无菌回收膝关节镜术后引流液体外培养自体间充质干细胞的可行性

In vitro culture of autologous mesenchymal stem cells from the joint drainage fluid after knee arthroscopy:a feasibility study

背景:目前间充质干细胞在骨科中的应用前景极为广阔,尤其是对于治疗关节软骨缺损疾病表现出巨大的潜力,骨髓是间充质干细胞的主要来源,但常规髂骨穿刺法抽取骨髓,来源有限,技术要求严格,限制了其应用程度,因此,探寻新的方便有效的细胞来源具有重要意义。目的:探讨膝关节镜术后引流液体外培养自体间充质干细胞的可行性。方法:选取行膝关节镜手术患者8例,在关闭切口前置特制的无菌血袋,收集术后引流液,采用羟乙基淀粉沉淀法及密度梯度离心法分离培养间充质干细胞,观察细胞形态,绘制生长曲线,流式细胞仪鉴定表面标志物,进行三系诱导分化染色鉴定。结果与结论:(1)细胞呈梭形,贴壁生长,细胞增殖能力好,表面表达CD44、CD90、CD105、CD73,而不表达CD45;(2)通过标准方法诱导,间充质干细胞能向成骨细胞、脂肪细胞及软骨细胞分化,表明培养的细胞为间充质干细胞;(3)8例样本均培养出间充质干细胞,其细胞形态、增殖能力、细胞表型等无明显差异;(4)该结果表明,膝关节镜术后引流液可以培养出间充质干细胞,且细胞质量稳定。

BACKGROUND： Mesenchymal stem cells have a extreme prospect in orthopedics, which show great potential especially in the treatment of articular cartilage defect disease. Bone marrow is the main source of mesenchymal stem cells, and the iliac puncture is a conventional way to obtain bone marrow, but is restricted by the limited resources and strict technical requirements. Therefore, it is of great significance to explore new effective and convenient sources of mesenchymal stem cells. OBJECTIVE： To explore the feasibility of autologous mesenchymal stem cells derived from the joint drainage fluid after knee arthroscopy.METHODS： We selected eight patients who underwent arthroscopic surgery to collect joint drainage fluid by pre-made sterile blood bag before the wound closure. Precipitation with hydroxyethyl starch and density gradient centrifugation method were performed to isolate and culture mesenchymal stem cells from the joint drainage fluid. Cell morphology, growth curve, surface marker identification were observed and detected using flow cytometry. Then, adipogenic, chondrogenic and osteogenic differentiation of cells were induced and identified by oil red O, toluidine blue staining, and alizarin red staining, respectively. RESULTS AND CONCLUSION： The cultured cells were spindle-shaped, adherently grew and had good proliferation ability, which were positive for CD44, CD90, CD105 and CD73, but not for CD45. Under standard inductions, the cultured cells were induced to differentiate into osteoblasts, adipocytes and chondrocytes. Therefore, these cells were confirmed as mesenchymal stem cells. Mesenchymal stem cells were successfully isolated from the joint drainage fluid of eight patients and had no difference in cell morphology, proliferation and phenotypes. To conclude, the joint drainage fluid is an ideal source of mesenchymal stem cells with the guaranteed quality and quantity.