钟招明,张 宇,查丁胜,赵成毅,徐俊昌,陈建庭.成人黄韧带细胞的体外培养[J].中国脊柱脊髓杂志,2010,20(1):62-65.
成人黄韧带细胞的体外培养
中文关键词:  黄韧带  细胞培养  退行性变
中文摘要:
  【摘要】 目的:探讨成人黄韧带细胞的体外培养方法,为研究黄韧带退变的发病机制奠定基础。方法:采集成年胸腰椎骨折患者后路减压术中的黄韧带,应用胶原酶预消化组织块培养法分离黄韧带细胞,并传代培养;倒置相差显微镜下观察细胞从组织块内迁出时间、细胞形态和生长状态;传1、3、5代细胞培养1~8d,用四甲基偶氮唑盐比色法(MTT)测定其吸光度(OD)值,评价细胞增殖状况,并绘制细胞生长曲线;用免疫荧光染色法检测传3代细胞的波形蛋白和Ⅰ型胶原的表达。结果:在原代培养第10~14天,黄韧带细胞开始从组织块迁出,细胞呈多种形态,主要为梭形和多角形,细胞接近融合时呈涡流状生长。传1、3、5代细胞生长曲线呈S形,同一代细胞在不同时间点的OD值差异有统计学意义(P<0.001),同一时间点不同代次细胞的OD值无统计学差异(P>0.05),细胞代次与时间点间无交互效应,各代细胞增殖状况无统计学差异(F=0.283,P=0.957)。传3代细胞的波形蛋白和Ⅰ型胶原免疫荧光染色呈阳性。结论:胶原酶预消化组织块培养法能有效分离成人黄韧带细胞;体外培养的黄韧带细胞呈成纤维细胞样表型,细胞在传5 代以内生物学特性稳定。
In vitro culture of adult human ligamentum flavum cells
英文关键词:Ligament flavum  Cell culture  Degeneration
英文摘要:
  【Abstract】 Objective:To explore the culturing of human ligamentum flavum(HLF) cells in vitro.Method:HLF were harvested during surgery for thoracolumbar burst fractures.HLF cells were isolated by collagenase-predigested explants cultures.Under an inverted phase microscope,cells were examined for outgrowth,morphology and growth status.After 1-8 days′ culturing,for HLF cells at the first,third and fifth passage(P1,P3,P5),the 3-(4,5-dimethy thioazol-2-yl)-2,5-di-phenytetrazoliumromide(MTT) method was used to analyze the cell proliferation by measuring the optical density(OD) values,and cell growth curves were drawed.Expressions of vimentin and type Ⅰ collagen in cells of third passage were detected by immunofluorescence staining.Result:Within 10-14 days after explants,outgrowth of cells was observed from ligament tissue explants and became monolayer.Morphologically,HLF cell lines varied widely in appearance,and arranged mainly as spindle-shape and polygonal-shape.At subconfluence,cells showed vortex-like growth.S-shaped growth curve was shown in the P1,P3 and P5 cells.The OD values at different time points showed significant difference compared with the same passage cells(P<0.001).Significant differences in OD value was not found among the 3 different passage cells(P>0.05).No interaction effect was observed between cell passages and time points,the cell proliferation of the P1,P3 and P5 showed no significant difference(F=0.283,P=0.957).Immunofluorescence staining showed expression of vimentin and type Ⅰ collagen.Conclusion:HLF cells can be successfully isolated by collagenase-predigested explant cultures.HLF cells in primary culture shows fibroblast-like phenotype.Biological characteristics of cells within 5 generations are stable.
投稿时间:2009-10-09  修订日期:2009-11-04
DOI:10.3969/j.issn.1004-406X.2010.[quarter_id].62.[Num
基金项目:
作者单位
钟招明 南方医科大学南方医院脊柱骨病外科 510515 广州市 
张 宇  
查丁胜  
赵成毅  
徐俊昌  
陈建庭  
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