脱氧雪腐镰刀菌烯醇对机体免疫功能影响的实验研究
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摘要
目的：单端孢霉烯族毒素是一组结构相似的真菌次级代谢产物，具有明显的毒性效应。脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)又名呕吐毒素(vomitoxin VT)，是其中最常见的一种，其结构为3α,7α,15-三羟基-12,13-环氧单端孢霉-9烯-8酮，为雪腐镰刀菌烯醇的脱氧衍生物，主要由某些镰刀菌产生。在全世界范围内，粮食及其制品DON污染谷类的情况均非常普遍。研究表明，在我国食管癌高发区居民饮食中DON也是主要污染霉菌毒素之一。动物实验结果提示，DON可抑制生物大分子的合成，也可对免疫系统发挥一定的影响。目前有关DON对免疫功能影响的研究尚存在一些问题，即DON对免疫功能的影响多是在体外进行的，主要集中在淋巴细胞因子分泌方面，而且有关消除DON对免疫系统影响的研究甚少。机体的免疫系统是一个非常复杂的生物学系统，包括补体系统、细胞因子分泌、黏附分子系统、免疫细胞的凋亡与增殖以及抗原加工递呈等诸多方面。故进一步了解DON对免疫细胞的凋亡与增殖以及抗原加工递呈的影响和如何消除或降低DON对免疫系统的负面作用是非常必要的。本研究探讨了DON体内对小鼠胸腺细胞凋亡和增殖的影响并初步探讨了维生素C和核黄素体内对DON诱发小鼠胸腺细胞凋亡和增殖抑制的阻断作用；同时探讨了DON对体外培养的人外周血单个核细胞表面人白细胞抗原Ⅰ类分子（human leucocyte antigenⅠ,HLA-Ⅰ）以及抗原处理相关转运体－１（transporter associated with antigen processing, TAP-1）和低分子量蛋白多肽－２(low molecular weight polypeptides, LMP-2)表达的影响。旨在全面了解DON对机体免疫系统的影响，以期进一步分析DON的生物效应，揭示饮食DON暴露对机体的可能影响及其在我国食管癌高发区肿瘤发生发展中的可能作用。
方法：
１ 脱氧雪腐镰刀菌烯醇对小鼠胸腺细胞凋亡和增殖影响的检测分析
昆明小鼠48只随机分为对照组和实验组，实验组根据DON处理浓度又分为DON 0.5mg/kg、DON 1mg/kg、DON 2mg/kg、DON 4mg/kg和DON 8mg/kg组，共6组，每组８只实验动物。实验组和对照组动物分别腹腔注射0.2mL不同浓度的DON溶液和生理盐水。于注射后12 h，股动脉放血处死动物，取出胸腺组织，应用电镜观察、DNA琼脂糖凝胶电泳和流式细胞定量检测技术（FCM）检测不同浓度DON处理胸腺细胞凋亡和增殖情况。
２ 核黄素和维生素C预处理对DON诱导小鼠胸腺细胞凋亡和增殖抑制的影响。
昆明小鼠112只，随机分为对照组、DON （4mg/kg）组、核黄素（1.25mg/kg和10mg/kg）组、抗坏血酸(25mg/kg和100mg/kg)组、不同剂量核黄素（1.25mg/kg,2.5mg/kg,5.0mg/kg,和10.0mg/kg）预处理+ DON（4mg/kg）组和不同剂量抗坏血酸（25mg/kg,50mg/kg,75mg/kg,和100mg/kg）预处理+DON（4mg/kg）组，共14组，每组８只实验动物。核黄素、抗坏血酸组及预处理组分别按分组剂量灌喂核黄素和抗坏血酸，每日1次，每次0.5mL/只，连续5d；对照组和DON（4mg/kg）组灌喂等体积的生理盐水。于最后1次灌喂后12h，对照组、核黄素（1.25mg/kg和10mg/kg）组和抗坏血酸(25mg/kg和100mg/kg)组分别腹腔注射生理盐水0.2mL/只，其余各组实验动物分别腹腔注射DON溶液（4mg/kg.BW, 0.2mL /只）。12 h后，股动脉放血处死动物，取出胸腺组织，应用DNA琼脂糖凝胶电泳和流式细胞术检测方法进行凋亡和增殖的检测。
３ 体外培养的人外周血单个核细胞表面HLA－Ⅰ抗原检测
健康献血员抗凝静脉血200mL，采用聚蔗糖-泛影葡胺密度梯度离心法(ficoll-hypaque density gradient centrifugation）分离人外周静脉血单个核细胞，接种于含10%胎牛血清，105U/L青霉素，100mg/L链霉素的RPMI 1640培养基中，加入PHA至终浓度300μg/mL，37℃，5%CO2培养48h，更换不含PHA的新的培养基。随机分为实验组和对照组，实验组加入DON至终浓度分别为50ng/mL，100ng/mL，1000ng/mL和2000ng/mL，对照组加等体积的生理盐水。处理24h，更换不含DON的新的RPMI
1640培养基，继续培养24h，然后离心收集细胞。采用流式细胞定量检测技术（FCM）和蛋白印迹（Western Blotting）检测HLA-Ⅰ分子的表达情况。
４ 体外培养的人外周血单个核细胞TAP-1和LMP-2的mRNA及其蛋白表达的检测
人外周静脉血单个核细胞的分离培养和处理方法同前，离心收集细胞提取总RNA，应用反转录－聚合酶连反应（RT-PCR）方法检测体外培养的人外周静脉血单个核细胞（HPBMc）抗原处理相关转运体－１（TAP-1）和低分子量蛋白－２（LMP-2）mRNA的表达情况；应用流式细胞定量检测技术（FCM）检测TAP-1和LMP-2蛋白的表达情况。
结果：
１ 脱氧雪腐镰刀菌烯醇体内对小鼠胸腺细胞凋亡和增殖的影响
FCM DNA直方图表明，不同浓度DON处理12h后，小鼠胸腺细胞DNA直方图二倍体峰前均可见凋亡细胞特征性的亚二倍体峰。FCM定量检测结果表明，不同浓度DON处理后，各组小鼠胸腺细胞的平均凋亡百分率都明显高于生理盐水对照组（P<0.01），而且随着DON浓度的升高，凋亡百分率也相应升高，在0.5mg/kg到8mg/kg的浓度范围内，两者呈明显的正相关，直线回归方程为Y=5.75+0.98X ( r=0.788, P<0.01, n=8)。透射电
Objectives： Trichothecene mycotoxin is a group of structurally similar fungal secondary metabolites that are capable of producing a wide range of toxic effects. Deoxynivalenol (DON, vomitoxin), a most commonly seen trichothecene mycotoxins mainly produced by some Fusarium fungi, is the deoxyderivative of nivalenol. DON contamination of grains is a very common phenomenon worldwidely. DON is one of the predominating contaminating mycotoxins in the grains and foodstuffs in the high incidence area of esophageal cancer in China. Previous studies showed that DON could inhibit synthesis of biological macromolecules in animals and have some negative effects on immune system. Nevertheless, there are still some problems and doubts need to be further elucidated. Most previous experiments on immunotoxic effects of DON were carried out in vitro and mainly involved cytokine secretion. Few works on how to preclude immunotoxic effects of DON were carried out. It is known to all that immune system is a very complex biological system, including complement system, cytokine secretion, adhesion molecule, apoptosis and proliferation of immune cells and processing and transport of antigen. Therefore, to completely evaluate immunotoxic effects of DON, it is very important and necessary to further explore the effects of DON on apoptosis and proliferation of immune cells and processing and transport of antigen and how to how to preclude immunotoxic effects of DON.
To further elucidate the effects of DON on immunological function in vitro and in vivo and explore the putative effects of DON exposure on the carcinogenesis of esophageal cancer in the high incidence area, the following studies were carried out:
1. Effects of deoxynivalenol at different concentrations on apoptosis and proliferation of mouse thymocytes in vivo were studied with animal experiment, electron microscoPIc observation, DNA agarose gel electrophoresis and flow cytometric analyses.
2. Effects of riboflavin and ascorbic acid on the apoptosis and proliferation inhibition of thymocytes induced by DON in KM mice were studied with animal experiment, DNA agarose gel electrophoresis and flow cytometric DNA content analysis.
3. The effects of DON at different concentrations on HLA-Ⅰexpression of human peripheral blood mononuclear cells in vitra at protein level were studied with flow cytometric analyses and western blotting.
4. TAP-1 and LMP-2 expression of human peripheral blood mononuclear cells treated with DON at different concentrations were studied with reverse transcription-polymerase chain reaction(RT-PCR) and flow cytometric analysis.
Methods:
1 Determination of apoptosis and proliferation of thymocytes in KM mice treated with DON in vivo
48 KM mice were randomLy divided into 6 groups: control group, DON 0.5mg/kg, 1mg/kg, 2mg/kg, 4mg/kg and 8mg/kg group. Eight mice in each group. The mice were respectively treated with saline and different concentration DON solutions by intraperitoneal injection. All mice were killed by exsanguinations from femoral artery, 12h after intraperitoneal injection. Fresh thymus specimens were prepared for FCM single cell suspension, DNA abstraction and election microscoPIc specimen. The apoptosis and proliferation of mouse thymocytes treated with deoxynivalenol at different concentrations were determined with electron microscoPIc observation, DNA agarose gel electrophoresis and flow cytometric analyses.
2 Determination of thymocytes apoptosis and proliferation inhibition induced by DON after riboflavin and ascorbic acid
pretreatment in KM mice in vivo
118 KM mice were randomLy divided into 14 groups: control group, DON (4mg/kg) group, riboflavin (1.25mg/kg and 10mg/kg) groups, ascorbic acid (25mg/kg and 100mg/kg) groups, riboflavin pretreatment (1.25mg/kg, 2.5mg/kg, 5.0mg/kg and 10.0mg/kg)+DON (4mg/kg) groups, ascorbic acid pretreatment (25mg/kg, 50mg/kg, 75mg/kg and 100mg/kg) +DON (4mg/kg) groups, 8 mice in each group. The mice in riboflavin groups, ascorbic acid groups, riboflavin pretre
方法：
１ 脱氧雪腐镰刀菌烯醇对小鼠胸腺细胞凋亡和增殖影响的检测分析
昆明小鼠48只随机分为对照组和实验组，实验组根据DON处理浓度又分为DON 0.5mg/kg、DON 1mg/kg、DON 2mg/kg、DON 4mg/kg和DON 8mg/kg组，共6组，每组８只实验动物。实验组和对照组动物分别腹腔注射0.2mL不同浓度的DON溶液和生理盐水。于注射后12 h，股动脉放血处死动物，取出胸腺组织，应用电镜观察、DNA琼脂糖凝胶电泳和流式细胞定量检测技术（FCM）检测不同浓度DON处理胸腺细胞凋亡和增殖情况。
２ 核黄素和维生素C预处理对DON诱导小鼠胸腺细胞凋亡和增殖抑制的影响。
昆明小鼠112只，随机分为对照组、DON （4mg/kg）组、核黄素（1.25mg/kg和10mg/kg）组、抗坏血酸(25mg/kg和100mg/kg)组、不同剂量核黄素（1.25mg/kg,2.5mg/kg,5.0mg/kg,和10.0mg/kg）预处理+ DON（4mg/kg）组和不同剂量抗坏血酸（25mg/kg,50mg/kg,75mg/kg,和100mg/kg）预处理+DON（4mg/kg）组，共14组，每组８只实验动物。核黄素、抗坏血酸组及预处理组分别按分组剂量灌喂核黄素和抗坏血酸，每日1次，每次0.5mL/只，连续5d；对照组和DON（4mg/kg）组灌喂等体积的生理盐水。于最后1次灌喂后12h，对照组、核黄素（1.25mg/kg和10mg/kg）组和抗坏血酸(25mg/kg和100mg/kg)组分别腹腔注射生理盐水0.2mL/只，其余各组实验动物分别腹腔注射DON溶液（4mg/kg.BW, 0.2mL /只）。12 h后，股动脉放血处死动物，取出胸腺组织，应用DNA琼脂糖凝胶电泳和流式细胞术检测方法进行凋亡和增殖的检测。
３ 体外培养的人外周血单个核细胞表面HLA－Ⅰ抗原检测
健康献血员抗凝静脉血200mL，采用聚蔗糖-泛影葡胺密度梯度离心法(ficoll-hypaque density gradient centrifugation）分离人外周静脉血单个核细胞，接种于含10%胎牛血清，105U/L青霉素，100mg/L链霉素的RPMI 1640培养基中，加入PHA至终浓度300μg/mL，37℃，5%CO2培养48h，更换不含PHA的新的培养基。随机分为实验组和对照组，实验组加入DON至终浓度分别为50ng/mL，100ng/mL，1000ng/mL和2000ng/mL，对照组加等体积的生理盐水。处理24h，更换不含DON的新的RPMI
1640培养基，继续培养24h，然后离心收集细胞。采用流式细胞定量检测技术（FCM）和蛋白印迹（Western Blotting）检测HLA-Ⅰ分子的表达情况。
４ 体外培养的人外周血单个核细胞TAP-1和LMP-2的mRNA及其蛋白表达的检测
人外周静脉血单个核细胞的分离培养和处理方法同前，离心收集细胞提取总RNA，应用反转录－聚合酶连反应（RT-PCR）方法检测体外培养的人外周静脉血单个核细胞（HPBMc）抗原处理相关转运体－１（TAP-1）和低分子量蛋白－２（LMP-2）mRNA的表达情况；应用流式细胞定量检测技术（FCM）检测TAP-1和LMP-2蛋白的表达情况。
结果：
１ 脱氧雪腐镰刀菌烯醇体内对小鼠胸腺细胞凋亡和增殖的影响
FCM DNA直方图表明，不同浓度DON处理12h后，小鼠胸腺细胞DNA直方图二倍体峰前均可见凋亡细胞特征性的亚二倍体峰。FCM定量检测结果表明，不同浓度DON处理后，各组小鼠胸腺细胞的平均凋亡百分率都明显高于生理盐水对照组（P<0.01），而且随着DON浓度的升高，凋亡百分率也相应升高，在0.5mg/kg到8mg/kg的浓度范围内，两者呈明显的正相关，直线回归方程为Y=5.75+0.98X ( r=0.788, P<0.01, n=8)。透射电
Objectives： Trichothecene mycotoxin is a group of structurally similar fungal secondary metabolites that are capable of producing a wide range of toxic effects. Deoxynivalenol (DON, vomitoxin), a most commonly seen trichothecene mycotoxins mainly produced by some Fusarium fungi, is the deoxyderivative of nivalenol. DON contamination of grains is a very common phenomenon worldwidely. DON is one of the predominating contaminating mycotoxins in the grains and foodstuffs in the high incidence area of esophageal cancer in China. Previous studies showed that DON could inhibit synthesis of biological macromolecules in animals and have some negative effects on immune system. Nevertheless, there are still some problems and doubts need to be further elucidated. Most previous experiments on immunotoxic effects of DON were carried out in vitro and mainly involved cytokine secretion. Few works on how to preclude immunotoxic effects of DON were carried out. It is known to all that immune system is a very complex biological system, including complement system, cytokine secretion, adhesion molecule, apoptosis and proliferation of immune cells and processing and transport of antigen. Therefore, to completely evaluate immunotoxic effects of DON, it is very important and necessary to further explore the effects of DON on apoptosis and proliferation of immune cells and processing and transport of antigen and how to how to preclude immunotoxic effects of DON.
To further elucidate the effects of DON on immunological function in vitro and in vivo and explore the putative effects of DON exposure on the carcinogenesis of esophageal cancer in the high incidence area, the following studies were carried out:
1. Effects of deoxynivalenol at different concentrations on apoptosis and proliferation of mouse thymocytes in vivo were studied with animal experiment, electron microscoPIc observation, DNA agarose gel electrophoresis and flow cytometric analyses.
2. Effects of riboflavin and ascorbic acid on the apoptosis and proliferation inhibition of thymocytes induced by DON in KM mice were studied with animal experiment, DNA agarose gel electrophoresis and flow cytometric DNA content analysis.
3. The effects of DON at different concentrations on HLA-Ⅰexpression of human peripheral blood mononuclear cells in vitra at protein level were studied with flow cytometric analyses and western blotting.
4. TAP-1 and LMP-2 expression of human peripheral blood mononuclear cells treated with DON at different concentrations were studied with reverse transcription-polymerase chain reaction(RT-PCR) and flow cytometric analysis.
Methods:
1 Determination of apoptosis and proliferation of thymocytes in KM mice treated with DON in vivo
48 KM mice were randomLy divided into 6 groups: control group, DON 0.5mg/kg, 1mg/kg, 2mg/kg, 4mg/kg and 8mg/kg group. Eight mice in each group. The mice were respectively treated with saline and different concentration DON solutions by intraperitoneal injection. All mice were killed by exsanguinations from femoral artery, 12h after intraperitoneal injection. Fresh thymus specimens were prepared for FCM single cell suspension, DNA abstraction and election microscoPIc specimen. The apoptosis and proliferation of mouse thymocytes treated with deoxynivalenol at different concentrations were determined with electron microscoPIc observation, DNA agarose gel electrophoresis and flow cytometric analyses.
2 Determination of thymocytes apoptosis and proliferation inhibition induced by DON after riboflavin and ascorbic acid
pretreatment in KM mice in vivo
118 KM mice were randomLy divided into 14 groups: control group, DON (4mg/kg) group, riboflavin (1.25mg/kg and 10mg/kg) groups, ascorbic acid (25mg/kg and 100mg/kg) groups, riboflavin pretreatment (1.25mg/kg, 2.5mg/kg, 5.0mg/kg and 10.0mg/kg)+DON (4mg/kg) groups, ascorbic acid pretreatment (25mg/kg, 50mg/kg, 75mg/kg and 100mg/kg) +DON (4mg/kg) groups, 8 mice in each group. The mice in riboflavin groups, ascorbic acid groups, riboflavin pretre
引文
1 Zhang XH, Xie TX, Li SS, et al. Preventive detection of fungi and mycotoxins in corn from high risk area of esophageal cancer in Cixian county. Chin J Cancer Res, 1995, 7(3):172-176.
2 Zhang XH, Xie TX, Li SS, et al. Contamination of fungi and mytoxins in foodstuffs in high risk area of esophageal cancer. Biomed Environ Sci, 1998; 11(2) :140-146.
3 王会艳，孙旭明，张祥宏，等. 脱氧雪腐镰刀菌烯醇、黄曲霉毒素G1对体外培养的人外周血淋巴细胞凋亡影响的研究. 卫生研究，1999; 28: 102-104.
4 王会艳, 张祥宏，杨永滨，等. 脱氧雪腐镰刀菌烯醇对体外培养人外周血单核细胞增殖及肿瘤坏死因子－α分泌的影响. 卫生研究，2000; 29(6): 387-389.
5 左连富主编.流式细胞术样品制备技术. 第一版.北京：华夏出版社，1991，14-19.
6 Smith C.A, Williams G.T, Kongston R, Jenkinson E.J, Owen J.J.(1989). Antibodies to CD3/T cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature, 337: 181-184.
7 Pestka JJ, Yan D, King LE. Flow cytometric analysis of the effects of in vitro exposure to vomitoxin (deoxynivalenol) on apoptosis in murine T, B and IgA+ cells. Food Chem Toxicol, 1994; 32(12): 1125-1136.
8 Zhou HR, Harkema JR, Hotchkiss JA, et al. Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs. Toxicol Sci, 2000; 53(2): 253-263.
9 Zhou HR,Yan D, Pestka JJ. Differential cytokine mRNA expression in mice after oral exposure to the trichothecene vomitoxin (deoxynivalenol): dose response and tine course. Toxicol Appl Pharmacol, 1997; 144(2): 294-305
10 Thuvander A, Wilkman C, Gadhasson I. In vitro exposure of human lymphocytes to trichothecenes: individual variation in sensitivity and effects of combined exposure on lymphocyte function. Food chem toxicol, 1999; 37(6): 639-48
11 Berek L, Petri IB, Mesterhazy A, et al. Effects of mycotoxins on human immune functions in vitro. Toxicol In Vitro, 2001; 15(1): 25-30.
2 Zhang XH, Xie TX, Li SS, et al. Contamination of fungi and mytoxins in foodstuffs in high risk area of esophageal cancer. Biomed Environ Sci, 1998; 11(2) :140-146.
3 王会艳，孙旭明，张祥宏，等. 脱氧雪腐镰刀菌烯醇、黄曲霉毒素G1对体外培养的人外周血淋巴细胞凋亡影响的研究. 卫生研究，1999; 28: 102-104.
4 王会艳, 张祥宏，杨永滨，等. 脱氧雪腐镰刀菌烯醇对体外培养人外周血单核细胞增殖及肿瘤坏死因子－α分泌的影响. 卫生研究，2000; 29(6): 387-389.
5 左连富主编.流式细胞术样品制备技术. 第一版.北京：华夏出版社，1991，14-19.
6 Smith C.A, Williams G.T, Kongston R, Jenkinson E.J, Owen J.J.(1989). Antibodies to CD3/T cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature, 337: 181-184.
7 Pestka JJ, Yan D, King LE. Flow cytometric analysis of the effects of in vitro exposure to vomitoxin (deoxynivalenol) on apoptosis in murine T, B and IgA+ cells. Food Chem Toxicol, 1994; 32(12): 1125-1136.
8 Zhou HR, Harkema JR, Hotchkiss JA, et al. Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs. Toxicol Sci, 2000; 53(2): 253-263.
9 Zhou HR,Yan D, Pestka JJ. Differential cytokine mRNA expression in mice after oral exposure to the trichothecene vomitoxin (deoxynivalenol): dose response and tine course. Toxicol Appl Pharmacol, 1997; 144(2): 294-305
10 Thuvander A, Wilkman C, Gadhasson I. In vitro exposure of human lymphocytes to trichothecenes: individual variation in sensitivity and effects of combined exposure on lymphocyte function. Food chem toxicol, 1999; 37(6): 639-48
11 Berek L, Petri IB, Mesterhazy A, et al. Effects of mycotoxins on human immune functions in vitro. Toxicol In Vitro, 2001; 15(1): 25-30.