兔椎间盘髓核细胞体外生物学特性的实验研究

目的 对兔椎间盘髓核细胞进行体外培养,观察细胞的形态、表型及超微结构改变,研究其体外生物学特性.方法 取2周龄健康新西兰白兔椎间盘髓核组织,在含有15%灭活FBS的DMEM/F12培养液中培养,倒置相差显微镜下观察原代和传代细胞形态.分别在取材后、原代、第1代、第2代细胞培养期间,进行髓核细胞活力测定;爬片培养后进行甲苯胺蓝、HE、聚集蛋白聚糖番红O、Ⅰ型及Ⅱ型胶原免疫组织化学染色观察;MTT法绘制髓核细胞生长曲线,并行原代及第2代细胞透射电镜观察,对体外细胞的生物学特性进行研究.结果 倒置相差显微镜见原代髓核细胞呈类圆形,折光性较强;5 d开始有细胞贴壁,细胞呈多角形或短梭形;6～8 d细胞生长进入指数生长期;约17 d时,细胞长满瓶壁,可进行传代;随传代次数增加,细胞形态逐渐由多角形、短梭形向长梭形改变.髓核细胞活力测定在刚分离完成后细胞活力为95%～97%,原代培养期间为98%～100%,第1代培养期间仍能维持为100%,第2代细胞活力下降较为明显,为75%～80%.髓核细胞甲苯胺蓝染色呈强阳性:HE染色见细胞核、细胞质着色明显.第1代髓核细胞Ⅰ型胶原免疫组织化学染色呈阴性,Ⅱ型胶原免疫组织化学染色呈阳性,聚集蛋白聚糖番红O染色呈阳性;第2代细胞Ⅰ型胶原免疫组织化学染色呈阳性,Ⅱ型胶原免疫组织化学染色呈弱阳性,聚集蛋白多糖番红O染色着色较浅.MTT生长曲线与体外细胞培养时生长过程相符.透射电镜显示原代髓核细胞内线粒体少,胞质内有大量糖原颗粒,随传代次数增加,糖原颗粒减少,线粒体数量增多,细胞器开始肿胀.结论 明确了兔髓核细胞体外生物学特性变迁,为组织工程髓核的种子细胞研究提供了实验依据.

EXPERIMENTAL STUDY ON BIOLOGICAL FEATURE OF RABBIT INTERVERTEBRAL DISC NUCLEUS PULPOSUS IN VITRO

OBJECTIVE: To research the biological feature of intervertebral disc nucleus pulposus cells (NPCs) by observing cell morphous, phenotype and ultramicrostructure. METHODS: The NPCs from 2-week-old healthy rabbit were cultured in DMEM/F12 medium with 15% FBS. The cell biological features were observed by inverted phase contrast microscope, light microscope, electron microscope, cell vitality assay, cell growth curve and cells staining after harvest and during the periods of culturing the primary, the 1st passage and 2nd passage. RESULTS: The results of inverted phase contrast microscope showed that the primary passage adhered at 5 days, grew exponentially at 6-8 days, and were subcultured after covering the bottom at 17 days. The phenotype of the NPCs changed from polygon to long fusiform with passage increased; the vitality assay showed that there was about 95%-97%, 98%-100%, 100% and 75%-80% NPCs survived just after isolation from intervertebral disc, during the period of culturing the primary, the 1st passage and the 2nd passage, respectively. The toluidine blue staining of the NPCs was strongly positive, and HE staining showed clear cell nucleus and cytoplasm. The I collagen immunohistochemical staining showed negative results in the 1st passage, but II collagen immunohistochemical staining and safranin O staining showed positive results. However, the I collagen immunohistochemical staining showed positive result in the 2nd passage, and II collagen immunohistochemical staining and safranin O staining showed weakly positive results. The cell growth curve showed the same as the growth course of cell cultured in vitro. The results of TEM showed that there were many glycogen particles and less chondriosomes in the primary passage. With the increased passage, the glycogen particles decreased and the chondriosomes increased, and cell organ became swell. CONCLUSION: This study clarifies the biological feature of NPCs in vitro, providing the experimental basis for the seed cell research of the nuclues pulposus tissue.