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Alternative TitleMembrane Permeability of Human Pluripotent Stem Cells to Me2SO,Glycerol and 1,2-Propanediol
Thesis Advisor徐霞
Degree Grantor中国科学院研究生院
Degree Discipline生物工程
Keyword人胚胎干细胞   诱导多能干细胞   膜对冷冻保护剂渗透参数   水力渗透参数   冷冻保存
Abstract多能干细胞,人胚胎干细胞(hESCs)和诱导多能干细胞(hiPSCs),因具有无限的自我更新和向三个胚层组织细胞分化的能力,在再生医学方面具有巨大的潜能。但是目前多能干细胞面临的一个主要问题就是细胞在采用传统冻存方法长期储存之后复苏率极低和活性丧失,这极大地限制了细胞的商业化医疗应用。探究hESCs和hiPSCs对冷冻保护剂(二甲基亚砜,Me2SO,丙三醇,glycerol,1,2-丙二醇,1,2-propanediol)的膜渗透参数以及渗透过程的活化能有助于优化冷冻保存程序,提高细胞复苏率。本研究的主要结论如下: (1)hESCs和hiPSCs细胞的等渗体积(V0),分别为2006±196 μm3和2524±195 μm3,非渗透体积分别为0.32V0和0.42V0。 (2)确定了在8,19,24和30oC下,hESCs和hiPSCs分别对10%,20%Me2SO,10%glycerol和1,2-propanediol的细胞膜渗透参数包括细胞膜对水渗透系数(Lp)和细胞膜对冷冻保护剂的渗透系数(Ps),参数分析表明hESCs和hiPSCs之间具有生物物理学差异。 (3)确定了hESCs和hiPSCs的细胞膜渗透参数和温度之间的依赖关系,水渗透过程活化能大小说明hESCs和hiPSCs在保护剂导入过程中的水分运输很可能不是通过膜蛋白介导机制完成的。 (4)比较了hESCs和hiPSCs在预测最佳冷冻速率和传统慢速冻存下的细胞复苏率和克隆生长情况。实验结果表明预测最佳冷冻速率并未提高所有冻存条件下的细胞复苏率,由于基础低温生物学模型本身的局限性可能导致最佳冷冻速率的预测值与真实值有所偏差。在Me2SO,glycerol和1,2-propanediol作为冷冻保护剂条件下,1,2-propanediol不利于hESCs和hiPSCs克隆形成。
Other AbstractDue to the unlimited capacity of self-renewal and ability to differentiate into derivatives of three germ layers, human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have great potential in regenerative medicine. A major challenge we are facing during the long-term storage of human pluripotent stem cells with conventional slow cooling method is the low cell recovery rate after cryopreservation which cannot meet the requirements for future clinical applications. Evaluating the cell membrane permeability and the corresponding activation energy of hESCs and hiPSCs for water and different cryoprotective agents (CPA), including dimethyl sulfoxide (Me2SO), 1,2-propandiol and glycerol, is important for facilitating the development of cryopreservation protocol to enhance cell recovery after cryopreservation. The main results are listed below: (1) The isotonic volumes of hESCs and hiPSCs were 2006 ± 196 μm3 and 2524 ± 195 μm3, and the osmotically inactive volumes of hESCs and hiPSCs determined using the Boyle-van’t Hoff model were 0.32V0 and 0.42V0, respectively. (2) The membrane permeability was assessed from the volume changes of cells exposed to Me2SO, 1,2-propanediol and glycerol at the temperatures ranging from 8oC to 30oC, the results of which suggested the biophysical differences between hESCs and hiPSCs. (3) Their activation energy for water and CPAs extrapolated from the Arrhenius relationship indicated that the water transport was probably not through the channel-mediated mechanism. (4) Comparing the cell recovery rate and colony development of hESCs and hiPSCs cryopreserved using the conventional slow cooling rate to that using the predicted optimal cooling rate, the results showed that the predicted optimal cooling rate did not always bring benefits to cell recovery, probably caused by the constrains of the fundamental cryobiological model which result in the deviation of the optimal cooling rate from the true value. Moreover, no colony were observed for both hESCs and hiPSCs after the cryopreservation using 1,2-propanediol.
Document Type学位论文
Recommended Citation
GB/T 7714
许艳青. 人多能干细胞对二甲基亚砜,丙三醇和1,2-丙二醇的膜渗透性能的研究[D]. 中国科学院研究生院,2014.
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