牙周膜肌成纤维细胞功能特点的体外研究

Functions of human periodontal myofibroblast in vitro

目的体外实验研究牙周膜肌成纤维细胞(MFB)的作用特点。方法体外培养人牙周膜成纤维细胞(hPDLFs),采用转化生长因子-β1(TGF-β1)诱导成纤维细胞向MFB转化。MFB为实验组,以hPDLFs为对照,连续培养两组细胞,按0、12、24、48、72 h共5个时间点终止培养收样。免疫细胞化学检测MFB标记物α-平滑肌肌动蛋白(α-SMA)的表达情况,检测实验组纤维粘连蛋白(FN)以明确细胞间接触作用情况。逆转录聚合酶链式反应(RT-PCR)检测α-SMA mRNA、胶原(Col)ⅠmRNA、ColⅢmRNA的表达情况,免疫印迹法检测α-SMA、ColⅠ蛋白的表达情况,用以比较两组细胞分泌细胞外基质情况。结果实验组α-SMA持续稳定表达,从0 h开始表达均显著高于对照组(P<0.001);细胞间FN阳性表达,提示MFB之间有细胞突触相互连接。从24 h开始,实验组ColⅠ、ColⅢ的表达均显著高于对照组(P<0.001)。结论牙周膜MFB持续高表达α-SMA并且可能通过FN相互作用。MFB具有大量分泌细胞外基质的能力。

Objective To investigate the functions of human periodontal myofibroblast （MFB） in vitro. Methods Human periodontal fibroblast （hPDLFs） was eul^red and induced to MFB by transforming growth factor-β1 （TGF-131）. MFB was denoted as the experimental group, whereas the hPDLFs was the control group. The groups were continuously cultured and harvested at 0, 12, 24, 48, and 72 h. The MFB marker a-smooth muscle actin （ct-SMA） was examined by immunocytochemistry. The expression of fibronectin （FN） between MFB was examined by immunocytochemistry to detect the MFB contact relationship. The mRNA expression levels of α-SMA, collagen （Col） Ⅰ, and Col Ⅲ were measured by reverse transcription-polymerase chain reaction （RT-PCT） to analyze extracellular matrix secretion. The protein expression levels of α-SMA and Col I were also assessed by Western blot. Results The experimental group had significantly higher α-SMA expression than the control group at 0 h （P〈0.001）. A positive expression of FN was found between MFB. The experimental group had significantly higher expression levels of Col I and Col Ⅲ than the control group at 24 h （P〈0.001）. Conclusion Human periodontal MFB pre- sents a continuous, high expression of a-SMA. MFB could interact through FN. MFB is significantly capable of extracelhilar matrix secretion.