Takeuchi H, Kubota S, Murakashi E, Fukada T, Hashimoto S, Takigawa M, Numabe Y
Department of Periodontology, School of Life Dentistry at Tokyo, Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo, Japan.
J Periodontal Res. 2009 Apr;44(2):161-9. doi: 10.1111/j.1600-0765.2008.01093.x. Epub 2008 Feb 6.
Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro.
Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription-polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy.
In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization.
The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue.
结缔组织生长因子（CCN2/CTGF）在伤口愈合及牙周组织细胞外基质调节中发挥重要作用。然而，转化生长因子-β1水平改变与CCN2/CTGF之间的功能关系在人牙龈成纤维细胞和牙周膜细胞中尚未得到广泛研究。本研究在体外调查了转化生长因子-β1对人牙龈成纤维细胞和牙周膜细胞中CCN2/CTGF基因表达的影响。
从正常牙周组织分离细胞，并在含10%胎牛血清的杜氏改良 Eagle 培养基/F12中培养。亚汇合细胞在血清饥饿条件下维持24小时，然后用含0.5%胎牛血清的杜氏改良 Eagle 培养基/F12（对照）以及0.1、1、5或10 ng/mL的转化生长因子-β1处理24、48或72小时。通过逆转录-聚合酶链反应测量转化生长因子-β1对CCN2/CTGF mRNA表达的影响。使用酶联免疫吸附测定法定量分析CCN2/CTGF蛋白。利用免疫荧光显微镜观察CCN2/CTGF蛋白在人牙龈成纤维细胞和牙周膜细胞中的亚细胞分布。
在人牙龈成纤维细胞和牙周膜细胞中，在有转化生长因子-β1存在的情况下，CCN2/CTGF mRNA和CCN2/CTGF蛋白的表达均以剂量和时间依赖性方式显著增加。此外，免疫荧光分析表明，对CCN2/CTGF的免疫反应性显示出蛋白质定位的颗粒状模式。
转化生长因子-β1在人牙龈成纤维细胞和牙周膜细胞中诱导CCN2/CTGF mRNA和蛋白的表达。这些结果表明CCN2/CTGF在伤口愈合和牙周组织再生中发挥重要作用。
