| Cholangiocarcinoma is the most common kind of biliary malignant tumor. Its morbidity has increased year by year. Because of its special anatomical position, most patients have been in the late stage when found tumor, and lose the radical surgery opportunity. The resection rate and5years survival rate are far from satisfaction. Cholangiocarcinoma is not sensitive to chemotherapy and radiotherapy, some other non-operative treatment is also difficult to get ideal effect. There is still lack of the early diagnosis method and comprehensive treatment of cholangiocarcinoma. Deepening our understanding of the tumor’s biology characteristics, exploring the development intrinsic mechanism, and seeking for effective. early diagnosis and treatment methods to improve the curative effect of cholangiocarcinoma can improve the prognosis of patients with cholangiocarcinoma.T lymphoma invasion and metastasis inducing factor1is considered to be a proto-oncogene, and closely related to the tumor’s transfer ability. Tiam1can regulate the cytoskeleton, induce ruffling, and adjust the affinity of adhesion molecules, to promote cancer cell’s proliferation and migration function. Tiaml is highly expressed in lymphoma, pancreatic cancer, breast cancer, bladder cancer and lung cancer and other kinds of tumors, and its expression level is closely related to the tumor’s invasion and metastasis ability.The most important biology behavior of cholangiocarcinoma is invasion of peripheral tissue, lymph node and distant metastasis. Although the expression of Tiaml gene in various tumors and their relationship have been reported by some scholars, Tiaml gene expression in cholangiocarcinoma and its function have not yet been reported. Therefore, this research combines clinical data analysis, RNA interference silencing genes, and in vivo and vitro experiment, observes the Tiam1gene’s role in cholangiocarcinoma through patients, molecular, cellular and animals, and discusses its mechanism, to provide a new gene target for the diagnosis and treatment of cholangiocarcinoma. Outline:Chapter1:to observe the expression differences of Tiam1gene in cholangiocarcinoma tissue and normal tissue of the bile duct, then to discuss the relationship of expression level with tumor differentiation, invasion and metastasis ability.Chapter2:to construct the lentiviral vector which targets at interfering Tiam1gene expression, then to verify the interference effect for the next function experiment;Chapter3:to use the lentiviral vector which targets at interfering Tiam1gene expression to infect RBE cells, then to observe the invasion and metastasis abilities change.Chapter4:to establish nude mouse planting tumor model of stable interference Tiaml gene, and to observe the nude mouse planting tumor biological behavior change after the silence of Tiam1gene. Chapter1Expression and Its Significance of Tiaml in Human Cholangiocarcinoma Tissue and CellObjective:to observe the expression differences of Tiaml gene in cholangiocarcinoma tissue and normal tissue of the bile duct, to discusse the relationship of expression level and tumor differentiation, invasion and metastasis ability, and to observe the expression of Tiaml gene in cholangiocarcinoma cells.Methods:by using immunohistochemical method to detect Tiam1expression in83cases of cholangiocarcinoma tissue and25cases of normal bile duct tissue, to collect cholangiocarcinoma patients clinical data, to analyze the relationship between Tiaml gene expression and cholangiocarcinoma patients clinical and pathological features, and by using the Real-time PCR to detect Tiaml expression in cholangiocarcinoma cell QBC939and RBE.Results:the positive rate of Tiaml protein expression in cholangiocarcinoma tissue is obviously higher than that in the normal bile duct tissue (P<0.001). Cholangiocarcinoma Tiaml protein expression was correlated with the differentiation degree, TNM stage, lymph node metastasis (P<0.05), while has no significant correlation with gender, age, metastases (P>0.05). Tiaml mRNA has expressions in QBC939and RBE cells, and is higher in RBE cells.Conclusion:(1) Tiaml expression in cholangiocarcinoma tissues were expressed significantly higher, and increased with the rise of malignancy degree of the carcinoma;(2) Tiaml expression in RBE cells is higher, so choose REB cells for next experiment. Chapter2Production, Packaging and Virus Titer Detection of Ientiviral Vector of Tiaml-shRNAObjective:to screen effective Tiaml-siRNA sequence, construct recombinant lentiviral vector of Tiam1-shRNA, and detect the titer of virus then package virus.Methods:design the siRNA sequence according to the Tiam1target gene sequence, construct the restructure shRNA shuttle plasmid, and cotransfect293T cell to package virus; assess transfection efficiency by observing the expression of reporter gene after the RBE cell infection by Tiaml-shRNA lentiviral vector; evaluate interference efficiency by detecting the expression of Tiam1using Real-time PCR; construct satisfactory lentiviral vector and packaging.Results:the recombinant plasmid DNA sequencing results show that it is the same sequence as the one of the expected DNA, so the reorganization succeeds. The transfection efficiency rate after the RBE cell infection by Tiaml-shRNA lentiviral vector is above80%, and the interference efficiency rate by detecting the expression of Tiam1using Real-time PCR is more than90%. Tiam1-shRNA lentiviral vector construction succeeds, in which the titer of virus detected was1x109TU/mL.Conclusion:Tiaml-shRNA lentiviral vector was constructed successfuly, which can effectively transfect RBE cells, and effectively down-regulate RBE cell Tiam1gene expression. Chapter3Effects of Tiaml Gene Expression Inhibition by RNAi on Cholangiocarcinoma Cell Biological CharacteristicsObjective:to study the effects of Tiam1targeted inhibition on cholangiocarcinoma cell proliferation and migration ability.Methods:RBE cells are divided into three groups. The experimental group and the control group were transfected by Tiaml-shRNA lentiviral vector and shRNA negative control lentiviral vector, and nothing is done with blank group. Detect the Tiam1gene expression by Real-time PCR. By using Cell cycle experiment and MTT experiment detect cell proliferation activity, and by using transwell experiment detect cell migration vitality.Results:The Real-time PCR detection showed that Tiam1expression level in experimental group was significantly lower than that in control group and blank group (p<0.05), which indicated that Tiaml-shRNA had silenced Tiaml gene effectively. Cell cycle experiment showed that S phase proportion of experimental group was significantly lower compared with control and blank group (p<0.05), indicating that cell proliferation was inhibited after down-regulating of Tiam1gene expression. MTT experiment results suggested that the overall growth rate in experimental group was significantly reduced compared with it in control and blank group (p<0.05), indicating that the cancer cell proliferation activity was inhibited by Tiaml gene expression inhibition. Transwell testing results showed that the experimental group transfer rate was significantly lower than those of the control group and the blank group (p<0.05), indicating that targeted inhibition of Tiaml gene expression can significantly inhibit RBE cell migration ability.Conclusion:the silence of Tiaml can inhibit the cholangiocarcinoma cell proliferation and migration activity. Chapter4Effect of RNAi Inhibited Tiaml Expression on Planted Subcutaneous Tumors of Nude MiceObjective:To explore the growth inhibition effects of RNAi inhibited Tiam1expression on planted subcutaneous tumors in human cholangiocarcinoma tumor-bearing nude mice.Methods:the nude mice were divided into3groups:the experimental group, the control group and the blank group, and they were subcutaneous planted with Tiam1-shRNA lentiviral vector infected RBE cells, shRNA negative lentiviral vector infected RBE cells and RBE cells. Establish cholangiocarcinoma nude mouse subcutaneous planting tumor model, and calculate tumor-formation rate. The volumes of planted subcutaneous tumors were measured per3days after tumor-formation and then make tumors growth curves. Mice were euthanized at21days, then weigh the tumor. Detect nude mouse transplantation tumor Tiaml gene expression by Real-time PCR.Results:the nude mouse subcutaneous planting tumor observation was established successfully after5days, and tumor-formation rate is100%. The nude mouse planting tumor growth rate of experimental group was significantly lower than the control group and the blank group (P<0.05); The end point weight was also significantly lower than those of the control group and the blank group (P<0.05). Real-time PCR detection results showed that the experimental group nude mouse transplantation tumor Tiaml gene expression was significantly lower than that in the control group and the blank group (P<0.05).Conclusion:Tiaml-shRNA lentiviral vector can effectively silence Tiaml gene after21days progress by planting in the nude mouse body. After RNA interferring the silenced Tiaml genes it can restrain nude mouse planting tumor growth. |
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