T-2毒素在正常和低营养状态下对大鼠膝关节骺板及干骺端骨组织生长发育的影响

目的 观察T-2毒素在正常与低营养状态下对大鼠膝关节骺板及股骨、胫骨干骺端骨组织生长发育的影响,为探讨大骨节病的可能致病因素及早期干预治疗提供实验依据.方法 3周龄Wistar大鼠90只,雌雄各半,体质量60～70 g,将大鼠按体质量、性别随机分为3组:对照组(普通饲料喂养)、T-2毒素+普通饲料组、T-2毒素+低营养饲料组,每组30只.T-2毒素1.0 mg/kg,每周5次,灌胃给药,连续4周.观察大鼠毛发、活动度、体质量变化.在第1、2、4周后,取左侧膝关节骺软骨和股骨、胫骨干骺端(股骨远端、胫骨近端),HE和Masson染色后,光镜下观察大鼠骺板软骨细胞形态和骺板基质中胶原的含量;应用Image-Pro Plus 6.0软件,分析胫骨干骺端骨小梁体积分数.结果 对照组大鼠毛发色泽光亮,活动良好,T-2毒素+普通饲料组及T-2毒素+低营养饲料组大鼠毛发色泽暗,活动减少.对照组、T-2毒+普通饲料组、T-2毒素+低营养饲料组大鼠在第1、2、4周的体质量分别为[(81.0±6.2)、(79.0±5.1)、(77.0±7.5)g],[(101.8±6.7)、(97.0±6.8)、(93.0±53)g],[(151.1±15.7)、(126.5±11.9)、(106.5±11.5)g];体质量在第2、4周组间比较差异有统计学意义(F值分别为9.72、41.65,P均＜0.05),其中第4周组间两两比较差异有统计学意义(P均＜0.01).光镜下,在第2周,T-2毒素+普通饲料组、T-2毒素+低营养饲料组大鼠骺板肥大带软骨细胞出现骺软骨细胞坏死;在第4周,细胞坏死程度加重,其中T-2毒素+低营养饲料组细胞坏死较为明显.T-2毒素+低营养饲料组大鼠骺板基质中胶原着色明显变淡,表明基质中胶原含量明显减少.图像分析显示,大鼠胫骨干骺端骨小梁体积分数在第2、4周,组间比较差异有统计学意义(F值分别为5.4、29.6,P＜0.05);其中T-2毒素+低营养饲料组[(0.55±0.12)%、(0.21±0.08)%]与对照组[(0.67±0.09)%、(0.51±0.14)%]比较差异有统计学意义(P均＜0.01).结论 在正常营养状态下,T-2毒素可导致大鼠骺板软骨肥大层细胞坏死,骺板胶原含量降低,干骺端骨小梁形成障碍;在低营养状态下,T-2毒素可导致大鼠骺板细胞坏死和于骺端成骨障碍更为显著,但这种表现是否与大骨节病的发生有关,还有待研究.

Effect of T-2 toxin on growth and development of rat knee epiphyseal plate and metaphyseal bone in normal and low nutritional status

Objective To observe the effect of T-2 toxin on growth and development of rat epiphyseal plate of left knee and metaphyseal bone of femur and tibia in normal and low nutritional status, to find out possible pathogenic factors of Kashin-Beck disease and provide experimental basis for early intervention. Methods Ninety 3-week-old Wistar rats, weighing 60 - 70 g, were randomly divided into three groups: control group(general feed), T-2 toxin + general feed group, T-2 toxin + low nutrition feed group, thirty rats in each group with equally sex ratio. T-2 toxin (1.0 mg/kg) was administered orally 5 times a week via a gavage needle for 4 weeks. The change of hair, activity and body weight was observed. After 1, 2, 4 weeks, the epiphyseal plate of left knee and metaphyseal bone of femur and tibia (including distal femur and proximal tibia) were collected. Specimens were processed with HE and Masson staining. The morphology of chondrocytes and matrix collagen content in epiphyseal plate was observed. Trabecular bone volume fraction in tibial metaphyseal bone was analyzed by Image-Pro Plus 6.0 software. Results In the control group, rats were in good movement and hair with light, but in T-2 toxin + general feed group and T-2 toxin + low nutrition feed group, rats were found with reduced activities and hair with dark color. Body weights(g) of the control group, the T-2 toxin + general feed group and the T-2 toxin + low nutrition feed group were 81.0 ± 6.2, 79.0 ±5.1, 77.0 ± 7.5, respectively, by the end of first week; 101.8 ± 6.7, 97.0 ± 6.8, 93.0 ± 5.3, respectively, by the end of second week; 151.1 ± 15.7, 126.5 ± 11.9, 106.5 ± 11.5, respectively, by the end of fourth week. There was significant difference in groups by second week and the fourth week (F = 9.72, 41.65, all P ＜ 0.05 ). There was significant difference among multi-groups by the fourth week(all P ＜ 0.01 ). Under light microscope, at the second weeks, coagulative necrosis of chondrocytes was found in hypertrophic zone in the two groups with T-2 toxin; at the fourth weeks, cell necrosis increased. Masson staining showed collagen staining in the two groups with T-2 toxin significantly turned to clear pale coloration, indicating that the collagen matrix was significantly reduced. Image analysis showed there was significant difference in groups at the second and fourth week(F= 9.72, 41.65, all P＜ 0.05)in tibial metaphyseal trabecular bone volume fraction. There was significant difference between T-2 toxin + low nutrition feed group[(0.55 ± 0.12)％, (0.21 ± 0.0)％] and control group[(0.67 ± 0.09)％, (0.51 ± 0.14)％] by the second and fourth week(all P ＜ 0.01 ). Conclusions Under normal nutritional status, T-2 toxin can induce hypertrophic epiphyseal cartilage necrosis, collagen content decreased in epiphyseal plate, metaphyseal trabecular bone formation disorders; in the low nutritional status, T-2 toxin can lead to rat epiphyseal necrosis and significant metaphyseal bone disorder, but whether the performance is related to Kaschin-Beck disease needs to be studied further.