维甲酸调控IGF-2基因对软骨细胞促凋亡的作用

目的 探讨维甲酸对软骨细胞凋亡的影响及细胞内IGF-2的表达规律. 方法 1月龄雄性中国白兔1只,体重约500 g.取兔双膝关节股骨髁软骨,采用胰酶消化法体外培养白兔软骨细胞.取第2代软骨细胞,培养液内加入终浓度为1×10-6 mol/L的全反式维甲酸(all-trans-retinoic acid,ATRA)25μL(实验组),作用24 h;对照组加入25μL DMEM液作为对照.采用细胞免疫组织化学法观察软骨细胞中Ⅱ型胶原的分泌变化,流式细胞仪检测软骨细胞凋亡率,RT-PCR半定量分析软骨细胞中IGF-2 mRNA的表达,Western blot印迹杂交鉴定软骨细胞中IGF-2蛋白质表达. 结果 实验组ATRA抑制了软骨细胞中Ⅱ型胶原的表达.流式细胞仪检测实验组软骨细胞凋亡率为21%±2%,较对照组(5%±1%)增加了5倍;RT-PCR检测实验组IGF-2 mRNA的表达为0.2±0.1,较对照组(0.8±0.2)降低75%;Western blot印迹杂交分析发现,实验组软骨细胞IGF-2的表达为0.3±0.1,较对照组(0.7±0.2)降低了57%;各指标组间比较差异均有统计学意义(P＜0.05). 结论 维甲酸可能通过抑制软骨细胞靶基因IGF-2的表达,负性调节软骨细胞分泌和增殖功能,促进软骨细胞凋亡.

PRO-APOPTOTIC EFFECT OF RETINOIC ACID ON CHONDROCYTE THROUGH REGULATION ON GENE EXPRESSION OF IGF-2

OBJECTIVE: To investigate the effect of retinoic acid (RA) on cell apoptosis and gene regulation of IGF-2 in chondrocyte. METHODS: One 1-month-old Chinese rabbit weighted 500 g was used in this experiment. The chondrocyte from rabbit knee were cultured by enzyme digestion. Twenty-five microL all-trans-retinoic acid (ATRA) (1 x 10(-6) mol/L) were added in the media of cultured chondrocyte for 24 hours as experimental group, while 25 microL DMEM were added as control group. The secretion of collagen II was observed by immunohistochemistry method, cell apoptosis was detected by flow cytometry, IGF-2 mRNA and protein expression in chondrocyte were detected by RT-PCR and Western blot analysis. RESULTS: The expression of collagen II was down-regulated by ATRA in the experimental group. The cell apoptosis in chondrocyte exposed to ATRA at 1 x 10(-6) mol/L was 21% +/- 2%, which increased 5 times compared with the control group (5% +/- 1%). The IGF-2 mRNA and protein level in the experimental group were decreased 75% and 57%, respectively, compared to the control group. There were significant difference between the experimental group and control group in each index (P < 0.05). CONCLUSION: RA may down-regulate the secretion and cell proliferation, but up-regulate the cell apoptosis in chondrocyte. The apoptotic effect may carry out through inhibiting the IGF-2 expression of chondrocyte.