红细胞负载海藻糖可能的作用机制
红细胞对海藻糖的负荷明显依赖于胞外海藻糖溶液的浓度。细胞内外的渗透压不平衡能促进细胞对海藻糖的摄取，随着胞外海藻糖浓度的增加，胞内海藻糖量也增加。红细胞对海藻糖的摄取未发现有饱和依赖性。与之不同的是，人血小板对海藻糖的摄取有浓度依赖性。Wolkers等［18］在实验中发现：37℃孵育，胞外海藻糖浓度在0-35 mmol/L时，血小板对海藻糖的吸收率成直线上升。当胞外海藻糖浓度达到75 mmol/L时，海藻糖的摄取率反而下降，负荷血小板对激动剂的反应能力下降。小分子物质如葡萄糖、赤藻糖醇、乙烯二醇、甘油等可通过易化扩散通过红细胞胞膜，而尿素、硫脲等物质通过被动扩散完成跨膜转运，其浓度梯度作为主要推动力。Satpathy等［24］在实验中发现：当胞外海藻糖浓度低于600 mmol/L时，红细胞对海藻糖没有明显的摄取。这说明其摄取机制可能并不是易化扩散，因为易化扩散可以在很低的胞外介质浓度时发生。但当胞外海藻糖浓度大于600 mmol/L时，红细胞对其的摄取开始增加，似乎又表明海藻糖的跨膜转运能量有部分是来源于细胞内外的渗透压差，增加细胞内外海藻糖的浓度梯度看来是海藻糖跨膜转运中首先出现的推动力。由阿利纽斯作图法（Arrhenius plot）计算所得的转运海藻糖所需的活化能是16.05±1.5 kcal/mol。Weith等［25］报道，赤藻糖醇经易化扩散通过人红细胞胞膜时，需要的活化能为21.5±0.7 kcal/mol。当胞外海藻糖浓度固定（且足够高）时，红细胞在4℃和37℃孵育条件下，胞内海藻糖量是不同的，有理由认为：海藻糖的跨膜转运不单是经易化扩散，而是伴有膜脂的向温性换位。红细胞对海藻糖的摄取明显依赖于孵育温度，而后者能影响红细胞胞膜的流动性。4℃条件下，红细胞对海藻糖的摄取率很低，37℃时，摄取率明显增加，这个结果同温度降低引起红细胞膜磷脂有序性升高的现象是一致的。红细胞膜在14℃时由于膜磷脂的“溶解”，出现相变的低协同效应，另一个协同效应出现在34℃，由富含胆固醇和神经鞘磷脂区域的“溶解”所引起。红细胞对海藻糖最大的摄取量（胞内海藻糖浓度大约为55 mmol/L）发生在37℃，此时的红细胞膜磷脂处于液晶态［26］，这个温度略高于使神经鞘磷脂微区解链或“漂移”的温度（34℃）［27］。 Satpathy等［24］推测“漂移”效应可能参与了37℃时红细胞膜对海藻糖的转运，导致了此温度条件下胞膜对海藻糖的较高摄取量。苯甲醇实验进一步证实了海藻糖的转运与红细胞胞膜的物理性质有关。如果海藻糖的跨膜转运由脂质相变所介导，那么细胞膜的液化剂将增加低温下的海藻糖的摄取。苯甲醇是一种膜液化剂，分隔红细胞膜引起胞膜相变温度降低。 Satpathy等［24］在实验中，将红细胞置于800 mmol/L的海藻糖溶液中于4℃孵育22小时，从而使胞内海藻糖浓度明显增加。增加胞膜流动性可提高胞内海藻糖浓度表明，改变细胞膜的物理状态在海藻糖跨膜转运过程中发挥一定的作用。
【参考文献】
1 Newman YM, Ring SG, Colaco C. The role of trehalose and other carbohydrates in biopreservation. Biotechnol Genet Eng Rev,1993; 11:263-294
2 Beattie GM, Crowe JH, Lopez AD, et al. Tehalose: a cryprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage. Diabetes, 1997； 46: 519-523
3Garcia-de-Castro A, Tunnacliffe A. Intracellular trehalose improves osmotolerance but not desiccation tolerance in mammalian cells. FEBS Letters, 2000； 487: 199-202
4Chen T, Acker JP, Eroglu A, et al. Beneficial effect of intracellular trehalose on the membrane integrity of dried mammalian cells. Cryobiology, 2001； 43: 168-181
5Pellerin-Mendes C, Million L, Marchand-Arvier M, et al. In vitro study of the protective effect of trehalose and dextran during free-zing of human red blood cells in liquid nitrogen. Cryobiology, 1997；35: 173-186
6 Wolkers WF, Walker NJ, Tabin F, et al. Human platelets loaded with trehalose survive freeze-drying. Cryobiology, 2001；43: 79-87
7 Kawai H, Sakurai M, Inoue Y, et al. Hydration of oligosaccharides: anomaious hydration ability of trehalose. Cryobiology, 1992； 29:599-606
8 Magazu S, Branca C, Faraone A, et al. Comparison of disacchaide solution across glass transition. Physica B, 2001； 301:126
9 周俊, 刘景汉. 血小板冻干保存的研究进展. 中国实验血液学杂志，2004; 12 : 542-545
10 Sussich F, Skopec C, Brady J, et al. Reversible dehydration of trehalose and anhydrobiosis: from solution state to an exotic crytal?. Carbohydrate Research, 2001, 334: 165-176
11 Leslie SB, Israeli E, Lighthart B, et al. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol， 1995； 61： 3592-3597
12 Esteves MI, Quintilio W, Sato RA, et al. Stabilisation of immunoconjugates by trehalose. Biotechnol Lett， 2000； 22: 417-420
13 Crowe JH, Oliver AE, Hoekstra FA, et al. Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification. Cryobiology, 1997； 35: 20-30
14 Hincha DK, Hellwege EM, Meyer AG, et al. Plant fructans stabilize phosphatidylcholine liposomes during freeze-drying. Eur J Biochem， 2000； 267: 535-540
15 Crowe JH. Anhydrobiosis: an unsolved problem. American Naturalist, 1971； 105:563-573
16 Crowe JH, Oliver AE, Hoekstra FA, et al. Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification. Cryobiology, 1997, 35: 20-30
17 Oliver, Jamil K, Crowe JH, et al. Loading MSCs with trehalose by endocytosis. Cell Preservation Technol, 2004; 2: 35-44
18 Wolkers WF, Looper SA, Fontanilla RA, et al. Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets. Biochim Biophys Acta, 2003； 1612: 154-163
19Eroglu A, Russo MJ, Biaganski R, et al. Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nat Biotechnol, 2000； 18: 163-167
20Guo N, Puhlev I, Brown DR, et al. Trehalose expression confers desiccation tolerance on human cells, Nat Biotechnol, 2000; 18: 168-171
21Puhlev I, Guo N, Brown DR, et al. Desiccation tolerance in human cells, Cryobiology, 2001; 42: 207-217
22 Katkov II. Electroporation of cells in application for cryobiology: summary of 20 year experience. Probl Cryobiol, 2002; 2: 3-8
23 Shirakashi R, Kostner CM, Muller KJ, et al. Intracellular delivery of trehalose into mammalian cells by electropermeabilization. J Membr Biol, 2002; 189: 45-54
24 Satpathy GR, Torok Z, Bali R, et al. Loading red blood cells with trehalose: a step towards biostabilization. Cryobiology, 2004; 49: 123-136
25Weith JO. Effects of hexoses and anions on the erythritol permeability of human red cells. J Physiol, 1971; 213: 435-453
26 Wolkers WF, Crowe LM, Tsvetkova NM, et al. In situ assessment of erythrocyte membrane properties during cold storage. Mol Membr Biol, 2002; 19: 59-65
27 Moore DJ, Sills RH, Patel N, et al. Conformational order of phospholipids incorporated into human erythrocytes: an FTIR study. Biochemistry, 1996; 35: 229-235
特发性血小板减少性紫癜的细胞免疫异常研究新进展
用Midi MACS方法从白血病患者分选CD34＋/CD123＋细胞