应用大鼠血清建立体外软骨细胞退变模型

目的:应用大鼠血清建立大鼠体外软骨细胞退变模型。方法:取出生24 h SD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,取原代细胞进行实验。软骨细胞分别用含10%胎牛血清的DMEM(对照组)、含50 ng/mL 白细胞介素(IL)-1β+10%胎牛血清的DMEM(IL-1β组)、含2.5%大鼠血清的DMEM(2.5%血清组)及含5.0%大鼠血清的DMEM(5.0%血清组)中培养。培养24 h后,观察细胞形态变化,MTT法检测各组细胞的增殖情况,蛋白质印迹法检测增殖细胞核抗原的表达和Ⅱ型胶原及MMP-13的表达,实时定量PCR检测退变相关基因ADAMTS5、MMP-9、Aggrecan和SOX-9的表达。结果:两血清组和IL-1β组的软骨形态均由原来的多角形变成长梭形,且两血清组和IL-1β组均促进软骨细胞的增殖,下调转录因子SOX-9和上调基质降解酶MMP-13、MMP-9、ADAMTS5的表达。结论:大鼠血清具有促进软骨退变的作用,可用于建立体外软骨退变模型。
Abstract: Objective: To establish a model of chondrocyte degeneration in vitro. Methods: Chondrocytes were isolated from articular cartilages of newly born SD rats by digestion with typeⅡ collagenase. The chondrocytes were cultured with H-DMEM medium containing 10%FBS, 50 ng/mL IL-1β+10%FBS, 2.5% rat serum and 5% rat serum, respectively; and the chondrocytes at passage one were used in the experiments. The morphology changes were investigated under phase contrast microscope after chondrocytes were treated with rat serum and IL-1β. Proliferation of chondrocytes was detected by MTT method. The protein expression levels of PCNA, typeⅡ collagen and MMP-13 were examined by Western blotting. The levels of ADAMTS5, MMP-9, Aggrecan and SOX-9 mRNA were detected by real-time PCR. Results: The cell morphology was changed from polygon to spindle in both rat serum groups and IL-1β group, and the proliferation of chondrocytes in these groups was much higher than that in control group. The results showed that the expression levels of typeⅡ collagen, Aggrecan and SOX-9 decreased while the expression levels of MMP-13, MMP-9 and ADMATS5 were up-regulated in rat serum and IL-1β-treated groups compared with control group. Conclusion: The results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro. Key words: Interleukin-1beta Chondrocytes Rats, wistar Serum Cell proliferation Cartilage diseases Cells, cultured