体外培养软骨细胞凋亡与caspase-3表达的实验研究

APOPTOSIS OF FETUS CHONDROCYTES CULTURED IN VITRO AND EXPRESSION OF CASPASE-3

目的 探讨软骨细胞体外培养过程中凋亡发生以及 caspase- 3的表达。 方法 采用 Annexin 和TU NEL 法检测第 1～ 4代软骨细胞在体外正常培养体系中第 3天和第 7天的凋亡率 ;RT- PCR和 Western blot分析caspase- 3表达水平 ;EL ISA检测 caspase- 3蛋白酶活性。 结果 第 1～ 4代软骨细胞在体外培养过程中均存在不同程度的凋亡现象 ,各代次软骨细胞第 7天的凋亡率 15 .7%± 0 .3% ,明显高于第 3天的 8.9%± 0 .6 % ,有统计学意义 (P<0 .0 1)。caspase- 3m RNA和蛋白质在整个培养过程中都有表达 ,随着时间延长表达上调 ,第 7天的表达水平高于第 3天。体外培养 7天后 caspase- 3被激活 ,可检测到分子量为 2 0 ku的裂解片段。caspase- 3蛋白酶活性随着培养时间延长也逐渐增高 ,与细胞凋亡程度基本一致。 结论 软骨细胞在体外培养过程中存在凋亡 ,caspase- 3参与了凋亡事件并发挥着重要作用。

Objective To investigate apoptosis of chondrocytes cultured in vitro and related expression of caspase 3. Methods Apoptosis of chondrocytes were detected by flow cytometry analysis and TUNEL staining. The expression of caspase 3 was determined by RT PCR and Western blot, and caspase 3 protein activity was determined by ELISA. Results Apoptosis was observed in chondrocytes cultured in vitro from passage 1 to passage 4 at various degrees. The percentage of apoptosis of chondrocytes on day 7 was much higher than that on day 3 (15.7%±0.3% vs 8 9%±0.6%, P< 0.01). caspase 3 mRNA and protein expressed in chondrocytes during whole culture process. Along with the culture time extension in vitro, caspase 3 expression and protein activity up regulated, coincident with apoptosis of chondrocyte. caspase 3 was activated and a fragment of 20 kDa was detected after 7 days of culture. Conclusion caspase 3 is involved in apoptosis of chondrocytes cultured in vitro.