【摘要】 目的通过检测孕期地塞米松暴露（PDE）致雌性胎大鼠软骨病理变化和地塞米松处理软骨细胞后基因表达的影响,探讨地塞米松影响软骨发育的可能机制。方法 （1）在体实验:健康Wistar雌性大鼠自然受孕,PDE组于孕9～20 d（GD9～20）每天1次sc给予地塞米松0.2 mg·kg_-1。GD20处死母鼠,收集雌性胎大鼠软骨样本。HE和番红O染色观察软骨组织形态;实时定量PCR技术检测软骨组织的蛋白聚糖、木糖基转移酶Ⅰ（XylT-Ⅰ）、葡萄糖醛酸转移酶Ⅰ（GlcAT-Ⅰ）、半乳糖基转移酶Ⅰ（GalT-Ⅰ）、胰岛素样生长因子1(IGF_-1)、IGF_-1受体(IGF_-1R)、蛋白激酶B（AKT）和活化蛋白1(AP_-1)mRNA表达;免疫组化法检测XylT-Ⅰ,GlcAT-Ⅰ,GalT-Ⅰ,IGF_-1,AKT和AP_-1蛋白表达。（2）离体实验:另取GD20 Wistar雌性胎大鼠膝关节制备软骨细胞,分别用地塞米松(100和500 nmol·L_-1),IGF_-1(100μg·L_-1),IGF_-1R抑制剂NVP-AEW541(1μmol·L_-1),AKT抑制剂MK-2206(1μmol·L_-1)和AP_-1抑制剂SR_-11302(1μmol·L_-1)处理24 h。DMB染色法检测软骨细胞上清糖胺聚糖（GAG）含量;实时定量PCR法检测软骨细胞XylT-Ⅰ,GlcAT-Ⅰ,GalT-Ⅰ,IGF_-1R,AKT和AP_-1 mRNA表达;Western蛋白印迹法检测XylT-Ⅰ,GlcAT-Ⅰ和GalT-Ⅰ蛋白表达水平。结果 （1）在体实验:HE和番红O染色结果显示,PDE组雌性胎大鼠关节软骨细胞数减少,表层软骨细胞排列紊乱,软骨基质染色着色度浅,着色不均一;实时定量PCR和免疫组化结果显示,与正常对照组相比,PDE雌性胎大鼠膝关节组织的XylT-Ⅰ,GlcAT-Ⅰ,GalT-Ⅰ和IGF_-1/AKT/AP_-1通路相关蛋白和基因表达降低（P<0.05,P<0.01）。（2）离体实验:与正常对照组相比,地塞米松组雌性胎大鼠软骨细胞上清液GAG含量降低（P<0.05）,XylT-Ⅰ,GlcAT-Ⅰ,GalT-Ⅰ和IGF_-1/AKT/AP_-1通路相关蛋白的mRNA表达水平降低（P<0.05）;与地塞米松500 nmol·L_-1组相比,IGF_-1可使上述基因mRNA表达水平升高（P<0.05,P<0.01）。与正常对照组相比,IGF_-1 100μg·L_-1处理组XylT-Ⅰ,GlcAT-Ⅰ,GalT-Ⅰ蛋白和mRNA表达水平升高,IGF_-1R/AKT/AP_-1各抑制剂处理组XylT-Ⅰ,GlcAT-Ⅰ和GalT-Ⅰ蛋白和mRNA表达水平降低（P<0.05,P<0.01）;与单独SR_-11302组相比,SR_-11302+IGF_-1处理后,XylT-Ⅰ,GlcAT-Ⅰ和GalT-Ⅰ蛋白和mRNA表达水平变化不明显。结论孕期暴露PDE可致雌性胎大鼠软骨发育不良,其发生机制可能与IGF_-1/AKT/AP_-1通路低基础活性介导糖基转移酶低表达所致软骨基质合成不足有关。更多还原

【Abstract】 OBJECTIVE To investigate the effect of prenatal dexamethasone exposure（PDE）on chondrogenesis of female fetal rats and those of dexamethasone treatment on gene expression of chondrocytes,and to explore the underlying mechanism.METHODS（1）In vivo,Wistar female rats were conceived naturally,and the PDE group was sc given dexamethasone 0.2 mg·kg^-1 once a day for9-20 d after gestation（GD9-20）.Pregnant rats were sacrificed on GD20,and cartilage samples of female fetal rats were collected.The morphology of cartilege was observed by HE and saffranin O staining.The expressions of aggrecan,O-xylosyltransferaseⅠ（Xyl T-Ⅰ）,β1,3-glucuronosyltransferaseⅠ（Glc AT-Ⅰ）,β1,4-galactosyltransferase 7,（GalT-Ⅰ）,insulin-like growth factor-1（IGF-1）,IGF-1 receptor（IGF-1R）,protein kinase B（AKT）and activator protein 1（AP-1）mRNA in cartilage tissues were examined by realtime quantitative PCR（RT-PCR）.The expressions of Xyl T-Ⅰ,Glc AT-Ⅰ,Gal T-Ⅰ,IGF-1,AKT and AP-1were detected by immunohistochemistry.（2）In vitro,GD20 Wistar female fetal rats were used to extract chondrocytes from fetal knee joints.Dexamethasone(100 and 500 nmol·L^-1),IGF-1(100μg·L^-1),IGF-1R inhibitor NVP-AEW541(1μmol·L^-1),AKT inhibitor MK-2206(1μmol·L^-1)and AP-1 inhibitor SR-11302(1μmol·L^-1)were treated for 24 h.The content of glycosaminoglycan（GAG）in the cell supernatant was detected by DMB staining.The expressions of XylT-Ⅰ,GlcAT-Ⅰ,GalT-Ⅰ,IGF-1R,AKT and AP-1mRNA were detected by RT-PCR.The expressions of XylT-Ⅰ,GlcAT-Ⅰand GalT-Ⅰprotein were detected by Western blotting.RESULTS（1）In vivo the results of HE and saffranin O staining showed that the number of articular chondrocytes of female fetal rats in PDE group was reduced,the arrangement of surface chondrocytes was disorderly,and staining of cartilage matrix became lighter and uneven.The protein and m RNA expression of cartilage,Xyl T-Ⅰ,Glc AT-Ⅰ,Gal T-Ⅰand proteins related to IGF-1/AKT/AP-1 pathway were reduced in PDE group compared with normal control group（P<0.05,P<0.01）.（2）In vitro compared with the normal control group,the content of GAG in the supernatant of chondrocytes of female fetal rats was decreased（P<0.05）,and the mRNA expressions of XylT-Ⅰ,GlcAT-Ⅰ,GalT-Ⅰand proteins related with IGF-1/AKT/AP-1 pathways were deceased（P<0.05）.Compared with the dexamethasone 500 nmol·L^-1group,IGF-1 100μg·L^-1increased the mRNA expression levels of the above proteins（P<0.05,P<0.01）.Compared with the normal control group,the protein and mRNA expressions of XylT-Ⅰ,GlcAT-Ⅰand GalT-Ⅰin IGF-1 treatment group were increased,while those in IGF-1R/AKT/AP-1 inhibitor treatment groups were decreased（P<0.05,P<0.01）.Compared with the SR-11302 group,there was no significant change in the protein and m RNA expression levels of Xyl T-Ⅰ,GlcAT-Ⅰand GalT-Ⅰafter co-treatment with IGF-1.CONCLUSION PDE induceds chondrodysplasia in female fetal rats,which is related to the insufficiency of cartilage matrix synthesis induced by decreasing expression of glycosyltransferases mediated by low basic activity of IGF-1/AKT/AP-1 pathway.更多还原