不同低温保护剂对皮肤组织桥粒芯糖蛋白1表达影响的比较

COMPARATIVE STUDY OF THE EFFECT OF DIFFERENT CRYOPROTECTANTS ON DESMOGLEIN 1 IN HUMAN SKIN

目的比较两种不同低温保护剂对皮肤组织桥粒芯糖蛋白1(desmoglein1,Dsg1)低温保存后表达的影响,为寻求皮肤组织低温保护剂的优良配方提供实验依据。方法取烧伤患者整形术后自愿捐献所余大腿部刃厚皮片5块,于取皮后4h内进行实验。根据冷冻时添加保存剂不同,将皮片分为3组。A组(n=2):0.5mol/L的海藻糖/DMSO;B组(n=2):传统低温保护剂DMSO/丙二醇;置于—196℃液氮中分别储存7、21d后复温取材进行实验;C组(n=1):新鲜刃厚皮片,不作任何保护剂处理。对各组皮片行免疫组织化学染色观察,RT-PCR方法检测皮片Dsg1基因水平。结果免疫组织化学染色见储存7d后,A、B组Dsg1蛋白染色均呈棕黄色,分布较均匀,表皮基底细胞表达信号与C组相似;A、B组吸光度(A)值分别为0.285±0.006、0.284±0.004;与C组0.287±0.008比较差异无统计学意义(P>0.05)。储存21d后,A组阳性细胞表达无明显减弱,B组强度减弱;A、B组A值分别为0.282±0.004、0.275±0.005,B组与A、C组比较差异均有统计学意义(P<0.05)。RT-PCR检测示C组、储存7dA、B组及储存21dA、B组Dsg1基因表达A值分别为0.814±0.012、0.810±0.012、0.803±0.008、0.806±0.008及0.782±0.013。储存后21dB组和A、C组比较差异有统计学意义(P<0.05),余时间点组间比较差异均无统计学意义(P>0.05)。结论海藻糖与DMSO联合应用对皮肤组织Dsg1的保护作用优于DMSO/丙二醇。

Objective To compare the effects of two different cryoprotectants on human desmoglein 1 （Dsg 1）, and to provide experimental basis for the optimization of cryoprotectant. Methods Five donated thin split-thickness skin grafts were used, and the experiment was conducted within 4 hours after skin grafts harvest. The skin grafts were divided into 3 groups： group A （n=2） in which skin grafts were immersed in 0.5mol/L trehalose/DMSO; group B（n=2） in which skin grafts were immersed in DMSO/propanediol; group C（n=1） in which fresh skin graft received no further treatment. Groups A and B were stored in 196℃ liquid nitrogen for 7 and 21 days, respectively, and then underwent experiment. Immunohistochemistry staining observation was performed on each group, RT-PCR method was used to detect the expression of Dsg 1 in skin. Results The immunohistochemistry staining showed that the protein in groups A and B was stained brown-yellow and distributed evenly 7 days after cryopreservation; the expression signal of epidermal basal cell was similar to that of group C; absorbance （A） value of groups A, B and C was 0.285±0.006, 0.284±0.004 and 0.287±0.008, respectively, suggesting there was no significant difference between groups A and B and group C （P 〉0.05）. At 21 days after cryopreservation, the expression of positive cells in group B decreased; no obvious decrease was observed in group A, A value of groups A and B was 0.282±0.004 and 0.275±0.005, respectively, indicating there was a significant difference between group B and groups A and C （P 〈 0.05）. RT-PCR detection showed that A value of groups A and B at 7 days after cryopreservation was 0.810±0.012 and 0.803±0.008, respectively; A value of groups A and B at 21 days after cryopreservation was 0.806±0.008 and 0.782±0.013, respectively; and the A value of group C was 0.814±0.012, indicating there was significant difference between group B and groups A and C at 21 days after cryopreservation （P 〈 0.05）, and no significant differences among groups were noted at other time points （P 〉0.05）. Conclusion Trehalose/DMSO is better than traditional cryoprotectant DMSO/propanediol in protecting Dsg 1 of human skin.