Cathepsin K在人软骨细胞体外培养去分化过程中的表达研究

Cathepsin K expression in dedifferentiation of human chondrocytes cultured in vitro

目的通过基因芯片技术研究在体外培养的去分化人软骨细胞的基因改变，从中筛选出目标基因，并对其在各代软骨细胞中的表达进行研究。方法取体外培养的P1代和去分化的P4代软骨细胞进行8300点基因芯片的DNA微阵矩杂交，筛选出差异最明显的基因，并通过RT-PCR和Western-blot方法检测各代软骨细胞中该基因的表达水平。结果体外培养的P1、P4代软骨细胞间共140个基因差异表达，其中Cathepsin K基因表达差异水平最明显，P4与P1的比值平均为29．72，RT-PCR显示在596bp的条带处，随传代次数增加逐渐增亮，半定量分析P4／P1为3．21，差异有统计学意义（P〈0．05）。Western-blot检测显示在相对分子质量为40000的蛋白条带随体外传代蛋白浓度增加，灰度分析P4／P1为3．39，差异有统计学意义（P〈0．05）。结论基质降解半胱氨酸蛋白酶Cathepsin K在去分化软骨细胞中表达强烈，提示其与软骨细胞去分化过程中的基质降解有关。

Objective To study the expressions of dedifferentiation-related genes in human chondrocytes cultured in vitro by microarray analysis, and the expression of the target gene selected in the process of dedifferentiation. Methods The differences in gene expressions in normal （P1） and dedifferentiated （P4） human chondrocytes cultured in vitro were analyzed by microarray with a set of 8300 human genes. Then the most dedifferentially expressed gene was analyzed by RT-PCR and Western Blot. Results Of all the different gene expressions between normal and dedifferentiated chondrocytes, a total of 140 differentially expressed genes were identified. The gene, cathepsin K, was found to be the most differentially expressed one. Its ratio of P4 to P1 was about 29.72. Up-regulations of cathepsin K mRNA and protein expression were observed in the dedifferentiation process of chondrocytes cultured in vitro by RT-PCR and Western-blot, and their ratios of P4 to P1 were about 3.21 and 3.39 respectively. Conclusion Strong expression of cathepsin K, the cysteine proteinase, in dedifferentiated chondrocytes suggests that it may play an important role in matrix degradation in the dedifferentiation process.