二甲基亚砜和乙二醇对玻璃化保存大鼠坐骨神经的协同保护作用

Synergistic protective effect of dimethyl sulfoxide and ethylene glycol on rat sciatic nerves preserved by vitrification preservation

目的探讨冷冻保护剂二甲基亚砜(DMSO)、乙二醇(EG)单独及联合应用对周围神经玻璃化保存的影响。方法取30只3月龄雄性SD大鼠双侧坐骨神经,制成长约15 mm神经段,在含不同冷冻保护剂的玻璃化溶液中-80℃保存4周:空白组(不含冷冻保护剂)、10%DMSO组、20%EG组、10%DMSO+20%EG组(每组n=15)。透射电镜观察神经组织超微结构,Calcein-AM和PI双染色激光扫描共聚焦显微镜(LSCM)检测神经组织生物活性,Western blotting检测神经组织Caspase-3、Caspase-8表达。取玻璃化保存4周后的各组坐骨神经,于37℃、5%CO2DMEM(含l0%胎牛血清)中培养7 d,RT-PCR、Western blotting检测培养神经表达的神经生长因子(NGF)和胶质源性神经营养因子(GDNF)。结果坐骨神经玻璃化保存4周,透射电镜显示:空白组神经纤维脱髓鞘变和轴质萎缩严重,而10%DMSO组、20%EG组、10%DMSO+20%EG组神经纤维脱髓鞘变和轴质萎缩均较轻(以10%DMSO+20%EG组最轻)。LSCM检测显示:空白组神经纤维绿色荧光较弱,而红色荧光较强、分布较广;10%DMSO组、20%EG组神经纤维绿色荧光较强、红色荧光较弱;而10%DMSO+20%EG组神经纤维绿色荧光最强、红色荧光最弱。Western blotting检测显示:玻璃化保存后坐骨神经Caspase-3、Caspase-8的表达,空白组与10%DMSO组、20%EG组、10%DMSO+20%EG组比较,10%DMSO+20%EG组与10%DMSO组、20%EG组比较,差异均具有统计学意义(P<0.05);但10%DMSO组与20%EG组间差异无统计学意义(P>0.05)。RT-PCR、Western blotting检测显示:培养神经NGF、GDNF的表达,空白组与10%DMSO组、20%EG组、10%DMSO+20%EG组比较,10%DMSO+20%EG组与10%DMSO组、20%EG组比较,差异均具有统计学意义(P<0.05);但10%DMSO组与20%EG组间差异无统计学意义(P>0.05)。结论 10%DMSO、20%EG对-80℃玻璃化保存的坐骨神经具有保护作用,能提高玻璃化保存后神经组织的生物活性,且二者联合应用具有协同作用。

Objective To investigate the effects of application of cryoprotectants（CPA）,dimethyl sulfoxide（DMSO） and/or ethylene glycol（EG）,on the vitrification preservation of sciatic nerve.Methods Bilateral sciatic nerves of 30 3-month-old male SD rats were harvested and prepared into nerve segments of 15 mm.Nerve segments were preserved in the vitrification solution of different cryoprotectants at-80℃ for 4 weeks and divided into the blank control group（no cryoprotants）,10% DMSO group,20%EG group,and 10% DMSO＋20% EG group（n=15）.The ultrastructure of sciatic nerves was observed by TEM.The biological activity of sciatic nerves was detected by LSCM after Calcein-AM and PI double staining.Expressions of Caspase-3 and Caspase-8 in sciatic nerves were tested by Western blotting.The sciatic nerves preserved for 4 weeks were cultured by DMEM（containing 10%fetal bovine serum） in an incubator with 5% CO_2 at 37℃ for 7 d.Expressions of NGF and GDNF were detected by RT-PCR and Western blotting.Results After sciatic nerves were preserved for 4 weeks,the results of TEM showed that demyelination and axoplasmic atrophy of the blank control group were severe and those of other groups were mild（those of the 10% DMSO＋20% EG group were the mildest）.The results of LSCM showed that there were weak green fluorescent and strong red fluorescent in the blank control group,strong green fluorescent and weak red fluorescent in the 10% DMSO group and 20% EG group,and the strongest green fluorescent and the weakest red fluorescent in 10% DMSO＋20% EG group.The results of Western blotting indicated that the differences of expressions of Caspase-3 and Caspase-8 of sciatic nerves being preserved for 4 weeks between the blank control group and other groups,and the 10%DMSO＋20% EG group and other groups（except the blank control group） were statistically significant（P〈0.05）.But the difference between the 10% DMSO group and 20%E G group was not statistically significant（P〉0.05）.Results of RT-PCR and Western blotting showed that the differences of expressions of NGF and GDNF of cultured nerves between the blank control group and other groups,and the 10% DMSO＋20% EG group and other groups（except the blank control group） were statistically significant（P〈0.05）.But the difference between the 10% DMSO group and 20% EG group was not statistically significant（P〉0.05）.Conclusion The 10%DMSO or 20% EG can protect the sciatic nerves preserved by vitrification preservation at—80℃ and improve the biological activity of the sciatic nerves after vitrification preservation.The combination of 10% DMSO and 20% EG has the synergistic effect.