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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 effect of two different hypothermia protective agents on the expression of desmoglein1 (Dsg1) in skin after cryopreservation, and to provide an experimental basis for seeking an excellent formula of hypothermia protective agent for skin tissue. Methods After burned patients, 5 pieces of remaining thin blade of thighs were donated voluntarily after plastic surgery, and the experiment was conducted within 4 hours after the skin was taken. According to different additives when frozen, the skin will be divided into 3 groups. Group A (n = 2): 0.5mol / L trehalose / DMSO; Group B (n = 2): traditional cryoprotectant DMSO / propylene glycol; placed in liquid nitrogen at -196 ℃, respectively, Samples were taken for experiment; Group C (n = 1): fresh blade thick skin, without any protective agent. Immunohistochemical staining was performed on the skin of each group, and the Dsg1 gene level was detected by RT-PCR. Results Immunohistochemical staining showed that the staining of Dsg1 protein in group A and group B was brown and stained evenly, and the signal of basal cell in epidermis was similar to that of group C. The absorbance (A) values of group A and group B were 0.285 ± 0.006 , 0.284 ± 0.004 respectively. There was no significant difference with 0.287 ± 0.008 in C group (P> 0.05). After 21 days of storage, the positive expression of A in group A was not significantly weakened and the intensity of group B was weakened. The values of A in group A and B were 0.282 ± 0.004 and 0.275 ± 0.005, respectively, and there was significant difference between group B and group A and group C <0.05). RT-PCR results showed that the A value of Dsg1 gene expression in group C, storage 7dA, group B and storage 21dA were 0.814 ± 0.012, 0.810 ± 0.012, 0.803 ± 0.008, 0.806 ± 0.008 and 0.782 ± 0.013, respectively. There was a significant difference between 21dB group and A and C groups after storage (P <0.05), but there was no significant difference between the two groups (P> 0.05). Conclusion The protective effect of trehalose in combination with DMSO on dermal tissue Dsg1 is better than DMSO / propylene glycol.