不同直径的培养皿对三维牙周膜细胞团培养的影响

Effect of different diameters of culture dish on three-dimensional periodontal ligament cell pellet cultivation

目的通过一系列方法评价不同直径的培养皿对三维牙周膜细胞团培养的影响。方法对人牙周膜细胞进行分离,培养和鉴定。用含有维生素C的DMEM培养液分别将牙周膜细胞培养于直径为3.5cm、6cm及10cm的培养皿内,2周后,从皿底部刮取细胞层,分别放于离心管中继续培养1周,以形成密实的细胞团。然后,分别通过形态学检测、荧光染色、MTT以及PCR 4种方法来检测不同细胞团里的细胞活性和牙周再生相关基因表达。结果不同细胞团的活性和其它特征差别较明显,其中直径为3.5em培养皿培养出的细胞团相比另外两组有更好的细胞活性和更高的相关基因表达。结论直径为3.5cm的培养皿培养的细胞团相对更适合用来培养三维牙周膜细胞团,应用于牙周组织再生。

Objective The aim of the study was to assess the effect upon various aspects of periodontal ligament cell pellet culture influenced by the diameter of the culture dishes used. Methods Human periodontal ligament （PDL） cells were isolated, cultured and examined. Cell proliferation and adhesion were examined. PDL cells were cuhured in dif- ferent diameters（3.5cm,6cm, lOcm） of the culture dishes respectively in DMEM medium with ascorbic acid. After 2 weeks, the cells in the dishes could form different sheets which could detached from the culture plate, then the sheets was cultured in a tube to produce a compact cell pellet. After the cell pellets were mature, a variety of techniques were applied to assess the viability of periodontal ligament cell pellets, including histological analysis, fluorescent assessment, MTY assay and polymerase chain reaction （PCR） to detect two target gene expressions in the cultured PDL cells. Results The viability and some other special features of PDL cell pellets cultured in culture dishes of different sizes are distinct. PDL cells from culture dishes with small diameters showed high viability and a elevated expression level of two target gene. Conclu- sions The results indicated PDL cell pellets cultured in 3.5cm diameter dishes possessed the highest viability compared with that cultured in 6 cm or 10 cm diameter dishes. Accordingly, a proper diameter for three-dimensional periodontal liga- ment cell pellet probably should be applied to the periodontal tissue reconstruction.