T-2毒素对体外培养软骨细胞炎性细胞因子、CD44及整合素的影响  

Effects of T-2 toxin on inflammatory cytokines, CD44 and integrin in chondrocytes cultured in vitro

作　　者：任海娟 领兄 曹艳红[2] Ren Haijuan;Ling Xiong;Cao Yanhong(Department of Medical Records and Statistics,Tongde Hospital of Zhejiang Province,Hangzhou 310012,China;Institute for Kaschin-Beck Disease Control,Center for Endemic Disease Control,Chinese Center for Disease Control and Prevention,Harbin Medical University,Harbin 150081,China)

机构地区：[1]浙江省立同德医院病案与统计科,杭州310012 [2]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》2021年第7期530-534,共5页Chinese Journal of Endemiology

摘　　要：目的观察T-2毒素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及整合素表达的影响。方法取1~2日龄SPF级Wistar乳鼠2只制备原代软骨细胞,进行体外培养,采用甲苯胺蓝染色法进行软骨细胞鉴定,细胞增殖/毒性检测试剂盒(CCK-8)法检测不同剂量T-2毒素(0、2、4、6、10、12、20 ng/ml)染毒24、48、72 h对软骨细胞的毒性作用,根据细胞存活率确定终浓度为0(对照)、2、4、6、8 ng/ml的T-2毒素作用48 h为后续实验条件。采用酶联免疫吸附测定(ELISA)法检测细胞上清液白细胞介素(IL)-6、IL-1β、肿瘤坏死因子(TNF)-α及CD44含量;蛋白免疫印迹(Western blot)法检测软骨细胞整合素α5、β1蛋白表达水平。结果在相同作用时间下,不同剂量组间软骨细胞存活率比较,差异均有统计学意义(F=130.759、258.250、123.337,P均<0.01)。作用48 h,不同剂量T-2毒素组软骨细胞上清液中IL-6、IL-1β、TNF-α、CD44含量比较,差异均有统计学意义(F=10.613、4.805、2.943、12.395,P<0.01或<0.05)。其中,2、4、6、8 ng/ml组IL-6含量均高于对照组(P均<0.05);6、8 ng/ml组IL-1β含量均高于对照组和2 ng/ml组,且6 ng/ml组明显高于4 ng/ml组(P均<0.05);6、8 ng/ml组TNF-α含量均低于对照组(P均<0.05);2、4、6、8 ng/ml组CD44含量均低于对照组(P均<0.05)。作用48 h,不同剂量T-2毒素组软骨细胞整合素α5、β1蛋白表达水平比较,差异均有统计学意义(F=4.635、4.376,P均<0.05)。其中,6、8 ng/ml组整合素α5蛋白表达水平均明显低于对照组(P均<0.05),整合素β1蛋白表达水平均明显高于对照组(P均<0.05)。结论T-2毒素可能通过上调IL-6、IL-1β和整合素β1的表达,同时下调TNF-α、CD44和整合素α5的表达,破坏软骨细胞与细胞外基质的平衡,造成软骨细胞损伤。Objective To investigate the effects of T-2 toxin on expression of inflammatory cytokines,human leukocyte differentiation antigen(CD)44 and integrin in chondrocytes cultured in vitro.Methods Primary chondrocytes from SPF Wistar rats aged 1 to 2 days were isolated and cultured in vitro,and the chondrocytes were identified by toluidine blue staining.The effects of different dose of T-2 toxin(0,2,4,6,10,12,20 ng/ml)on proliferation of chondrocytes for 24,48 and 72 h were detected via the cell counting kit-8(CCK-8)method.According to the cell survival rate,T-2 toxin concentrations of 0(control),2,4,6 and 8 ng/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)-αand CD44 in cell supernatant were detected via the enzyme linked immunosorbent assay(ELISA).The protein expression levels of integrinα5 and integrinβ1 in chondrocytes were detected by Western blotting.Results At the same exposure time,there were significant differences of the survival rate of chondrocytes between different dose groups(F=130.759,258.250,123.337,P<0.01).At 48 h after exposure,there were statistically significant differences in IL-6,IL-1β,TNF-αand CD44 contents in the culture supernatant of chondrocytes between different dose of T-2 toxin groups(F=10.613,4.805,2.943,12.395,P<0.01 or<0.05);among them,the IL-6 levels of 2,4,6 and 8 ng/ml groups were higher than that of control group(P<0.05);the IL-1βlevels of 6,8 ng/ml groups were higher than that of control and 2 ng/ml groups,and the 6 ng/ml group was higher than that of 4 ng/ml group(P<0.05);the TNF-αlevels of 6,8 ng/ml groups were lower than that of control group(P<0.05);the CD44 levels of 2,4,6 and 8 ng/ml groups were lower than that of control group(P<0.05).At 48 h after exposure,there were statistically significant differences in integrinα5 and integrinβ1 protein expression levels between different dose of T-2 toxin groups(F=4.635,4.376,P<0.05).Among them,the protein expression levels of integ

关 键 词：软骨细胞 T-2毒素 炎性细胞因子 白细胞分化抗原44 整合素 

分 类 号：R68[医药卫生—骨科学]