兔椎间盘髓核细胞体外退变模型的建立

Establishment of the model of degenerative rabbit nuleus pulposus cells ex vivo

目的建立兔椎间盘髓核细胞体外退变模型。方法体外分离培养兔椎间盘髓核细胞,并传至第5代,细胞被分成原代细胞组和第5代退变细胞组,用细胞爬片进行HE染色,流式细胞术测定细胞周期,RT-PCR测定Ⅰ、Ⅱ型胶原和聚合素的mRNA水平的表达,化学和免疫组织化学的方法定量测定DNA、糖胺聚糖和Ⅱ型胶原的含量。结果退变细胞由原代细胞的多角形退变成纤维细胞样的梭形,原代细胞处于S期细胞显著多于退变细胞;退变细胞的Ⅱ型胶原和聚合素的表达显著下降,而Ⅰ型胶原的表达显著升高(P<0.01);退变细胞的DNA含量及糖胺聚糖和Ⅱ型胶原与DNA的比值较原代细胞显著下降(P<0.01)。结论兔椎间盘髓核细胞传至第5代会发生显著退变,主要功能基质mRNA水平的表达和含量显著的下降;Ⅰ型胶原显著升高,有退变为纤维细胞的趋势。

Objective To establish the model of degenerative rabbit nuleus pulposus cells ex vivo. Methods Primary rabbit nucleus pulposus cells were obtained and passaged to generate 5. These ceils were divided into 2 groups of primary group （GO） and geaerate 5 group（G5）. The differences between them were observed by HE staining, flow cytometry, RT-PCR, chemistry and immunohistochernistry. Results The shape of GO looked like multangular while that of G5 like spindle under revert microscope. The number of GO cells in S stage was significantly more than that of G5 cells. The expressions of type Ⅱ collagen and aggecan in GO in mRNA level were significantly lower than those in G5, while type Ⅰ collagen in GO was significantly higher than G5 （P〈 0. 01 ）. Containning of DNA and the ratio of GAG or type Ⅱ collagen to DNA were higher in GO than those in G5 （P〈0. 01）. Conclusion GO cells are degenetated significantly after passsaged to G5. Main functional extracellular matrix expression is significantly decreased, but type Ⅰ collagen increased with a degenerative trend of fibro-like cells.