骨组织超低温保存中CPA导入和洗脱过程的传递现象
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摘要
超低温保存技术是一种长时间保存细胞和组织的有效方法，在许多领域都得到了广泛的应用。而临床医学的发展和需求，使得工程组织和生物活体器官的超低温保存成为了当今生物医学工程研究的最前沿的研究课题。目前在全世界每年进行器官移植手术中，骨移植的数量仅次于输血居第二位。但对于异体骨移植，如何实现长期保存就成为了问题的关键。若能将超低温保存技术成功用于保存治疗大体积骨缺损所需的自体骨或异体骨，则可使骨缺损的临床治疗更加便利、大大减少病人的痛苦和损失。因此骨组织的超低温保存有着辉煌的发展前景和广泛的应用价值。
本实验首先对骨细胞和骨组织的体外培养进行了研究，确定所研究对象的准确性，并保证实验前期实验材料的活性。
其次对实验的关键技术进行了交叉检验。通过核磁共振技术和渗透压技术对冷冻保护液中冷冻保护剂的浓度进行交叉检验；同时用荧光双重标记法和台盼兰据染法对骨组织内的细胞的成活率进行交叉检验。实验结果表明，渗透压测定浓度的方法是一种既简洁又准确的表征溶液中主要溶质浓度的方法；台盼兰据染法在正确操作的前提下可以得到很准确的CPA处理后的骨细胞的成活率。
同时还考察了冷冻保护剂在骨组织中的导入和洗脱过程对细胞和组织活性的影响。选取了两种冷冻保护剂的三种不同的浓度，考察了冷冻保护剂种类、浓度、操作温度、导入或洗脱方案以及后期培养等因素对骨细胞成活率的影响。实验结果表明：冷冻保护剂在导入过程中对细胞或组织的损伤主要为渗透损伤和毒性损伤，受二者共同作用存在一个最佳导入时间和最佳导入方案。洗脱方案的确定也由这二者的共同作用的影响；连续式导入或洗脱之后的细胞成活率要高于分步导入或洗脱；甘油渗透性要比DMSO差，但其毒性作用也比DMSO小；甘油渗透性受温度影响比较大，DMSO毒性受温度影响比较大。
最后，对骨组织内的主要细胞，成骨细胞进行了超低温保存过程中的冷冻和复温过程的考察。通过改变降温速率和复温速率对三种冷冻保护剂的保护作用进行了实验。结果表明，玻璃化冻存效果要明显优于非玻璃化冻存。非玻璃化冷冻保护剂在冷冻过程中存在部分玻璃化现象，但不能体现出玻璃化的优势。玻璃化冷冻保护剂也只有在冷却速率达到一定数值时才能实现以玻璃化为主的部分玻璃化，或者完全玻璃化。随冷却速率的增大，玻璃化冷冻保护剂的玻璃化能力越来越显著；VS1无论是在玻璃化温度方面，完全玻璃化能力方面，玻璃化形态(有无低温断裂)方面，对成骨细胞的整体保护作用方面，在最佳降温速率适用范围方面还是在对组织超低温保存的适用性方面，均优于7mol／L的DMSO溶液，只有在反玻璃化方面要逊于7mol／L的DMSO溶液；玻璃化冷冻保护剂的反玻璃化现象与低温断裂以及降温过程形成的冰晶都有着密切的联系，并且反玻璃化的程度随着复温速率的增大而减小。
Cryopreservation is a technique by which cells and tissues can be long-term preserved. With the more rapid development of medical science and the more urgent demand of clinic treatment, the cryopreservation of engineered tissue and live organ becomes the most advanced and promising research field of biomedical engineering. The data shows that around the world, the number of bone transplant operation is the second large one only less than blood transfusion. It will facilitate the clinic therapy and lessen the patients' pain if the autograft and heterograft of bone used for large-area bone loss can be successfully cryopreserved. Therefore the cryopreservation of bone tissue has a promising future and an extensive practical value.
At the beginning of the research of this thesis, osteoblasts and bone tissue is cultured in-vitro and the main techniques used in the experiment are cross-checked. By cross-checking the concentration of cryoprotective agent (CPA) and the viability of cells within bone tissue, we conclude that osmotic pressure technique and Typan blue stain method can precisely reflect two major parameters above.
Meanwhile, the loading and removing process of cryopreservation are studied by choosing three different concentrations of two CPAs. Type and the concentration of CPA, operating temperature, loading and removing protocol and post-culture is set as parameters to check their effects on the characteristics of the whole process. Results show that the osmosis damage and the toxicity damage are two major factors for the loss of cells' viability, that continuous loading and removing is better than intermittent ones, that glycerol's osmosis ability is worse than DMSO, but its toxicity is less than DMSO and that the effect of temperature change on the glycerol's osmosis ability is much more than on DMSO's, but its effect on DMSO's toxicity is much more than on glycerol's.
Finally, osteoblasts, the major cells in bone tissue, are freezed and thawed. By using three kinds of CPAs and changing the cooling rate and thawing rate, we conclude that for osteoblasts' cryopreservation, vitrification overmatches the crystallization. Non-vitrified CPA can partially vitrify, but can not show the superiority of vitrification, that with the increase of the cooling rate, vitrified CPA's ability to vitrify shows more greatly, that for two kinds of vitrified CPA studied in this experiment, VS1 is superior to 7mol/L DMSO in almost all aspects except the ability to overcome the devitrification, that the phenomena of the devitrification is closely related to the ice crystal appeared while freezing and the cryo-fragmentation and that the devitrification is blocked and finally prevented with the increasing of the thawing rate.
本实验首先对骨细胞和骨组织的体外培养进行了研究，确定所研究对象的准确性，并保证实验前期实验材料的活性。
其次对实验的关键技术进行了交叉检验。通过核磁共振技术和渗透压技术对冷冻保护液中冷冻保护剂的浓度进行交叉检验；同时用荧光双重标记法和台盼兰据染法对骨组织内的细胞的成活率进行交叉检验。实验结果表明，渗透压测定浓度的方法是一种既简洁又准确的表征溶液中主要溶质浓度的方法；台盼兰据染法在正确操作的前提下可以得到很准确的CPA处理后的骨细胞的成活率。
同时还考察了冷冻保护剂在骨组织中的导入和洗脱过程对细胞和组织活性的影响。选取了两种冷冻保护剂的三种不同的浓度，考察了冷冻保护剂种类、浓度、操作温度、导入或洗脱方案以及后期培养等因素对骨细胞成活率的影响。实验结果表明：冷冻保护剂在导入过程中对细胞或组织的损伤主要为渗透损伤和毒性损伤，受二者共同作用存在一个最佳导入时间和最佳导入方案。洗脱方案的确定也由这二者的共同作用的影响；连续式导入或洗脱之后的细胞成活率要高于分步导入或洗脱；甘油渗透性要比DMSO差，但其毒性作用也比DMSO小；甘油渗透性受温度影响比较大，DMSO毒性受温度影响比较大。
最后，对骨组织内的主要细胞，成骨细胞进行了超低温保存过程中的冷冻和复温过程的考察。通过改变降温速率和复温速率对三种冷冻保护剂的保护作用进行了实验。结果表明，玻璃化冻存效果要明显优于非玻璃化冻存。非玻璃化冷冻保护剂在冷冻过程中存在部分玻璃化现象，但不能体现出玻璃化的优势。玻璃化冷冻保护剂也只有在冷却速率达到一定数值时才能实现以玻璃化为主的部分玻璃化，或者完全玻璃化。随冷却速率的增大，玻璃化冷冻保护剂的玻璃化能力越来越显著；VS1无论是在玻璃化温度方面，完全玻璃化能力方面，玻璃化形态(有无低温断裂)方面，对成骨细胞的整体保护作用方面，在最佳降温速率适用范围方面还是在对组织超低温保存的适用性方面，均优于7mol／L的DMSO溶液，只有在反玻璃化方面要逊于7mol／L的DMSO溶液；玻璃化冷冻保护剂的反玻璃化现象与低温断裂以及降温过程形成的冰晶都有着密切的联系，并且反玻璃化的程度随着复温速率的增大而减小。
Cryopreservation is a technique by which cells and tissues can be long-term preserved. With the more rapid development of medical science and the more urgent demand of clinic treatment, the cryopreservation of engineered tissue and live organ becomes the most advanced and promising research field of biomedical engineering. The data shows that around the world, the number of bone transplant operation is the second large one only less than blood transfusion. It will facilitate the clinic therapy and lessen the patients' pain if the autograft and heterograft of bone used for large-area bone loss can be successfully cryopreserved. Therefore the cryopreservation of bone tissue has a promising future and an extensive practical value.
At the beginning of the research of this thesis, osteoblasts and bone tissue is cultured in-vitro and the main techniques used in the experiment are cross-checked. By cross-checking the concentration of cryoprotective agent (CPA) and the viability of cells within bone tissue, we conclude that osmotic pressure technique and Typan blue stain method can precisely reflect two major parameters above.
Meanwhile, the loading and removing process of cryopreservation are studied by choosing three different concentrations of two CPAs. Type and the concentration of CPA, operating temperature, loading and removing protocol and post-culture is set as parameters to check their effects on the characteristics of the whole process. Results show that the osmosis damage and the toxicity damage are two major factors for the loss of cells' viability, that continuous loading and removing is better than intermittent ones, that glycerol's osmosis ability is worse than DMSO, but its toxicity is less than DMSO and that the effect of temperature change on the glycerol's osmosis ability is much more than on DMSO's, but its effect on DMSO's toxicity is much more than on glycerol's.
Finally, osteoblasts, the major cells in bone tissue, are freezed and thawed. By using three kinds of CPAs and changing the cooling rate and thawing rate, we conclude that for osteoblasts' cryopreservation, vitrification overmatches the crystallization. Non-vitrified CPA can partially vitrify, but can not show the superiority of vitrification, that with the increase of the cooling rate, vitrified CPA's ability to vitrify shows more greatly, that for two kinds of vitrified CPA studied in this experiment, VS1 is superior to 7mol/L DMSO in almost all aspects except the ability to overcome the devitrification, that the phenomena of the devitrification is closely related to the ice crystal appeared while freezing and the cryo-fragmentation and that the devitrification is blocked and finally prevented with the increasing of the thawing rate.
引文
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[2]．薛庆善．体外培养的原理与技术．科学出版社，2001：129．
[3]．华泽钊，任禾盛．低温生物医学技术．科学出版社，1994：1-2．
[4]. H Fansa, F Lassner, P H Kook, G Keilhoff, W Schneider. Cryopreservation of peripheral nerve grafts. Muscle Nerve, 2000,23:1227-1233
[5]. W J M Mass, W R Leeman, J P Groten, J J M Van De Sandt. Cryopreservation of precision-cut rat liver slices using a computer-controlled freezer. Toxicology in Vitro 2000,14:523-530.
[6]. A Guillouzo, L Rialland, A Fautrel, C Guyomard. Survival and function of isolated hepatocytes after cryopreservation. Chemico-Biological Interactions, 1999,121:7-16.
[7]. A P Li. Overview:hepatocytes and cryopreservation-a personal historical perspective. Chemico-Biological Interactions 1999,121:1-5.
[8]. G Vajta. Vitrification of the oocytes and embryos of domestic animals. Animal Reproduction Science, 2001,60-61:357-364.
[9]. P F Watson. The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science, 2000,60-61:481-492.
[10]. S Menoret, M Jean, L Tesson, J soulillou, I Anegon, B Charreau. Optimization of cryopreservation procedures for rat embryos. Transplantation Proceedings 1999,31: 1531-1532.
[11]. J M Shaw, A Oranratnachai, A O Trounson. Fundamental cryopreservation oocytes and ovarian tissue. Theriogenology, 2000,53:59-72.
[12]. W V Hok. Fundamental aspects of sperm cryobiology:the importance of species and individual differences. Theriogenology, 2000,53:47-58.
[13]. J M Shaw, S L Cox, A O Trouson, G Jenkin. Evaluation of the long-term function of cryopreserved ovarian grafts in the mouse, implications for human applications. Molecular and Cellual Endocrinology, 2000,161:103-110.
[14]. N R Mtango, M D Varisanga, Y J Dong. The effect of prefreezing the diluent portion of the straw in a step-wise vitrification process using ethylene glycol and polyvinylpyrrolidone to preserve bovine blastocysts. Cryobiology, 2001,42: 135.
[15]. L L Kuleshova, J M Shaw, A O Trounson. Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation. Cryohiology, 2001, 43:21.
[16]. Y Udoh, H Yanaga, Y Tai, K Y Inoue. Long-term viability of cryopreserved cultured epithelial grafts. Burns, 2000,26:535-542.
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