关节软骨细胞传代培养后细胞骨架与糖胺多糖合成变化的研究

Changes in cytoskeleton and glycosaminoglycan synthesis in passaged articular chondrocytes

目的观察关节软骨细胞随传代细胞骨架（CSK）及糖胺多糖（GAG）合成量的变化，并探讨两者关系。方法雄性8月龄新西兰白兔8只，空气栓塞处死，无菌酶解法分离双膝关节软骨细胞并行单层培养，取原代（P0）及传1、2代（P1、P2）的软骨细胞，分别接种于底部置有圆形盖玻片的24孔板中爬片并于细胞贴附后固定。以免疫荧光抗体对各代软骨细胞CSK染色，利用激光扫描共聚焦显微镜观察各代软骨细胞CSK形态并分别测定其CSK蛋白荧光强度。待每代细胞融合后，换无血清培养液，并于换液后12、24、36、48、60h分别抽取上清液以阿尔新蓝法检测GAG浓度。结果软骨细胞随传代其中间纤维网状结构变松散且核周密集分布减少，而在细胞周边形成的微管突起增多。软骨细胞CSK蛋白荧光强度测定结果显示，P1软骨细胞肌动蛋白荧光强度较P0升高，差异有统计学意义（P〈0．05），而P0与P2、P1与P2差异均无统计学意义（P〉0．05）；波形蛋白和微管蛋白荧光强度随传代逐渐下降，P0、P1、P2软骨细胞间两两比较差异均有统计学意义（P〈0．05）。软骨细胞在各个时间点的上清液GAG浓度均随传代逐渐下降，P0、P1、P2软骨细胞间两两比较差异均有统计学意义（P〈0．05）。结论软骨细胞随传代其CSK主要成分发生形态及蛋白表达强度的改变，同时其合成基质之一的GAG合成量也随之下降，表明软骨细胞传代后特性的变化，且二者可能存在一定关联。

Objective To observe the changes in cytoskeleton （CSK） and glycosaminoglycan （GAG） synthesis following passage culture of articular chondrocytes and the correlation between CSK and GAG. Methods Eight male New Zealand White rabbits （8-month-old） were sacrificed by air embolism. After the chondrocytes from their knee joints were isolated by enzymolysis method, monolayer culture was performed. The ehondrocytes of primary passage （PO） and passages 1 ＆ 2 （P1, P2） were inoculated into 24-well plates with round cover slips put at the bottoms. Cell climbing slices were fixed after attachment of chondrocytes. The CSK proteins, actin, vimentin, tubulin and vinculin were stained by immunofluorescence antibody on P0, P1 and P2 cell climbing slices, respectively. The CSK morphology was observed by laser confocal scanning microscopy and the fluorescence intensities of CSK proteins were detected by the fluorescence intensity software. The medium was changed for each generation after cell fusion and the GAG con- centrations in the supernatants were measured at 24, 36, 48, 60 h after medium change by alcian blue method. Results The intermediate filament networks became loosen and the dense distributions surrounding the nucleus decreased; more microtubule processes formed at the cell periphery with passage. The fluorescence intensity of actin of P1 chondrocytes was significantly increased than that of P0 （ P 〈 0. 05）, but there were no such significant differences between P0 and P2 or between P1 and P2 （ P 〉 0. 05）. The fluorescence intensities of vimentin and tubulin were significantly decreased with passage respectively, and there were such significant differences between any two of P0, P1 and P2 （ P 〈 0. 05）. The GAG concentrations in the supematants were significantly decreased with passage at each time point, and there were such significant differences between any two of P0, P1 and P2 （ P 〈 0. 05）. Conclusions Passage culture of articular chondrocytes may lead to changes in morphology and protein expression intensity of the main components of CSK, and accordingly to decreased synthesis amount of GAG, one of the extracellular matrix of chondrocytes, indicating the changed characteristics of chondrocytes after passage and a certain correlation between CSK and GAG.