成人黄韧带细胞的体外培养

In vitro culture of adult human ligamentum flavum cells

中文关键词:  黄韧带  细胞培养  退行性变

英文关键词:Ligament flavum  Cell culture  Degeneration

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。

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.