To Detect Peripheral Blood Regulatory T Cells Of CD4~+ CD25~+ FOXP3 In Oral Cancer Patients Before And After Operation And To Show Their Clinical Significance

Objective: Oral cancer is one of the most common malignant tumor, which ranked sixth in malignant tumors in human body(lower than lung, stomach, breast, colorectal and cervical cancer), in recent years, the worldwide incidence of oral cancer has increased year by year, the number of cases emerging each year more than 500 thousand. China is most populous country, has also been divided into areas of high incidence of oral cancer, like tongue cancer, gum cancer, buccal cancer and so, its causes have not yet entirely clear, may be caused by a variety of comprehensive factors, such as mental factors, environmental factors and immune factors, etc. At present, the immunological factors that has been the close attention of many scholars. The study confirmed that the immune function in both early and late oral cancer patients are likely decreased than normal population immune function decrease, and more significant decline in late cases. Therefore, the body's immune function in cancer occurrence and development of the process has some reference value. Recent studies have found that a class of immune function of T cells , called CD4~+ CD25~+ regulatory T cells(regulatory T cells,Tregs), which plays an important role in organisms antineoplastic immune and it can lower the body's immune response system to reduce the host antineoplastic immune response, so it has became a hot spot in recent years. There studies have shown that FOXP3 mainly express in the activated CD25~+CD4~+ T lymphocyte surface, which plays a crucial role in CD25~+CD4~+ Tregs'development and function. This study was designed to accurately detect oral cancer patients before and after operation in peripheral blood T lymphocytes and expression of the subtypes of CD4~+CD25~+ Treg expression by flow cytometry techniques, mainly through the analysis of oral cancer patients'expression level of CD4 ~+ CD25~+, CD4~+CD25~+ FOXP3 to cellular immunity in patients with oral cancer relationship with preoperative and postoperative oral cancer patients of their immune status and clinical significance, to provide a theoretical basis for oral cancer immunotherapy.Methods: Take fresh venous blood 100μl eight copies, were respectively added 10μl FITC labeled CD3 monoclonal antibodies, 10μl FITC labeled CD4 monoclonal antibodies, 10μl PE labeled CD8 monoclonal antibody and CD56 monoclonal antibodies, 10μl PE labeled Fas monoclonal antibodies, 10μl FITC labeled CD4 monoclonal antibody and PE labeled Fas monoclonal antibodies, 10μl PE labeled FasL monoclonal antibodies and FITC labeled CD4 monoclonal antibody and 10μl PE labeled FasL monoclonal antibodies, avoid light incubated at room temperature for 1 hour, add red blood cells lysis for 5 minutes, add PBS to wash hemoglobin and in 1mlPBS to suspended again, to detect each T lymphocyte phenotypes accounts for a percentage of total lymphocytes, the percentage of CD4~+FasL~+ and CD4~+Fas~+ T lymphocyte, the proportion of FasL~+ and Fas~+ T lymphocyte, and calculate the ratio of CD4~+/CD8~+cells by flow cytometry. In another 2 copies of 100μl of fresh venous blood, were respectively added 20μl FITC labeled CD4 monoclonal antibodies and APC labeled CD25 monoclonal antibody, avoid light incubated at room temperature for 1 hour, add red blood cells lysis for 5 minutes, add PBS to wash hemoglobin and in 1mlPBS to suspended again, added 10μl PE labeled FOXP3, avoid light incubated at room temperature for 1 hour, to detect the proportion of CD4~+CD25~+, CD4~+CD25~+FOXP3 T lymphocyte.Results:1 The changes in immune parameters before and after surgery of oral cancer patients1.1 The proportion of CD3~+, CD4~+, CD8~+ and CD56~+T lymphocytes in peripheral blood of preoperative oral cancer patients were 56.43±9.44%, 29.54±4.88%, 31.42±10.95%, 21.43±6.03%, the ratio range of CD4~+/CD8~+ is 0.91±0.32; the proportion of CD3~+, CD4~+, CD8~+ and CD56~+T lymphocytes in peripheral blood of healthy control group were 68.81±6.55%, 42.50±7.56%, 24.77±6.25% and 11.78±2.87%, the ratio range of CD4~+/CD8~+ is 1.73±0.56. 1.2 To collect again the peripheral blood a week after surgery in oral cancer patients and to detect the proportion of CD3~+, CD4~+, CD8~+ and CD56~+T lymphocytes in peripheral blood, the results were 67.65±2.64%, 41.05±4.92, 25.06±3.38% and 15.81±2.31%, the ratio range of CD4~+/CD8~+ is 1.59±0.18. The expression of CD3 in postoperative patients is obviously higher than preoperative, the result was statistically significant(P﹤0.05), compared with healthy control group has no statistically significant(P>0.05), we can think that the the difference in expression of CD3 compared with the control group was not significant; the expression of CD4 in postoperative patients is obviously higher than preoperative, the result was statistically significant(P﹤0.05), but was not statistically significant compared with healthy control group(P>0.05); the expression of CD8 in postoperative patients is lower than preoperative, the result was statistically significant(P﹤0.05), compared with healthy control group has no statistically significant(P>0.05); the expression of CD56 in postoperative patients is lower than preoperative, the result was statistically significant(P﹤0.05), also has no statistically significant compared with healthy control group(P>0.05).2 The changes in Fas and FasL from peripheral blood before and after surgery of oral cancer patientsThe proportion of CD4~+FasL~+, CD4~+Fas~+, FasL~+ and Fas~+T lymphocytes in oral cancer in preoperative peripheral blood were 22.65±7.02%, 29.22±5.19%, 0.60±0.24% and 36.05±6.28%, after operation they were 28.80±7.53%, 24.58±7.25%, 0.92±0.34% and 37.93±5.65%, the expression of CD4~+FasL~+, FasL~+ and Fas~+ in postoperative patients is obviously higher than preoperative(P﹤0.05); 34.95±6.40%,65.34±3.43% CD4~+Fas~+ is obviously lowher than preoperativ(P﹤0.05).3 The changes in CD4~+CD25~+ and CD4~+CD25~+FOXP3 T lymphocytes from peripheral blood before and after surgery of oral cancer patients3.1 The proportion of CD4~+CD25~+ and CD4~+CD25~+FOXP3T lymphocytes in peripheral blood of preoperative oral cancer patients were 30.93±3.55% and 65.34±3.43%. The expression of CD4~+CD25~+ and CD4~+CD25~+FOXP3T lymphocytes in postoperative patients is obviously lower than preoperative, the result was statistically significant(P﹤0.05), but is higher than healthy control group(P﹤0.05).3.2 The relationship between different clinical factors and the expression of CD4~+CD25~+and CD4~+CD25~+FOXP33.2.1 The expression of CD4~+CD25~+and CD4~+CD25~+FOXP3 in oral cancer patients with peripheral blood is higher than normal control group(P﹤0.05) , which expression level in periodⅢ~+Ⅳpatients is obviously higher than periodⅠ~+Ⅱ(P﹤0.05); the expression of CD4~+CD25~+ and CD4~+CD25~+FOXP3 in oral cancer patients with lymph node metastasis is higher than the patients without lymph node metastasis(P﹤0.05).3.2.2 There was no correlation between The factors of age, gender and smoking have no correlation with the expression of CD4~+CD25~+ and CD4~+CD25~+FOXP3 in oral cancer patients.Conclusion:1 Compared with healthy people, the expression level of helper T lymphocy- tes in oral cancer patients is reduced and the inhibitory T lymphocyte is increased, the body's immune function all have reduce in different degrees, excision the tumor by surgery, can partly remove the inhibition of the immune function. So, the operation for oral cancer patients is effective.2 For oral cancer patients, the expression level of Fas and FasL in peripheral blood of oral cancer patients can be seen as a immunological indicator to evaluate the immune function in the oral cancer patients'treatment.3 For oral cancer patients, the expression level of CD4~+CD25~+ and CD4~+CD25~+FOXP3 in peripheral blood of oral cancer patients may be associated with tumor's periods and lymph node metastasis.4 One week after operation, the expression level of CD4~+CD25~+FOXP3 in peripheral blood of oral cancer patients is reduced than preoperative, to show that the degree of immune suppression is reduced without tumor and immune function may restore somewhat.5 The expression level of CD4~+CD25~+ and CD4~+CD25~+FOXP3 in peripheral blood can be seen as a clinical monitoring indicators to oral cancer patients.