大鼠肋软骨细胞体外培养及老化对基质代谢的影响

Culture of Rat Costochondral Chondrocytes and Effect of Aging on Matrix Catabolism of Cultured Chondrocytes

目的建立大鼠肋软骨细胞(RCC)分离、培养的方法,探讨其老化过程中细胞外基质及其相关基因的变化,为软骨细胞增殖分化的调控和软骨组织工程修复的研究建立实验基础。方法显微解剖出大鼠软骨,酶消化法获得分散的单个RCC。观察单层培养的不同代数RCC的细胞形态变化;采用免疫细胞化学方法检测不同代数RCC蛋白多糖和Ⅱ型胶原的表达;RT-PCR从mRNA水平分析Ⅱ型胶原、蛋白聚糖及蛋白聚糖酶-1、2(Aggrecanse-1,2)基因表达量。结果本实验分离的RCC呈多角形或圆形,第4代细胞形态发生变化;第1代到第3代细胞蛋白聚糖逐渐减少,第4代明显下降;Ⅱ型胶原(Collagen)含量随着细胞的老化逐渐减少;Aggrecanse-1前3代无明显变化,第4代明显上升,而Aggrecanse-2则无明显改变。结论本研究所建立的分离培养方法可以获得高纯度、高活性的软骨细胞,前3代RCC保持了在体软骨细胞的表型,是研究软骨细胞生物活性的良好材料。

Objective To establish the methods for rat costochondral chondrocyte （RCC） separation and culture, and investigate their gene and matrix change in the course of cell aging, thereby provide experimental bases for studying the regulation of chondrocyte proliferation and differentiation. Methods RCC were obtained by microdissection and digestion, and cultured in monolayer. Morphological changes of the serial passage of RCC were observed. The cellular aggrecan and collagen type Ⅱ expression were detected by immune-histochemistry and RT-PCR method. Results The morphology of primary cultured RCC was round or polygon. In this experiment, the fourth passage RCC changed morphology. The expression of aggrecan was high before the third passage, and then became significantly low in the fourth passage. The expression of aggrecan collagen type Ⅱ became lower in the cell aging. The mRNA expression of aggrecanse-1 became significantly high in the fourth passage, while the aggrecanse-2 changed no difference. Conclusion The separation and culture methods adopted in this study can obtain high pure and viable RCC. The preceding three passage RCC maintains their in vivo phenotype, they are idle experimental materials for studying the regulation of chondrocyte.