The Primary Study Of Applying Protein Chip Identifying Different Inflammation Cytokines Of Human Tongue Squamous Cancer Cell Lines And The Expression Of MIP-3α In Human Tongue Squamous Cell Carcinoma Tissue

[Background&Objective]Squamous cell carcinoma of the tongue (TSCC) is one of the most common cancer, accounting for approximately25%-40%of the oral cavity squamous cell carcinoma (OSCC). In recent decades, although tongue squamous cell carcinoma treatment in clinic is very common, the tongue squamous cell carcinoma survival rate increase is not obvious, seeing from that the operation is given priority to, auxiliary preoperative and postoperative radiotherapy or chemotherapy. Surgical trauma results in an oral dysfunction for patients. Radiation and chemotherapy are easy to produce more complications, and the prognosis of OSCC patients is poorer.5-year overall survival rates are still low, hovering at around50%. For patients, tongue squamous cell carcinoma that often results in different kinds of disorders, such as speeching, swallowing disorders and facial deformity, which seriously impact patients’ quality of life. TSCC is one of the major diseases which endanger seriously the human life and health in the world. Therefore, improving tongue squamous carcinoma treatment effect, which is one of the most difficult problems in the world urgently needs human continuous research and seek for the safer and more effective treatment method.However, the recurrence and the metastasis of tumors have complicated processes of multiple factors, by the characteristics of tumor cells, tumor microenvironment and tumor cells and together with the surrounding environment influencing each other. Normal cells and their surrounding tissue environment have a kind of dynamic balance, the combined action can regulate cell activity, decide to cell proliferation, differentiation, apoptosis and secretion and expression of cell surface related molecules. But the process of tumor mutations is a vicious cycle of continuously breaking the balance. The tumor microenvironment is a complicated set of system, consisting of the tumor cells itself, mesenchymal cells, capillaries, tissue fluid, and a small amount of infiltrating cells. Interstitial cells and tumor cells can have the mutual induction and interaction, which produces a lot of inflammatory cytokine, growth cytokines, cell chemokines and substrate degradation enzyme, which is helpful for tumor proliferation, metastasis, invasion and anti-apoptosis.As early as in1979, Lord put forward the concept of the tumor microenvironment, which refers to the internal environment of the tumor generating process. In the tumor microenvironment, immune inflammatory reaction, hypoxia and acidosis, interstitial high-pressure formation, generating a large number of growth factors and proteolytic enzymes and so on constitute the biological characteristics of microenvironment. These characteristics have important influences on tumor proliferation and invasion, migration, adhesion, and the formation of new-generated blood vessels and lymphatic vessels, and they are important causes of continuous malignant transformation and metastasis in tumor.Not hard to find, many aspects of research on tumor microenvironment are saturated with the relationship between tumor and inflammation. The relationship between tumor and inflammation has become a new direction of cancer research. And the research on tumor inflammation microenvironment has become the hotspot of current cancer researches. Some scholars have listed the tumor inflammation microenvironment as the seventh great features of tumor after the self-sufficiency of growth signals, insensitivity of growth inhibitory signal, escape of apoptosis, unlimited replication, persistent angiogenesis ability, and organization of invasion and metastasis.So the targeted treatment of tumor related inflammation can transform and promote tumor inflammatory infiltratior, or prevent the inflammation cell from migrating into tumor foci. They can also adjust promoting tumor microenvironment into inhibiting tumor microenvironment, enhance specific acquired immune response, and inhibit metastatic spread. Although tumor inflammatory microenvironment has numerous targets, it is the first step to pick out the important molecular targets from numerous targets, and it is the key to determine the targets to find out the changes of various inflammatory factor levels in the microenvironment.Inflammatory cytokines associated with cancer have different performances in different types of cancers. As for a particular organization and a specific type of cancer, the certainty of specific inflammatory factors is very important. Traditional immune and histological techniques have restricting factors that it needs numerous required samples, and can’t detect a large number of antigen-antibody indexes and can’t reflect a variety of factors of complex changes in the pathogenic process. This study wants to through a method of high throughput, high efficiency, precise directly screen out inflammatory factors of different changes between tongue squamous cancer cells and normal oral mucosa epithelial cells, in order to further explore different inflammatory factors’influences on tumor biological behavior and provide the premise for the mechanism research, to lay the foundation for finding the ideal treatment targets in the tumor inflammation microenvironment.With smooth implementation of the human genome project (HGP), proteomics that use the performer of life-protein as the research object has become more and more important, and there are the conception and development of fast, efficient, parallel, high-throughput proteomic testing new technology--protein chip technology. Protein chip can analyze changes of thousands of proteins at the same time, to make studying protein function at the whole genome level become possible. Anti-inflammatory factor chip is a kind of functional protein chip, it can integrate cytokines associated with inflammatory into a chip, and it can detect the changes of the content of inflammatory cytokines that may be associated with certain diseases or environmental factors in biological samples.This study screen out and analysis of the expression and the significance of different inflammatory factors between tongue squamous cell cancer cell lines with different invasive ability, and between normal oral mucosa epithelium and lines, through the application of protein chip technology (anti-inflammatory factor chip) And combining with literature and using immunohistochemical method to detect differences in inflammatory cytokines expression in tongue squamous cell carcinoma tissues, and further to explore its clinical significance.The research content is divided into two chapters:Chapter one the primary study of applying the protein chip to identify the different inflammation cytokines of human tongue squamous cell carcinoma cell lines Chapter two the expression of MIP-3a in human tongue squamous cell carcinoma and its related clinical significance.Chapter one The primary study of applying the protein chip to identify the defferent inflammation cytokines of human tongue squamous cell carcinoma cell linesObjective Screening different inflammatory factors is to further explore different inflammation factors’influences on tumor biological behavior and to provide the premise for the mechanism research, to lay the foundation for finding the ideal treatment targets in tumor microenvironment.Methods The method was that culturing outside tongue cancer cells CAL-27, UM-1, Tca-8113and human oral epithelial cells, and after digestion respectively extracting extracellular and intracellular proteins, and then handling these on the glass chips with clean, blocking, and incubation, and then adopting the laser scanner GenePix4000B to scan the chip, and the software Axon GenePix Pro6.0to extra the chip data, and the software AAH-CYT-G5to analyse these expression datas of80obtained kinds of cell inflammatory cytokines. The upregulation cytokine signal value took greater than200, greater than2.0cytokine in ratio as the inclusion criteria, and the down-regulation cytokine signal value took greater than200, less than0.66cytokine in ratio as the inclusion criteria,by Pairwise comparison between tongue squamous cell carcinoma cell lines(UM-1,CAL-27,Tca-8113) and human normal oral mucosa epithelial cells, and between high invasive cell lines(UM-1,CAL-27)and low invasive cell lines(Tca-8113).And then choosing the different cytokines, including3kinds of different significant inflammatory cytokine. Using ELISA to re-detect the expression of the protein, the obtained result from the re-detection was been proceeding the statistical analysis, for the futher confirm the antibody array results.Results According to the inclusion criteria, choosing12different kinds of inflammatory cytokines from80kinds of detected cytokines. Compared between human normal oral mucosa epithelial cells and squamous cell carcinoma, IL-1(3,IL-6, IL-8, TNF-a in intracellular sample expressed higher, and MCP-4expressed lower, Osteoprotegerin in extracellular sample expressed higher; Compared with high and low invasive cell lines, RANTES, IL-6, IL-8, TNF-a in intracellular sample expressed higher, GFBP-4, TGF-β3expressed lower, and IP-10, MIP-1β, MIP-3a in extracellular sample expressed higher. The expression quantity of CAL-27and UM-1by ELISA, and IP-10, MIP-1β, and MIP-3a in extracellular sample all expressed higher, comparing with Tca8113. There was a significant difference in statistics (P<0.05). The result was consistent with chip screening.Chapter two the expression of MIP-3a in human tongue squamous cell carcinomaObjective Applying the immunohistochemical method to detect the expression of the different inflammatory cytokines MIP-3α in human tongue squamous cell carcinoma tissues, and further to explore its clinical significance.Methods Collecting46clinico-pathologically characterized TSCC paraffin-embedded tissues that were selected in Guangdong Provincial Stomatological Hospital from January2006to December2012. Immunohistochemistry-the method of SABC was used to investigate the expression of MIP-3α in tongue cancer tissue, and further to explore the relationship beween the expression of MIP-3a and tongue squamous cell carcinoma clinical staging, and pathological grading, and lymph node metastasis.Results1. As for MIP-3a, its positive reaction ratio in tongue cancer tissue was higher than that in nomal tongue tissue. The positive rated ot MIP-3α in the normal tongue tissues and the carcinoma of tongue tissues were respectively30%(6/20) and78%(36/46). And the expression of MIP-3a shows increasing in the tongue cancer tissue. There is a significant difference between the two groups (x2=14.030,P<0.05).2. The positive reaction of the MIP-3α maily located in the intracytoplasm, while some parts located in cell nucleus which perform buffy particle.Tongue squamous cell carcinoma cases of intracytoplasm in epithelial cells and/or cell nucleus of the expression of MIP-3a dyeing obviously, performing yellow changes. The epithelial cells in normal tongue mucosa and/or cell nucleus showed tan yellow, and light dyeing and less on the number of dyeing cells.3. The expression of MIP-3α had correlations with lymph node metastasis and clinical stage (P<0.05), but associated without tongue cancer patients histological differentiation (P>0.05).Conclusion1. The results demonstrate that different cytokines expression in different tongue cancer cell line can be relatively accurately screened via cytokine antibody array technology, and these cytokines may have certain connection with tongue cancer cell biological behaviors.2. The expression of the MIP-3a in OSCC tumor cells is found, and it is higher than that in nomal tongue tissue. There were related to lymph node metastasis and clinical stage (P<0.05), but associated without the pathologic grading of tongue squamous carcinoma of the tongue cancer patients.