血清化血小板裂解液替代胎牛血清对牙周膜细胞膜片形成的影响

Replacement of fetal bovine serum with serum- converted platelet lysate in the preparation of periodontal ligament cell sheet

目的:观察血清化血小板裂解液(PLS)替代胎牛血清(FBS)对牙周膜细胞(PDLCs)体外扩增和膜片形成的影响。方法:首先从人全血白膜中分离出血小板富集物,利用冻融法与钙离子激活法联合制备PLS;然后分别用含100 g/L的PLS(实验组)和100 g/L FBS(对照组)培养液对PDLCs进行体外培养和成膜诱导,分别采用MTT、RT-PCR和HE染色法检测两组细胞的增殖、成膜以及膜片中成膜相关基因/蛋白的表达水平。结果:MTT检测结果显示,与对照组相比,实验组可支持PDLCs的体外扩增,二者的生长曲线无差异(P>0.05);成膜诱导培养10 d后,RT-PCR检测显示,实验组的膜片中除Collagen I的表达水平显著低于对照组(P>0.05)外,另两种成膜相关基因(Integrinβ1、Fibronectin)的表达水平与对照组相比均无显著差异(P>0.05);组织学观察显示,两组细胞膜片的形态和厚度亦无显著性差异(P>0.05)。结论:PLS可以替代FBS用于牙周膜细胞的体外培养和膜片诱导。

AIM： To evaluate the possibility of replacing fetal bovine serum（ FBS） with serum- converted platelet lysate（ PLS） in the ex- vivo expansion of periodontal ligament cells（ PDLCs） and the cell sheet formation.METHODS： PLS was prepared from buffy- coat platelet concentrate using a combination of freezing- thawing method and calcium- activating method. 100 g / L PLS（ PLS group） and 100 g / L FBS（ FBS group） were respectively used during the culture and sheet- induction of PDLCs. The growth curve,sheet formation,and the expression level of genes and proteins associated with sheet- formation were examined by MTT assay,RT- PCR and HE staining respectively.RESULTS： Cell proliferation in the 2 groups showed no difference（ P〉0. 05）. After 10 days of cell sheet formation culture,RT- PCR showed that the expression of collagen I was reduced in PLS group（ P〉0. 05）. The expression of integrin β1 and fibronectin, genes related to cell sheet formation showed no difference between the 2 groups（ P〉0. 05）. No significant difference was noticed in the shape and thickness of cell sheets between the 2 groups（ P〉0. 05）. CONCLUSION： PLS can substitute FBS in supporting the expansion and sheet formation of PDLCs.