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Experimental Study on the Effects of Sterigmatocystin on the Cytokines of Immune Cells in Vivo

Author: ZhangYing
Tutor: ZhangXiangHong;XingLingXiao
School: Hebei Medical University
Course: Pathology and Pathophysiology
Keywords: Sterigmatocystin peripheral blood mononuclear cell peritoneal macrophage cell cytokine RT-PCR ELISA
CLC: R735.1
Type: Master's thesis
Year: 2008
Downloads: 104
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Abstract


Objective: Sterigmatocystin (ST), being the secondary metabolite of Aspergillus verdicolor and Aspergillus nidulans etc, is a carcinogenesis mycotoxin. Studies show that ST is one of the most common seen mycotoxin contaminanted in grain all over the world. It could be detected even in the carpet dust from damp dwellings. Works in China have revealed that ST has been one of the predominating mycotoxins in the grains and foodstuffs in the high incidence area of esophageal cancer since 1970s.There has been growing concern regarding on the effects of mycotoxins on immunological function, especially on the expression and secretion of some cytokines. Cytokines act as a kind of“language”in the signal communication among the immune cells and in the regulation of immune response. Thus, study of the cytokines of peripheral blood mononuclear cells and peritoneal macrophages is of great importance in the study of immune function.Up to now, the studies on ST have been mainly focused on the mechanism of toxin-producing, contamination status and carcinogenic effect, few works involved in the putative effects of ST on immune function. Our preliminary study showed that ST could not only inhibit the secretion of IL-12 of murine peritoneal macrophage cells and the secretion of IL-2 of human peripheral blood mononuclear cells (HPBMc), but also could induce or inhibit the expression of IL-2, IFN-γand IL-4 of murine spleen cells in vitro. All these studies showed that ST has some effects on the immune function.The previous researches were carried out in vitro. Few studies about the effect of ST on immune cells in vivo were seen up to now. The effects of ST on the expression of TNF-α, IL-6 and IL-12 mRNA of the peripheral blood mononuclear cells and the murine peritoneal macrophage cells, and the effects of ST on the protein level of TNF-αand IL-6 in the serum in BALB/c mice after single injection intraperitoneally were studied with RT-PCR and ELISA method respectivly this study. The aim of this study is to further explore the effects of ST on immune cells and reveal the putative roles of ST contamination in the high incidence area of esophageal cancer in our country.Methods1 Experimental animal and treatmentNinety six male BALB/c mice were randomly divided into three groups: ST treatment group, solvent control group and saline control group. Mice in the three groups were injected intraperitoneally with ST (3000ug/kg), DMSO dilution and normal saline (all in same volume) respectively at the beginning of the experiment. The mice in each group were respectively sacrificed 2h, 6h, 12h and 24h after ST treatment.2 Isolation of the peripheral blood mononuclear cellsThe blood was collected in the sterile eppendorf containing Na3C8H5O7.2H2O for anticoagulation. The peripheral blood mononuclear cells were isolated from the blood by Ficolly-Hypaque density gradient centrifugation.3 Isolation of the serumThe blood was collected in the sterile eppendorf and then centrifuged after clotting. The supernatant was harvested and stored at -80℃.4 Culture of the murine peritoneal macrophage cellsFollowing enucleation of the eyeball, BALB/c mice were washed in 75% ethanol. Irrigation of peritoneal cavity was performed with 10ml ice-cold in incomplete RPMI-1640. Emigrated cell in abdominal cavity were counted and adjusted to 5×106 cells/ml in RPMI-1640 culture medium containing 10% fetal calf serum, streptomycin (100ug/ml), penicillin (100U). Macrophage cells were allowed to adhere for 2h in 37℃5%CO2.5 Abstraction and quantitation of the total RNAThe total RNA was abstracted by single-step method with guanidinium isothiocyanate. The integrity of the total RNA was identfied at 90V on 1% agarose gels. The UV Spectro- photometer was used for the quantitation of the total RNA.6 The expression of cytokine mRNA detected by RT-PCRThe effects of ST on the expression of TNF-α, IL-6 and IL-12 of the peripheral blood mononuclear cells and the murine peritoneal macrophage cells were determined with RT–PCR method. The relative cytokines expression was calculated as the ratio between the density of cytokines and that of GAPDH by BIO-LD densitometric image analyzer.7 The serum level of cytokines detected by ELISATNF-αand IL-6 ELISA detection kits made by Jingmei Biotechnology Company Limited was applied to determine the effect of ST on the level of TNF-αand IL-6 in the serum.8 StatisticsData from these studies were analyzed by one-way analysis of variance (ANOVA) and bivariate correlation. The SPSS 12.0 was employed for all calculations. The results were expressed as means±SD.Results1 The effects of ST on the expression of TNF-α, IL-6 and IL-12 mRNA in the peripheral blood mononuclear cells (PBMC) in vivo1.1 The effects of ST on the expression of TNF-αmRNA in the peripheral blood mononuclear cellsRT-PCR products were detected by 1.5% agarose gel electrophoresis and analyzed quantitatively by BIO-LD. The result showed that there was no significant difference in the expression of TNF-αat mRNA level between the saline group and DMSO group. TNF-αmRNA expression in ST 2h, 6h, 12h and 24h group (0.28±0.04, 0.16±0.04, 0.21±0.06, 0.22±0.05 respectively) was much lower than that in corresponding DMSO group (0.38±0.03, 0.41±0.03, 0.42±0.05, 0.37±0.06, P<0.01). The most significant inhibiting effect was seen in ST 6h group.1.2 The effects of ST on the expression of IL-6 mRNA in the peripheral blood mononuclear cellsNo significant differences were found between the saline group and DMSO group. IL-6 mRNA expression level in ST 2h and 6h group (0.75±0.09 and 1.38±0.20) was significantly higher than those in corresponding DMSO group(0.54±0.13 and 0.48±0.08, P<0.01). The most significant inducing effect was seen in ST 6h group. The expression level of IL-6 mRNA begins to decrease from 12h ST treatment group, and reaches the lowest in ST 24h group(0.25±0.05), which was dramatically lower than that in DMSO 24h group(0.55±0.09,P<0.01).1.3 The effects of ST on the expression of IL-12 mRNA in the peripheral blood mononuclear cellsThere was no significant difference between the saline group and DMSO group. The relative expression of IL-12p35 mRNA in the ST groups at different time points was 0.33±0.06, 0.29±0.07, 0.30±0.04, 0.35±0.05 respectively , which was significantly lower than that in corresponding DMSO group( 0.66±0.10, 0.69±0.13,0.71±0.05,0.73±0.16,P<0.01). There were no significant differences between the ST groups.The expression of IL-12p40 mRNA was not detected in all the above groups.2 The effects of ST on the level of TNF-αand IL-6 in serum 2.1 The effects of ST on the TNF-αlevel in serum of miceELISA analysis showed that the TNF-αlevel in each ST treated group was decreased significantly compared with the DMSO 2h、6h、12h、24h group{(11.43±0.60),(12.17±1.04),(11.69±1.53),(12.43±1.40)pg/ml ,P<0.01}. The serum level of TNF-αin ST 2h and 6h group was (9.14±0.58) pg/ml and (8.57±0.57) pg/ml respectively, but it could not be detected at 12h and 24h. There is a negtive relationship between the serum TNF-αlevel and ST treat time (r = -0.905, P<0.01).2.2 The effects of ST on the IL-6 level in serum of miceELISA analysis showed that all the serum level of IL-6 in ST treated groups was significantly lower than that in the corresponding DMSO group {(10.63±1.58), (11.63±1.52), (12.25±1.26), (10.90±1.14) pg/ml, P<0.01}. The serum level of IL-6 in ST treated group at 2h and 6h was (7.32±1.10) pg/ml and (7.00±1.00) pg/ml. It could not be detected at ST 12h and 24h treated group. A significant negative correlation could be found between ST treat time and the level of TNF-αin serum(r = -0.933, P<0.01).3 The effects of ST on the expression of TNF-α,IL-6, IL-12 mRNA in the murine peritoneal macrophage cells3.1 The effects of ST on the expression of TNF-αmRNA in the murine peritoneal macrophage cellsRT-PCR analysis showed that there was no significant difference in expression of TNF-αmRNA between the saline group and the DMSO group. The relative expression of TNF-α mRNA in ST(2h、6h、12h、24h) group was 0.37±0.08, 0.34±0.06, 0.15±0.01, 0.13±0.03, which was all significantly lower than that in the solvent group at the same time point(0.51±0.06,0.52±0.04,0.54±0.10,0.53±0.10,P<0.05).There was a negtive correlation between ST treatment time and the expression of TNF-αmRNA(r = -0.833, P<0.01).3.2 The effects of ST on the expression of IL-6 mRNA in the murine peritoneal macrophage cellsNo significant differences in expression of IL-6 mRNA were found between the saline group and DMSO group. The ratio of IL-6 mRNA expression in the ST mice was 0.21±0.05,0.29±0.05,0.27±0.06,0.24±0.06 respectively for 2, 6, 12 and 24h group,which was significantly lower than that in DMSO group respectively(P<0.01). But no significant differences were found among the different ST groups at the time range from 2h to 24h.3.3 The effects of ST on the expression of IL-12 mRNA in the murine peritoneal macrophage cellsAn inhibiting effect on the expression of IL-12p35 mRNA was found among the ST treated groups from the time range 2h to 24h.The expression of IL-12p40 mRNA was not detected in all above groups.Conclusion1 ST could inhibit the expression of TNF-αin the peripheral blood mononuclear cells of mice administered intraperioneally with ST3000ug/kg from 2h to 24h after ST treatment, especially in ST 6h group.2 The effects of ST on IL-6 mRNA expression vary with the time after ST treatment. ST could induce the expression of IL-6 mRNA at 2h and 6h, but inhibition effect was seen at 24h.3 ST could inhibit the expression of IL-12p35 mRNA at all ST groups from 2h to 24h after treatment.4 ST could decrease TNF-αand IL-6 level in serum of mice, and the effects were more obvious with the time prolonged.5 ST could inhibit the expression of TNF-α,IL-6 and IL-12p35 mRNA in the murine peritoneal macrophage cells in vivo at the time range from 2h to 24h. The inhibiting effect on the expression of TNF-αmRNA intensified gradually as time prolonged.6 All these data suggested that except for its carcinogenic effects, ST exposure might have certain negative effects on function of immune cells via the moduating effects on the mRNA and protein level of the cytokines.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Esophageal tumors
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