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Objective: China is a high incidence of esophageal cancer this year, the incidence of esophageal cancer is rising year by year. In order to study the mechanism of esophageal cancer, the laboratory of the high incidence of esophageal residents dietary toxins pollution monitoring of up to 10 years of aflatoxin, sterigmatocystin and deoxynivalenol sickle bacteria enol esophageal cancer development, first discovered three toxins can affect the function of the immune cells, to reduce antigen processing presenting related molecules expression, and the role of deoxynivalenol Fusarium enol more obvious. The enol of Fusarium deoxynivalenol (deoxynivalenol, DON), also known as vomitoxin (vomitoxin, VT) contamination of DON is extremely prominent in some of our digestive tract tumors, such as a high incidence area of ??gastric cancer and esophageal cancer, the detection rate of DON and detectable amount in the first of mycotoxin contamination. Some studies have shown, DON can significantly affect the immune function of humans and animals. The study further found that sesquiterpene structure of DON is the origin of the suppression of the immune function, which can inhibit the process of transcription, translation and protein synthesis, DON through the body's normal immune regulation jammer and enhance immune function. Therefore, DON on the immune function of both positive and negative impact, not only can suppress the body's immune response, but also can induce autoimmune reaction. Studies have found that, of DON can suppress immune cell proliferation and induce apoptosis, can activate macrophages, T cells produce proinflammatory cytokines, helper T cells produce cytokines. Normal expression of HLA-I molecules and antigen processing transport molecules is to ensure that the premise of the body's normal immune function. DON role in normal esophageal squamous epithelium, the expression of the immune-related molecules, at home and abroad are rarely reported. In this study, the use of cell culture, immunocytochemistry, Western blot, RT-PCR method to detect DON (1000μg / L) treatment 6h, 12h, 24h, 36h hours after primary cultured human normal esophageal epithelial cells of HLA-abc, the TAP -1, LMP-2 and CNX protein and mRNA expression analysis of the DON process at different times affect antigen presentation time dependent expression of related molecules, DON exposed to further reveal the possible negative effects on human immune function, as well as in our esophageal The high incidence of tumor development possible role. Method: 1 normal esophageal epithelial cells in primary culture under sterile conditions, the esophageal mucosal tissue containing penicillin 200U/ml streptomycin 200μg/ml PBS solution repeatedly washed until no trace of blood so far, and to cut off the excess blood vessels of the submucosa 25g / L neutral protease (Dispase) and connective tissue, placed in digestion 24h forceps mucous layer torn off, put 25g / L trypsin digestion for 20-30min, 37 ℃ and subsequently adding the same amount containing 1640 medium 10% fetal bovine serum terminate digestion, wind and percussion into a single cell suspension, washed twice with PBS solution to 1500r/min centrifugal 5min K-SFM serum-free medium (0.1-1.0 ) × 106 / cm2 were seeded in 25cm2 flasks placed in 37 ℃ 5% CO2 incubator. When the cells adherent medium was changed for the first time, after the medium was changed every 2-3 days time. 2 normal human esophageal epithelial cells identified under an inverted microscope observation of cell morphology and growth, the cells seeded broad-spectrum CK Immunocytochemistry identify cell sources. 3 experimental groups and deal with normal esophageal epithelial cell growth covered the bottom of the bottle 70% -80%, were randomly divided into a treatment group and a control group. The treatment group plus DON (1mg/ml) to a final concentration of 1000μg / L, reaction time was 6h, 12h, 24h, 36h, control group, an equal volume of saline. Trypsinized cells were collected by centrifugation or detect the expression of each indicator cells seeded. 4 total cellular RNA extraction, identification and quantification of total cellular RNA using Trizol method. 1% agarose gel electrophoresis integrity. UV spectrophotometer quantitative. 5 RT-PCR using RT-PCR method to detect DON handling different times normal esophageal squamous cells of HLA-abc, TAP-1, LMP-2, CNX mRNA expression of affect. The gel analysis software (BIO-LD) for quantitative analysis of each group expressed as the ratio to the target gene and internal reference gene GAPDH relative expression levels of the target gene. 6 total cell protein extraction and quantitative applications precooled Cell Lysates added to the collected cells, the extraction of total cellular protein. The protein extracted using UV spectrophotometer quantification. 7 Western blot (Western Blotting) extraction of total cellular protein by Western blot to detect DON treatment at different times of normal esophageal epithelial cells of HLA-abc, TAP-1, LMP-2 and CNX protein expression levels. Immunocytochemistry SP 8 immunocytochemical staining cells seeded in strict accordance with the instructions, DAB color under the microscope. 9 Statistical analysis of the data were expressed as mean ± standard deviation (x ± s) SPSS13.0 statistical software for one-way ANOVA analysis (analysis of variance, ANOVA), the SNK method pairwise comparisons. Results: 1 primary cultures of normal human esophageal epithelial cell morphology observed and identified by the enzymatic digestion of a single cell suspension was inoculated into glass flasks, K-SFM serum-free medium, 1-2 days of single cells assembled into cell mass ,3-4 days adherent cells into round, oval and polygon volume contours unclear, transparent cytoplasm, the nucleus is large and clear, cell fusion, tightly packed, was typical paving stone-like arrangement (Fig.3). Less pollution, fibroblasts, fibroblast cells decreased with time, finally disappeared. The Immunocytochemistry display 90% of the cells showed a broad-spectrum CK positive, that cultured cells esophageal squamous cell. 2 DON treatment at different times of normal human esophageal epithelial cells of HLA-abc, TAP-1, LMP-2 and CNX expression 2.1 DON handle different expression of HLA-abc affect Western blot showed: normal esophageal epithelial cells by DON (1000μg / L) treatment, 6h-36h treated HLA-abc relative expression level of the protein compared with the control group was first increased and then decreased. 6h and 12h treatment group was higher level, 24h and 36h treatment group than the control group level, 12h of the treatment group compared to the control group with a significant difference (P lt; 0.05), and the rest of the group and the control group compared There was no significant difference. RT-PCR results show that: the normal human esophageal epithelial cells treated by DON (1000μg / L), 6h-36h treated HLA-a mRNA relative expression were higher than the level, with the time, the treatment group HLA- a mRNA relative expression level decreased to normal levels. Where 6h, 12h and 24h treatment group compared to the control group with a significant difference (P lt; 0.05), 36h group compared with the control group, no statistically significant difference. The normal human esophageal epithelial cells by DON (1000μg / L) treatment, 6h-36h treatment group of HLA-bmRNA the relative expression levels of higher than levels, with time, the treatment group HLA-b mRNA relative expression level gradually decline. 6h, 12h and 36h group compared with the control group differences were significant (P lt; 0.05), 24h group compared with the control group, no significant differences. The normal human esophageal epithelial cells 6h-36h treated HLA-c mRNA relative expression level showed a trend first and then decreased by DON (1000μg / L) treatment, with time, the treatment group relative expression levels of some decline. 6h treatment group was significantly lower than the level of the control group (P lt; 0.05), 12h and 24h treatment group was significantly higher than the control level (P lt; 0.05), 36h compared with the control group no significant difference. 2.2 DON process at different times of TAP-1 expression Western blot showed: normal human esophageal epithelial cells via of DON (1000μg / L), 6h-36h group TAP-1 protein relative expression level and the control group compared to increased first and then decreased to below the level of the control group, the the 6h treatment group was higher level ,12-36h processing each treatment group compared with the control group had no significant difference. RT-PCR results show that: the normal human esophageal epithelial cells by DON (1000μg / L) after treatment, compared to 6h-36h treated TAP-1 mRNA relative expression level of the control group was first decreased and then increased the trend. 6h treatment group was significantly lower than the level of the control group (P lt; 0.05), 12h-36h treatment group were significantly higher than the control level (P lt; 0.05). 2.3 DON LMP-2 expression Western blot showed: normal human esophageal epithelial cells treated by DON (1000μg / L), 6h-36h treatment group relative expression levels of LMP-2 protein than the control handle different time group level and has a significant difference (P lt; 0.05). RT-PCR results show: the normal human esophageal epithelial cells treated by DON (1000μg / L), 6h-36h treated LMP-2 mRNA relative level of expression were higher than that of the control group, 12h and 36h of the treatment group and the control group. compared with a significant difference (P LT; 0.05). The 2.4 DON dealing with different time expression of CNX Western blot showed: normal human esophageal epithelial cells by DON (1000μg / L) after treatment, compared to 6h-36h treatment group CNX relative expression level of the protein with the control group was the first rose high then decreased with time, the treatment group relative expression amount gradually decreased to normal levels, which 12h treatment group reached the highest point and the significantly higher level (P lt; 0.05), the rest of the treatment group and the control groups showed no significant difference. RT-PCR results show that: the normal esophageal epithelial cells 6h-36h treatment group relative CNX mRNA expression were higher than the level of DON (1000μg / L) treatment, with time, the treatment group relative expression levels decline, 12h and 36h group compared with the control group there were significant differences (P LT; 0.05). Conclusion: an enzymatic digestion method plus K-SFM serum-free medium of cultured human normal esophageal epithelial cell growth, cell fusion fast, the fibroblast contamination substantially eliminate, the high purity cells ,10-12 cells can be covered with bottom 70% -80%, this cultured cells can be cryopreserved, recovery and passaging, the best method of esophageal epithelial cells in primary culture obtained in this experiment. 2 in the range of process 6h-12h, DON (1000μg / L) in the protein and mRNA levels promote human esophageal epithelial cells in HLA-abc promote the expression of TAP-1, LMP-2 and CNX expression at the mRNA level . Processing within 12h-36h, DON (1000μg / L) of HLA-abc, LMP-2, CNX protein and mRNA and TAP-1 mRNA role significantly weakened. 3 processing within 6h-36h, DON (1000μg / L) of human normal esophageal epithelial cells in TAP-1 protein had no significant effect. 4 processing within 6h-36h, DON (1000μg / L) can affect normal human esophageal epithelial cells in HLA-Ⅰ molecules and their associated molecules of protein and mRNA expression, but with different processing time impact, the change is more complex . Short time DON performance to promote the role, as the treatment time, the role of DON waning. DON aspects of the disorder caused by antigen-presenting HLA-I class.
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