优化底物酶及冻存剂组成对软骨细胞增殖力及活力的影响

[目的]本文研究的目的在于找到软骨细胞分离的合适方法及优化冰冻保存软骨组织的配方。[方法]来源于骨关节炎膝关节置换术的软骨标本被用于本次试验,采用300或400 u/ml II型胶原酶(CTII-1或2)进行软骨细胞体外分离,保存在不同组成的冷冻剂中,进行梯度冷却并保存在液氮中48 h,随后进行软骨细胞活力及增殖潜力的测定。[结果]CTII-1较CTII-2更能分离出高活力的软骨细胞(P<0.05),并且300或400 u/ml的底物酶浓度比较合适。10%DMSO+90%FCS是一种比较合适的冰冻保护剂,能够最大程度保留软骨细胞的增殖潜力与活力,并且毒副作用小。[结论]通过相关的优化措施诸如软骨细胞体外分离底物酶以及冰冻保护剂的组成,从关节软骨组织中分离具有高增殖潜力的活力软骨细胞是一种可行的方法。

Effect of optimized substrate enzyme and composition of cryoprotectant agent on the proliferation and viability of human articular chondrocytes

[Objective] To optimize methods of human articular chondrocytes isolation and composition of cryoprotectant agent(CPA).[Methods]Human articular cartilage specimens from osteoarthritis patients were used in this study.The articular chondrocytes were isolated with 300 or 400 u/ml type II Collagen enzyme(CTII-1 or 2),preserved in different composition of cryoprotectant agents,step cooled and stored in liquid nitrogen for up to 48 hours and finally determined in vitro for proliferative potential and cell viability.[Results]The results showed that CTII-1 was more effective than CTII-2 in isolating articular chondrocytes(P<0.05) and 300 or 400 u/ml CTII-1 was optimal substrate enzyme concentration.10% Dimethyl Sulfoxide(DMSO) in 90% fetal calf serum(FCS) was a successful CPA for chondrocyte cryopreservation to the greatest degree of retaining proliferative potential and cell viability and reducing side effects.[Conclusion]By optimizing of appropriate factors,for instance cryoprotectant agent and isolation protocol,it is a feasible method to isolate viable chondrocytes with high proliferative capacity from human articular cartilage tissue.