【作者】 张颖；

【导师】 张祥宏； 邢凌霄；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 目的:杂色曲霉素（sterigmatocystin, ST）是致癌性的真菌毒素,是杂色曲霉（Aspergillus versicolor）、构巢曲霉（Aspergillus nidulans）等真菌产生的毒性代谢产物。在粮食和饲料中的污染非常普遍,甚至在潮湿居所的地毯灰尘中也可检出杂色曲霉素的污染。研究表明,在我国食管癌高发区居民饮食中ST也是主要污染霉菌毒素之一。众所周知,恶性肿瘤的发生除致癌物的直接作用外,机体的免疫状态也发挥着非常重要的作用。近年来,一些真菌毒素对人或动物的免疫机能,特别是对细胞因子分泌和表达的影响引起了学术界的关注。由于细胞因子在免疫细胞间充当信号联络作用,是免疫细胞在功能上相互联络的"语言"之一。通过这种"语言",整个免疫系统才能协调一致。因此,研究外周血单个核细胞及腹腔巨噬细胞所产生的细胞因子的作用是揭示免疫应答本质的一种重要手段。目前国内外有关ST的研究多侧重于产毒的分子机制、污染现状和致癌性研究方面,而有关ST对机体免疫机能影响的研究较少。本研究组既往对ST的免疫调节作用进行了一系列的研究,研究结果表明ST可以抑制体外培养的小鼠腹腔巨噬细胞IL-12的表达与分泌;可以诱导或抑制小鼠脾细胞IL-2、IFN-γ和IL-4的表达与分泌;并且ST还可以对体外培养的人外周血单个核细胞（human peripheral bloodmononuclear cells, HPBMc）培养上清中IL-2的分泌有一定抑制作用,提示ST对机体的免疫功能具有一定的影响。但是,上述研究均为体外分离培养免疫细胞进行的研究,从动物整体水平上的研究尚很少。因此,本研究采用半定量RT-PCR方法探讨了腹腔注射ST对小鼠外周血单个核细胞和小鼠腹腔巨噬细胞的TNF-α、IL-6和IL-12在mRNA水平的影响,并用ELISA方法研究ST对小鼠血清中TNF-α及IL-6含量的影响,旨在从免疫角度探讨我国食管癌高发区粮食中常见优势污染霉菌毒素-ST对机体免疫系统的可能影响,揭示ST的暴露在我国食管癌高发区肿瘤发生发展中的可能作用。方法:1实验分组及动物处理选用健康雄性BALB/c小鼠96只,随机分为3组,分别为ST处理组、溶剂对照组和对照组。按照处死时间的不同,又将上述各组分为2h、6h、12h和24h四组。各ST处理组小鼠均经腹腔单次注射ST 3000μg/kg,溶剂对照组和对照组动物分别腹腔注射同等容量的溶剂（DMSO生理盐水溶液）和生理盐水。2小鼠外周血单个核细胞的分离小鼠摘眼球取血,用3.2%柠檬酸三钠（Na₃C₈H₅O₇.2H₂O）抗凝,采用聚蔗糖-泛影葡胺密度梯度离心法分离小鼠外周血单个核细胞。3小鼠血清的制备小鼠摘眼球取血,待血凝固后离心取上清,-80℃保存备用。4小鼠腹腔巨噬细胞的分离将小鼠眼球放血处死后,浸泡于75%酒精,用10 ml冰冷的无血清RPMI-1640培养液冲洗腹腔,无菌收集腹腔渗出细胞。计数细胞后,按5×106cells/mL接种于含105U/L青霉素、100mg/L链霉素的RPMI-1640培养液中,37℃、5% CO₂培养2小时以使巨噬细胞贴壁。5细胞总RNA的提取、鉴定及定量采用异硫氰酸胍一步法提取细胞总RNA。1%琼脂糖电泳鉴定其完整性。紫外分光光度计进行定量。6 RT-PCR检测细胞因子mRNA的表达检测ST在不同处理时间对小鼠外周血单个核细胞及腹腔巨噬细胞TNF-α、IL-6和IL-12 mRNA表达的影响。采用凝胶分析软件（BIO-LD）对凝胶进行定量,各组以目的基因与内参照基因GAPDH量的比值表示目的基因相对表达量。7 ELISA检测血清中细胞因子的水平采用晶美生物工程（北京）有限公司的小鼠TNF-α、IL-6定量试剂盒,检测ST处理不同时间对小鼠外周血中TNF-α和IL-6水平的影响。8统计采用SPSS12.0软件进行相关分析与单因素方差分析（analysis of variance, ANOVA）,数据用x±s表示。结果:一ST对小鼠外周血单个核细胞TNF-α、IL-6和IL-12 mRNA表达的影响1 ST对小鼠外周血单个核细胞TNF-αmRNA表达的影响RT-PCR产物经1.5%琼脂糖凝胶电泳及定量分析表明,与生理盐水对照组相比,各溶剂对照组小鼠外周血单个核细胞TNF-αmRNA相对表达量无明显不同。ST 2h、6h、12h和24h处理组TNF-αmRNA相对表达量分别为0.28±0.04,0.16±0.04,0.21±0.06和0.22±0.05,均明显低于其对应的溶剂对照组（0.38±0.03,0.41±0.03,0.42±0.05和0.37±0.06,P<0.01）,其中以ST 6h处理组TNF-αmRNA的表达降低最明显（P<0.05）。2 ST对小鼠外周血单个核细胞IL-6 mRNA表达的影响半定量RT-PCR检测小鼠外周血单个核细胞IL-6 mRNA水平的结果表明,IL-6 mRNA的表达在各生理盐水对照组与溶剂对照组细胞之间无明显差异。而ST处理组与其相应的溶剂对照组比较IL-6 mRNA的表达均有一定差异,随ST处理时间不同对IL-6 mRNA的表达影响也不同。ST 2h、6h处理组IL-6 mRNA的相对表达量（0.75±0.09,1.38±0.20）均高于溶剂对照组（0.54±0.13,0.48±0.08,P<0.01）。但ST 12h、24h处理组表达呈降低趋势,ST 24h处理组的相对表达量为0.25±0.05,明显低于相应的溶剂对照组（0.55±0.09,P<0.01）。3 ST对小鼠外周血单个核细胞IL-12 mRNA表达的影响与生理盐水对照组相比,各溶剂对照组细胞IL-12p35 mRNA相对表达量无明显不同。ST 2h、6h、12h和24h处理组IL-12p35 mRNA的相对表达量分别为0.33±0.06,0.29±0.07,0.30±0.04和0.35±0.05,均明显低于相应溶剂对照组（0.66±0.10,0.69±0.13,0.71±0.05,0.73±0.16,P<0.01）。但在ST处理组之间其表达无明显改变。在空白对照组、溶剂对照组、ST处理组均未检测到小鼠外周血单个核细胞IL-12p40 mRNA的表达。二ST对小鼠外周血血清中TNF-α和IL-6水平的影响1 ST对小鼠血清中TNF-α水平的影响ELISA检测小鼠血清中TNF-α水平的结果表明,ST2h、6h、12h、24h处理组与相应的溶剂对照组的TNF-α水平{（11.43±0.60）、（12.17±1.04）、（11.69±1.53）、（12.43±1.40）pg/ml}相比,TNF-α水平均明显降低（P<0.01）。ST 2h和6h处理组TNF-α血清水平分别为（9.14±0.58）pg/ml和（8.57±0.57）pg/ml,而在ST 12h和24h处理组均未检测到TNF-α。ST处理组小鼠血清中TNF-α水平,随时间的延长而逐渐降低,二者呈负相关（r = -0.905, P<0.01）。2 ST对小鼠血清中IL-6水平的影响ELISA检测小鼠血清中IL-6水平的结果表明,ST2h、6h、12h、24h处理组与相应的溶剂对照组{（10.63±1.58）、（11.63±1.52）、（12.25±1.26）、（10.90±1.14）pg/ml}相比,IL-6水平均明显降低（P<0.01）。在ST 2h和6h处理组IL-6的水平分别为（7.32±1.10） pg/ml和（7.00±1.00） pg/ml,ST12h和24h处理组均未检测到IL-6蛋白的分泌。ST处理组小鼠血清中IL-6水平随时间的延长而逐渐降低（r = -0.933,P<0.01）。三ST对小鼠腹腔巨噬细胞TNF-α、IL-6及IL-12 mRNA表达的影响1 ST对小鼠腹腔巨噬细胞TNF-αmRNA表达的影响RT-PCR产物经1.5%琼脂糖凝胶电泳及定量分析表明,各溶剂对照组的小鼠腹腔巨噬细胞TNF-αmRNA的表达与生理盐水对照组相比无差异。ST 2h、6h、12h、24h处理组TNF-αmRNA的相对表达量依次为0.37±0.08,0.34±0.06,0.15±0.01 , 0.13±0.03 ,均明显低于相应的溶剂对照组（0.51±0.06,0.52±0.04,0.54±0.10,0.53±0.10,P<0.05）。ST处理组小鼠腹腔巨噬细胞TNF-αmRNA的表达随时间的延长逐渐降低（r = -0.833, P<0.01）。2 ST对小鼠腹腔巨噬细胞IL-6 mRNA表达的影响与生理盐水对照组相比,各溶剂对照组细胞IL-6 mRNA相对表达量无明显改变。不同时间ST处理组小鼠腹腔巨噬细胞IL-6 mRNA的相对表达量均比相应的溶剂对照组显著降低（P< 0.01）,但各ST处理组间IL-6 mRNA相对表达量无统计学差异。3 ST对小鼠腹腔巨噬细胞IL-12 mRNA表达的影响IL-12p35 mRNA相对表达量在溶剂对照组与生理盐水对照组之间没有明显变化。各ST处理组小鼠腹腔巨噬细胞IL-12p35 mRNA的相对表达量均较其相应的溶剂对照组显著降低。在空白对照组、溶剂对照组、ST处理组均未检测到小鼠腹腔巨噬细胞IL-12p40 mRNA的表达。结论:1单次给予小鼠腹腔注射ST 3000ug/kg后,在2-24h范围内ST对小鼠外周血单个核细胞TNF-αmRNA的表达主要为抑制作用,其中以6h处理组的抑制最为显著。2 ST对小鼠外周血单个核细胞IL-6 mRNA表达的影响随处理时间的不同而不同,在处理2h和6h,ST对IL-6 mRNA的表达具有一定的诱导作用,而随着ST处理时间延长至24h,表现为明显的抑制作用。3 ST可以显著抑制小鼠外周血单个核细胞IL-12p35 mRNA表达。4 ST可以降低小鼠外周血血清中TNF-α及IL-6的水平,且作用随时间的延长而加强。5 ST处理可以抑制小鼠腹腔巨噬细胞TNF-α、IL-6及IL-12p35 mRNA的表达,其中ST对TNF-αmRNA的抑制作用随时间的延长而加强。6本研究结果提示,ST除其致癌性外,ST还可能通过细胞因子的改变对机体免疫系统产生不利影响。更多还原

【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.更多还原