Detection Of CD4⁺CD25^highCD127^low Regulator T Cells In Periperal Blood From The Patients With Cervical Cancer And Its Significance

Cervical cancer is the most common gynecologic malignancy which occurs at the junction of cervix squamous cell and cervical columnar epithelial cells.The incidence of cervical cancer is No.2 in malignant tumors and the prevalence rate among female cancer is No.1. Although the cause of cervical cancer has not yet fully understood, a large number of epidemiological data and research show that the incidence of bacterial and viral infections, birth injuries, substances causing to cancer, sexual disorders and a variety of geographical and environmental factors cause the disease, and occurrence of cervical cancer, development and cancer are closely related to immune function.Regulatory T cells are a group of inhibitory effect of the negative control cells. According to their different cell surface markers, Treg cells can be divided into CD4+Treg cells, CD8+Treg cells, NKT Treg cells and double-negative Treg cells, and CD4+Treg cells has been the immunology research focus. Some studies found that Treg cells played a very important protective role in maintaining the body's own immune stability, prevention of autoimmune diseases and organ transplant rejection, but the Treg cells in cancer patients was able to weaken the body's anti-tumor immune response and reduced tumor immune treatment. So some scholars have vividly compared Treg cells to tumor escape immune system surveillance of the "invisibility cloak."Although both CD25 and Foxp3 are the molecular markers in CD4+Treg cells, but the effect of T cells also expressed CD25, and in vitro studies have shown that only high expression of CD25 in CD4+Treg cells have immune regulation.Because of intro-celluar specifically expression of Foxp3, Treg cells need to be fixed and perforated when detected, but it can not be used to live Treg cells and for subsequent functional studies. CD127 specifically low expressed in CD4+Treg cells, and it has a negative correlation with Foxp3 expression in tumor tissue and peripheral blood without destruction of the cell membrane. So we used CD4+CD25highCD127lowTreg cells as a standard screening not only ensure the integrity of Treg cells but also avoid the effect of T cells and other non-Treg cell interference.Although studies have shown that large number of Treg cells were found in cervical cancer tissue and draining lymph nodes, and some studies reports increased expression of the Treg cells in peripheral blood of gastric cancer, esophageal cancer and prostate cancer patients, the expression of CD4+CD25highCD127lowTreg Treg cells in peripheral blood of the cervical cancer patient as well as relations with the clinical stage have rarely reported.The study detected CD4+CD25highCD127lowTreg cells in different clinical stages of cervical cancer patients with CD4+T lymphocytes, CD8+T lymphocytes and NK cells by flow cytometry, and explore clinical significance between CD4+CD25highCD127lowTreg cells and the development ofcervical.1 Objector1.1 Experimental group:Experimental group:All patients come from the Third Affiliated Hospital of Zhengzhou University and confirmed by biopsy. There are 19 cases of squamous cell carcinoma and 2 cases of mucinous adenocarcinoma, aged 34 to 65 years, mean (47±9) years of age. Exclude heart, brain and liver, and kidney disease. All patients have not done anti-tumor therapy nearly a month and have not used immunostimulants nearly 3 months. All patients staged with International Union Obstetrics and Gynecology (FIGO,2000) revised clinical staging,10 cases were stageⅠ,7 cases were stageⅠand 4 cases were stageⅢ.1.2 Healthy control group:Physical examination in our hospital were randomly selected 11 healthy women, aged 30 to 68 years. There is no statistical significance about age between healthy control group and experimental group.2 Methods2.1 Specimen collection:Collecteing 2ml peripheral venous blood use EDTA anticoagulant when fasting in the morning. Conserve the blood in 20-25℃room temperature and stained by the within 6h. White blood cell count before staining, (3-10)×109/L is appropriate range. When the white blood cell count increased,we diluted blood with phosphate buffered saline to the appropriate range.2.2 Specimen processing:Low-speed vortexing to mix whole blood and mark the test tube. Then added 100ul whole blood and some immuno-fluorescence antibodies according to reagent instructions and low-speed vortex mixing. Dark reaction at room temperature 20min, then added 1ml hemolytic agent. Low-speed vortex mixing at room temperature before dark reaction for 10min.300g centrifugation for 5min, supernatant discard, then added 1m sheath fluid 1.300g centrifugation for 5min and supernatant discard. Low-speed vortex mixing, and finally adding 500ul sheath fluid, then immediately detected on the machine.2.3 Flow cytometry:According to the operating manualof flow cytometry, CD4+CD25highCD127lowTreg cells, CD4+T lymphocytes, CD8+T lymphocytes and NK cells in the peripheral blood were detected by flow cytometry.3 Statistical analysisSPSS 16.0 was used for studies. The data was calculated using mean±SD. The mean of each group was compared by t test treatments. The data was calculated using Pearson correlation analysis.The test level is a=0.051 CD4+CD25highCD127lowTreg cells determination:Compared with those of healthy group,the frequency of CD4+CD25highCD127low Treg in the peripheral blood of cancer patients showed a significant increase (P<0.05).2 The relationship between CD4+CD25highCD127lowTreg and the level of tumor clinical stage:Compared with the healthy group and patients of stageⅠ,the frequency of CD4+CD25highCD127low Treg in patients of stage(Ⅱ+Ⅲ) was significantly higher(P<0.05,both).3 CD4+CD25highCD127lowTreg cells in different histological types of cervical carcinoma patients:Compared with those of healthy group, the frequency of CD4+CD25highCD127lowTreg in the peripheral blood of patients with cervical adenocarcinoma and squamous cell carcinoma is significantly higher (P<0.05). There is no significantly difference about the the frequency of CD4+CD25highCD127lowTreg between adenocarcinoma and squamous cell carcinoma patients. (P>0.05).4 CD4+CD25highCD127lowTreg cells in peripheral blood of patients with lymph node metastasis:Compared with those of healthy group, the frequency of CD4+CD25highCD127lowTreg either in the peripheral blood of patients without lymph node metastasis or lymph node metastasis is significantly higher (P<0.05). There is no significantly difference about the the frequency of CD4+CD25highCD127lowTreg between the two gropes. (P>0.05).5 CD4+CD25highCD127lowTreg cells in peripheral blood of patients with different levels of squamous cell carcinoma:Compared with those of healthy group, the frequency of CD4+CD25highCD127lowTreg either in the peripheral blood of patients with squamous cell carcinoma in situ or in progress is significantly higher (P<0.05). There is no significantly difference about the the frequency of CD4+CD25highCD127lowTreg between the two gropes. (P>0.05).6 CD4+CD25highCD127lowTreg cells in peripheral blood of patients with differentiation of squamous cell carcinoma:Compared with those of healthy group, the frequency of CD4+CD25highCD127lowTreg either in the peripheral blood of patients with middle differentiation squamous cell carcinoma or low differentiation is significantly higher (P<0.05). There is no significantly difference about the the frequency of CD4+CD25highCD127lowTreg between the two gropes. (P>0.05).7 NK cells determination:Compared with those of healthy group,the frequency of NK cells in the peripheral blood of cancer patients showed a significant decrease (P<0.05).8 The relationship between NK cells and the level of tumor clinical stage: Compared with the healthy group,the frequency of NK cells in patients of stage(Ⅱ+Ⅲ) was significantly lower(P<0.05).9 CD4+T, CD8+T cell levels and CD4+T/CD8+T cell ratio of change:There is no significantly difference about the the frequency of CD4+T cells, CD8+T cells between healthy group and gynecologic malignancy patients. (P>0.05).10 The relationship between CD8+T cells and the level of tumor clinical stage: There is no significantly difference about the the frequency of CD8+T cells among healthy group,stage I and stage(Ⅱ+Ⅲ) (P>0.05).11 The relationship between CD4+CD25highCD127lowTreg and the NK cell: There was negative relation between the level of CD4+CD25highCD127lowTreg and that of NK cells(P<0.05). As the expression level of CD4+CD25highCD127lowTreg rise, the expression levels of NK cells will down. 1 Compared with those of healthy group,the frequency of CD4+CD25highCD127lowTreg in the peripheral blood of cervical cancer patients showed a significant increase. CD4+CD25highCD127low regulator T cells may be responsible for immune suppression by inhibiting NK cells2 It is supposed that the changes of CD4+CD25highCD127lowTreg may be helpful for diagnosis and treatment of cervical cancer.