犬脂肪干细胞膜片的构建及其复合富血小板纤维蛋白后的成骨能力

目的 初步研究犬脂肪干细胞(adipose-derived stem cell,ADSCs)膜片的构建及其生物学特性对膜片复合富血小板纤维蛋白(platelet-rich fibrin,PRF)后成骨能力的影响.方法 取犬腹股沟处的脂肪进行消化,分离培养成脂肪干细胞进行相关检测.将P3代细胞加入含抗坏血酸的成膜培养液培养2周以上,构建脂肪干细胞膜片,采用HE染色及扫描电镜对膜片进行形态学检测.提取静脉血PRF,分为细胞膜片复合PRF的实验组和膜片对照组,2组分别成骨诱导后进行ALP染色和茜素红染色,并于诱导后第3、5天,采用RT-PCR检测成骨相关基因ALP、Runx2的表达.结果 体外分离培养脂肪干细胞,成功构建干细胞膜片.光镜下可见细胞极性排列紧密,细胞外基质分泌.扫描电镜可见膜片表面光滑,细胞紧密排列.HE染色可见蓝染细胞紧密相连,细胞外大量基质.成骨诱导后,ADSCs膜片复合PRF的实验组ALP表达及茜素红染色钙结节形成量明显优于ADSCs膜片对照组,实验组ALP、Runx2基因mRNA表达高于对照组.结论 成功构建犬脂肪干细胞膜片,膜片复合PRF后具有更强的成骨能力.

Preparation of adipose-derived stem cell sheet and its osteogenic effects after combining with platelet-rich fibrin

Objective To prepare cell sheet containing adipose-derived stem cells (ADSCs),and investigate its biological characteristics and effect on osteogenic differentiation after combination with plateletrich fibrin (PRF).Methods The fat tissue samples were harvested from the groin of beagle dogs,and then the ADSCs were isolated by collagenase digestion and cultured in vitro.After the obtained cells were identified,the cells at passage 3 were cultured in the medium containing ascorbic acid for over 2 weeks to prepare ADSCs cell sheet.HE staining and scanning electron microscopy were used to observe the morphology of the produced cell sheets.PRF was collected from the whole blood of dogs.The cell sheets were assigned to the PRF group and the control group.Alkaline phosphatase (ALP) and alizarin red staining were employed to observe the sheets after osteogenesis induction,and RT-PCR was adopted to detect the expression of osteogenesis genes,including ALP and Runx2 in 3 and 5 d after induction.Results ADSCs were isolated and cultured in vitro,and cell sheets were prepared successfully.Optical microscopy displayed that the cells were in polar elongation and close arrangement,with large amount of extracellular matrix.Scanning electron microscopy showed that the surface of cell sheets was smooth and the cells were tightly arranged.HE staining showed the blue-stained cells were closely arranged,and there were numerous extracellular matrix.After osteogenetic induction,the expression level of ALP and formation of calcium nodule with alizarin red staining were better in the PRF group than the control group.And the mRNA levels of ALP and Runx2 in the PRF group were significantly higher than the control group.Conclusion A ADSCs sheet is successfully prepared,and it will have stronger osteogenic differentiation when combined with PRF.