| ObjectiveAs a threat to human health, esophageal cancer is one of the common malignant tumors, which is involved in accumulation of multi-factor, multi-stage, multi-gene mutation and interactions of complex process in oncogenes, tumor suppressor genes and proteins at the molecular level. In this study, immune cells, immune-regulating molecules and cells were investigated in the tumor microenvironment of esophageal cancer, in order to determine the immunopathological factors in the development of esophageal cancer and make a more effective immunotherapy for esophageal cancer.Methods1. Peripheral blood (PB) samples were obtained from 60 patients with pathologically confirmed esophageal squamous cell carcinoma (ESCC). Mononuclear cells from PB samples were isolated by Ficoll density-gradient centrifugation from studied subjects. The mRNA levels of Th1 type transcription factor T-bet and Th2 type transcription factor GATA-3 were detected by realtime quantitative PCR (RT-qPCR). The expression of Th1 type cytokines IFN-γ, IL-2 and Th2 type cytokines IL-4, IL-10 was measured by enzyme linked immunosorbent assay (ELISA). Investigate the relations between expression of T-bet/GATA-3 and cytokines of Th1/Th2 type and clinicopathologic factors.2. A total of 60 radiotherapy patients with ESCC were randomly assigned equally into the single radiotherapy group and Aidi group (Aidi injection applied by intravenous drip during radiotherapy). The mRNA levels of Th1 type transcription factor T-bet and Th2 type transcription factor GATA-3 were detected by RT-qPCR. The expressions of IFN-γ, IL-2 IL-4 and IL-10 were measured by ELISA. Analyze the differences of expression of transeription factors and cytokines and clinical efficacy between the groups.3. The population of NK.γδT cells in the PBMCs collected from 35 patients with ESCC and 18 healthy volunteers were evaluated by flow cytometric analysis. The expression of ULBP3 was investigated by RT-qPCR and immunohistochemistry.4. The population of Th17 cells in tumor tissues, tumor-free tissues and PB collected from 58 patients with ESCC was evaluated by flow cytometric analysis. The expressions of RORyt and IL-17a mRNA were measured by RT-qPCR.5. The population of CCR6+ Th17 cells in tumor tissues, tumor-free tissues and PB collected from 37 patients with ESCC was evaluated by flow cytometric analysis. The mRNA levels of CCL20 in tumor tissues and matched tumor-free tissues were measured by RT-qPCR and immunohistochemistry. Mononuclear cells isolated from tumor tissues from 3 patients randomly were cultured in Transwell system to investigate the chemotactic activity of CCL20.6. The ratios of Treg and Th17 in PB, tumor-free and tumor tissues of patients with ESCC were detected by flow cytometry, the Treg/Th17 ratio change were analyzed. The expression levels of transcription factors RORyt and FoxP3 mRNA were detected by real time PCR technology. The relation of Treg/Th17 changes and clinical pathological indicators was analyzed. The expression levels of transcription factors RORyt and FoxP3 mRNA were detected by RT-qPCR technology,Results1. Compared with the healthy controls, the expressions of Thl type transcription factor T-bet and cytokines IFN-γ, IL-2 in ESCC patients were significantly lower, while the expressions of Th2 type transcription factor GATA-3 and cytokines IL-4, IL-10 were significantly higher (P<0.01). The radio of T-bet/GATA-3 in patients with ESCC was significantly lower than that of healthy controls (0.36±0.15 vs.14.03±6.02, P<0.01). The levels of IFN-γ(r= 0.709) and IL-2 (r= 0.753) had positive correlation with the radio of T-bet/GATA-3, while IL-4 (r =-0.552) and IL-10 (r=-0.569) had negative correlation with it.2. In the single radiotherapy group, compared with before radiotherapy, the expressions of T-bet and cytokines IFN-γ, IL-2 were significantly lower (P<0.01), the expressions of GATA-3 and cytokines IL-4, IL-10 was significantly higher (P<0.01) after radiotherapy. After therapy in Aidi group, the expressions of T-bet, IFN-γand IL-2 were slightly lower, GATA-3, IL-4 and IL-10 were slightly higher, however, there was not a significance between them. There was statistically significance between the expression of transcription factors and cytokines in the single radiotherapy group and Aidi group. These data suggested that Aidi can inhibit the Th1/Th2 shift.3. The proportion of NK cells as a percentage of total lymphocyte cells in PBMCs of ESCC patients was (22.14±12.43)%, slightly higher than that of healthy controls (19.11±6.99)%(P>0.05). However, the level of NK cells in 0-Ⅱstage was significantly higher than that inⅢ-Ⅳstage (24.35%±12.74% vs.14.63%±5.35%, P<0.01). The level ofγδT cells was lower in ESCC patients than healthy controls (2.80%±1.49% vs. 5.73%±4.75%, P<0.05). However, there was no significant difference between the level ofγδT cells and TNM stage. The expression of ULBP3 was up-regulated in ESCC microenvioment.4. The levels of Th17 cells, RORyt and IL-17a mRNA in tumor tissues, tumor-free tissues and PB were significantly higher than that in healthy controls (tumor> tumor-free> PB> healthy control, P<0.05). The proportion of Th17 cells in PBMCs of ESCC patients had no significant difference with patient sexes, ages, tumor sizes (P>0.05), but was associated with lymph node metastasis factors and clinical stages (P< 0.01).5. The levels of Th17 cells in tumor tissues were significantly higher than that in tumor-free tissues and PB (P<0.05). The mRNA level of CCL20 in tumor tissues were significantly higher than that of tumor-free tissues, moreover, the level of Th17 cells was positively correlated with CCL20 mRNA level. Directly chemotactic activity of CCL20 from tumor-derived Th17 cells was detected by Transwell system in vitro.6. Th ratios of Treg and Th17 cells, and FoxP3 and RORyt mRNA expression in peripheral blood, tumor-free and tumor tissues of patients with ESCC in the progress stage were increased (TIL> NIL> PBMC), the difference between the groups had statistics significance (P<0.05), but the comparison between Treg/Th17 ratio and the ratio of Foxp3/RORγt in TIL, NIL and PBMC were no statistically significant (P >0.05).7. The Treg/Th17 ratios in peripheral blood of patients with ESCC had no relation with patients' age, sex, tumor location, lesion length, degree of differentiation and depth of invasion (P>0.05), but had relation with lymph node metastasis and pTNM stage (P<0.05).Conclusions1. Th1/Th2 shift maybe one of the mechanisms of tumor immune escape.There were significant correlations between the Th1/Th2 shift and the tumor CT staging and lesion extent, but there was not significant relation to the lesion location, pathological type and differentiation.2. Radiotherapy promoted Th1/Th2 shift in ESCC. Aidi injection during radiotherapy inhibited the Thl/Th2 shift, and elevated the immune function in organism decreased the toxic-adverse of effert of radiotherapy and improved life quality. Our study showed the advantages of integrative Chinese and Western medicine treatment in immunotherapy.3. The differentiation of Th1/Th2 cells was controlled by T-bet,GATA-3 in ESCC patients.4. The levels of Th17 cells, RORyt and IL-17a mRNA were up-regulated in ESCC microenvironment. Through RORyt and IL-17a, Th17 cells may contribute to the progression of ESCC, CCR6-CCL20 axis plays an important role in the Th17 cells infiltrating into the tumor tissues.5. The Treg/Th17 cell expression in patients with ESCC in progress stage was imbalanced and Foxp3/RORyt expression imbalance may be one of the mechanisms of Treg/Th17 cell imbalance.
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