两步冷冻法对周围神经雪旺细胞生物活性的影响

目的探讨两步冷冻法不同冷冻温度对周围神经雪旺细胞(Schwanncell,SC)生物活性的影响。方法雌性SD大鼠80只,切取双侧坐骨神经2cm,随机分为8组,每组10只20条坐骨神经。1组为新鲜对照组,不作任何处理;另7组为实验组,采用两步冷冻法分别降温至-20、-30、-40、-50、-60、-70和-80℃,保存2h后转入液氮中(-196℃)保存48h,取出后在37℃水浴箱中快速复温1min,消化收集各组SC,经Calceim-AM荧光染色,作流式细胞仪分析,求出各组细胞平均荧光强度,再经共聚焦显微镜直接观察SC荧光强度,进一步判断SC的生物活性。结果经流式细胞仪检测各组SC荧光强度为新鲜对照组242.5220±9.5684,-20℃组168.6770±10.2070,-30℃组214.9920±8.3291,-40℃组235.5260±9.2805,-50℃组222.4340±8.5155,-60℃组217.4090±9.5157,-70℃组132.3760±13.4597,-80℃组108.1320±16.0331;-40℃组荧光强度较其它实验组强,差异有统计学意义(P<0.01)。共聚焦显微镜观察各组SC生物活性,并作荧光强度分析:新鲜对照组143.7000±5.5678,-20℃组119.7000±5.1651,-30℃组121.3000±4.3474,-40℃组139.7000±5.0122,-50℃组121.0000±4.5461,-60℃组118.4000±4.9261,-70℃组81.2000±5.1164,-80℃组79.0000±5.7164;新鲜对照组SC生物活性较各实验组强,-40℃组SC生物活性较其它实验组强,差异均有统计学意义(P<0.01)。结论两步冷冻法均能较好保存SC生物活性,以-40℃组SC生物活性最好。

AN EFFECT OF THE TWO-STEP FREEZING METHOD ON THE SCHWANN CELL BIOLOGICAL ACTIVITY IN THE PERIPHERAL NERVE OF THE RAT

OBJECTIVE: To investigate an effect of different temperature cryopreservation of the two-step freezing method on the Schwann cell biological activity in the peripheral nerve of the rat. METHODS: Eighty female SD rats were randomly divided into 8 groups of 10 rats each. One was the control group and 7 were the experimental groups. Two 2-cm-long sciatic nerve segments were respectively taken from both legs of each rat. In the control group, the sciatic nerve segments did not undergo the treatment of cryopreservation; however, in the 7 experimental groups, the sciatic nerve segments respectively underwent the different temperature cryopreservation of the two-step freezing method at -20 degrees C, -30 degrees C, -40 degrees C, -50 degrees C, -60 degrees C, -70 degrees C and -80 degrees C. The sciatic nerve segments were cryopreserved for 2 hours,and then placed into the liquid nigrtrogen at -196 degrees C. After 48 hours of storage, the nerve segments were thawed quickly in the 37 degrees C water bath box for 1 minute. Then, the sciatic nerve segments each group were harvested. The cells of the sciatic nerve were incubated with Calcein-AM for 15 minutes. The average fluorescence intensity of the cells was measured by the flow cytometry. The nerve fibers were also incubated with Calcein-AM for 15 minutes. The fluorescence intensity of the cells was analyzed by the confocal fluorescence microscope. The Schwann cell biological activity intensity was measured. RESULTS: The fluorescence intensity in the -40 degrees C group was the strongest and the Schwann cell biological activity in this group was the best among all the groups (P < 0.01). The fluorescence intensity in the 8 groups measured by the flow cytometry was as follows: 242.5220 +/- 9.5684 in the control group, 168.6770 +/- 10.2070 in the -20 degrees C group, 214.9920 +/- 8.3291 in the -30 degrees C group, 235.5260 +/- 9.2805 in the -40 degrees C group, 222.4340 +/- 8.5155 in the -50 degrees C group, 217.4090 +/- 9.5157 in the -60 degrees C group, 132.3760 +/- 13.4597 in the -70 degrees C group, and 108.1320 +/- 16.0331 in the -80 degrees C group. The fluorescence intensity detected by the confocal fluorescence microscope was as follows: 143.7000 +/- 5.5678 in the control group, 119.7000 +/- 5.161 5 in the -20 degrees C group, 121.3000 +/- 4.3474 in the -30 degrees C group, 139.7000 +/- 5.0122 in the -40 degrees C group, 121.0000 +/- 4.5461 in the -50 degrees C group, 118.4000 +/- 4.9261 in the -60 degrees C group, 81.2000 +/- 5.1164 in the -70 degrees C group, and 79.0000 +/- 5.7164 in the -80 degrees C group. CONCLUSION: The Schwann cell biological activity treated by the two-step freezing method can be preserved and the activity is cryopreserved best at -40 degrees C.