脱氧雪腐镰刀菌烯醇对人胚胎膝关节软骨细胞的毒性及作用机制  

The cytotoxic effect and injury mechanism of deoxynivalenol on articular chondrocytes in human embryo

作　　者：侯海峰[1] 李金平[1] 丁国永[1] 叶文静[2] 焦鹏[2] 李群伟[1] 

机构地区：[1]泰山医学院流行病学研究所,泰安271000 [2]泰山医学院生命科学研究所,泰安271000

出　　处：《中华预防医学杂志》2011年第7期629-632,共4页Chinese Journal of Preventive Medicine

基　　金：山东省自然科学基金（Y2008C59）;山东省教育厅资助课题（J07E06）

摘　　要：目的探讨脱氧雪腐镰刀菌烯醇（DON）对人胚胎膝关节软骨细胞的毒性及作用机制。方法取原代培养人胚胎膝关节软骨，用DMEM／F12培养基进行培养和传代，在培养基中加入DON，建立以下DON染毒浓度：0．1、0．2、0．4、1．0μg／ml组和空白对照组。在处理72h后进行相关研究指标的检测：分别用ELISA法检测培养上清基质金属蛋白酶13（MMP-13）、前列腺素E2（PGE2）含量，硝酸还原酶法检测上清液的一氧化氮（NO）含量，流式细胞术法检测软骨细胞凋亡情况、软骨细胞内诱导型一氧化氮合酶（iNOS）和Ⅱ型胶原表达的情况，RT—PCR法分析iNOSmRNA和Ⅱ型胶原mRNA的表达。结果DON组凋亡率为6．78％-19．05％，高于对照组的1．20％，凋亡率随DON浓度增高而增加（F：174．761，P〈0．05）。DON组上清液NO含量为20．8～40．7μmol／L，高于对照组的10．2μmol／I。（F：91．966，P〈0．05）；MMP．13含量为0．25～0．56μmol／L，高于对照组的0μmol／L（F=78．420，P〈0．05）；PGE2含量为3．2—20．6μmol／L，高于对照组的1．6μmol／L（F=276．453，P〈0．05）。DON组软骨细胞iNOS表达强度为14．8％～56．8％，高于对照组7．1％（F=214．614，P〈0．05）。高剂量DON组（0．4μg／ml和1．0μg／ml）1I型胶原表达强度分别为56．7％和52．7％，低于对照组的62．2％（F=5．134，P〈0．05）。DON组软骨细胞内iNOSmRNA表达的吸光度值（4值）为1．07～1．33，高于对照组的0．62（F=8．358，P〈0．05）。高剂量DON组（0．4、1．0μg／ml）软骨细胞内Ⅱ型胶原mRNA表达量分别为0．83和0．82，低于对照组的1．14（F=7．887，P〈0．05）。结论DON导致人软骨细胞合成NO增加，并通过NO调节MMP-13和PGE2等促进Ⅱ型胶原等软骨基质的降解和软骨毒性损伤；DON可促进软骨细胞的凋亡。Objective This study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol （DON）on articular chondrocytes in human embryo. Methods Articular cartilage cells were isoIated from knees of human embryo and cultured in DMEM/F12 medium. The cells of the 4th generation were divided into five groups and incubated with varying concentrations of DON as the followings ： control group and group with DON of 0. 1,0. 2,0. 4, 1.0 p^g/ml. The effects of DON were observed 72 hours after incubation. Cell apoptosis was assayed by flow cytometry （FCM） with Annexin V-FITC/PI staining; MMP-13 and PGE2 were detected by ELISA kits; NO was measured by Griess assay with spectrophotometer. Inducible nitric oxide synthase （iNOS） and collagen Ⅱ in cells were detected by FCM. The expression levels of iNOS, mRNA and collagen Ⅱ mRNA were measured with RT-PCR. Results The rates of cell apoptosis in DON groups were 6. 78% - 19.05% ,which were significantly higher than that in control （ 1.20% , F = 174. 761, P 〈 0. 05 ）. The levels of NO in DON groups were 20. 8 - 40. 7μmol/L,which were significantly higher than that in control （ 10. 2 μmol/L, F = 91. 966, P 〈 0. 05 ）. The levels of MMP-13 in DON groups were 0. 25 -0. 56 μmol/L, which were significantly higher than that in control（0 μmoL/L, F = 78.420, P 〈 0. 05 ）. The levels of PGE2 in DON groups were 3.2 - 20. 6μmol/L, which were significantly higher than that in control（ 11.6 μmol/L,F = 276. 453,P 〈 0. 05 ）. The proportions of cells with positive iNOS in DON groups were 14. 8% - 56. 8% which were significantly higher than that in controls （7.1% ,F = 214. 614,P 〈 0. 05 ）. The proportions of cells with positive collagen Ⅱ in groups with DON of 0.4 μg/ml and 1.0 μg/ml were 56. 7% and 52. 7% , which were significantly lower than that in control（62.2% ,F =5. 134 ,P 〈0. 05）. The relative absorbance values of iNOS mRNA in DON groups were 1.07 - 1.33 ,which were significantly higher than that in control（0.

关 键 词：软骨细胞 毒性试验 分子作用机制 脱氧雪腐镰刀菌烯醇 

分 类 号：R684.1[医药卫生—骨科学] R726.8[医药卫生—外科学]