牙周膜干细胞膜片的构建及其生物学特性的研究

Construction of periodontal ligament stem cell sheet and characterization of its biological properties

目的:探讨牙周膜干细胞膜片的构建及其生物学特性。方法:采用胶原酶消化人牙周膜组织,获得牙周膜干细胞(Periodontal ligament stem cells,PDLSCs),经体外鉴定、扩增后构建PDLSCs细胞膜片,并通过倒置显微镜、HE染色、扫描电镜(SEM)对PDLSCs细胞膜片形态学进行检测。此外,细胞膜片厚度及膜片中细胞密度也被检测。结果:PDLSCs成功被分离、培养、鉴定,并且PDLSCs体外培养2周后,获得白色膜状PDLSCs细胞膜片。倒置显微镜下观察显示,细胞复层生长,细胞呈典型的纺锤状。体式显微镜下观察显示,细胞与细胞之间紧密连接。组织学观察显示,细胞与细胞之间存在着大量细胞外基质(Extracellular matrix,ECM)。扫描电镜下观察膜片表面显示,细胞在膜片上充分伸展,细胞之间紧密连接。PDLSCs细胞膜片厚度由起初的(48±3.2)μm到3周后的(69±3.4)μm,但在第2周细胞厚度(64±3.3)μm,变化最明显。细胞膜片中的细胞密度检测显示连续培养3周后仍然有大量具有活性的细胞存在,然而细胞密度在第2周时最大。结论:本研究证明我们已经成功探索出一套稳定、可靠的PDLSCs细胞膜片构建方法,为利用PDLSCs细胞膜片修复、再生牙周组织及牙周缺损提供了一定的技术保证。

AIM： To construct periodontal ligament stem cell（PDLSC） sheet and characterize the biological property of the constructed sheet.METHODS： PDLSCs were obtained by collagenase digestion of human periodontal ligament tissues.After identification,PDLSCs were subcultured to construct PDLSC sheet.Cell sheet was examined by inverted microscope,HE staining and scanning electron microscope.PDLSC sheet thickness and cell density were assayed.RESULTS： After 2 weeks in vitro culture,a white membranaceous PDLSC sheet formed.Under inverted microscope,it was shown that PDLSCs grew in multilayer and retained their fibroblastic spindle shape.Histological examination indicated abundant extracellular matrix（ECM） existed among the cells.By scanning electron microscopy,it was found that PDLSCs expanded adequately on the surface of PDLSC sheet and PDLSCs connected closely.Cell sheet thickness varied from an initial 48±3.2 μm to 69±3.4μm at 3 weeks.The cells in the cell sheet were shown to be viable during the 3-week culture period,with the maximum cell number in the second week.CONCLUSION： We successfully established a stable and reliable method for constructing PDLSC sheet,which provided technological foundation for future studies in regeneration and repair of periodontal tissue destruction.