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

Isolation and identification of nucleus pulposus mesenchymal stem cells from tissues removed by percutaneous endoscopic lumbar discectomy

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。

AIM： To explore a novel method to isolate human nucleus pulposus mesenchymal stem cells （hNP- MSCs） in vitro and to identify their biological characteristics. METHODS ： The explant culture method was employed to i-solate hNP-MSCs from nucleus pulposus tissue obtained by percutaneous endoscopic lumbar discectomy （PELD）. The iso-lated cells were passaged for purification and cultured in vitro followed by morphological observation. The cell proliferation ability was detected by CCK-8 assay. Growth curves of the cells were drawn and surface antigens were detected by flow cy-tometry. The cells at the 3rd ？ 6th passages were induced for adipogenic,osteogenic and chondrogenic differentiation, and examined by oil red 0 staining, alizarin red staining and Alcian blue staining. RESULTS ： The cells with self-renewal were obtained from nucleus pulposus tissue obtained by PELD. The results of flow cytometry analysis revealed that the cells were positive for CD29, CD44, CD90, CD73 and CD105, but negative for CD34 and CD45. The proliferative capacity was con-sistent with the growth characteristics of MSCs and multilineage differentiation potential was identified. CONCLUSION ： A novel method to efficiently isolate and culture hNP-MSCs, PELD combined with explant culture method, was established,which would promote the study of regenerative medicine based on hNP-MSCs.