Experimental Study On The Effects Of Deoxynivalenol On The Antigen Presentation Molecule And Apoptosis Of Humam Esophageal Squamous Cells In Vitro

Objective: Deoxynivalenol (DON, vomitoxin) is a type B trichothecene. This mycotoxin commonly found in corn and other cereals grains used worldwide in animal feed and human foods. DON had been listed in the most dangerous food pollutant by United Nations Food Agriculture Organization (FAO) and World Health Organization (WHO). The research on DON should take first place.The research revealed that DON was the main fungeal toxins which could be detected in the food of the people living in higher incidence areas of oesophagus cancer in China. Although the toxicity of DON was weaker than other mycotoxins, DON was the most common pollution in the corn and the pollution level is higher than other mycotoxins which were produced by F. culmorum . Some research report DON was to coexist and coact with other fungi toxins. They had adverse impact on human health. DON had an extensive cellular toxicity, inhibiting synthesizing of big molecule;promoting the apoptosis of immunity cell and suppressed proliferation as well. The secreting of various cytokines were suppressed or stimulated by DON. It also influenced the activity of various kinase inside the cell, to produce adverse impact on human immunologic function. Some authors reported that DON was related to esophageal cancer,IgA nephritis,Keshan disease and Kaschin-Beck disease occurred in human.As we all to known, the occurrence of the malignant tumor was highly related with the immunologic derangement of the patients besides the influence of cancerogen. The normal expression of HLA-abc molecular was important to maintain anti-tumor immunity function. The research showed that the lost and lower expression of HLA-abc were detected the losing and low expression of HLA-abc in various malignant tumors. The previous research revealed DON induced the apoptosis of mouse thymocyte and human peripheral blood mononuclear cells, and inhabited the process of antigen presentation in the level of mRNA and protein. This phenomenon contributed to the failure of immunity surveillance and the occurrence of tumor. But there was little study about the effects of DON on esophageal squamous cell at home and abroad.The aim of this research was to further explore the effects of DON on the expression of HLA-abc, TAP-1 and LMP-2 in esophageal squamous cells. This research was studied with western blot, RT-PCR, cell culture, immunocytochemistry and FCM method respectivly to detect the expression of HLA-abc, TAP-1, LMP-2, ERK and p-ERK,and apoptosis of esophageal squamous cells. This research revealed the putative roles of DON contamination and the cancerogenic mechanism of DON in the high incidence area of esophageal cancer in our country.Methods:1 Epithelial cell isolation and characterizationThe esophagus was rinsed well in 4℃1640 containing antibiotics and brought back to laboratory. The epithelium was mechanically separated from the connective tissue and blood vessel, then cut to chips (the diameter is about 2mm). Coating the FBS on the bottom of 25 ml culture bottle, then we put chips on the bottom of the bottle ; and putting little culture medium on it and adding to 2ml within 48 hours. The medium contained DMEM:F12(Sigma) modified by the addition of 15% fetal calf serum. The cells were incubated at 37°C with 5% carbon dioxide. The epithelium can cover the bottom of the bottle within 21 days.2 The observation of human primitive esophagus epithelial cellObserving the shape and the growth condition of cells with inverted microscope, the characteristic of epithelial cells were identified by CKpan.3 Grouping and treatmentDON was added to culture flasks when human esophagus epithelial cells were in the exponential growth phase to obtain final DON concentrations of 0, 50, 100, 1000 and 2000μg /L. Then the cells in different groups were incubated with different concentrations of DON (final concentrations of DON 50, 100, 1000 and 2000μg /L) for 24h.The cells were harvestd, centrifuged and collected for FCM, RT-PCR and Western blotting.4 RNA extraction, identify and quantitationTotal RNA from human esophagus epithelial cells was isolated with guanidinium isothiocyanate(GITC) based on the method of Chomczynski and Sacchi, 1987P.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-PCRTotal RNA was reverse transcribed using reverse transcriptase with oligo dT at 43°C for 1.5h to synthesize cDNA.Then the PCR was carried out in a complex mixture such as cDNA,dNTP,Taq DNA polymerase,primers and so on, denaturation for 5 mins, 94℃5 0s, 56℃3 0s, 72℃30s, 30 circulations. After amplification, each sample was analyzed by 1.5% agarose gel electrophoresis for 35 mins and visualized by ethidium bromide staining.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 BlottingFifty micrograms of protein were electrophoretically separated using a 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with primitive monoclone antibodies and secondary monoclone antibodies. Immunoreactive bands were visualized using ECL.8 ImmunocytochemistryIn order to test the morphological observations resulted from a change of HLA-abc on protein level, the immunocytochemistry procession was also carried out according to manufacturer's instructions on ABC reagent kit. In each slide, the positive cells counted were performed independently by three experimenters blind to treatment condition in at least 10 high power fields.9 FCMIn order to detect the apoptosis and HLA-abc molecule, FCM was used according to manufacturer's instructions. The cells were fixed with 70% ethanol and 2% formaldehyde respectively for the apoptosis and the detection of HLA-abc. 10 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.Results:1 The morphology and identifition of human primary esophagus epithelial cells cultured in vitroThe cells cultured with DMEM:F12 displayed cobblestone morphology with inverted microscope, the cells were oval and polygon, and the shape of cells were big and pure cytoplasm with nuclei located in the central. The cells covered the flasks within 21 days. The tissue explants could be implanted in a new flask for several times with a better growth condition of regenerate epithelial cells. 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 which were cultured in 1640 were bigger, feeble and more fibroblast. The result of immunocytochemisty(ICC) using CKpan antibodies presented that 90% of cells showed positive staining.2 The effect of DON on the expressions of HLA-abc, TAP-1 and LMP-2 in humam esophageal squamous cells in vitro2.1 The effect of DON on the expressions of HLA-abc mRNA and protein in human esophagus epithelial cells Twenty four hours after DON treatment, the analysis ofRT-PCR showed that no significant differences were found in the expression of HLA-a mRNA (P>0.05).The ICC staining revealed that the percentages of positive cells in DON (50, 100, 1000 and 2000μg /L) groups were 78.75±10.16%, 70.20±11.69%, 46.70±4.55% and 44.85±7.11% respectively. Comparison of control group (81.90±6.30%), the percentages in DON (100, 1000 and 2000μg /L) groups were predominantly decreased (P<0.05).The FCM revealed that the FI in DON (50, 100, 1000 and 2000μg /L) groups were 0.98±0.06, 0.90±0.09, 0.89±0.04 and 0.94±0.07 respectively. Comparison of control group(1.04±0.04), the FI in DON (100, 1000 and 2000μg /L) groups were predominantly decreased (P<0.05). Furthermore, a negative correlation was found between the expression of HLA-abc protein and the concentration of DON( r=-0.634, n=3, P<0.05)2.2 The effecte of DON on the expressions of TAP-1 mRNA and protein in human esophagus epithelial cellsTwenty four hours after DON treatment, the analysis of RT-PCR showed that no significant differences betweten DON groups and conctrol (P>0.05).The result of Western blotting showed that the expression of TAP-1 protein were 0.47±0.22, 0.28±0.16, 0.24±0.18 and 0.21±0.19 in DON treated groups (50, 100, 1000 and 2000μg /L) respectively. Compared with control group (0.88±0.49), the expression of TAP-1 protein in DON (100, 1000 and 2000μg /L) groups were statistically decreased (P<0.05). Furthermore, a negative correlation was found between the expression of TAP-1 and the concentration of DON (r=-0.595, n=3, P<0.01).2.3 The effect of DON on the expression of LMP-2 mRNA and protein in human esophagus epithelial cells in vitroTwenty four hours after DON treatment, the analysis of RT-PCR showed that the expression of LMP-2 mRNA in DON (100μg /L) group was significantly increased than control group (P<0.05).The result of Western blotting showed that the expression of LMP-2 in DON (100μg /L) group was significantly increased (P<0.05). 3 The effect of DON on the apoptosis, ERK and p-ERK proteion in humam esophageal squamous cells in vitro3.1 The effect of DON on the apoptosis of humam esophageal squamous cellsThe FCM revealed that the apoptosis rates in DON(50, 100, 1000 and 2000μg /L) groups were 6.69±3.57, 9.76±4.52, 14.68±2.26 and 19.15±1.72 respectively. Comparison of control group (5.29±2.00), the apoptosis rates in DON (100, 1000 and 2000μg /L) groups were remarkably increased (P<0.05). Furthermore, a positive correlation was found between the apoptosis rate and the concentration of DON(r=0.885, n=3, P<0.01).3.2 The effect of DON on ERK and p-ERK protein in human esophagus epithelial cellsThe result of Western blotting showed that there were no significant difference on the expression of ERK and p-ERK protein between DON groups and control.Conclusion1 The human esophagus epithelial cells display a cobblestone morphology with inverted microscope, characteristic of epithelial cells cultured in DMEM:F12 with modified tissue explant method, which is a convenient and safe cultural method for human esophagus epithelial cells with high purity and abundant yield.2 DON could decrease the expression of HLA-abc protein, but have no effect on HLA-a mRNA of human normal esophageal epithelial cells.3 DON could decrease the expression of TAP-1 protein with no impact TAP-1 mRNA of human normal esophageal epithelial cells.4 DON (100μg /L) could increase the expressions of LMP-2 protein and mRNA of human normal esophageal epithelial cells.5 The mechanism of DON on the antigen presentation molecule is complicated involving in transcription, translation and post-translation in human esophagus epithelial cells in vitro.6 DON could induce apoptosis of human esophagus epithelial cells in a dose-effect mode, but have no effect on the expression of p-ERK protein. So, the result suggests that p-ERK maybe dose not involve in the prosess of apoptosis induced by DON in human esophagus epithelial cells.