细胞冻存对髁突软骨细胞去分化特性的影响研究

Study on the effect of cryopreservation on condylar cartilage cells

目的研究细胞冻存处理对髁突软骨细胞去分化特性的影响。方法分离并体外培养2周龄新西兰大白兔双侧髁突软骨细胞,将P1代冻存处理15 d后传代培养。采用倒置相差显微镜观察比较正常传代培养的P2代髁突软骨细胞(对照组)和经冻存处理后传代的P2代髁突软骨细胞(实验组)间的细胞形态;通过荧光定量PCR和Western免疫印迹技术检测和分析两组间软骨细胞标志物COL2、COL10、Sox9和Aggrecan基因和蛋白水平的表达差异,并使用SPSS13.0软件包对数据进行统计学分析。结果倒置相差显微镜观察发现实验组P2代髁突软骨细胞会出现一定程度的形态学改变;但是并未发现实验组和对照组之间COL2、COL10、Sox9和Aggrecan的表达有统计学意义的差异;实验组和对照组软骨标志基因的表达均会较P1代出现降低。结论体外培养髁突软骨细胞存在去分化现象,但细胞冻存处理并不会加速其去分化进程,所以体外培养的髁突软骨细胞可以通过冻存技术来实现髁突软骨细胞的暂存。

Objective To study the effect of cryopreservation on the dedifferentiation of the condylar cartilage cells. Methods Con- dylar cartilage was aseptically dissected from the temporomandibular joint of 2 week old New Zealand white rabbit. The condylar carti- lage cells were subcultured and analyzed after culturing in vitro to the 1st passage and then frozen for 15 days as the experimental group. The cytoactive of the 2nd passage ceils of the experiment group and that of the control group were assessed by inverted phase contrast microscope. Quantitative real-time PCR and Western blot technique were used to analyze the expression of Collagen Type I1 , Collagen Type X, Sox9 and Aggrecan. One-way ANOVA was performed using SPSS13.0 software package. Results The 2nd passage of the experimental group did have morphological changes, but Quantitative real-time PCR and Western blot both showed that there was no significant difference in the expression of Collagen Type 11 , Collagen Type X, Sox9 and Aggrecan between the experiment group and the control group. Both the experiment group and the control group would have a lower expression of those cartilage specific genes than the 1 st passage cells. Conclusions Cell cryopreservation will not accelerate the dedifferentiation progress of condylar cartilage cells during subcultivation. That means the condylar cartilage cells cultured in vitro can be stored by cryopreservation.