SDF-1/NELL-1双基因转染促进脂肪干细胞体外成骨△

SDF-1 and NELL-1 co-transfection promotes osteogenesis differentiation of rabbit adipose stem cells in vitro

摘要:

[目的]观察腺病毒载体转染基质细胞衍生因子-1(stromal cell-derived factor-1, SDF-1)和尼尔样-1 型分子(Nellike molecule-l, NELL-1) 基因对兔脂肪干细胞 (adipose stem cells, ADSCs) 体外成骨分化能力的影响。[方法] 自兔分离培养 ADSCs，分为单纯细胞组 (original cell control, OCC)、空质粒转染组 (empty vector control, EVC)、SDF-1 组、NELL-1 组和 SDF-1/NELL-1 组，给予相应体外处理。成骨诱导 14 d 后，茜素红染色检测钙结节形成及碱性磷酸酶活性，Western blot 检测和 RT-PCR 检测相应蛋白与 mRNA 表达水平。[结果]单纯 SDF-1 或 NELL-1 转染，或 SDF-1/NELL-1 两者共同转染均显著增加目标基因的蛋白表达水平(P＜0.05)，特别是共同转染同时增加两种基因标目蛋白的表达。茜素红染色钙结节计数和 ALP 活性检测方面，SDF-1 组、NELL-1 组和 SDF-1/NELL-1 组均显著高于 OCC 组和 EVC 组(P＜0.05)，其中，SDF-1/NELL-1 组最高。 RT-PCR 检测方面，SDF-1 组、NELL-1 组和 SDF-1/NELL-1 组的 OPN 和 OCN mRAN 表达水平高于 OCC 组和 EVC 组 (P＜ 0.05)，其中 SDF-1/NELL-1 组的 OPN 和 OCN mRAN 表达水平最高。[结论] SDF-1、NELL-1 单独转染，或两者共转染 ADSCs 均可获得目标蛋白的高表达，以上两种基因单独转染，特别是两种基因共同转染，可显著增强 ADSCs 的体外成骨。

Abstract:

[Objective] To explore the effect of adenovirus-mediated stromal cell-derived factor-1 (SDF-1) and Nel-like molecule-l (NELL-1) genes transfection on osteogenic differentiation of the rabbit adipose stem cells (ADSCs) in vitro. [Methods] ADSCs isolated from rabbit adipose tissue were divided into 5 groups, including the original cell control (OCC) group, the empty vector control (EVC) group, the SDF-1 transfected group, the NELL-1 transfected group and the SDF-1/NELL-1 transfected group, which received corresponding treatment involving transfection by adenovirus vectors carrying different target genes. Subsequently, alizarin red staining for calcium nodule formation and alkaline phosphatase (ALP) assay were conducted 14 days of osteoinductive culture, additionally, western blot and RT-PCR assays were done to detect expression levels of related proteins and mRNA of genes. [Results] Both SDF-1 and NELL-1 gene transfections, as well as the co-transfection of them significantly increased the expressions of target proteins, especially the co-transfection. In terms of calcium nodule accounts with alizarin red staining and ALP activity assay, the SDF-1 group, the NELL-1 group and the SDF-1/NELL-1 group proved significantly greater than the OCC and EVC groups (P＜0.05) , with the co-transfection group the highest among the 5 groups. In terms of RT-PCR assays, the SDF-1 group, the NELL-1 group and the SDF-1/NELL-1 group had significantly greater expression of mRNAs of osteopontin (OPN) and osteocalcin (OCN) than the OCC and EVC groups (P＜0.05) , among which the co-transfection group was the highest. [Conclusion] ADSCs transfected by both SDF-1 and NELL-1 genes, as well as co-transfection of them do highly express the target proteins, which considerably enhance osteogenesis in vitro, especially the adenovirus-mediated co-transfection of the two genes.