The Mechanism Study Of Notch1 Participation In Srcasm Down-regulation Activation Src Tyrosine Kinase In Esophageal Squmous Cell Carcinoma

The esophageal cancer is infective in worldwide. Our country has a high incidence of esophageal carcinoma. The mortality of esophageal carcinoma accounts for 21.8% in malignant tumor mortality of our country. The esophageal carcinoma deaths account for 40% of the total cancer deaths in Henan province. On average, the esophageal carcinoma deaths per year in Henan province are about 25,000 people. The esophageal carcinoma arises from the basal layer cell of esophageal mucosa. In the high incidence region of esophageal carcinoma including our country, the mainly pathologic type is squamous cell carcinoma, and it approximately accounts for 95%. Obviously, with esophageal squamous cell carcinoma as the research object has the notable practical significance. Although after decades of research, we think the occurrence of esophageal carcinoma has the relationship of nitrosamines compounds, the lack of trace elements and the genetic predisposition factors, but the pathogenesis is still not clear, and it also causes the treatment effect is not satisfactory. The esophageal lining is protected by a stratified squamous epithelium which is non-keratinized stratified squamous. Esophageal squamous epithelium is self-renewing tissue that maintains a stable integument by carefully regulating epithelial cell growth and terminal differentiation. The mechanisms controlling the switch from a differentiated to a proliferative state in esophageal epithelium are the key point events to understanding the esophageal epithelial disorders, especially the origin of esophageal carcinoma. The relevant research provides the possibility for us to explore the mechanism of esophageal carcinoma, and may also provide the theory basis of esophageal carcinoma gene therapy.The Src family tyrosine kinases (SFKs) is including at least nine closely similarity non-receptor tyrosine kinases. They are respectively named Src, Fyn, Yes, Lck, Hck, Lyn, Blk, Yrk and Fgr. Structurally, SFKs are highly related to one another and contain conserved structural elements among family members. These elements include the N-terminal Src homology4 (SH4) domain, the SH3 domain, the SH2 domain, a linker sequence, the tyrosine kinase domain, and the Cterminal tail. The N-terminal domain, SH4, serves as a site for myristoylation and thus targets SFKs to the cytoplasmic membrane. The SH3 domain binds amino acid sequences rich in proline residues. This domain is critical for Src activity, intracellular localization, and the recruitment and binding of Src substrates. The SH2 domain binds to short motifs containing phosphotyrosines. Together, the SH2 and SH3 domains cooperate in regulating SFK catalytic activity. In the inactive conformation, Src contains a phosphorylated tyrosine at position 527 in humans, which interacts with its own SH2 domain. This positions the SH3 domain to interact with the proline-rich linker domain and keeps Src in a tightly bound inactive state. Upon dephosphorylation of tyrosine 527, intramolecular interactions are destabilized, ultimately resulting in the autophosphorylation of tyrosine 416. This series of events then allows the openingof the molecule and frees the SH2 and SH3 domains to interact with signal molecule.Because of historical reasons, SFKs in carcinoma research focused on birds and mammals fibroblasts and human blood system cells. Recently, elevated SFKs protein expression have been observed in some kinds of human carcinomas, such as colon carcinoma, breast carcinoma, and non-small cell lung cancer. In theory, increased SFKs activity in tumors could be caused by activating mutations and/or impairment of down-regulatory mechanisms. However, the SFKs activation mutations in these tumors are rare, so the impaired downregulation of activated SFKs is perhaps the main mechanism to account for the high level of SFKs in these kinds of tumors. Srcasm is a recently described substrate and negative regulator of Src-family tyrosine kinases. Notch1 is a typeⅠtransmembrane proteins, and it may be involved in the mechanism of Srcasm downregulation Src tyrosine kinases in human skin squamous cell carcinoma.Which role does the activation Src tyrosine kianse (Tyr416) play in the esophageal carcinoma occurrence and development? Whether does the mechanism of Srcasm downregulation Tyr416 exist in the esophageal squamous cell carcinoma. Whether does the Notch1 involve in the mechanism of Srcasm downregulation Src tyrosine kinases in human esophageal squamous cell carcinoma. This study attempts to explore this question, discovered the relationship between the Tyr416 and the esophageal carcinoma, and reveals the possible negative regulatory mechanism of it.This paper is divided into three parts of the content.The first part:introduction of experimental materials and methodsSolutions are prepared according to the formulas in the< Experimental Directory of Medical College of University of Pennsylvania>.Methods:immunohistochemical staining, immunofluorescence, bacterial transformation, plasmid extraction, extraction of protein, plasmid transfection, western blotting. The second part:The relationship study between activation Src tyrosine kinases, Srcasm and Notch1 and the esophageal squamous cell carcinoma.Aim:Observe the expression of Tyr416, Srcasm and Notch1 in esophageal squamous cell carcinoma, and explore the relationship between them.Methods:1. Detect the Tyr416, Srcasm and Notch1 protein expression in esophageal squamous cell carcinoma by immunohistochemical staining.2. Observe the Tyr416, Srcasm and Notch1 protein expression in esophageal squamous cell carcinoma by tissue immunofluorescence.3. Observe the Tyr416, Srcasm and Notch1 protein expression in esophageal squamous cell carcinoma tissue by western-blotting.4. Observe the Tyr416, Srcasm and Notch1 protein expression in human esophageal squamous cell carcinoma TE1 cell line and esophageal epithelial EPC2-htert cell line by immunofluorescence.Results:1. Tyr416 protein level in esophageal squamous cell carcinoma tissue is higher than that in normal esophageal mucosa tissue, the differences are statistically significant.2. Tyr416 level in esophageal squamous cell carcinoma TE1 and TE9 cell lines are higher than that in normal esophageal mucosa epithelium EPC2-htert and EPC1-htert cell lines.3. Srcasm protein level in esophageal squamous cell carcinoma tissue is lower than that in normal esophageal mucosa tissue, the differences are statistically significant.4. Srcasm level in esophageal squamous cell carcinoma TE1 and TE9 cell lines are lower than that in normal esophageal mucosa epithelium EPC1-htert and EPC2-htert cell lines.5. Notch1 protein level in esophageal squamous cell carcinoma tissue is lower than that in normal esophageal mucosa tissue, the differences are statistically significant.6. Notch1 level in esophageal squamous cell carcinoma TE1 and TE9 cell lines are lower than that in normal esophageal mucosa epithelium EPC1-htert and EPC2-htert cell lines.Conclusion:1. The Tyr416 protein level is high in esophageal squamous cell carcinoma, and is low in normal esophageal mucosa tissue. This pattern of Tyr416 suggests that the Tyr416 plays an important role in the development of esophageal squamous cell carcinoma.2. The Srcasm protein level is low in esophageal squamous cell carcinoma, and is high in normal esophageal mucosa tissue. This pattern of Srcasm suggests that it may act as a negative regulator in the development of esophageal squamous cell carcinoma.3. The Notch1 protein level is low in esophageal squamous cell carcinoma, and is high in normal esophageal mucosa tissue. This pattern of Notch1 suggests that it may act as a negative regulator in the development of esophageal squamous cell carcinoma. The Notch1 receptor anchored on the cell membrane, it may be an important upstream molecule in the development of esophageal squamous cell carcinoma. The third part:The mechanism study of Srcasm negatively regulates the activation Src tyrosine kinases in the esophageal squamous cell carcinomaAim:To explore the negative control mechanism of activation Src tyrosine kinases in the esophageal squamous cell carcinoma and the possible signal molecules.Methods:1. Human pcDNA3.1-Srcasm plasmid DNA carrier transfects and the human Srcasm adenovirus vector (Ad-Srcasm) infects the human esophageal squamous cell carcinoma TE1 cell line. According to the results, we can select a better experimental way.2. Human pcDNA3.1-Srcasm plasmid DNA carrier transfects into the esophageal squamous cell carcinoma TE1 cell line. To observe the interaction between the Srcasm and activation Src tyrosine kinases.3. Human pcDNA3.1-Notch1 plasmid DNA carrier transfects into the esophageal squamous cell carcinoma TE1 cell line. To observe the interaction between the Notch1 and Srcasm.Results:1. After transfect the Human pcDNA3.1-Srcasm plasmid DNA carrier into the esophageal squamous cell carcinoma TE1 cell line, the results show that Srcasm protein expression level rises up.2. After infect the Human Srcasm adenovirus (Ad-Srcasm) vector into the esophageal squamous cell carcinoma TE1 cell line, the results show that Srcasm protein expression level rises up.3. A densitometric analysis of western blot data after standardization toβ-actin signals shows that the Srcasm protein level in transfection group is higher than that in infection group.4. In dose-dependent assay, with the gradually increased Srcasm protein expression level, the Tyr416 protein expression gradually decreased.5. After transfect the pcDNA3.1-Notch1 plasmid DNA into the human esophageal squamous cell carcinoma TE1 cell line, Srcasm protein expression level significantly increased.Conclusion:1. The efficiency of transfection by pcDNA3.1-Srcasm plasmid DNA is higher than the infection by Ad-srcasm.2. Srcasm down-regulates the Tyr416 in human esophageal squamous cell carcinoma TE1 cell line.3. Notchl may be the upstream signal molecule of Srcasm in the human esophageal squamous cell carcinoma TE1 cell line.Conclusion of the dissertation:1. The activation Src tyrosine kinase is an important signal molecule in he development of esophageal squamous cell carcinoma.2. Srcasm can down-regulate the activation Src tyrosine kianse in human esophageal squamous cell carcinoma.3. Notch1 may act as a upstream signal molecule of Srcasm down-regulation activation Src tyrosine kinase in human esophageal squamous cell carcinoma.