC7901 cells. Conclusion: Twist siRNA vector was successfully constructed and could significantly decreased Twist expression in SGC7901 cells.3 Effects of Twist silence on cell growth and apoptosis of SGC7901 cells and its relative mechanism.Objective: to detect the Effects of Twist silence on cell growth and apoptosis of SGC7901 cells and its relative mechanism. Methods: Light microscope and electronic microscope were employed to observe the growth pattern and utra-structure of SGC7901 cells. CCK-8 kit was used to describe growth curve. PI staining and FACS were applied to detect the cell cycle. AnnexinV/PI staining, DAPI staining, TUNEL kit were employed to detect apoptosis. Enzyme activity of caspase-3,9 were detected by ELISA. Protein expression of p53, bcl-2 and bax were detected by FACS. Fluo-3/AM staining was used to detect the Ca2+ content in different cells. Results: Twist silence group cells showed smaller phenotype of atypia compared with control groups. There were many death cells in the culture medium. Under the electronic microscope, Twist silence group cells showed smaller cell volume, decreased cell microvillus, increased but shorter synapses, increased vacuoles in cell plasma, and expanded endoplasmic reticulum. Growth curve showed that Twist silence cells growth more slowly than control cells. Cell cycle analysis showed that Twist silence cells got a significantly higher proliferation index compared with control groups. Apoptosis analysis showed that Twist silence group had a significantly higher ratio of apoptosis cells compared with control groups. Enzyme activity of caspase-3, 9 was significantly higher than control groups. Expression of p53 and bax were significantly increased , but bcl-2 significantly decreased in Twist silence cells compared with control groups. Ca2+ content in Twist silence cells was significantly higher than that in control groups. Conclusion: Twist silence could change cell phenotype and ultra-structure. Twist silence could induce apoptosis significantly through the mitochondria mediated apoptosis pathway.4 Effects of Twist silence on cell migration, invasion and their related mechanisms.Objective: To investigate the effects of Twist silence on cell migration, invasion and their related mechanisms. Methods: Colony formation assay, adherence and spreading assay, cell injury migration assay, Boyden chamber assay, mouse renal capsule invasion assay and gelatin zymography were employed to detected the effects of Twist silence on cell migration, invasion and their related mechanisms. Results: Soft-agar colony formation assay showed that Twist silence cells formed more colonies significantly compared with control groups. There was a significantly higher number of adherent and spreading cells in Twist silence group compared with control groups. The relative migration distance of Twist silence cells was significantly smaller than control groups. Boyden chamber assay showed that the invasion depth and invasion cell number in Twist silence group were significantly shorter and smaller than control groups. In the renal capsule invasion assay, invasion depth of Twist silence group was significantly shorter than that of control groups. Gelatin zymography analysis showed that there was a significantly decreased expression of MMP-2 in Twist silence cells compared with control groups. Twist silence also inhibited MMP-2 activation significantly. Conclusion: Twist silence could significantly inhibit cell colony formation, adherence and spreading, migration on ECM, and invasion of SGC7901 cells in vitro and in vivo. Twist silence may inhibit cell invasion though down regulation of MMP-2 expression and activation.In summary, our study suggested that detection of Twist expression in gastric carcinoma could make contribution to clinical gastric carcinoma diagnosis and therapy. Twist RNAi technique could be a new way to deliver gastric carcinoma therapy.更多还原