| Cervical cancer is one of the serious threat to women's health, its morbidity is the second in malignant tumor. below the incidence of breast cancer in women. In recent ears, cervical cancer incidence in a clear upward trend, and the age of onset is becoming younger. At present, cervical cancer etiology is still not completely clear, and the incidence of cervical cancer is related to many factors. Low immunity and chronic cervicitis is thought to be important risk factors of cervical cancer. (?)nutrition, it is believed to be the associated factors of causing cervical cancer. Several studies have reported that the occurrence and development of tumor related with human papilloma virus (HPV) infection. Molecular biology research results show, more than 90% of the cervical cancer patients with human papilloma virus infection. In case of HPV virus persistence, together with other factors undermined the cervical epithelial cells, and the induction of immune response imbalance. At bresent, almost all epidemiological investigation showed that high-risk HPV infection is the main conditions in incidence of cervical cancer and precancerous lesions. In (?)tients with cervical cancer, immune microenvironment changes are pay more and more attention. In the long course of HPV infection, Immune function of the patients (?)d be affected. Therefore, in-depth study of the immune status in patients with (?)ical cancer has a positive meaning.In recent years, people pay a lot of attention to the CD4+ T cells in antitumor mune effect. Under stimulation of antigens, CD4+T cells are induced to differentiate into Thl, Th2, Th9, Th17, Tfh and Treg etc. IL-17-generated Th7 cells is dosely related to many immune inflammation, including various allergic and (?)immune diseases. Treg cell is a kind of immune suppressive T cell subsets, it plays an important role in the maintenance of immune stability, regulation of immune esponses and T cell anergy, and immune suppression makes tumor escape immune response, then causes immune unresponsiveness or immune tolerance to tumor cells. At the same time, patients with in vivo tumor microenvironment can produce a large number of specific chemokines to recruit immune cells, corresponding to tumor tissue, which produce large amounts of cytokines, growth (?)rs, such as lipid signal suppression of anti-tumor immune response, enhanced (?)or cell survival ability, promote tumor growth. Therefore, tumor immunotherapy has become a hotspot in tumor therapy. The relationship between CD4+T cell subsets or immune functional status in patients with cervical cancer and prognosis is still unclear in the present. Analysis of organism internal environment in patients, including immune cells, relevant cytokines or transcription factor changes, and possible mechanisms study for clinical monitoring and immune therapy may offer new thinking.1. Objective(1) To study the CD4+T cell subsets ratio in cervical cancer patients in peripheral blood and tumor tissue, and analyze its related transcription factor and cytokine expression level. At the same time, use Th17 cells as targets to investigate the Th17 cell surface chemokine receptors and tumor tissue in the corresponding ligand expression on Th17 cells, and analyze the relationship between Th17 cell and cervical cancer pathological staging.(2) Through the observation of Th17/Th1, Th17/Treg, Th17/Th1/Th2 balance state, to preliminary understand the immune microenvironment of cervical cancer patients, to study the cervical cancer occurrence and development regularity based on immune imbalance adjustment.(3) To study the relationship between TLR8 and the imbalance of CD4 positive cell subsets during the development of cervical cancer occur, through detecting the expression levels of TLR7, TLR8 and TLR9 in cervical cancer expression, and analyzing the relationship between TLR8 expression and Bcl-2 or VEGF.2. Methods(1) Clinical cases and specimen collection.(2) The flow cytometry was used to detect the Thl/Th2 ratio in peripheral blood and tumor tissue from patients with cervical cancer. And anti-CD3-APC, CD8-Percpcy5.5 and CD45-FITC were used to performe analysis of fluorescent antibody staining. The cells were stimulated by PMA and ionomycin before fluorescent antibody staining was carried out.(3) Detection of Th17cell in cervical cancer. Immunohistochemical analysis was used to detect the expression of IL-17 in tumor tissue from patients with cervical cancer.Th17 cell and Th17 cell surface chemokine CCR5, CXCR4and CCR6 expression were detected by flow cytometry. QRT-PCR was used to detect the mRNA levels of ROR y t and IL-17 from peripheral blood mononuclear cell in patients with cervical cancer.(4) Treg cell detection:Mononuclear cell membrane surface and intracellular fluorescent antibody staining were carried out using CD4+FoxP3+ and CD45+CD4 +FoxP3+ antibody respectively, and analysis by flow cytometry(5) Immunofluorescence assay was used to detect the expression of TLR8 in cervical cancer cell lines (Hela cells), and fluorescence microscopy was used to analyze fluorescent staining positive cells. Real-timePCR was used to detect the mRNA expression levels of TLR7,8,9 and Bcl-2. VEGF.3. Results(1) Thl cell from peripheral blood in patients with cervical cancer was decreased significantly, but Th2 cells had no significant change. The ratio of Th1/Th2 was significantly decreased in peripheral blood from patients with cervical cancer, but the ratio in tumor tissue had no significant change.(2) The results of Th17 assay1) Immunohistochemical analysis of IL-17expression in tumor tissues showed, with cervical cancer progression of disease. IL-17 in local tumor tissue expression levels in an upward trend.2) The expression levels of Th17 cell specific transcription factor ROR y t and the main cytokine IL-17 were significantly higher than those of healthy controls (P< 0.01, P< 0.05), especially in patients with advanced cervical cancer.3) In the peripheral blood and local tumor tissue from patients, Th17 cells ratio were both significantly higher than that from control group.4) Correlation analysis of Thl7and Thl cells in the peripheral blood of patients showed that there was no obvious correlation, whereas in the occurrence and development of tumor stage, Th17 and Thl cells in tumor tissue showed a negative correlation.5) Th17 cell surface chemokine receptor:The expressions of CCR5 and CXCR4 in PBMC and tumor tissues from patients were changed, but not statistically significant, and CCR6 expression was increased significantly. The results of RT-PCR detection of tumor tissue CCR5, CCR6 and CXCR4 corresponding ligands showed, CCR6 ligand CCL20 mRNA expression was significantly increased, while RANTES, CXCL12 expression was not significantly different.(3) The detection results of Treg cells1) The Treg cell ratio in peripheral blood and tumor tissue from patients were higher than those of healthy controls (P< 0.01).2) Thl7/Treg ratio in patients with advanced cervical cancer was significantly lower than that in early cervical cancer (P< 0.01):correlation analysis showed that Th17 negatively correlated with Treg (P< 0.05).(4) The results of TLR8 detection1) Real-timePCR results showed that the Hela cells expressed high TLR8 mRNA transcript levels; fluorescent antibody staining showed the similar results, the expression rate of nearly 100%, the TLR8 protein was localized in the cytoplasm of Hela cells.2) The expression levels of TLR7, TLR8, Bcl-2 and VEGF In cervical carcinoma tissues were significantly increased than those in the control group (P< 0.05). while the expression levels of TLR9 was no significantly difference between patients and healthy controls (P> 0.05). In tumor tissue, the changes of mRNA expression level of Bcl-2, VEGF were positively correlated with TLR8, and no correlation with TLR7.4. Conclusion(1) In peripheral blood of patients with cervical cancer Thl cell levels and Thl/Th2 ratio was significantly reduced, it suggested that the Thl cell mediated antitumor immunity was suppressed,it have close relationship between tumor occurrence and development. No significantly changes in the Th1/Th2 ratio was found In tumor tissue, it prompted that the overall immune state and local immunological microenvironment of cancer tissue are not fully consistent, may be associated with disease development of spatial and temporal differences in the draft or immune cell tissue migration ability.(2) Immunohistochemistry, QRT-PCR, flow cytometry and other detection methods shown that Th17 cells level was increased significantly in peripheral blood and tumor tissue from patients with cervical cancer. At the same time, Thl7cells in tumor tissue was significantly higher than that in peripheral blood, it may due to Thl7cells with high expression of chemokine receptor CCR6, while in the cancer tissue expressed high CCR6 ligand CCL20.(3) Treg cell level was increased In patients with cervical cancer, and in the cervical cancer later period, with the Treg elevated the levels of Th17 decreased, there was a significant negative correlation between them. Th17/Treg cell ratio was also significantly reduced it may be used as a immune index in clinical monitoring or prognosis.(4) Analysis of Toll like receptor on the surface of cervical cancer cells. The Detection of TLR8 expression on tumor cells was found, especially in Hela cell line. TLR7. TLR8 expression was significantly increased in cancer tissues from patients with cervical cancer, and there was a positive correlation between Bcl-2 or VEGF expression level and TLR8 mRNA level in tumor tissues. Tips for the expression of TLR8 and the incidence of cervical cancer development may exist certain relationship. |
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