人正常椎间盘髓核细胞体外不同培养代次的生物学性状分析：适应于组织工程椎间盘种子细胞的选择

Biological character analysis of nucleus pulposus cells of the human normal intervertebral disc at different passages in vitro:Choice of seed cells for tissue engineered intervertebral disc

背景:目前用作组织工程椎间盘研究的种子细胞主要来源于体外培养的髓核细胞,而体外培养的髓核细胞传至一定代次后即出现老化现象,老化的细胞失去生长能力不适于组织工程研究。目的:比较不同代次正常髓核细胞的形态学、增殖特性以及细胞的主要细胞外基质成分基因表达情况。方法:以酶消化法分离、培养人正常椎间盘髓核细胞,对不同代次髓核细胞采用光镜、电镜等形态学方法进行大体形态和超微结构观察,采用XTT实验检测不同代次细胞间的生长动力学差异,测定髓核细胞的活力和细胞Ⅱ型胶原及糖胺多糖mRNA表达。结果与结论:传3代之前细胞形态饱满,胞浆丰富,细胞呈多角形、短梭形,细胞内细胞器丰富,形态正常,线粒体含量少,核糖体含量多,说明细胞代谢旺盛,增殖能力强,生长曲线和XTT实验也提示传3代之前髓核细胞活性高,增殖能力强,细胞活力高,细胞外基质分泌旺盛。从传4代起,部分细胞形态逐渐向长梭形演化,逐渐表现为成纤维细胞的形态,说明细胞开始出现"返祖"现象,也称为老化现象,细胞的生长趋缓,传六七代后细胞均表现为长梭形,粗面内质网扩张,线粒体增多,细胞活力降低,细胞外基质明显减少,生长处于停滞状态。实验结果提示,体外培养条件下,人髓核细胞前3代细胞形态良好,增殖能力强,适合作为组织工程椎间盘的种子细胞,而细胞传4代后增殖能力逐渐减弱,不宜作为组织工程椎间盘的种子细胞。

BACKGROUND：Seed cells used for studying tissue engineered intervertebral disk are mainly derived from in vitro cultured nucleus pulposus.The in vitro cultured nucleus pulposus will affect aging at a certain passage.The aging cells lost growth ability,and were not suitable for the study of tissue engineering.OBJECTIVE：To compare the different passages of the normal nucleus pulposus cells of the morphology,proliferation characteristics and major extracellular matrix gene expression.METHODS：Human normal intervertebral disc nucleus pulposus cells were isolated and cultured using enzyme digestion method.Light microscopy,electron microscopy and other morphological methods were used to observe the general morphology and ultrastructure of the cells.XTT experiment was used to measure the differences in growth kinetics and expression of type Ⅱ collagen and glycosaminoglycan mRNA among different passages of cells.RESULTS AND CONCLUSION：Before the third passage,cells were rich in cytoplasm,presented polygonal,short spindle-shaped apperance,rich organelles in the cells,with normal morphology,less mitochondrial content and more ribosome content,which showed that the cells metabolism were exuberant vitality.Growth curve and the experimental XTT absorbance also showed that the cell proliferation was exuberant vitality,with high extracellular matrix secretion.The fourth passage of cells evolved to the long-spindle,and gradually showed fibroblast morphology,indicating cells started to present ＂throwback＂ phenomenon,also known as aging.Cell growth slowed down,the sixth and seventh passages appeared as spindle morphology,with the presence of rough endoplasmic reticulum,more mitochondria,growth stagnation,decreased cell viability and decreased extracellular matrix significantly.Results indicated that in vitro culture conditions,human nucleus pulposus cells before the third passage showed good cell morphology,proliferation ability,suitable as seed cells for tissue engineering of intervertebral disc,while the cell proliferation after fourth passage was gradually weakened and cannot be used as seed cells for tissue engineering of intervertebral disc.