目的:通过Fibronectin介导进行差别粘附法筛选出人黄韧带干细胞并进行鉴定,探讨其作为组织工程种子细胞的可行性。方法:从椎间孔镜或椎间盘镜微创手术中获取10例人黄韧带标本,机械-酶消化法联合获取原代细胞,扩增第二代后接种于Fibronectin包被过的培养瓶。流式细胞仪检测细胞表面特异性标志物。免疫组化方法测定干细胞表达相关特异性蛋白。使用干细胞诱导培养基(实验组)向成骨、成脂、成软骨多向诱导分化,使用常规生长培养基培养者作为阴性对照(对照组),分别行茜素红染色、油红O染色、阿利辛兰染色鉴定。PT-PCR检测成骨、成脂肪、成软骨基因表达。取同一患者的髂骨骨髓提取骨髓间充质干细胞,统计学分析黄韧带干细胞与骨髓间充质干细胞的细胞周期、增殖能力和干性基因表达差异。结果:原代细胞形态呈三角形或多角形,经Fibronectin介导粘附培养后形态比较均一,呈长梭形克隆群。流式细胞仪检测筛选后的黄韧带干细胞CD90、CD73阳性率>96.8%,CD105阳性率>95.9%,而stro-1为阴性。实验组茜素红染色呈强阳性,油红O染色可见大小不等脂滴呈红色,阿利辛兰染色大量蛋白聚糖聚集呈蓝色,对照组均为阴性。PT-PCR测定实验组成骨基因(OC、ALP、RUNX-2)、成脂肪基因(LPL、APP、PPAR2)、成软骨基因(COLⅡ、AGG、SOX9)显著上调,其中RUNX-2在对照组有低水平表达。免疫组化显示黄韧带干细胞和骨髓间充质干细胞均表达a-SMA、Ⅰ型胶原、纤连蛋白,但均不表达Ⅱ型胶原。两种干细胞均超过80%的细胞比例处于G0/G1期提示都有很强自我更新能力(P>0.05)。CCK-8检测不同时间点两种细胞的增殖能力,在第1~10天时OD值逐渐增加,第10~14天达稳定状态,二者有相似的增殖能力(P>0.05)。两种干细胞均表达干性基因NONAG、OCT-4、SOX2,组间差异无显著性(P>0.05)。结论:通过Fibronectin差别粘附筛选法可有效筛选纯化出黄韧带干细胞,其可向成骨、成脂、成软骨多向分化,为组织工程技术治疗退变椎间盘提供了新的种子细胞。 OBJECTIVE: To screen and identify stem cells of human Flavonoplasty by Fibronectin-mediated differential adhesion method and to explore their feasibility as seed cells for tissue engineering. Methods: Ten specimens of ligamentum flavum were obtained from minimally invasive surgery under the microscope of foraminifera or discectomy. The primary cells were harvested by mechanical-enzymatic digestion. The second passage was expanded and inoculated into Fibronectin-coated culture flasks. Flow cytometry to detect cell surface specific markers. Immunohistochemistry was used to determine the specific proteins related to stem cell expression. Induction of differentiation into osteoblasts, adipocytes and cartilage using stem cell-inducing medium (experimental group), using conventional growth medium as a negative control (control group), were performed with alizarin red staining, oil red O staining, Alixin Lan dyeing identification. PT-PCR detection osteogenic, adipogenic, cartilage gene expression. Bone marrow mesenchymal stem cells were extracted from the iliac bone marrow of the same patient, and the cell cycle, proliferation and dry gene expression of ligamentum flavum stem cells and bone marrow mesenchymal stem cells were statistically analyzed. Results: The primary cells were triangular or polygonal in shape. After Fibronectin-mediated adhesion culture, the morphology of the primary cells was relatively uniform and a long fusiform clone was formed. The positive rate of CD90 and CD73 in the ligamentum flavum cells screened by flow cytometry was> 96.8% and the positive rate of CD105 was> 95.9%, while stro-1 was negative. The alizarin red staining in the experimental group was strongly positive. Oil red O staining showed lipid droplets of varying sizes in red. Alicin-blue staining of a large amount of proteoglycan aggregated in blue and the control group was negative. The expressions of osteogenic genes (OC, ALP, RUNX-2), adipogenic genes (LPL, APP and PPAR2) and cartilage genes (COL Ⅱ, AGG and SOX9) were significantly increased in PTX- Low level of expression. Immunohistochemistry showed that both a-SMA, type I collagen and fibronectin were expressed in the ligamentum flavum stem cells and bone marrow mesenchymal stem cells, but neither type II collagen was expressed. The proportion of cells with more than 80% of both stem cells in G0 / G1 phase suggested strong self-renewal ability (P> 0.05). CCK-8 was used to detect the proliferation of the two cells at different time points. OD values ​​increased gradually from day 1 to day 10, and reached steady state from day 10 to day 14. Both of them had similar proliferative ability (P> 0.05). Both stem cells expressed NONAG, OCT-4 and SOX2, but there was no significant difference between the two groups (P> 0.05). Conclusion: Fibronectin differential adhesion screening method can effectively screen and purify the stem cells of the ligamentum flavum. The stem cells can differentiate into osteoblasts, adipocytes and cartilage, and provide new seed cells for tissue engineering to treat degenerated disc.