半胱氨酰天冬氨酸特异性蛋白酶-3和β-连环蛋白、糖原合成激酶-3β在体外培养保存关节软骨组织中的表达及其相关性

The expression and correlativity of cysteinyl aspartate - specific protease - 3, β - catenin and glyco- gen synthase kinase -3βon culture -storaged articular cartilage in vitro

目的体外培养保存关节软骨组织，探讨半胱氨酰天冬氨酸特异性蛋白酶-3（Caspase-3）和Wnt／β-连环蛋白（β-catenin）信号通路中关键蛋白表达规律及其相关性。方法选取20只健康成年新西兰大白兔，切取膝骨软骨柱100枚，随机分为5组（n=20）。对照组：取材2h内的新鲜软骨；实验组：分为4组，将软骨柱浸入MEME培养液中保存，第14、21、28、35天时各取一组检测指标。采用膜联蛋白V-异硫氰酸荧光素（Annexin V—FITC）细胞凋亡检测法测定凋亡率，Western blot检测Caspase-3、糖原合酶激酶-38（GSK-3B）表达，免疫组织化学法检测β-catenin表达。结果随着保存时间延长，软骨细胞凋亡率逐渐增加，Caspase-3、GSK-3β表达逐渐上调，各组间差异均有统计学意义（P〈0．05），各组软骨细胞凋亡率为：对照组（6．04±1．09）％、14d（12．75±0．40）％、21d（21．90±0．57）％、28d（30．44±0．79）％、35d（40．70±0．43）％，Caspase-3表达量为：0．078±0．032、0．193±0．026、0．327±0．041、0．510±0．023、0．719±0．063，GSK-3β表达量为：0．162±0．025、0．242±0．019、0．493±0．012、0．717±0．020、0．905±0．011；免疫组织化学结果显示，细胞质与细胞核中B-catenin阳性染色逐渐增强，组间差异有统计学意义（P〈0．05）；细胞凋亡率与Caspase-3、GSK-3β、β—Catenin表达呈高度正相关（r=0．695、0．702、0．682，P〈0．01）；Caspase-3与GSK-3β、β—catenin表达呈高度正相关（r=0．713、0．661，P〈0．01）。结论体外培养保存关节软骨组织过程中，软骨细胞凋亡伴有Wnt／β-catenin信号通路关键蛋白β—catenin和GSK-3β表达的显著上调。

Objective To explore the expression and correlativity of Cysteinyl aspartate - specific protease （Caspase） -3 and key protein in the signaling pathway of Wnt/13 -catenin in the process of culture - storaged articular cartilage in vitro. Methods One hundred osteochondral plugs, which randomly divided into five groups （ n = 20 ）, were harvested from the knee of 20 healthy adult New Zealand white rabbits. The fresh articular cartilage was as control group. The experimental groups were stored in culture medium of MEME, the results were measured on the 14th, 21st, 28th, and 35th day. Annexin V -fluoresceine isothiocyanate （FITC） was applied to detected the apoptosis of chondrocyte, The expression of Caspase -3, glycogen synthase kinase -3β（GSK -3β） were detected by Western blotting, and immunohistochemistry method to observed the expression of β- catenin, Results The chondrocyte apoptosis increased as the culture time elapsed, while the expression of Caspase - 3, GSK - 3β gradually increased. There are statistical differences at each interval （P 〈0. 05）. The chondrocyte apoptosis rate in each group were as follows： （6.04 ± 1.09）%, （12.75 ±0.40）%, （21.90 ±0.57）%, （30.44 ±0.79）%,（40. 70 ±0.43）%. The expression of Caspase - 3 were as follows： 0. 078 ± 0. 032, 0. 193 ± 0. 026, 0.327 ±0.041,0.510±0.023, 0.719 ±0.063. The expression of GSK - 3β were as follows： 0. 162 ± 0. 025, 0. 242 ± 0. 019, 0. 493 ± 0. 012, 0. 717 ± 0. 020, 0. 905 ± 0. 011. Immunohistochemical results revealed positivity of β-catenin staining in the cytoplasm and nucleus increased gradually. There are statistical differences at each interval （P 〈 0. 05）. The chondrocyte apoptosis is highly positively correlated with the expression of Caspase - 3, GSK - 3β and β- catenin （ r = 0. 695, 0. 702, 0. 682, P 〈 0. 01 ）. Caspase - 3 is highly positively correlated with GSK - 3βand β- catenin （ r = 0. 713, 0. 661 P 〈 0. 01 ）. Conclusion The results suggest that culture -storaged articular cartilage in vitro chondrocyte apoptosis is accompanied by the remarkably up - regulated of the expression of β- catenin and GSK - 3β in the signaling pathway of Wnt/β- catenin.