| Esophageal cancer located around the world, which is the world's ninth largest malignant disease, and the incidence of esophageal cancer is different in different countries. Esophageal cancer has a high incidence in China, which accounted for 21.8% of cancer mortality. The average annual deaths of Henan province due to esophagus Cancer is approximately 25,000 of which accounted for about 40% of the total cancer deaths of. Esophageal cancer derives from esophageal epithelium. Squamous cell carcinoma accounting for about 95% of all esophageal cancers in the high-incidencing areas of China, So the study of esophageal squamous cell carcinoma has obviously practical significance. Despite decades of research we believe that the incidence of esophageal cancer is related with such factors as nitrosamine compounds, the lack of trace elements, smoking, reflux disease and others, but its precise mechanism is still unclear, this is the main factors why the outcomes are not satisfactory at present. The regulatory mechanism for which the state of esophageal epithelium differentiation state converts to the proliferation state is a significant key to unlock the esophageal epithelium disease, especially in research on the pathogenesis of esophageal cancer, relevant research make it possible to reveal the mechanism of esophageal cancer.SRC family tyrosine kinases (SFKs), including at least nine kinds of highly similar structure of non-receptor tyrosine kinases, namely Src, Fyn, Yes, Lck, Hck, Lyn, Blk, Yrk, Fgr.Structural analysis showed that SFKs are highly similar family members. The structure contains conserved structural units, including the N-Src homology domain 4 (SH4), homology domain 3 (SH3), homology domain 2 (SH2), connected sequence, kinase domain and carboxyl-terminal domain. N-terminal and the SH4 domain contains the myristoylation site in the cell membrane.SH3 domain can bind to proline-rich sequence of amino acids, and Play a key role in the activity of the kinase, intracellular localization, recruitment and integration of kinase substrate. The main role of SH2 domain is binding the structure unit of phosphotyrosine. In short, SH3 and SH2 domain played a pivotal role in regulating kinase activity.Study of SFK has focused on the fibroblasts of birds and mammals, and aspects of human hematopoietic system cells. Recently, studies have shown that the abnormal increase of SFKs protein levels is related to the occurrence of human colon cancer, prostate cancer, non-small cell lung cancer and other tumors of epithelial origin. However, relevant studies focus on the relationship between Src and cancer, other family members involved less. Recent studies have shown that Fyn is also a very important signal molecule in mitosis and cell cycle regulator, which participate in regulating cell growth, proliferation, adhesion and other functions, and plays a significant role in the signal transduction pathway in tumor formation. Theoretically, the mutant of kinase activity and the damage of negative regulation is the possible mechanism of higher level of SFKs in tumor tissue, but SFKs activity mutations in these tumors are rare, so the damage of negative regulation may be the main mechanism. Relevant research may reveal their pathogenesis. What role does the Src family tyrosine kinase Fyn plays in the development of esophageal squamous cell carcinoma? What is the possible negative regulation mechanism in esophageal squamous cell carcinoma? This study explores this issue, trying to find the relationship between the Src family tyrosine kinase and esophageal squamous cell carcinogenesis and reveal the possible mechanism of negative regulation.The thesis is divided into the following three parts:Part I the experimental materials and methods introducedReagent method references "Guide to molecular cloning". The main experimental methods include:immunohistochemistry, immunofluorescence methods, organization immunofluorescence method, Western blot method, adenovirus infection, bacterial transformation, the extraction of plasmid DNA, plasmid transfection, the extraction of tissue and cell protein and so on.Part II the relationship between the esophageal squamous cell carcinoma and the Src family tyrosine kinases Fyn and Tom1L1Objective:To observe the expression of Src family tyrosine kinases Fyn and Tom1L1 in esophageal squamous cell carcinoma, and explore the the relationship between the carcinogenesis and the development of the esophageal squamous cell carcinoma and the Src family tyrosine kinases Fyn and Tom1L1.Methods:1. Observation the expression of Src family tyrosine kinases Fyn and Tom1L1 in esophageal squamous cell carcinoma tissues with the Immunohistochemical method. 2. Observation the expression of Src family tyrosine kinases Fyn and Tom1L1 in esophageal squamous cell carcinoma tissues with the organization immunofluorescence method.3. Observation the expression of Src family tyrosine kinases Fyn and Tom1L1 in esophageal squamous cell carcinoma tissues with the Immunoblotting method.4. Observation the expression of Src family tyrosine kinases Fyn and Tom1L1 in TE1 cell lines of human esophageal squamous cell carcinoma and EPC2-htert cell lines of esophageal epithelial cells with the Immunofluorescence method.Results:1. The expression of Src family tyrosine kinase Fyn is high in esophageal squamous cell carcinoma and low in normal esophageal mucosa.2. The expression of Src family tyrosine kinase Fyn in cell lines TE1 and TE9 of human esophageal squamous cell carcinoma is high and low in cells EPCl-htert and EPC2-htert of esophageal epithelial.3. The expression of Src family tyrosine kinase Fyn is higher in esophageal squamous cell carcinoma tissues than in normal esophageal mucosa, P<0.01, significant difference.4. The expression of Tom1L1 in esophageal squamous cell carcinoma and high in normal esophageal mucosa epithelium.5. The expression of Tom1L1 is low in cell lines TE1 and TE9 of human esophageal squamous cell carcinoma and high in cells EPC1-htert and EPC2-htert of normal esophageal epithelium.6. The expression of Tom1L1 in esophageal squamous cell carcinoma is lower than in the tissue of normal esophageal mucosa, P<0.05, significant difference. Conclusion:1. The expression of Src family tyrosine kinase Fyn is high in esophageal squamous cell carcinoma and low in normal esophageal mucosa, suggesting that it plays an important role in the development process in esophageal squamous cell carcinoma.2. The expression of Tom1L1 in esophageal squamous cell carcinoma and high in normal esophageal mucosa epithelium, suggesting that Tom1L1 is the possible negative regulation mechanism in the carcinogenesis and development of esophageal squamous cell carcinoma.PartⅢTom1L1 negative regulating role of the Src family tyrosine kinase Fyn in the esophageal squamous cell carcinoma and its mechanism.Objective:Explore the negative regulation mechanism of Src family tyrosine kinase Fyn in esophageal squamous cell carcinoma.Methods:1. Transfect and infect Human pcDNA3.1-Tom1L1 plasmid DNA vector and human Tom1L1 adenovirus vector (Ad-Tom1L1) into cell lines TE1 of human esophageal squamous cell carcinoma, and screen better methods.2. Transfect Human pcDNA3.1-Tom1L1 plasmid DNA vector in cell lines TE1 into human esophageal squamous cell carcinoma, and observe their effect to Src family tyrosine kinase Fyn.3. Transfect Tom1L1 plasmid DNA vector into cell lines TE1 of human esophageal squamous cell carcinoma, give different doses of doxycycline, and observe the interaction. Results:1. After transfecting Human pcDNA3.1-Tom1L1 plasmid DNA vector by liposome into TE1 cell line of human esophageal squamous cell carcinoma, expression of Tom1L1 was tested increased by Western blot.2. After infecting People Tom1L1 adenovirus vector (Ad-Tom1L1) into TE1 cell line of human esophageal squamous cell carcinoma, expression of Tom1L1 was tested increased by Western blot.3. Tom1L1 protein levels was higher in pcDNA3.1-Tom1L1 plasmid DNA transfection compared with Ad-Tom1L1 adenovirus infection, tested by Quantity-one software measurement gray and standardized by theβ-actin.4. With the increasing transfection dose of pcDNA3.1-Tom1L1 plasmid DNA, the expression of TE1 cells Tom1L1 protein gradually increased, while Fyn expression is gradually reduced.5. Transfect the same dose of pcDNA3.1-Tom1L1 plasmid DNA, but give different doses of doxycycline, and it shows that with the doxycycline dose increasing, the expression of Fyn protein reduced significantly.Conclusion:1. For Human esophageal squamous cell carcinoma cell lines,the transfection efficiency of TE1 pcDN A3.1-TomlLl plasmid DNA is higher than that of human Tom1L1 adenovirus vector (Ad-Tom1L1) infection.2. Tom1L1 plays a negative regulation role on Fyn in Human esophageal squamous cell carcinoma TE1 cell lines.3. Doxycycline plays a coordinate role in cell lines TE1 Tom1L1 negative regulation of Fyn in esophageal squamous cell carcinoma. Full Text Conclusion:1. Src family tyrosine kinase Fyn is an important signaling molecule in the mechanism of esophageal squamous cell carcigenesis.2. Tom1L1 plays a negative regulation role on the Src family tyrosine kinase Fyn in esophageal squamous cell carcinoma.3. Doxycycline plays a coordinate role in Tom1L1 negative regulation of Fyn in esophageal squamous cell carcinoma.
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