The role of periodontal ligament cells during experimental tooth movement

对人拔牙中分离的牙周韧带（periodontal ligament， PDL）细胞进行克隆分析，建立同源单细胞衍生的PDL细胞培养方法，并阐明PDL细胞在正畸牙齿运动中的作用。人PDL从前磨牙或第三磨牙切除，因正畸原因取出，切碎，置于含有10%胎牛血清的Dalbecco改良鹰培养基（DMEM）培养皿中。10 ~ 14 d后，从PDL组织中延伸出成纤维细胞和圆形细胞。20 d后，圆形细胞完全包围PDL组织，成纤维细胞在圆形细胞外延伸。显微镜下还观察到其他形状的细胞。所有细胞在15 - 20次传代培养中在形态和细胞生长方面都失去了自身的潜能。接下来的实验建立了一种新的从单细胞衍生的PDL细胞中分离克隆的方法。用胶原酶和胰蛋白酶对新鲜的人PDL组织成功分离出单个PDL细胞，细胞损伤最小。虽然上述方法分离的细胞多数为成纤维细胞，但也分离出不同形状的细胞。部分细胞进行细胞角蛋白抗体免疫染色。这些细胞被认为来自马拉塞兹上皮细胞。我们还观察到trkA，一种神经营养因子受体，在Malassez上皮细胞中表达。trkA在PDL原代培养中的表达研究正在进行中。我们也正在分析单细胞衍生细胞的免疫学和分子特性。此外，我们还将单细胞衍生细胞的多个克隆皮下移植到裸鼠体内，以评估其体内分化能力。我们还研究了PDL细胞在大鼠牙齿运动中的作用。这些观察报告已提交给国际期刊，其中一些已被接受。少

Clonal analysis of periodontal ligament (PDL) cells isolated from human extracted teeth was performed for establishing a culture method of homologous, single cell derived PDL cells, and for elucidating roles of PDL cells in orthodontic tooth movement.Human PDL was excised from a premolar or a third molar, which were extracted from orthodontic reasons, minced and placed onto culture dish with Dalbecco's modified eagle medium (DMEM) containing 10 % fetal bovine serum. After 10 to 14 days fibroblastic cells were extended and then round-shaped cells were extended from PDL tissue. After 20 days round-shaped cells surrounded PDL tissue completely and fibroblastic cells were extended outside of round-shaped cells. Other shaped cells were also observed in microscopy. All cells lost their own potentials within 15 to 20 subcultures in morphologically and in cellular growth. Next experiments were done for establishing a novel isolation method for clone from a single-cell derived PDL cell. Treatme … More nts with collagenase and trypsin to fresh human PDL tissue were successfully isolated a single PDL cells with minimum cell damage. Although a majority of cells isolated by above method was fibroblastic cells, different shaped cells were isolated. Some cells were stained immunologically for cytokeratin antibody. These cells were thought to be from Malassez epithelial rest. We also observed that trkA, a neurotrophin receptor, was expressed at epithelial cells in Malassez epithelial rest. Then the study for trkA expression in primary PDL culture is in progress. We are also in progress for analyzing immunological and molecular properties of single-cell derived cells. Furthermore, several clones form single-cell derived cells were transplanted subcutaneously into nude mice for evaluating their differentiation capacities in vivo. We have also investigated the roles for PDL cells during tooth movement in rat. The reports of these observations are submitted to the international journals, and some of them have been accepted. Less