软骨细胞老化过程中胞外基质表达水平的改变

Age-related changes of the expression of extracellular matrix in in vitro cultured chondrocytes

目的研究猪耳软骨细胞在体外培养传代过程中,细胞老化相关基因和胞外基质相关基因表达水平的改变。方法取体外培养猪耳软骨细胞,通过光镜,对各代细胞的形态学变化进行观测。RT-PCR检测各代细胞中bcl-2、端粒酶、Ⅰ型胶原和Ⅱ型胶原在mRNA表达水平上的改变。结果体外培养软骨细胞随着老化的进程,逐渐显现衰老和凋亡的特征。凋亡抑制基因bcl-2和控制细胞分裂的端粒酶的表达显著下降(P<0.01)。软骨细胞的特征性基质分子Ⅱ型胶原在第3代时表达水平有显著下降(P<0.01),降至原代细胞的5.47%±1.04%,在第4代几乎不再表达。而Ⅰ型胶原在第5代时,有显著上升(P<0.01);在第9代时又显著下降(P<0.01)。结论猪软骨细胞经体外培养,由第4代起逐渐发生去分化,丧失软骨细胞的表型。同时,细胞的增殖分裂能力亦逐步减退,细胞凋亡现象明显增加。

Objective To investigate the gene expression changes of senescence associated proteins and extracellular matrix(ECM) in in vitro cultured porcine chondrocytes during cell aging. Methods The morphological assessment of chondrocytes in different passages was proceeded by light microscopy. The mRNA transcriptional regulation of bcl 2, telomerase, pro collagen Type Ⅰ and Type Ⅱ was examined by RT PCR. Results The chondrocytes in vitro became aged with increased culture time, which demonstrated decreased gene expression of bcl 2 and telomerase. The expression of pro collagen Type Ⅱ , the marker ECM molecule of chondrocytes, decreased significantly at passage 3(P< 0.01) and almost did not express at passage 4. Additionally, gene expression of pro collagen Type Ⅰ increased significantly at passage 5(P< 0.01) and then decreased at passage 9(P< 0.01). Conclusion The dedifferentiation of porcine chondrocytes cultured in vitro begins at passage 4 with the loss of chondrocyte phenotype. At the same time, cells gradually stop proliferation and become apoptotic.