新型抗冻剂海藻糖对低温保存皮肤组织α-辅肌动蛋白表达的实验研究

目的 比较海藻糖与传统低温保护剂对皮肤组织a-辅肌动蛋白(a-actinin)低温保存后表达的影响,进一步揭示海藻糖的低温抗冻机制.方法 实验共进行6次,每次取自愿捐献烧伤患者整形术后所余大腿部刃厚皮片5块,根据冻存时添加保存液不同,将皮片随机分为5组,每组各1块.T/D组添加0.5 mol/L海藻糖/DMSO,完全浸没皮片;D/P组DMSO/丙二醇;D/K组DMSO/无血清角质细胞培养基;DMEM组DMEM;置于-196℃液氮中储存14 d后复温进行实验;对照组刃厚皮片不作任何处理.对各组皮片进行免疫组织化学染色观察,RT-PCR方法 检测皮片a-actinin基因水平,皮肤内琥珀酸脱氢酶(succinate dehydrogenase,SDH)定量测定法检测皮肤活力.结果 对照组、T/D组、D/P组、D/K(组及DMEM组吸光度(A)值分别为27.50±7.92、18.40±5.81、13.10±5.11、11.50±4.54及5.30±2.14.对照组与T/D组比较差异无统计学意义(P＞0.05),与其他组比较差异均有统计学意义(P＜0.05).RT-PCR检测对照组、T/D组、D/P组、D/K组及DMEM组a-actinin基因表达A值分别为0.816±0.134、0.723±0.245、0.564±0.265、0.245±0.071及0.148±0.048.对照组与T/D、D/P组比较,差异无统计学意义(P＞0.05);与D/K、DMEM组比较,差异均有统计学意义(P＜0.05).对照组、T/D组、D/P组、D/K组及DMEM组SDH活性值分别为18.2±3.7、12.3±3.6、10.2±2.4、7.3±2.1及5.7±1.5.对照组与T/D组比较差异无统计学意义(P＞0.05),与其他组比较,差异均有统计学意义(P＜0.05).结论 海藻糖对低温保存皮肤组织a-actinin的保护作用在其抗冻机制中发挥了重要作用.

INVESTIGATION ON THE EFFECT OF TREHALOSE ON α-ACTININ IN CRYOPRESERVED HUMAN SKIN

OBJECTIVE: To compare the effect of trehalose with that of different traditional cryoprotectants on human skin and to detect the new protection mechanism of trehalose in hypothermia. METHODS: The skins to be cryopreserved were first treated with DMSO/Propyleneglycol (D/P group), trehalose/DMSO (T/D group), DMSO/serumfree keratinocyte medium (D/K group), DMEM (DMEM group), respectively, so as to be compared with fresh skin (control grouop). Then the histological structure of skin of different groups was observed and analyzed by pathological technology (SP immunohistochemistry, DAB staining). Furthermore, the influence of trehalose on alpha-actinin at gene level with RT-PCR was investigated. The viability of skin in 5 respective groups was evaluated by using succinate dehydrogenase (SDH). The experiments were carried out 14 days after cryopreservation. RESULTS: The results of immunohistochemistry showed that A values of control group, T/D group, D/P group, D/K group and DMEM group were 27.50 +/- 7.92, 18.40 +/- 5.81, 13.10 +/- 5.11, 11.50 +/- 4.54 and 5.30 +/- 2.14, respectively. There was no significant difference between control group and T/D group (P > 0.05), but control group was significantly different from the other groups (P < 0.05). The results of PCR studies showed that A values of control group, T/D group, D/P group, D/K group and DMEM group were 0.816 +/- 0.134, 0.723 +/- 0.245, 0.564 +/- 0.265, 0.245 +/- 0.071 and 0.148 +/- 0.048, respectively. Control group was not significantly different from T/D group and D/P group (P > 0.05), but was significantly different from D/K group and DMEM group (P < 0.05). The results of SDH showed that A valuse of control group, T/D group, D/P group, D/K group and DMEM group were 18.2 +/- 3.7, 12.3 +/- 3.6, 10.2 +/- 2.4, 7.3 +/- 2.1 and 5.7 +/- 1.5, respectively. There was no significant difference between control group and T/D group (P > 0.05), while control group was significantly different from the other groups (P < 0.05). CONCLUSION: The results suggest that cryopreservation protocol-trehalose/DMSO is better than the traditional cryoprotectant for cryopreservation on alpha-actinin of human skin.