【摘要】 目的:观察反式维甲酸(TRA)对体外培养大鼠软骨细胞的生长增殖、形态和软骨退变相关基因表达的影响。方法:取出生24hSD大鼠关节处软骨,Ⅱ型胶原酶多次消化后获得原代软骨细胞,取P1代细胞进行实验。对照组软骨细胞于体积分数为10%的胎牛血清(FBS)的高糖DMEM培养基中进行培养,而TRA组是在对照组培养基的基础上加入10μmolTRA进行培养。培养24h后,观察细胞形态变化,MTT法检测两组细胞的增殖情况,Western Blot检测Ⅱ型胶原和MMP13的表达,DMMB法测定细胞中GAG的含量,定量PCR检测ADAMTS5,MMP9,A-can,Sox9,COLX和ALP的表达。结果:TRA组的软骨形态由原来的多角形变成长梭形,且TRA组抑制软骨细胞的增殖,同时下调基质蛋白Ⅱ型胶原,A-can,GAG含量及转录因子Sox9表达,上调MMP13,MMP9,ADAMTS5,COLX及ALP的表达。结论:TRA具有促进软骨退变的作用,可用于建立体外软骨退变模型。更多还原

【Abstract】 Objective:To investigate the effects of trans retinoic acid(TRA)on the chondrocyte’proliferation,morphology,and the expression levels of cartilage degeneration associated gene in rat chondrocytes in vitro.Methods:Chondrocyte was cultured from articular cartilages of newly born(within 24 h)SD rats,and digested with typeⅡ collagenase.Chondrocyte at passage one was used for the experiment.The chondrocyte was cultured with H-DMEM medium containing10% FBS in Control group,and H-DMEM medium containing 10% FBS plus 10μmol trans retinoic acid in TRA group.The morphology change was investigated under phase contrast microscope after chondrocyte were treated with trans retinoic acid for 24 h.Chondrocytes’ proliferation was detected by MTT method,and the protein expression levels of typeⅡcollagen and MMP13 were examined by Western-blot.The GAG contents in chondrocytes were detected by DMMB method.The mRNA levels of ADAMTS5,MMP9,A-can,Sox9,ALP and ColX were detected by real-time PCR.Results:The cell morphology was changed from polygon to fibro-like chondrocytes in TRA group compared to control group,and the proliferation of chondrocytes in TRA group were inhibited.The results showed that the expression levels of typeⅡcollagen,A-can,GAG and Sox9 were decreased while the expression levels of MMP13,MMP9,ADMATS5,COLX and ALP were up-regulated in TRA group compared with control group.Conclusion:It was proved that TRA could be used as an efficient inducer in establishing OA model in vitro which induces chondrocytes’ degeneration.更多还原