DON对体外培养软骨细胞炎性细胞因子、CD44、MMP-13及整合素表达的影响  

Effects of DON on the expression of inflammatory cytokines,CD44,MMP-13,and integrin in cultured chondrocytes

作　　者：领兄 任海娟 曹艳红[1] Ling Xiong;Ren Haijuan;Cao Yanhong(Institute for Kaschin-Beck Disease Control and Prevention,Center for Endemic Disease Control,Chinese Center for Disease Control and Prevention,Harbin Medical University,Harbin 150081,China)

机构地区：[1]哈尔滨医科大学中国疾病预防控制中心地方病控制中心大骨节病防治研究所,150081

出　　处：《中华地方病学杂志》2020年第3期178-183,共6页Chinese Journal of Endemiology

摘　　要：目的观察脱氧雪腐镰刀菌烯醇(DON)对体外培养软骨细胞炎性细胞因子、人类白细胞分化抗原(CD)44、基质金属蛋白酶(MMP)-13、整合素表达的影响。方法提取1~2日龄Wistar乳鼠原代软骨细胞,采用甲苯胺蓝染色法进行软骨细胞鉴定,CCK-8法检测DON(剂量分别为0.0、0.1、0.2、0.4、0.8μg/ml)染毒24、48、72 h软骨细胞增殖情况,根据细胞存活率选取0.00(对照)、0.05、0.10、0.15、0.20μg/ml DON用于后续实验,确定作用时间为48 h。采用酶联免疫吸附试验(ELISA)法检测细胞培养上清液白细胞介素(IL)-6、IL-1β、肿瘤坏死因子(TNF)-α、CD44及MMP-13含量;蛋白免疫印迹(Western blot)法检测软骨细胞CD44和整合素α2、α5、β1蛋白表达水平。结果随着DON剂量和作用时间增加,软骨细胞存活率逐渐降低,存在浓度和时间依赖性(P均<0.01)。细胞培养上清液IL-6含量组间比较差异有统计学意义(H=13.425,P<0.01),其中0.10μg/ml组[中位数(四分位数间距):256.89(191.02,477.58)pg/ml]显著高于对照组[10.37(0.00,119.13)pg/ml,P<0.05];IL-1β和TNF-α含量组间比较差异无统计学意义(F=0.881、1.317,P均>0.05);0.10、0.15、0.20μg/ml组CD44含量[(0.87±0.21)、(0.85±0.24)、(0.77±0.17)pg/ml]明显低于对照组[(1.06±0.19)pg/ml,P均<0.05];MMP-13含量组间比较差异有统计学意义(F=7.947,P<0.01)。CD44和整合素α2、α5、β1蛋白表达水平组间比较差异有统计学意义(F=5.737、6.562、6.074、4.476,P<0.05或<0.01)。结论DON可能通过增加IL-6,MMP-13,整合素α2、β1表达和抑制CD44、整合素α5表达,破坏细胞与细胞外基质间的平衡,造成软骨损伤。Objective To observe the effects of deoxynivalenol(DON)on the expression of chondrocyte inflammatory cytokines,cluster of differentiation(CD)44,matrix metalloproteinase(MMP)-13 and integrin in vitro.Methods Primary chondrocytes from Wistar rats aged 1 to 2 days were isolated,and the chondrocytes were identified by toluidine blue staining.The effects of DON(dose groups of 0.0,0.1,0.2,0.4,0.8μg/ml)on proliferation of chondrocytes were detected by CCK-8 method for 24,48 and 72 h.According to the cell survival rate,the DON concentrations[0.00(control),0.05,0.10,0.15,0.20μg/ml]were selected for subsequent experiments,and the exposure time was 48 h.The contents of interleukin(IL)-6,IL-1β,tumor necrosis factors(TNF)-α,CD44 and MMP-13 in the cell culture supernatant were detected by enzyme linked immunosorbent assay(ELISA)kit.The expression levels of CD44 and integrin subunitsα2,α5,andβ1 protein in chondrocytes were detected by Western blotting.Results The survival rate of chondrocytes decreased with the increase of DON concentrations and time(P<0.01).There were statistically significant differences in IL-6 content between different groups(H=13.425,P<0.01),and 0.10μg/ml group[256.89(191.02,477.58)pg/ml]was significantly higher than that of the control group[10.37(0.00,119.13)pg/ml,P<0.05].There were no statistically significant differences in IL-1βand TNF-αcontents between the groups(F=0.881,1.317,P>0.05).The CD44 contents in 0.10,0.15,and 0.20μg/ml groups[(0.87±0.21),(0.85±0.24),(0.77±0.17)pg/ml]were lower than that in the control group[(1.06±0.19)pg/ml,P<0.05].There were statistically significant differences in MMP-13 expression between the groups(F=7.947,P<0.01).There were statistically significant differences in protein expression of CD44,integrin subunitsα2,α5 andβ1 between the groups(F=5.737,6.562,6.074,4.476,P<0.05 or<0.01).Conclusion DON may disrupt the balance between cells and extracellular matrix by increasing the expression of IL-6,MMP-13,integrin subunitsα2 andβ1,and inhibiting the expres

关 键 词：软骨细胞 脱氧雪腐镰刀菌烯醇 炎性细胞因子 白细胞分化抗原44 基质金属蛋白酶-13 整合素 

分 类 号：R6[医药卫生—外科学]