深低温冷冻肌腱细胞活性的研究

目的研究深低温冷冻方法对肌腱细胞活性的影响,比较程序性降温和普通深低温冷冻法对腱细胞活性的影响.方法纯种SD大鼠24只（出生21 d）,随机分为3组,取双侧跟腱.新鲜肌腱对照组（A）,常规深低温冷冻组（B）,程序性降温深低温冷冻组（C）.采用相同的方法对3组肌腱细胞进行细胞培养.相差显微镜观察原代和传代后细胞的生长,绘制细胞的生长曲线,考察细胞的活性;对细胞进行成纤维细胞染色、胶原染色和对细胞进行形态观察（扫描电镜）;水解法定量分析细胞培养基中羟脯氨酸浓度的变化,检测细胞合成胶原的能力.结果原代细胞培养时A组细胞的生长速度快于B组和C组（P＜0.01）,C组细胞的生长速度快于B组（P＜0.01）,这种生长速度的差异在细胞传代后消失.细胞的形态学和组织学符合成纤维细胞形态.3组细胞培养基中羟脯氨酸浓度变化的差异无统计意义（P＞0.05）.结论经深低温冷冻处理的肌腱中仍存在具有活性的腱细胞,但数量显著少于新鲜肌腱中活细胞的数量.应用计算机控制程序性慢速降温方法处理的肌腱其活细胞的数量有所提高,但仍低于新鲜肌腱中活细胞的数量.

Study of the effect of cryopreservation on the viability of tenocytes

To investigate the effect of cryopreservation on the viability of tenocytes, and compare the difference between normal cryopreservation and programmed cryopreservation. Methods Twenty-four 21-day-old Sprague-Dawley rats were randomly divided into 3 groups, control group (A) , normal cryopreservation group (B), and programmed cryopreservation group (C) . Tenocytes from the 3 groups were cultured with the same method. Primary and secondary passage cells were observed under phase microscope. The growth curve of the cells was obtained and cell viability investigated. Cell morphology was studied by using Masson II staining, flbroblast staining and scanning electron microscope. Analysis of the concentration changes of hydrolysis hydroxyproline in the culture medium was carried out to evaluate the ability of the tenocytes to synthesize collagen. Results In primary generation passage the tenocytes in group A grew more quickly than the cells in group B and group C (P<0.01), and the cells in group C grew faster than the cells in group B (P<0.01) . The difference of cell viability in the primary passage disappeared in the second passage. Morphology of the cells in all groups were that of fibroblasts. The cells in three groups had the same ability to synthesize collagen. Conclusion There are viable tenocytes left after cryopreservation of the tendon. However the number of viable cells is less than that of fresh tendons. Cryopreservation, but the alive cell population less than those in fres h tendon. Programmed cryopreservation can preserve more viable tenocytes than normal cryopreservation.