从脊柱内镜手术摘除组织中分离髓核间充质干细胞及其生物学特征鉴定

发布时间：2018-10-11 10:56

【摘要】：目的:利用经皮内镜下腰椎间盘切除术获取来源明确的髓核组织,结合组织块培养法高效分离培养人髓核间充质干细胞(human nucleus pulposus mesenchymal stem cells,hNP-MSCs),并鉴定其生物学特征。方法:收集6例腰间盘突出症手术摘除的髓核组织,利用组织块培养法分离培养。取第3~6代生长良好的细胞用于实验,采用CCK-8检测增殖能力;用流式细胞术检测细胞表面标志物;并分别向成骨、成脂和成软骨方向诱导分化,用油红O染色、茜素红染色及阿利新蓝染色对分化结果进行检测。结果:成功从椎间盘内镜摘除的髓核组织中分离培养具有增殖能力的细胞,流式细胞术检测显示细胞高表达CD29、CD44、CD90、CD73和CD105等间充质干细胞抗原,不表达造血干细胞标志CD34和CD45。生长曲线显示符合正常间充质干细胞增殖特征。茜素红染色、阿利新蓝染色及油红O染色均呈阳性,说明分离培养的细胞具有向成骨、成软骨和成脂肪诱导分化的能力。结论:首次结合椎间盘内镜微创手术和组织块培养法,体外高效分离培养了具有自我更新能力和多向分化潜能的hNP-MSCs。
[Abstract]:Aim: to study the biological characteristics of human mesenchymal stem cells (human nucleus pulposus mesenchymal stem cells,hNP-MSCs) isolated from nucleus pulposus by percutaneous endoscopic discectomy. Methods: six cases of lumbar disc herniation were collected and cultured by tissue mass culture. The 3 ~ (th) ~ (th) passage well growing cells were used for the experiment. The proliferative ability was detected by CCK-8, the cell surface markers were detected by flow cytometry, and the cells were induced to differentiate into osteogenesis, fat-forming and cartilage formation, respectively, and oil red O staining was used. Alizarin red staining and alisin blue staining were used to detect the differentiation results. Results: the proliferative cells were successfully isolated from the nucleus pulposus tissues removed by endoscopy of intervertebral disc. Flow cytometry showed that the cells highly expressed mesenchymal stem cell antigens such as CD29,CD44,CD90,CD73 and CD105, but did not express the markers CD34 and CD45. of hematopoietic stem cells. The growth curve was consistent with the proliferation characteristics of normal mesenchymal stem cells. Alizarin red staining, alisin blue staining and oil red O staining were positive, which indicated that the cells isolated and cultured had the ability to differentiate into osteogenesis, cartilage and fat. Conclusion: in combination with endoscopic minimally invasive disc surgery and tissue mass culture for the first time, hNP-MSCs. with self-renewal and multipotential differentiation was isolated and cultured in vitro.
【作者单位】：暨南大学基础医学院生物化学与分子生物学系;暨南大学附属第一医院骨科;仁伯爵综合医院澳门医学科技研究协会;暨南大学理工学院生物材料系;
【基金】：中央高校基本科研基金(No.21615436) 广东科技项目基金(No.2016B090913004);广东科技项目基金(No.201508020035) 暨南大学附属第一医院科研培育专业基金项目(No.511005024)
【分类号】：R681.5

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