海藻糖对低温保存的气管组织细胞活力的影响

Effects of trehalose on tracheal tissue and cell viabilities after cryopreservation

目的探讨海藻糖对低温保存的气管组织细胞活力的影响。方法新鲜配制两组保存液,其中:Ⅰ组LPD+DMSO,Ⅱ组LPD+DMSO+海藻糖(0.10mol·L-1)。切取SD大鼠气管后立即分别放入含上述两种溶液的冻存管,在程序降温仪降至-80℃后投入液氮中保存,分别在1,15,30,60,120d后对气管组织体外培养,并加入3HTdR做标记,检测细胞掺入率。结果采用3HTdR体外组织培养,低温保存后Ⅰ组气管3HTdR掺入率(88.72%～78.21%)明显高于Ⅱ组(80.99%～70.75%),这种趋势可长时间持续(共120d)。结论含有海藻糖的LPD溶液在低温下对气管组织细胞有明显的保护作用;3HTdR体外组织培养的方法可以比较客观地反映了器官保存后的活力。

Objective To detect the effects of trehalose on tracheal tissue and cell viabilities after cryopreservation. Methods Inbred male Sprague-Dawley (SD) rats were sacrificed with intraperitoneal injection of ketamine(150mg·kg~ -1).The tracheas were removed and immersed immediately in the freezing medium of low potassium dextran (LPD) solution containing with 10% dimethylsulfoxide(DMSO)(Group Ⅰ) and containing with 10% DMSO and 0.10 mol L~ -1 trehalose (Group Ⅱ) respectively. A sterile plastic tube containing a 1-cm-long trachea was filled with the freezing medium, sealed, and frozen to -80℃ at rate of -1℃ per minute in a programmable freezer. Then the tube was stored in liquid nitrogen(-196℃) for different periods of preservation(1,15,30,60,120d,n=5). For viability assessment, the specimen was thawed at 37℃ in an incubator and rinsed with physiologic saline solution 10 times. Next, each specimen was trimmed for equal weight(100mg) and incubated in 3 mL of RPMI1640 medium containing 10% fetal bovine serum at 37℃ for 24 hours. Then the tracheas were labeled with 3μCi~ 3H-TdR for 16 hours. The specimens were hydrolyzed in 80% formic acid(0.1mL) and incubated at 100℃ for 2 hours. At last, the solution was counted by liquid scintillation counter. Results The average rate of ~ 3H-TdR incorporation in group Ⅱ(88.72%~78.21%) were obvious higher than that in group Ⅰ(80.99%~70.75%) after cryopreservation. The protective function was observed all the time tested when the supplement of trehalose was added into the incubation medium. Conclusions LPD solution containing with 10% DMSO and 0.10mol·L~ -1 trehalose could protect the trachea viability after a 4-months cryopreservation and its protective effect was better than that with 10% DMSO only. The measurement of ~ 3H-TdR incorporation is a reliable index as a marker of tracheal viability.