贴壁法联合纤维连接蛋白差别黏附法分离纯化鉴定大鼠纤维环源干细胞

Isolation,purification and identification of rat annulus fibrosus-derived stem cells by adherent method combined with fibronectin differential adhesion method

背景:目前,细胞疗法治疗椎间盘退变多以骨髓间充质干细胞为主。但是,使用骨髓间充质干细胞作为纤维环再生的种子细胞存在修复部位形成异位骨化和畸胎瘤的危险。因此,寻找一种新的纤维环组织工程种子细胞对椎间盘退变的治疗将具有巨大的经济和社会意义。目的:通过贴壁法联合纤维连接蛋白差别黏附筛选法分离纯化大鼠纤维环源干细胞,观察纯化效果及细胞的生物学特性。方法:从SD大鼠椎间盘中获取纤维环组织,机械-酶消化法获取原代纤维环源干细胞,通过贴壁法联合纤维连接蛋白差别黏附法纯化纤维环源干细胞,在显微镜下观察细胞形态变化及增殖情况,应用细胞免疫荧光技术和qPCR技术检测干细胞表面标志物的表达,对筛选后的细胞进行多系诱导分化和特征性基因检测。结果与结论:①纯化后细胞生长良好,形态多以角形和星形多突起细胞为主,具有较好的增殖能力;②细胞膜表面抗原CD73、CD90、CD105呈阳性表达,CD45、CD34呈阴性表达;③经特定诱导后,细胞可以成功分化为成骨细胞、成软骨细胞及成脂细胞;④成骨诱导后Collagen-Ⅰ、Runx2、成软骨诱导后Collagen-Ⅱ、Sox-9和成脂诱导后PPAR-γ、LPL各特征性基因均在细胞中呈高表达,与诱导前比较,差异有显著性意义(P<0.05);⑤结果表明,贴壁法联合纤维连接蛋白差别黏附法能够有效筛选、纯化大鼠纤维环源干细胞,所得的细胞生物学性能良好,具有较好的增殖及多向分化潜能。

BACKGROUND:At present,bone marrow mesenchymal stem cells are the main seed cells used in cell therapy for the treatment of intervertebral disc degeneration.However,the use of bone marrow mesenchymal stem cells as seed cells for the regeneration of fibrous rings is at risk of heterotopic ossification and teratoma at the repair site.Therefore,it is of great economic and social significance to find a new kind of seed cells for tissue engineering of annulus fibrosus for the treatment of intervertebral disc degeneration.OBJECTIVE:To isolate and purify rat annulus fibrosus-derived stem cells by adherent method combined with fibronectin differential adhesion screening method,and to observe its purification effect and biological characteristics.METHODS:Annulus fibrosus tissue was obtained from a SD rat intervertebral disc.Primary annulus fibrosus-derived stem cells were obtained by the mechanical-enzymatic digestion method.Annulus fibronectin differential adhesion method was used to purify annulus fibrosus-derived stem cells.Morphological changes and proliferation of cells were observed through a microscope.The expression levels of stem cell markers were detected by immunofluorescence technique and qPCR.The screened cells were subjected to multi-lineage cell differentiation and characteristic gene detection.RESULTS AND CONCLUSION:(1)The purified cells grew well,and most of them were angular and star-shaped multi-process cells,which had good proliferation ability.(2)Cells were positive for cell membrane surface antigens CD73,CD90 and CD105,while negative for CD45 and CD34.(3)After specific induction,cells could successfully differentiate into osteoblasts,chondroblasts and lipoblasts.(4)Collagen-I,Runx-2 after osteogenic induction,Collagen II,Sox-9 after chondrogenic induction,and PPAR-γand LPL after lipogenic induction were highly expressed in cells,and the difference was significant compared with that before induction(P<0.05).(5)These findings confirm that the adherent method combined with fibronectin differential adhesion method is effective enough to screen,isolate and purify rat annulus fibrosus-derived stem cells,and has good cell biological properties,good proliferation ability and multiple differentiation potential.