自噬诱导剂海藻糖在动脉内皮细胞冻存过程中的作用

The Role of Autophagy Inducer Trehalose in the Cryopreservation of Arterial Endothelial Cells

摘要: 目的：探讨海藻糖在动脉内皮细胞冻存过程中的保护作用。方法：通过在传统细胞冻存液中添加一定浓度的海藻糖对人主动脉内皮细胞进行液氮深低温冻存，根据cck-8结果将冻存液分为对照组(A组)，50 mmol∙L⁻¹海藻糖组(B组)以及100 mmol∙L⁻¹海藻糖组(C组)，运用台盼蓝染色法观察复苏后细胞的存活率，免疫印迹检测自噬标志性蛋白LC3B，Beclin-1、P62的表达情况，以及CCK-8检测细胞复苏后的增殖活性。结果：人主动脉内皮细胞经6个月液氮冻存后，细胞计数结果显示与A、C组相比较，B组活细胞比例明显增多(F = 7.50, P < 0.05)；免疫印迹结果表明与A、C组相比，B组自噬标志性蛋白LC3B，beclin-1表达水平明显上升(F_LC3B = 36.88, F_beclin-1 = 135.6, P < 0.05)，P62表达较对照组下降(F = 163.1, P< 0.05)。复苏后细胞增殖活性显示随时间的增加，细胞增值率逐步升高且B组增殖率较A、C组高(F_{24 h} = 14.14, F_{48 h} = 19.95, P < 0.05)。结论：低浓度海藻糖可在一定程度上有效提升冻存后细胞的活性，这种作用可能是通过促进自噬的发生而实现的。

Abstract: Objective: To explore the protective effect of trehalose during the cryopreservation of arterial endothelial cells. Methods: Human aortic endothelial cells were cryopreserved in liquid nitrogen by adding a certain concentration of trehalose to the traditional cell cryopreservation solution. According to the results of CCK-8, the cryopreservation solution was divided into the control group (A), the 50 mmol/L trehalose group (B), and the 100 mmol/L trehalose group (C). Trypan blue staining method was used to observe the survival rate of resuscitated cells, and western-blot to detect the autophagy marker protein LC3B, Beclin-1 and P62 expression, and CCK-8 to test the cell proliferation activity after resuscitation. Results: After cryopreservation in liquid nitrogen for 6 months, the cell count showed that compared with group A and C, the proportion of viable cells in group B increased significantly (F = 7.05, P < 0.05); the result of western-blot showed that compared with A and C, the expression of autophagy marker proteins LC3B and Beclin-1 in group B increased (F_LC3B = 36.88, F_beclin-1 = 135.6, P < 0.05), and the expression of P62 decreased (F = 163.1, P < 0.05). After resuscitation, the cell proliferation activity showed an increase over time, and proliferation rate of group B was higher than the other groups (F_{24 h} = 14.14, F_{48 h} = 19.95, P < 0.05). Conclusion: Low-concentration trehalose can effectively enhance cell viability after cryopreservation, and this effect may be achieved by promoting the occurrence of autophagy.