Experimental Study On The Expression Of The Antigen Esentation Molecule Of Humam Esophageal Squamous Cells Treated By Deoxynicalenol For Different Time In Vitro

Objective:Hebei Taihang mountain area is one of the high incidence of esophageal cancer in the world, and esophageal cancer has been seriously affected the local social and economic development. To change this situation, from the 90 years since the last century, we had monitored and analysised the fungi and toxins in the food of the residents in Hebei Cixian for 10 years. Three high contamination rate of mycotoxins-aflatoxins,sterigmatocystin and deoxynivalenol were selected to simulate the mycotoxins exposure of the residents. They discussed the possible mechanism of these three fungal toxins in the process of esophageal cancer,and revealed sterigmatocystin and deoxynivalenol can reduce the expression of antigen presentation-related molecules for the first time.The structure of deoxynivalenol(DON), also known as vomitoxin (VT).In our high incidence of gastric cancer and esophageal cancer, the detection rate and amount of DON are the highest,up to 170.22 ng / kg . Recent studies have found that DON can inhibit the immune function of animals, and induce T cells , B cells and IgA cells in thymus, spleen and intestinal mucosa to apoptosis. There were studies that showed DON could inhibit transcription, translation and protein synthesis through the sesquiterpenes tructure and enhance the immune function by interfering the normal immune regulation. In vivo, DON could not only inhibit the body's immune response to pathogens, also could induce the autoimmune reactions. In addition, DON could induce apoptosis of the immune cells and inhibit their proliferation, and super-induce helper T cells to produce cytokines, activate macrophages and pro- inflammatory cytokines. Normal expression of HLA-I molecules and processing and transporting of the endogenous peptides is the important part to maintain the body's immune surveillance. Some studies suggested that some carcinogenic environmental factors can reduce HLA-I molecule expression in the surface of normal cells, so that the cells may transformat into malignant cells by escaping form the immune surveillance. However, the role of DON in the expression of immune-related molecules of human normal esophageal squamous epithelium has been reported rarely at home and abroad.In this study, cell culture, immunocytochemistry, Western blot, and RT-PCR were used to detect the expression changes of HLA-abc, TAP-1, LMP -2 and CNX protein of normal primary cultured esophageal squamous cells treated by DON for 6, 12, 24 and 36 hours.To discuss the possible effect of DON exposure for long time on the immune function, and further reveal the possible role of DON exposure in tumourgenesis of esophageal cancer in high incidence area of esophageal cancer in China.Methods:1 Primary culture of human esophageal epithelial cellsUnder sterile conditions, the esophageal mucosa was rinsed repeatedly with PBS solution penicillin 200U/ml and streptomycin 200μg/ml, until there was no blood.Cut off the excess blood vessels and connective tissue of submucosa, then digested the mucosa in Dispase (25g/L) for 24h.After that , removed the mucosa with eye tweezers, and put it into trypsin (25g/L) at 37℃for 20-30min, then added an equal amount with 1640 serum containing 10% fetal bovine to stop the digestion. Blowed into a single cell suspension and centrifugal 1500r/min 5min, then washed twice with PBS solution. Finally, added K-SFM serum-free medium and incubated in 25cm² culture flask according to (0.1-1.0)×10⁶A / cm², at 37℃with 5% CO₂. Did not changed the medium until the cells adherent, after that it was changed every 2-3 days.2 The observation of primitive esophagus epithelial cellsObserved the shape and the growth conditions of cells with inverted microscope and identified the characteristic by CKpan.3 Group and treatmentRandomly, the esophageal epithelial cells were divided into treatment and control groups when 70%-80% of bottom was covered by cells. DON (1mg/ml)was added to the treatment groups, the final concentration was 1000μg / L and reaction times were 6h,12h,24h, 6h.The same amount of saline was added into the control groups. The cells were harvestd, centrifuged and collected for RT-PCR and Western blotting.4 RNA extraction, identify and quantitationThe total RNA was isolated from the cultured esophagus epithelial cells using Trizol method. The integrity of total RNA was identfied at 90V on 1% agarose gels containing EB (0.5μg/ml).The UV Spectro-photometer was used for the quantitation of the total RNA.5 RT-PCRHLA-abc, TAP-1, LMP-2 and CNX mRNA expression of human esophageal squamous cells were detected by RT-PCR and analysised by gel analysis software (BIO-LD) .6 Protein extraction and quantitationThe cells were collected and splited in total cell lysates. The protein concentration was determined using Coomassie brilliant blue (CBB) reagent.7 Western BlottingThe total cellular protein was isolated and the expression of HLA-abc,TAP-1, LMP-2 and CNX protiens were detected using Western blot method.8 Immunocytochemistry analysisThe expression of HLA-abc, TAP-1, LMP-2 and CNX were detected in esophagus epithelial cells using immunohistochemistry SP method which carried out strictly according to the instructions and observed under the microscope.9 StatisticsData from these studies were analyzed by one-way analysis of variance (ANOVA) and bivariate correlation. The SPSS 13.0 was employed for all calculations. The results were expressed as means±SD.Results1 The morphology and identifition of human primary esophagus epithelial cells cultured in vitroThe single cells gathered into cell clusters after 1-2 days and adhered in 3-4 days. The cells were oval and polygon, and the shape of cells were big and pure cytoplasm with nuclei located in the central,showing a typical "cobblestone"-like morphology(Fig.3). The cells grew fast with little fibroblasts pollution,and with the culture time, fibroblasts gradually decreased and finally disappeared. The epithelial cells were stained positive for the epithelial cell marker cytokeratin, which verified that the cells were human esophagus epithelial cell. The epithelial cells were stained positive for the epithelial cell marker cytokeratin, which verified that the cells were human esophagus epithelial cell. The epithelial cells were stained positive for the epithelial cell marker cytokeratin, which verified that the cells were human esophagus epithelial cell.2 The time effect of DON(1000μg/L) on the expressions of HLA-abc, TAP-1, LMP-2 and CNX in normal human esophageal squamous cells in vitro.2.1 The time effect of DON (1000μg/L) on the expressions of HLA-abcThe results of Western blot showed that DON(1000μg/L) could affect the expression of HLA-abc protein with a tendency of increasing first and then decreasing compared with control group within 36 hours.6h-12h treatment groups were higher than control groups and 24-36h treatment groups were lower. Furthermore, 12h group was significantly higher compared with control group (P<0.05), while the other groups had no significant differences.The results of RT-PCR showed that DON(1000μg/L) could affect the expression of HLA-a mRNA with a tendency of increasing compared with control group within 36 hours.The expression of treatment groups gradually decreased as the treatment time. Furthermore, 6h , 12h and 24h treatment groups were significant increased compared with control groups (P<0.05). The expression of HLA-b mRNA also had a increased trend within 36 hours. The expression of treatment groups gradually decreased as the treatment time. Furthermore, the differences were more obvious in 6h, 12h and 36h DON treatment groups (P<0.05). The expression of HLA-c mRNA with a tendency of decreasing first and then increasing within 36 hours. The expression of treatment groups gradually decreased as the treatment time. Moreover, 6h treatment group was significantly lower (P<0.05) and 12h and 24h treatment groups were significantly higher than control groups(P<0.05).2.2 The time effect of DON(1000μg/L) on the expressions of TAP-1The results of Western blot showed that DON(1000μg/L) could affect the expression of TAP-1 protein with a tendency of increasing first and then decreasing within 36 hours. 6h treatment group was higher and 12h-36h treatment groups were lower than control groups. But each treatment and control group showed no significant difference.The results of RT-PCR showed that DON(1000μg/L) could affect the expression of TAP-1 mRNA with a tendency of decreasing first and then increasing within 36 hours.6h treatment group was significantly lower than control group (P<0.05) ,12-36h treatment groups were significantly higher (P<0.05).2.3 The time effect of DON(1000μg/L) on the expressions of LMP-2The results of Western blot showed that DON(1000μg/L) could affect the expression of LMP-2 protein with a tendency of increasing within 36 hours. Each treatment group was significantly higher than control group(P<0.05).The results of RT-PCR showed that DON(1000μg/L) could affect the expression of LMP-2 mRNA with a tendency of increasing within 36 hours. 12h and 36h treatment groups were significant higher (compared with the control groups P <0.05).2.4 The time effect of DON (1000μg/L) on the expression of CNXThe results of Western blot showed that DON(1000μg/L) could affect the expression of CNX protein with a tendency of increasing first and then decreasing within 36 hours. 12h treatment group was significantly higher (P<0.05), the other treatment groups were lower than the control groups and no significant.The results of RT-PCR showed that DON(1000μg/L) could affect the expression of CNX mRNA with a tendency of increasing within 36 hours and the expression of treatment groups gradually decreased as the treatment time. Each treatment group was higher than the control group and 12h and 36h groups were significant (P<0.05).Conclusion:1 In the serum-free medium K-SFM using enzyme digestion method, the cells grew as well as the former, covered 70%-80% area of culture flask after only 10-12 days. Moreover, the cells can be used for cryopreservation, recovery and passage.It was the best method we had found.2 The expression of HLA-abc,LMP-2,CNX proteins and mRNAs, TAP-1 mRNA were increased from 6hs to12hs in esophageal squamous cells by DON(1000μg/L). While the promotion gradually weakened from 24hs to 36hs.3 The expression of TAP-1 protein showed no significant changed from 6hs to 36hs in esophageal squamous cells treated by DON(1000μg/L).4 DON (1000μg/L) could effect the expressions of HLA-I molecule and its associated proteins and mRNA in normal human esophageal squamous cells in vitro from 6hs to 36hs.The influences were so complex that they could change as the treatment time.The role of DON was mainly to promote within a short time, while the promotion gradually weakened as the treatment time. DON could cause disorders of HLA-I molecule antigen presentation aspects.