Identification of the unknown periodontal ligament-related molecules

为了确定未知的牙周膜相关分子，我们进行了以下研究。人牙周膜细胞软骨分化诱导和转化生长因子-β-3作用后肌腱/韧带标记物表达的变化多种肌腱/韧带标记物可能在牙周韧带和肌腱/韧带的愈合和维持恒定性中发挥作用。此外，转化生长因子-β(转化生长因子-β)促进肌腱愈合。在本研究中，我们用高效液相色谱法检测了软骨分化诱导和转化生长因子-β-3处理后肌腱/韧带标记物表达的变化。当高效液相聚集成微球，与含有转化生长因子-β3的软骨分化培养液孵育时，未检测到软骨细胞样陷窝的形成。而Periostin、牙周膜相关蛋白-1(PLAP-1)、Biglycan和Decorin基因的表达水平较高，而生长分化因子-…与对照组相比，More-5(GDF-5)和软骨低聚蛋白(CoMP)基因表达水平较低。此外，在整个培养过程中，硬化轴基因的表达没有变化。在单层高效液相培养中，转化生长因子β-3可促进Periostin、PLAP-1、Biglycan、Score和Comp的表达，而抑制GDF5的表达，而该细胞因子对Decorin的表达无影响。这些结果表明，这些肌腱/韧带标记物分子在高效液相色谱中的表达受该条件的调控。这会导致软骨生成。以及转化生长因子-β-3的刺激。HPLC中存在侧群细胞的特征。大量Hoechst 33342染料注入的侧群(SP)中存在各种类型的干细胞。骨髓中的SP细胞表达多种间充质干细胞(MSC)标志物，并高度富含MSC活性。在本研究中，我们确定了从高效液相色谱分离的SP细胞的特性。用Hoechst 33342染色后，用流式细胞术分离SP细胞和非SP(主要群体：MP)细胞。高效液相色谱法中SP细胞的出现频率平均为细胞总数的0.07%。检测SP细胞和MP细胞的基因表达谱、PDL标志物基因表达、细胞增殖活性和多向分化能力。SP细胞中MSC标志基因CD73的表达水平高于MP细胞，而MP细胞中以HPL为特征的胞外分子基因(I/III型胶原、PLAP-1、Periostin、Decorin)表达水平较高。此外，SP细胞比MP细胞具有更高的细胞增殖活性。但向成骨细胞和脂肪细胞分化的活性在两种细胞类型之间没有差异。因此，MSC样细胞可能不会在高效液相中的SP组分中得到富含。较少

To identify the unknown periodontal ligament-related molecules, we performed the following studies.1. The changes of the tendon/ligament markers expression by induction of chondrogenic differentiation and TGF-β3 treatment in human periodontal ligament cells (HPLC)Many tendon/ligament marker molecules may play a role in healing and maintenance of constancy of periodontal ligament as well as tendon/ligament. Further, transforming growth factor-β(TGF-β) stimulates tendon healing. In the present study, we assessed the changes of the tendon/ligament markers expression by induction of chondrogenic differentiation and TGF-β3 treatment in HPLC. When HPLC were aggregated into micropellets and incubated with chondrogenic differentiatin medium with TGF-β3, chondrocyte-like lacunae formation was not detected in HPLC pellets. However, the expression levels of periostin, periodontal ligament associated protein-1 (PLAP-1), biglycan and decorin genes were higher, but growth and differentiation factor- … More 5 (GDF-5) and cartilage oligomeric protein (COMP) gene expression were lower levels, as compared with these pellets cultured with control medium. Further, scleraxis gene expression did not change throughout the culture periods. In monolayer cultures of HPLC, TGF-β3 stimulated expression of periostin, PLAP-1, biglycan, scleraxis and COMP, but suppressed GDF-5 expression, while, this cytokine had no effect on decorin expression. These results demonstrate that the expression of these tendon/ligament marker molecules in HPLC is modulated under the conditions. which induce chondrogenesis. as well as by the TGF-β3 stimulation.2. Characteristics of side population cells existing in HPLC.The various types of stem cells exist in side population (SP) effused strongly Hoechst 33342 dye. The SP cells in bone marrow express various mesenchymal stem cell (MSC) markers and are highly enriched for MSC activity. In the present study, we determined the characteristics of SP cells isolated from HPLC. SP cells and non-SP (main population : MP) cells were isolated from HPLC stained with Hoechst 33342 using flowcytometry. The frequency of SP cells existed in HPLC was mean 0.07% of total cells. Then, the gene expression profile, PDL-marker gene expression, activity of cell proliferation and multi-lineage differentiation were determined in SP cells and MP cells. Expression of CD73, a MSC marker gene, in SP cells was higher levels than that in MP cells, while MP cells expressed higher levels of extracellular molecule genes characterized in HPL (type I/III collagen, PLAP-1, periostin, decorin). Further, SP cells had higher cell proliferation activity than MP cells. However, activities of differentiation into osteoblasts and adipocytes were not different between the 2 cell types. Therefore, MSC-like cells may not be enriched in the SP fraction of HPLC. Less