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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。
OBJECTIVE: To investigate the method of culturing adult ligamentum flavum cells in vitro and lay a foundation for studying the pathogenesis of the degeneration of the ligamentum flavum. Methods: The ligamentum flavum in the decompression of adult patients with thoracolumbar vertebral fractures was collected. The ligamentum flavum cells were isolated by collagenase pre-digestion method and subcultured. The cells were removed from the tissue blocks under inverted phase contrast microscope. Cell morphology and growth status. Cells of passage 1, passage 3 and passage 5 were cultured for 1-8 days. The absorbance (OD) value was measured by MTT assay to evaluate cell proliferation and cell growth Curve. The expression of vimentin and type I collagen in the third passage cells was detected by immunofluorescence staining. Results: From day 10 to day 14 of primary culture, cells of the ligamentum flavum began to migrate from the tissue mass. The cells appeared in various forms, mainly fusiform and polygons. The cells grew vortex-like when they were close to each other. The cell growth curves of passage 1, passage 3, passage 5 were S-shaped. There was significant difference in OD value between the same generation of cells at different time points (P <0.001). There was no significant difference in the OD value of different passage cells at the same time point P> 0.05). There was no interaction between the generation of cells and the time point. There was no significant difference in the proliferation of cells (F = 0.283, P = 0.957). The third generation cells of vimentin and type Ⅰ collagen immunofluorescence staining. CONCLUSION: Collagenase pre-digestion method can effectively isolate human ligamentum flavum cells. The cultured cells of the ligamentum flavum have a fibroblast-like phenotype, and the cells are stable in biological characteristics in the 5th passage.