Hou Hai-Feng, Li Jin-Ping, Ding Guo-Yong, Ye Wen-Jing, Jiao Peng, Li Qun-Wei
Institute of Epidemiology, Taishan Medical College, Taian 271000, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2011 Jul;45(7):629-32.
This study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol (DON) on articular chondrocytes in human embryo.
Articular cartilage cells were isolated 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 µg/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.
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 - 40.7 µmol/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 3.2-20.6 µmol/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 (0.62, F = 8.358, P < 0.05). The levels of collagen II mRNA in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 0.83 and 0.82, which were significantly lower than that in control (1.14, F = 7.887, P < 0.05).
DON could promote anabolism of NO in articular cartilage cells by which up-regulated the expression of PGE2 and MMP-13, which both promoted resolution of articular cartilage matrix such as collagen II. DON induced apoptosis in articular cartilage cells.
本研究旨在探讨脱氧雪腐镰刀菌烯醇（DON）对人胚胎关节软骨细胞的细胞毒性作用及相关损伤机制。
从人胚胎膝关节分离关节软骨细胞，在DMEM/F12培养基中培养。将第4代细胞分为五组，分别用不同浓度的DON孵育，如下：对照组以及DON浓度为0.1、0.2、0.4、1.0μg/ml的组。孵育72小时后观察DON的作用。采用Annexin V-FITC/PI染色通过流式细胞术（FCM）检测细胞凋亡；用ELISA试剂盒检测MMP-13和PGE2；用分光光度计通过Griess法测定NO。通过FCM检测细胞中的诱导型一氧化氮合酶（iNOS）和Ⅱ型胶原蛋白。用RT-PCR检测iNOS、mRNA和Ⅱ型胶原蛋白mRNA的表达水平。
DON组细胞凋亡率为6.78% - 19.05%，显著高于对照组（1.20%，F = 174.761，P < 0.05）。DON组NO水平为20.8 - 40.7μmol/L，显著高于对照组（10.2μmol/L，F = 91.966，P < 0.05）。DON组MMP-13水平为0.25 - 0.56μmol/L，显著高于对照组（0μmol/L，F = 78.420，P < 0.05）。DON组PGE2水平为3.2 - 20.6μmol/L，显著高于对照组（11.6μmol/L，F = 276.453，P < 0.05）。DON组iNOS阳性细胞比例为14.8% - 56.8%，显著高于对照组（7.1%，F = 214.614，P < 0.05）。DON浓度为0.4μg/ml和1.0μg/ml组Ⅱ型胶原蛋白阳性细胞比例分别为56.7%和52.7%，显著低于对照组（62.2%，F = 5.134，P < 0.05）。DON组iNOS mRNA的相对吸光度值为1.07 - 1.33，显著高于对照组（0.62，F = 8.358，P < 0.05）。DON浓度为0.4μg/ml和1.0μg/ml组Ⅱ型胶原蛋白mRNA水平分别为0.83和0.82，显著低于对照组（1.14，F = 7.887，P < 0.05）。
DON可促进关节软骨细胞中NO的合成代谢，上调PGE2和MMP-13的表达，二者均促进关节软骨基质如Ⅱ型胶原蛋白的分解。DON诱导关节软骨细胞凋亡。
