循环静水压力对维持兔软骨细胞表型的影响

Effects of cyclic hydrostatic pressure on the stability of chondrocyte phenotype in vitro

目的:本研究将兔软骨细胞放在静水压力负载装置中培养,并传代,施加不同频率和大小的静水压,观察其对兔软骨细胞表型的影响。方法:用酶消化法获取兔关节软骨细胞,分别在不同的压力和频率下作普通培养瓶贴壁的单层培养,并传代。40 kPa循环静水加压为实验组,40 kPa持续静水加压培养和常压培养作为对照。Ⅰ型胶原和Ⅱ型胶原免疫组化,苏木素套核染色,倒置显微镜观察摄像。以RT-PCR方法检测软骨细胞中蛋白聚糖、聚集蛋白聚糖、Ⅰ型胶原和Ⅱ型胶原mRNA的表达。结果:在循环静水压力下培养,软骨细胞有较高的细胞增殖率,培养至第7代未见明显表型改变,仍然表达Ⅱ型胶原、蛋白聚糖和聚集蛋白聚糖等。常压培养,软骨细胞传代至第5代以后,逐渐失去软骨细胞的特有表型。而持续静水压力培养的软骨细胞传代至第3代以后就开始逐渐失去软骨细胞的特有表型。结论:一定的循环静水压力能维持软骨细胞的合成分泌功能,有助于软骨细胞特有表型的稳定。

Objective： To investigate how to maintain the chondrocytes phenotype under cyclic hydrostatic pressure. Methods ： Isolated rabbit chondrocytes which were obtained by using protease digestion method were cultured in monolayer and propagated under cyclic hydrostatic pressure （ in experimental group）, sustained hydrostatic pressure, or normal pressure （ in control group）. The proliferation and the intracellular matrix of chondrocytes were observed under light microscopy. The expression of proteoglycan, aggrecan,collagen type Ⅰ and Ⅱ were detected with reverse transcription-polymerase chain reaction. Resuits： Under the cylcic hydrostatic pressure culture, the growth rate was higher than that of other groups without chondrocyte phenotype changing until cultured to the 7th passage. Under the sustained hydrostatic pressure culture, the chondrocytes changed phenotype gradually after cultured to the 3rd passage. Under the normal pressure culture, the chondrocytes changed phenotype after cultured to the 5th passage. Conclusion： Cyclic hydrostatic pressure system is a promising method for the maintenance of the chondrocytes phenotype.