全反式维甲酸对骺软骨细胞生物学行为及其功能影响的研究

Effects of All-Trans Retinoic Acid on the Biological Behavior and Function of Epiphyseal Chondrocytes

该研究主要探讨了体外高浓度全反式维甲酸(all-trans retinoic acid,ATRA)对SD大鼠骺软骨细胞生物学性状和功能的影响以及体内ATRA对SD大鼠胫骨生长板的影响。以SD大鼠骺软骨细胞为研究对象、ATRA为干预因素,采用CCK-8、细胞流式术、HE染色、Annexin V-FITC细胞凋亡流式检测术、Hoechst染色、细胞划痕、Transwell实验分别评估ATRA处理后细胞的增殖、周期、形态、凋亡及迁移情况,Western blot检测蛋白聚糖、Ⅱ型胶原、X型胶原等相关功能蛋白的变化;以3周雄性SD大鼠为实验对象,分为对照组、60 mg/kg·d ATRA组、80 mg/kg·d ATRA组,进行10天连续ATRA灌胃处理,测量每只SD大鼠灌胃第1天、第10天的头尾长,处理10天后对胫骨生长板进行HE染色。结果表明,ATRA作用SD大鼠骺软骨细胞后,增殖能力减弱且细胞周期被阻滞在S期(P<0.01),细胞形态由三角形、多边形变为长条状,凋亡的发生增多(P<0.01),迁移能力受到抑制(P<0.05)以及Western blot结果显示蛋白聚糖、Ⅱ型胶原、X型胶原等功能相关蛋白较对照组表达均明显降低(P<0.01);对SD大鼠进行ATRA灌胃处理后,与对照组比较,60 mg/kg·d ATRA组和80 mg/kg·d ATRA组的头尾长均变短(P<0.01);胫骨生长板HE染色显示,ATRA灌胃组的生长板变窄甚至闭合。该研究证实了体外高浓度ATRA能够对SD大鼠骺软骨细胞的增殖、迁移起抑制作用,同时能够诱导凋亡,降低相关功能蛋白的表达,在SD大鼠体内证实,过量ATRA可影响生长板软骨内成骨过程,最终使生长板部分或全部提前闭合,进而影响SD大鼠身长的增长。

This study investigated the effect of high concentration of ATRA(all-trans retinoic acid)in vitro on the biological characteristics and function of epiphyseal cartilage cells in SD rats and the effect of ATRA in vivo on tibia growth plate in SD rats.The epiphyseal chondrocytes of SD rats were studied and ATRA was used as the intervention factor.CCK-8,cytometry,HE staining,Annexin V-FITC apoptosis flow cytometry,Hoechst staining,cell scratches and Transwell experiment were used to evaluate the cell proliferation,cycle,morphology,apoptosis and migration after ATRA treatment.The changes of proteoglycan,collagen-Ⅱ and collagen-X related functional proteins were detected by Western blot.Male SD rats at 3-weeks were divided into control group,60 mg/kg·d ATRA group and 80 mg/kg·d ATRA group for continuous gavage for 10 days.The length of the head and tail of each SD rat was measured on the first day and the tenth day of gavage,and the tibia growth plate was stained with HE after 10 days of treatment.The results showed that,compared with the control group,the proliferation ability was weakened and the cell cycle was blocked in S phase(P<0.01).Compared with the control group,the occurrence of apoptosis increased(P<0.01)and the migration ability was inhibited(P<0.05).Western blot results showed that the expressions of proteoglycan,collagen-Ⅱ and collagen-X were significantly lower than those of the control group(P<0.01).Compared with the control group,the head and tail length of the ATRA treatment group were all shorter(P<0.01).HE staining of tibia growth plate showed that the growth plate in the ATRA gavage group was narrowed or even closed.This study confirmed that high concentration of ATRA could inhibit cell proliferation,inhibit cell migration,induce cell apoptosis,and reduce the expression of related functional proteins.Excessive ATRA has been shown to affect the endochondral osteogenesis of growth plates in SD rats.Finally,ATRA caused the epiphyseal plate to be partially or completely closed in advance,thus affecting the growth of body length of SD rats.