玻璃化冻存对兔关节软骨细胞存活率及生物学活性的影响

Effect of vitrification on survival rate and biological activity of rabbit articular chondrocytes

目的探讨玻璃化冻存技术对兔关节软骨细胞存活率及生物学活性的影响。方法对兔关节软骨细胞进行玻璃化冻存法(A组)、程序性降温法(B组)及梯度降温法(C组)低温保存,以新鲜未冷冻的兔关节软骨细胞作为对照组(D组),采用流式细胞仪检测各组兔关节软骨细胞存活率和凋亡率,通过改良Alcianblue染色沉淀法评估其生物活性改变。结果 A组兔关节软骨细胞存活率为(86.6±1.7)%,明显高于B组的(79.4±3.1)%以及C组的(62.1±8.4)%,差异有统计学意义(P<0.05);A组、C组的凋亡率[(7.9±3.2)%、(7.3±6.2)%]明显低于B组的(14.0±5.2)%,差异有统计学意义(P<0.05);A组上清液中糖胺多糖(GAG)浓度为(29.4±2.9)ng/mL,明显高于B组的(18.5±4.1)ng/mL以及C组的(19.4±6.3)ng/mL(P<0.05)。结论与传统程序性降温法、梯度降温法比较,玻璃化冻存法能显著提高冷冻保存复苏后兔关节软骨细胞的存活率,并能维持关节软骨细胞的生物活性,是一种较为理想的软骨细胞低温保存方法。

Objective To explore the effect of vitrification on survival rate, apoptosis rate and biological activity of articular chondrocytes in rabbits. Methods Rabbit articular chondrocytes were divided into four groups. In A, B, C group, they were cyopreserved by vitrification, procedural cooling and gradient cooling respectively, while fresh chondrocytes not be cooled were in control group （D group）. The survival rate and apoptosis rate of the rewarming articular chondrocytes in each group were measured by flow cytometer, and the biological activity were evaluated by modified Alcian blue staining. Results The survival rate of the articular chondrocytes in A group （86.6 ± 1.7）% was higher than that in B group （79.4% ± 3.1%） and C group （62.1% ± 8.4%）, the difference had statistical significance （P 〈0.05）; The apoptosis rate of the articular chondrocytes in A group and C group （7.9% ±3.2%,7.3% ± 6.2%） were lower than that in B group （14.0% ± 5.2%）, the difference had statistical significance （P 〈0.05）; Concentration of glycosaminoglycan （GAG） in supematant solution in A group was （29.4 ± 2.9） ng/mE, was higher than （18.5 ± 4.1） ng/mL in B group and （19.4 ± 6.3） ng/mL in C group （P 〈0.05）. Conclusion Compared with traditional methods such as procedural cooling and gradient cooling, vitrification is an ideal cryopreservation method which could not only increase the survival rate of rabbits articular chondrocytes after cryopreservation and rewarming, but also maintain the biological activity of the chondrocytes.