大鼠皮肤成纤维细胞在低密度培养条件下基质金属蛋白酶9和13基因表达的抑制

Suppression of matrix metalloproteinases 9 and 13 genes in rat skin fibroblasts through low density culture

为探索体外促进原代皮肤成纤维细胞转分化为肌成纤维细胞的培养条件及原代皮肤成纤维细胞转分化前后两种状态下经肿瘤坏死因子(tumor necrosis factor alpha,TNF-α)刺激,其MMP基因的表达情况。利用Wistar大鼠真皮分离出的原代成纤维细胞为模型,分别以高密度与低密度将细胞培养于塑料界面,通过Western Blot和q RT-PCR检测肌成纤维细胞的标志物α-平滑肌肌动蛋白(α-SMA)的表达量;同时经TNF-α刺激后,利用q RT-PCR和明胶酶谱检测高密度与低密度两种状态下MMP-9和MMP-13的表达及两种状态下MMP-9的活性。发现大鼠真皮中分离出的原代成纤维细胞在低密度培养条件下,可以有效地转分化为肌成纤维细胞,其标志物α-SMA显著上调;经肿瘤坏死因子刺激,成纤维细胞在低密度培养条件下MMP-9和MMP-13对TNF-α刺激的响应程度较高密度培养时显著降低。初步表明在大鼠真皮(dermis)中,原代成纤维细胞体外转分化为肌成纤维细胞且MMP-9和MMP-13在肌成纤维细胞中下调的现象是细胞密度依赖型。

The purpose of this study was to determine the in vitro condition for trans-differentiation of primary skin fibroblasts, and to prove the concept of MMP silencing in skin fibroblasts after TNF-α challenge.Primary dermal fibroblasts were isolated from Wistar rats and varied degrees of culture density on plastic were applied to establish the in vitro trans-differentiation. Western Blot and qRT-PCR analysis were used to detect the trans-differentiation markers alpha-smooth muscle actin （α-SMA）. The skin fibroblasts of the dermis and the trans-differentiated myofibroblasts were challenged by TNF-ct. Their response to TNF-α stimulation and the expression of MMP-9 and MMP-13 were determined by qRT-PCR analysis. Gelatinolytic zymogram was employed to analyze the activity of MMP-9 after the skin fibroblasts were challenged by TNF-α. Being cultured on plastic at low cell density, the primary fibroblasts from rat dermis were readily trans-differentiated to myofibroblasts and the expression of α-SMA was up-regulated. Compared with high density culture, the fibroblasts cultured at low density lost capacity of MMP-9 and MMP-13 expression, even treated by TNF-α.These results suggested that the concept of MMP-9 and MMP-13 down- regulation in myofibroblasts derived from rats skinis cell density dependent type.