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背景:胚胎干细胞的长期低温贮存成为胚胎干细胞研究和应用的重要环节。传统的低温贮存保护剂主要包含二亚基亚砜、甘油、动物血清等,对于许多细胞类型并不能有效保证细胞的存活率,甚至于损失了细胞的某些功能。目的:开发适用于小鼠胚胎干细胞的低温贮存试剂。设计:观察实验。单位:广州医学院从化学院生理教研室与科宇联合干细胞生物技术有限公司。材料:实验于2004-10/2005-09在科宇公司实验室完成。小鼠细胞系mES-1为军事医学科学院基础医学研究所细胞生物学研究室惠赠。方法:将mES-1维持培养收集的细胞低温贮存。实验以二亚基亚砜为主要的低温贮存保护剂。按低温保护液成分不同分4组:对照组低温保护液包含DMEM(高糖)、10%二亚基亚砜、10%胎牛血清、10mg/mL抗坏血酸、0.18mg/mL肌醇、0.44mg/mL叶酸,甘露糖组在对照组的基础上补充7.56mg/mL甘露糖,海藻糖组补充34.23mg/mL海藻糖,蔗糖组补充41.94mg/mL蔗糖。观察不同多羟基化合物对小鼠胚胎干细胞低温贮存后存活率和多向分化能力的影响。主要观察指标:胚胎干细胞集落形成率和拟胚体形成率。结果:①冻存后各低温保存剂组的胚胎干细胞集落形成率均比冻存前有显著性降低[对照组:(24.0±8.8)%,甘露糖组:(42.0±10.1)%,海藻糖组:(84.0±8.2)%,蔗糖组:(70.0±14.2)%,冻存前:(95.0±4.7)%,P均<0.05],其中海藻糖组的胚胎干细胞集落形成率明显高于其他低温保护剂组(P<0.05)。②海藻糖组的拟胚体形成率高于对照组和甘露糖组[(90.0±5.2)%,(80.0±6.9)%,(82.0±9.6)%,P均<0.05]。与蔗糖组相比,差异无显著性意义。结论:以海藻糖为核心低温保护剂成分可有效维持小鼠胚胎干细胞的自我更新能力和多向分化能力。
Background: Long-term low temperature storage of embryonic stem cells has become an important part of embryonic stem cell research and application. Traditional cryopreservatives mainly include diphosphinothiazole, glycerol, animal serum, and many other types of cells can not effectively ensure the survival rate of cells, and even lost some of the cell's function. Objective: To develop low temperature storage reagent suitable for mouse embryonic stem cells. Design: observation experiment. Unit: Guangzhou Medical College from the Department of Physiology, College of Chemistry and Joint Branch Stem Cell Biotechnology Co., Ltd. Materials: The experiment was completed in the laboratory of Keyu Company from October 2004 to September 2005. The mouse cell line mES-1 is a gift from the Cell Biology Laboratory of the Institute of Basic Medical Sciences, Academy of Military Medical Sciences. Methods: Cells harvested from maintenance culture of mES-1 were stored at low temperature. Two sulfoxides as the main low-temperature storage protective agent. According to the different components of cryoprotectant, the mice were divided into 4 groups according to different components: the control group of cryoprotective solution containing DMEM (high glucose), 10% diphosphinothione, 10% fetal bovine serum, 10mg / mL ascorbic acid, 0.18mg / / mL folic acid, mannose group supplemented with 7.56mg / mL mannose on the basis of the control group, trehalose added 34.23mg / mL trehalose, sucrose group supplemented 41.94mg / mL sucrose. To observe the effect of different polyhydroxyl compounds on survival rate and multi-directional differentiation ability of mouse embryonic stem cells after cryopreservation. MAIN OUTCOME MEASURES: Embryonic stem cell colony formation rate and embryoid body formation rate. Results: ①The colony formation rate of embryonic stem cells after cryopreservation was significantly lower than that before cryopreservation [control group (24.0 ± 8.8)%, mannose group: (42.0 ± 10.1)%, trehalose (84.0 ± 8.2)%, sucrose group (70.0 ± 14.2)%, pre-cryopreservation (95.0 ± 4.7)%, P <0.05 respectively). The rate of embryonic stem cell colonization in trehalose group was significantly higher than that in other groups Cryoprotectant group (P <0.05). ② The embryoid body formation rate in trehalose group was significantly higher than that in control group and mannose group [(90.0 ± 5.2)%, (80.0 ± 6.9)%, (82.0 ± 9.6)%, P <0.05 respectively]. Compared with the sucrose group, the difference was not significant. Conclusion: Trehalose as the core cryoprotective agent can effectively maintain the ability of self-renewal and multi-differentiation of mouse embryonic stem cells.