| Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck, which the most common is tongue squamous cell carcinoma (TSCC)(about25%-40%of OSCC), in a highly invasive growthway and lymph node metastasis. From the1960s to the present, the treatment of cancer has made significant progress, but the mortality of OSCC remained unchanged, the5-year survival rate is hovering around50%, recurrence and metastasis are also important prognostic factors. We know, however, tumor recurrence and metastasis is a complex process, which depends not only on the intrinsic characteristics of the tumor cells, but also depends on some factors in the tumor microenvironment.As early as1979, Lord proposed the concept of the tumor microenvironment. Refers to the environment within the tumor occurrence, tumor cells themselves, stromal cells, microvascular, tissue fluid and a small amount of infiltrating cells together to form a complex collection system. Mutual induction between stromal cells and cancer cells, is conducive to the proliferation and invasion of the tumor.However, the tumor microenvironment often exist chronic inflammation, which caused by infection, hypoxia, low pH, highly pressure characteristics, etc. Presence of a large number of growth factors, chemokines and a variety of proteolytic enzymes, produced by the immune cells in inflammatory response, are conducive to the proliferation, invasion, adhesion, angiogenesis, radiotherapy and chemotherapy resistanced, which promote the emergence and development of malignant tumors.Easily, many aspects of the study of the tumor microenvironment are permeated with the contact of the tumor and inflammation. The relationship between tumor and inflammation has become a general direction of cancer research. Tumor inflammatory microenvironment of cancer research has become a hot topic.In previous experiments, we used high-throughput and precise protein chip technology (anti-inflammatory factor chip) to screen different inflammatory cytokines between several human tongue carcinoma cells and normal oral mucosal epithelial cells. Compared with the low invasiveness of tumor cells and normal oral mucosal epithelial cells, the high invasive tumor cell lines highly express chemotactic factor macrophage inflammatory protein-1β (MIP-1β), macrophage inflammatory protein-3a (MIP-3a), interferon-inducible protein-10(IP-10). In human tongue squamous cell carcinoma tumor cells, the expression of CCR5, CCR6, CXCR3as the receptor of MIP-1β, MIP-3a, IP-10has not been reported before, the specific mechanisms of MIP-1β, MIP-3a, IP-10in tumor development process is unclear.MIP-1β, MIP-3a, IP-10are chemokine superfamily members, and can lead to the directional movement of cells. Now, the increasing number of studies found that the tumor cells can be autocrine certain chemokines, which can promote the growth, invasion and metastasis of tumor through the proliferation of tumor cells, tumor angiogenesis, the secretion of extracellular matrix proteases or inhibite the body’s anti-tumor immunity. Interactions of chemokines and their corresponding receptors play a wide range of physiological and pathological roles in anti-tumor immunity, tumor development and metastasis. In tongue squamous cell carcinoma, CCR5, CCR6, CXCR3research is relatively rare. The specific mechanism of MIP-1β, MIP-3α, IP-10in tumor occurrence and development process is unclear, In this study, by Western Blot and immunofluorescence staining to detect the expression of CCR5、CCR6、CXCR3in three different tongue squamous cell carcinoma and normal oral mucosal epithelial cells, by CCK-8assay to detect the influence of MIP-1β、MIP-3α、IP-10on proliferation of tongue squamous cell carcinoma CAL-27, by Annexin V/PI double staining flow cytometry assay to detect them influence on tumor cells apoptosis; Transwell and gelatin zymography assay to detect them influence on tumor cell invasiveness. The purpose is to explore the role of MIP-1β, MIP-3α, IP-10in the emergence and development of tongue squamous cell carcinoma.Research is divided into three chapters:Chapter Ⅰ:CCR5, CCR6, CXCR3expression and significance in three different human tongue squamous cells; Chapter Ⅱ:Effect of MIP-1β, MIP-3', IP-10on proliferation and apoptosis of human tongue squamous cell carcinoma cell line CAL-27; Chapter Ⅲ:Impact of MIP-1β, MIP-3a, IP-10on invasion of human tongue squamous cell carcinoma cell line CAL-27.Chapter Ⅰ:CCR5, CCR6, CXCR3expression in three different human tongue squamous cells1. Objective:In order to detect CCR5, CCR6, CXCR3protein expression and positioning in three different human tongue squamous cell carcinoma cells UM-1, CAL-27, Tca-8113, and to lay the foundation of study of MIP-1β, MIP-3a, IP-10on the biological behavior of tumor cells.2. Method:Take the logarithmic phase of growth of the cells used in the experiment. We detect CCR5, CCR6, CXCR3expression in three different human tongue squamous cell carcinoma cells by Western blot and Immunofluorescence staining.3. Result:Western blot analysis showed that CCR5, CCR6, CXCR3protein band expressed respectively in40,524kD,42,494kD,40,660kD were all visible. Immunofluorescence staining showed that CCR5, CCR6, CXCR3in three tongue squamous cell carcinoma cell membrane and cytoplasm were expressed.Chapter Ⅱ:Effect of MIP-1β, MIP-3a, IP-10on proliferation and apoptosis of human tongue squamous cell carcinoma cell line CAL-271. Objective:To observe the effect of proliferation and apoptosis of MIP-1β, MIP-3a, IP-10on human tongue squamous cell carcinoma cell CAL-27.2. Method:1. Taking the logarithmic phase growth cells, which synchronized by the serum-free medium for24h, we used CCK-8assay to detect the effect of experimental group, MIP-1β, MIP-3a, the IP-10(10ng/mL,20ng/mL,40ng/mL) on proliferation of CAL-27cell,at12h,24h,48h. Negative control with PBS, blank hole zeroing;2. Taking the logarithmic phase growth cells, which synchronized by the serum-free medium for24h, then we added MIP-1β, MIP-3a, IP-10(lOng/mL,20ng/mL,40ng/mL) co-cultured for24h. Annexin V/PI double staining flow cytometry detect the cell apoptosis; PBS group is set to a negative control. 3. Result:(1) Compared to the control group, at12h,24h, different concentrations of MIP-1β could promote proliferation of CAL-27cell (P values were less than0.05); at48h, MIP-1β stimulation concentration of lOng/mL,20ng/mL can promote proliferation of CAL-27cell (P values less than0.05), but the40ng/mL MIP-1β without promoting role (P>0.05). With the prolonged duration of action, refer to MIP-1β stimulation concentration of40ng/mL, its effect on the proliferation was weakening, and it no longer promote cell proliferation at48h; At24h,40ng/mL MIP-1β has a promoting apoptosis effect on the CAL-27cell (P>0.05), while10ng/mL,20ng/mL MIP-1β on CAL-27cell apoptosis have no significant effect (P>0.05).(2) Compared to the control group, at12h,24h,48h, lOng/mL,20ng/mL,40ng/mL MIP-3a can promote proliferation of CAL-27cell (P values less than0.05), but there was no significant between each concentration difference (P values were greater than0.05); At24h, lOng/mL,20ng/mL,40ng/mL MIP-3a have no significant effect on apoptosis of CAL-27cell (P values were greater than0.05).(3) Compared to the control group, at12h, different concentrations of IP-10can promote the proliferation of CAL-27(P<0.05), and the concentration was positively correlated; At24h, different concentrations of IP-10are able to promote CAL-27cell proliferation (P<0.01),but the relationship with concentration was wireless; At48h, only40ng/mL IP-10can promote the proliferation of CAL-27cell(P<0.05), lOng/mL,20ng/mL IP-10have no effect on the proliferation; With the extension of duration,the proliferation rate of CAL-27cells treated with lOng/mL and20ng/mL IP-10has been declining and distinguished from the control group at the48h. After simulating24hours, IP-10has pro-apoptotic role on CAL-27cells,40ng/mL IP-10induce apoptosis more significantly (P<0.05). Chapter Ⅲ:Impact of MIP-1β, MIP-3α, IP-10on invasion of human tongue squamous cell carcinoma cell line CAL-271. Objective:To observe the impact of invision of MIP-1β, MIP-3α, IP-10on human tongue squamous cell carcinoma cell CAL-27.2. Method:1. In this study, using Transwell assay to observe the invasion impact of80ng/mL MIP-1β, MIP-3α, IP-10on human tongue squamous cell carcinoma cell CAL-27. Negative control with PBS;2. Gelatin zymography assay is adopted to verify the invasive of chemokines, which influenced invasion of CAL-27cell, and detect the expression of MMP-2, MMP-9.3. Result:Transwell invasion assay results showed that:CAL-27cell invision rate of80ng/ml MIP-3a group and80ng/ml IP-10group was significantly higher than control group(P<0.05), but80ng/mL MIP-1β group and control group had no significant difference (P>0.05). The gelatin zymography experimental results show that:in cell culture supernatant of80ng/ml MIP-3a, IP-10group, the optical density value of MMP-2, MMP-9were significantly higher than control group(P<0.05);Conclusion:1. CCR5, CCR6, CXCR3Located in the cell membrane and cytoplasm expressed in three different human tongue squamous cell carcinoma cells;2. MIP-1β can promote proliferation of CAL-27cell, but high concentrations of MIP-1β also can promote apoptosis of CAL-27. With the prolonged action of of high concentration of MIP-1β, the weakening effect on the proliferation probably caused by MIP-1β inducing apoptosis; 3. MIP-3a can promote proliferation of CAL-27cell, whereas no significant effect on its apoptosis;4. IP-10not only can promote CAL-27cell proliferation but also can promote apoptosis. With the extension of the duration of action, CAL-27cell proliferation rate in decline, the decline may be caused by gradually assuming IP-10pro-apoptotic role;5. MIP-3a, IP-10can enhance invasiveness of human tongue squamous carcinoma CAL-27cell. |
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