| The part one:The distribution characteristicsof LAP+CD4+regulatory T cells in the peripheral blood of patients with colorectal cancerObjective To detect the distribution of LAP+CD4+regulatory T cells in the peripheral blood of patients with colorectal cancer and observe its relation with the clinic stage of colorectal cancer and pathological typeMethods 40 patients with colorectal cancer and 20 healthy volunteers were selected as the research subjects. Flow cytometry was used to investigate the proportion of LAP+CD4+regulatory T cells in CD4+T cells in the peripheral blood of patients with colorectal cancerand healthy volunteers. Clinical data of the subjects including gender, age, nation, preoperative level of carcino-embryonic antigen, tumor size, clinic stage of colorectal cancer were collected to investigate the relationship between the distribution of LAP+CD+Treg cells in peripheral blood and the clinical data in patients with colorectal cancer.Results The proportion of LAP+CD4+Tregs in patients with colorectal cancer was significantly higher than that of healthy control groups (9.35±2.87 vs 1.52±0.69)%, P< 0.001.The proportion of LAP+CD4+Tregs were greater in group with lymph node metastasis (11.41±2.02)% than group without lymph node metastasis (9.25±2.07)%,P< 0.05 and the proportion of LAP+CD4+Tregs was significantly higher in stage C and D (10.77±1.99)% than stage A and B (8.07±1.09)%. There was no difference in the proportion of LAP+CD4+Treg cells in different sex, age, race, tumor size and tumor differentiation degree (P> 0.05). According to the Pearson correlation analysis, there was significant correlation between the proportion of LAP+CD4+Tregs and the levels of CEA in patients with colorectal cancer.(r=0.543, P<0.05).Conclusion The proportion of LAP+CD+Treg cells in peripheral blood of patients with colorectal cancer was significantly higher than that of healthy controls, suggesting that it may be involved in the development of colorectal cancer. The proportion of LAP+CD+Treg cells was different in lymph node metastasis or not and different tumor stages, but it was not considered to be related to the severity of the disease.The part two:The extracorporal study of LAP+CD4+Regulatory T cells in peripheral blood of colorectal carcinoma patientsObjective Selecting LAP+CD4+Treg cells from peripheral blood of patients with colorectal cancer and detecting cytokine profile of LAP+CD4+Treg cells to investigate its role in the pathogenesis of colorectal cancer. At the same time,to analyze the effect of IL-8 on LAP-CD4+T cells in vitro environment.Methods Peripheral blood from 32 patients with colorectal carcinoma receiving surgical treatment in department of colorectal surgery of first affiliated hospital of Guangxi medical university from November 10,2014 to February 1,2015 was collected to isolate LAP+CD4+Treg and LAP-CD4+T by Manetic cell sorting system (MACS). The purity of LAP+CD4+Treg cells was analyzed by Flow cytometry (FCM) and the survival rate of these cells was measured by trypan blue staining. Luminex liquichip technology was applied to detect expression of IL-2, IL-4, IL-10, IL-17 and INF-y. The concentration of TGF-β was detected by ELISA. Flow cytometry was used, after adding IL-8 to LAP-CD4+T at culture 72h, to investigate the propotion of LAP+CD4+regulatory T cells in LAP-CD4+T cellsResults The purity of LAP+CD4+Treg cells and LAP-CD4+T cells obtained by FCM was (95.14±1.67)%, (95.32±2.41)% respectively. The motility rate of LAP+CD4+Treg cells and LAP-CD4+T cells obtained by trypan blue staining was (92.54±2.31)%, (93.14±2.35)% respectively.. The result of Liquichip and ELISA showed that LAP+CD4+Treg cells secreted more TGF-β and IL-10 than LAP-CD4+T cells (110.65±12.45 vs.72.86±10.87,t=9.13, P=0.000; 107.03±17.69 vs.64.19±14.15, t=7.76, P=0.000).The proportion of LAP+CD4+Tregs induced by IL-8 (1.51±0.26)% was significantly higher than that induced by anti-CD3,anti-CD28 and IL-2 (0.48±0.54)%(P<0.001).Conclusion Manetic cell sorting system (MACS), a way of keeping highly live after work, is an economical and useful way to isolate LAP+CD4+Treg cells. LAP+CD4+Treg cells may play an important role in tumor immune escape by secreting higher immunosuppressive factor IL-10 and TGF-p than that of LAP-CD4+T cells.LAP-CD4+T cells could be induced into LAP+CD4+Treg cells highly after adding IL-8, which may be a new target for immunotherapy in the future. |
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