摘要目的 观察胰岛素样生长因子-1受体(IGF-1R)在大骨节病(KBD)儿童和低硒条件下T-2毒素染毒大鼠软骨组织中的表达,以及IGF-1R抑制剂对人正常软骨细胞(C28/I2细胞)凋亡的影响,探讨IGF-1R在KBD发病中的作用.方法 收集陕西省KBD病区病死儿童(5例)和非KBD病区车祸死亡及先天6指畸形手术儿童(5例)指关节,取指关节软骨经免疫组织化学法(免疫组化)检测IGF-1R表达情况.选择32只雄性SD大鼠,体质量为60～80g,按体质量采用随机数字表法分为常规饲料(硒含量101.5 μg/kg)和低硒饲料(硒含量1.1 μg/kg)组,每组16只;饲养30 d后,将常规饲料组分为对照组和T-2毒素组(100 ng·kg-1·d-1),低硒饲料组分为低硒组和低硒+ T-2毒素组,每组8只,饲养30 d后取大鼠左侧膝关节经免疫组化检测软骨组织IGF-1R表达情况.体外培养C28/I2细胞,以T-2毒素0(对照)、6、12、24μg/L及分别加亚硒酸钠(+0.1 mg/L)处理72 h;同时,以IGF-1R抑制剂0(对照)、250、500、1 000 μg/L处理48 h,采用实时荧光定量PCR (Real-time PCR)和免疫印迹法(Western Blot)检测软骨细胞IGF-1R mRNA和蛋白表达水平,采用流式细胞术检测软骨细胞凋亡情况.结果 与对照组[(100.00±0.00)％、(100.00±0.00)％]比较,KBD组儿童关节软骨表、中层IGF-1R阳性细胞表达率[(72.71±4.75)％、(36.33±4.32)％]显著降低(t=12.852、32.650,P均＜0.01).与对照组[(100.00±0.00)％、(1000.00±0.00)％、000.00±0.00)％]比较,低硒、T-2毒素、低硒+T-2毒素组大鼠关节软骨中层[(20.83±2.69)％、(26.45±2.84)％、(20.34±1.82)％],深层[(33.55±5.66)％、(48.89±8.39)％、(25.51±7.50)％],及骺板软骨肥大层[(47.50±1.47)％、(28.66±3.58)％、(40.52±6.78)％]IGF-1R阳性细胞表达率显著降低(P均＜ 0.01).与对照组比较,各T-2毒素组C28/I2细胞IGF-1R mRNA和蛋白表达水平均明显降低(P均＜0.05).6、12、24 μg/L T-2毒素+0.1 mg/L硒组C28/I2细胞IGF-1R mRNA表达水平(1.95±0.35、2.44±0.17、2.40±0.15)显著高于相同浓度T-2毒素组(0.80±0.08、0.63±0.08、0.61±0.11,t=-12.259、-11.279、-13.371,P均＜0.05);6、12 μgg/L T-2毒素+0.1 mg/L硒组C28/I2细胞IGF-1R蛋白表达水平(1.67±0.70、1.07±0.26)显著高于相同浓度T-2毒素组(0.52±0.05、0.72±0.05,t=-25.977、-10.776,P均＜0.05).与对照组[(5.33±0.85)％、(4.03±1.15)％]比较,各IGF-1R抑制剂组C28/I2细胞早期凋亡率[(8.26±1.51)％、(13.00±0.72)％、(13.19±1.05)％],及500、1 000 μg/L IGF-1R抑制剂组晚期凋亡率[(8.50±0.71)％、(14.21±1.10)％]明显升高(P均＜0.05).结论 KBD儿童和低硒条件下T-2毒素染毒大鼠软骨组织中IGF-1R表达均减少.T-2毒素下调软骨细胞IGF-1R表达,加硒可以部分对抗T-2毒素对IGF-1R的合成抑制作用.IGF-1R下调可引起软骨细胞凋亡,对KBD软骨细胞凋亡可能具有重要作用.

abstractsObjective To observe the expression level of insulin-like growth factor-1 receptor (IGF-1R) in the cartilage tissue of children with Kaschin-Beck disease (KBD) and T-2 toxin-poisoned rats under low selenium condition,and the effect of IGF-1R inhibitor on apoptosis of human normal chondrocytes (C28/I2 cells),and to investigate the role of IGF-1R in the pathogenesis of KBD.Methods The knuckles of dead children (5 cases) in the KBD areas,car accident death and congenital 6 finger deformity operation children (5 cases) in non-KBD areas in Shaanxi were collected,the expression of IGF-1R in the articular cartilage was detected by immunohistochemistry.Thirty-two male Sprague-Dawley rats with a body mass of 60-80 g were selected,according to the body mass,they were divided into the routine feed group (selenium content:101.5 μg/kg) and the low-selenium feed group (selenium content:1.1 μg/kg) by random number table method,16 rats in each group.After 30 days of feeding,the routine feed group was divided into control group and T-2 toxin group (100 ng·kg-1·d-1),the low-selenium feed group was divided into low selenium group and low selenium + T-2 toxin group,8 rats in each group,the expression of IGF-1R in the articular cartilage of the left knee joint was detected by immunohistochemistry after 30 days of feeding.C28/I2 cells were cultured in vitro and treated with T-2 toxin 0 (control),6,12,and 24 μg/L,and each concentration of T-2 toxin was accompanied with sodium selenite (+ 0.1 mg/L) for 72 h.Meanwhile,IGF-1R inhibitor with 0 (control),250,500,and 1 000 μg/L was treated on C28/I2 cells for 48 h.The expression levels of IGF-1R mRNA and protein in chondrocytes were detected by Real-time PCR and Western blotting,and the apoptosis of chondrocytes was detected by flow cytometry.Results Compared with the control group [(100.00 ± 0.00)％,(100.00 ± 0.00)％],the expression rates of IGF-1R positive cells in articular cartilage surface and middle layers [(72.71 ± 4.75)％,(36.33 ± 4.32)％] of children in KBD group were significantly reduced (t =12.852,32.650,P ＜ 0.01).Compared with control group [(100.00 ± 0.00)％,(100.00 ± 0.00)％,(100.00 ± 0.00)％],the expression rates of IGF-1R positive cells in articular cartilage middle layer [(20.83 ± 2.69)％,(26.45 ± 2.84)％,(20.34 ± 1.82)％],deep layer [(33.55 ± 5.66)％,(48.89 ± 8.39)％,(25.51 ± 7.50)％],and the expression rates of IGF-1R positive cells [(47.50 ± 1.47)％,(28.66 ± 3.58)％,(40.52 ± 6.78)％] in the hypertrophic layer of the metaphyseal plate of rats in low selenium,T-2 toxin,and low selenium + T-2 toxin groups were significantly reduced (P ＜ 0.01).C28/I2 cells were cultured in vitro,compared with the control group,IGF-1R mRNA and protein expression levels in each T-2 toxin groups were significantly reduced (P ＜ 0.05).The expression levels of IGF-1R mRNA (1.95 ± 0.35,2.44 ± 0.17,2.40 ± 0.15) in 6,12,24 μg/L T-2 toxin + 0.1 mg/L selenium groups were significantly higher than those in T-2 toxin groups (0.80 ± 0.08,0.63 ± 0.08,0.61 ± 0.11,t =-12.259,-11.279,-13.371,P＜ 0.05).The expression levels of IGF-1R protein (1.67 ± 0.70,1.07 ± 0.26) in 6,12 μg/L T-2 toxin + 0.1 mg/L selenium groups were significantly higher than those in T-2 toxin groups (0.52 ± 0.05,0.72 ± 0.05,t =-25.977,-10.776,P ＜ 0.05).Compared with the control group [(5.33 ± 0.85)％,(4.03 ± 1.15)％],C28/I2 cells early apoptosis rates [(8.26 ± 1.51)％,(13.00 ± 0.72)％,(13.19 ± 1.05)％] in each of IGF-1R inhibitor groups,and late apoptosis rates [(8.50 ± 0.71)％,(14.21 ± 1.10)％] in 500,1 000 μg/L IGF-1R inhibitor groups were increased significantly (P ＜ 0.05).Conclusions The expressions of IGF-1R in the cartilage tissue of KBD children and T-2 toxin-poisoned rats under low selenium condition are decreased.T-2 toxin decreases the expression of IGF-1R in chondrocytes,and selenium can partly inhibit the effect of T-2 toxin on IGF-1R.Down-regulation of IGF-1R causes chondrocyte apoptosis,and it may play an important role in KBD chondrocyte apoptosis.