玻璃化冷冻法保存兔腰椎间盘的实验研究

An experimental study on the vitrification preservation of rabbit intervertebral disc

目的观察玻璃化冷冻法保存兔腰椎间盘的效果。方法选取30只新西兰大耳白兔,随机分为玻璃化冷冻组、常规低温冷冻组和新鲜髓核细胞组,每组10只,将取下的腰椎间盘标本通过玻璃化冷冻法与常规低温冷冻法分别冻存30 d,复温洗脱后,利用MTT比色法测量两组腰椎间盘纤维环及髓核细胞的相对活性,利用台盼蓝拒染法检测其细胞存活率。结果 MTT比色法和台盼蓝拒染法所得结果基本一致,玻璃化冷冻组和常规低温冷冻组兔腰椎间盘髓核细胞存活率和相对活性均低于新鲜髓核细胞组,玻璃化冷冻组的兔腰椎间盘髓核细胞存活率和相对活性均优于常规低温冷冻组,其差异具有统计学意义(P<0.05)。结论玻璃化冷冻法保存兔腰椎间盘髓核细胞存活率及细胞活性高,保存效果优于常规低温冷冻法。

Objectives To observe the effect of preservation of the rabbit intervertebral disc by vitrification. Methods Thirty New Zealand white rabbits were randomly divided into vitrification preservation group, cryopreservation group and fresh nucleus pulposus cells group, with ten rabbits in each group. The specimens of the lumbar intervertebral disc were frozen by vitrification or cryopreservation respectively for 30 d. Then, the complex was removed from storage and rapidly thawed in a 37 ℃shaking water bath. MTT colorimetric method was used to measure the relative activity of two groups of lumbar intervertebral disc annulus and nucleus pulposus cells, and cell survival rate was detected by using the method of the trypan blue dye exclusion assay. Results The final results of MTT colorimetric method were basically in agreement with the results of trypan blue dye exclusion assay. The survival rate and relative activity of the rabbit lumbar disc nucleus pulposus cells in the vitrification preservation group and cryopreservation group were less than those in the fresh nucleus pulposus cells group. The survival rate and relative activity of the rabbit lumbar disc nucleus pulposus cells in the vitrification preservation group were better than those in the cryopreservation group, and the difference was statistically significant（P 0.05）.Conclusion The intervertebral disc preserved by vitrification had higher survival rate and better activity than the cryopreservation group.