卵母细胞Cryotop法玻璃化冷冻的研究

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I
摘要
卵母细胞的低温保存技术是人类辅助生殖领域的重要工具，通过对健康的卵
母细胞进行低温保存，将能够为需要的人提供更多的生育机会。此外，家畜胚胎
体外规模化生产、核移植研究、转基因动物等研究对卵母细胞的需求日益增加，
卵母细胞的低温保存技术可以为家禽胚胎工程技术的开展提供充足的材料来源，
同时它也是优质家畜保种和珍稀濒危野生动物保护的一条新途径。
Cryotop 技术是目前诸多卵母细胞玻璃化冷冻方案中最有效的方法之一。然而，
Cryotop 方案中样品所能够达到的最高冷冻速率有限，要实现玻璃化必须采用高浓
度的冷冻保护剂，这使得卵母细胞经 Cryotop 法玻璃化冷冻后的发育潜能显著低于
对照组。因此提高 Cryotop 冷冻中样品的冷冻速率，降低冷冻保护剂的浓度，对提
高卵母细胞玻璃化后的存活率和发育潜能将会有很大的帮助。本文以提高样品在
Cryotop 冷冻中所能实现的冷冻速率为主要目标，从传热学角度对卵母细胞 Cryotop
法玻璃化冷冻方案进行了分析和优化，并按照优化后的 Cryotop 方案进行了猪 GV
期卵母细胞的玻璃化冷冻的初步试验。具体研究内容如下：
1. 通过数值模拟计算，分析了四种具有代表性的卵母细胞玻璃化冷冻方法（弹
射法，OPS 法，QMC 法和 Cryotop 法）的传热特性，得出 Cryotop 方案优于其它
三种方案；进而对 Cryotop 冷冻方案进行了优化计算，结果表明随着载板厚度的减
小、冷冻保护液体积的减小、冷源温度的降低及对流换热系数的增大，细胞的冷
冻速率均随之增加。
2. 搭建了一套快速测温系统，通过实验测量得出了样品液滴在不同 Cryotop
载板厚度、不同冷源温度及添加纳米颗粒的几种情况下所能达到的冷冻速率。载
板厚度为 0.06mm、0.08mm 和0.10mm 的Cryotop 在液氮中实现的冷冻速率分别为
77093.0
3683.7
K/min
、71559.6
2210.5
K/min
、63142.9
2711.1
K/min
，结果
表明随着载板厚度的减少，样品液滴的冷冻速率会随之增加；Cryotop 载板厚度为
0.10mm 时样品液滴在浆态氮中实现的冷冻速率为 78151.3
2750.5
K/min
，结果表
明在浆态氮中所实现的冷冻速率比在液氮中高 23.8%；添加了浓度为 0.1%的三种
粒径（20nm、40nm、60nm）的纳米颗粒后，样品液滴在载板厚度为 0.10mm 的
Cryotop 中所实现的的冷冻速率分别为 62076.1
578.2 K/min、61562.8
508.8
K/min、61355.4
517.5 K/min，结果表明在 Cryotop 方案中冷冻保护剂中添加 0.1%
的纳米颗粒对样品冷冻速率没有显著性影响。
3. 采用模拟计算与冷冻速度快速测量相结合的方式，研究了 Cryotop 模型在
冷冻过程中的对流换热系数，得出在 Cryotop 法冷冻的模拟计算中所需要的对流换
II
热系数范围为 6000
KmW 2
<h<8000
KmW 2
；采用差示扫描量热仪（DSC）测
量出了冷冻保护剂和添加了三种粒径（20nm、40nm、60nm）纳米颗粒的冷冻保护
剂的结晶放热量，经过计算得出四种冷冻保护剂实现玻璃化转变所需冷冻速率阈
值范围，与实验测得的冷冻速率相比较，得出四种冷冻保护剂在卵母细胞的 Cryotop
方案中均能满足玻璃化转变的要求。
4. 采用 Cryotop 法对猪 GV 期卵母细胞进行了玻璃化冷冻实验，Cryotop 载板
厚度为 0.06mm、0.08mm、0.10mm 时，得出卵母细胞复温后的存活率分别为
%5.3%7.71
、
%5.69.3%6
、
4.9%70.7%
，结果表明随着载板厚度的减小，所
得的细胞复温后的存活率没有显著性变化。在冷冻保护剂终添加浓度均为 0.1%的
三种粒径（20nm、40nm 和60nm）的纳米颗粒，再次进行了 Cryotop 法猪 GV 期
卵母细胞玻璃化冷冻实验，得出复卵母细胞温后的存活率分别为
%0.103.0%8
、
%8.113.4%8
、
%0.102.0%8
，对照组的结果为
%0.61.3%7
，结果表明添加纳
米颗粒的冷冻保护剂组复温后卵母细胞的存活率高于未添加纳米颗粒的冷冻保护
剂组；三种不同粒径的纳米颗粒冷冻保护剂组所得到的复温后卵母细胞的存活率
没有显著性差异。
关键词：卵母细胞 玻璃化 Cryotop 数值模拟 快速测温 纳米颗粒
III
ABSTRACT
Oocyte cryopreservation is an important tool for human assisted reproduction. It
would be able to provide more reproductive opportunities for people who needed. In
addition, the technology can provide adequate sources for animal embryos in vitro
large-scale production、nuclear transplantation research、transgenic animals and other
studies on oocyte. And it is a new way for protection of high-quality livestock and
endangered wildlife.
Cryotop is one of the most effective way of oocyte vitrification. However, the
developmental potential of oocyte vitrification by Cryotop is significantly lower than
the result of control group. The main cause is that the highest freezing rate achieved by
Cryotop is limited, and consequently high concentrations of cryoprotectants(CPA) are
needed to achieve vitrification, which could reduce the survival and developmental
potential of oocyte. This study was performed to increase the freezing rate in Cryotop,
the Cryotop method was analyzed and opitimized from the heat transfer point of view.
The pig GV oocytes were vitrified by the the optimized Cryotop method. Following
work has been carried out:
1. The heat transfer characteristics of four representative oocyte vitrification
methods (ejection method, OPS method, QMC method and Cryotop method) were
studied by numerical simulation, the results showed the Cryotop method is superior than
other three methods. Then the Cryotop method was optimized by numerical simulation,
the results showed that as the carrier board thickness decreased, the volume of
vitrification solution decreased, the cold source temperature decreased and convective
heat transfer coefficient increased, the freezing rates of cells increased.
2. A set of fast temperature measurement system was built, the freezing rates of
sample in different conditions were measured by the system. When the thickness of
Cryotop were 0.06mm、0.08mm and 0.10mm, the freezing rates of the sample in liquid
nitrogen were
K/min 、
K/min 、
2711.1 63142.9
K/min, respectively. When the thickness of Cryotop was 0.10mm, the freezing rates of
the sample in slurry nitrogen was
2750.5 78151.3
K/min
,which was 23.8% higher
than in liquid nitrogen. When the thickness of Cryotop was 0.10mm and three kinds of
nanoparticles (20nm, 40nm, 60nm) were added in CPA, the freezing rates of the sample
in liquid nitrogen were
578.2 62076.1
K/min 、
508.8 61562.8
K/min 、
IV
517.5 61355.4
K/min, respectively. Nanoparticles had no significant effect on the
sample freezing rate in Cryotop.
3. With the combination of simulation and rapid freezing rate measurement, the
convective heat transfer coefficient of Cryotop used in the simulation was studied, its
range is about
KmW8000<h<KmW 6000 22
. With the differential scanning
calorimeter, the heat of crystallization of four kinds of CPA were measured, and the
threshold of freezing rate needed in vitrification were calculated, the results showed that
four kinds of CPA all could realize vitrification in Cryotop method.
4. The Pig GV oocytes were vitrified by Cryotop. When the thickness of Cryotop
were 0.06mm 、0.08mm and 0.10mm, the survival of vitrified oocytes were
%5.3%7.71
、
%5.69.3%6
、
4.9%70.7%
, respectively. There was no significant
difference between groups. When three kinds of nanoparticles (20nm, 40nm, 60nm)
were added in CPA, the survival of vitrified oocytes in three nanoparticle groups and
control group were
%0.103.0%8
、
%8.113.4%8
、
%0.102.0%8
、
%0.61.3%7
，
respectively. The oocyte survival rates of nanoparticle groups were higher than control
group and there was no significant difference between three nanoparticles groups.
Keyword: Oocytes, Vitrification, Cryotop, Numerical Simulation,
Rapid temperature measure, Nanoparticles
V
目 录
摘要................................................................................................................................... I
ABSTRACT ....................................................................................................................III
第一章 绪 论...............................................................................................................1
§1.1 卵母细胞的结构及功能 ...................................................................................... 1
§1.2 卵母细胞低温保存 .............................................................................................. 1
§1.2.1 卵母细胞低温保存的意义和背景................................................................1
§1.2.2 卵母细胞的低温保存方法............................................................................2
§1.3 国内外卵母细胞玻璃化冷冻保存技术的现状 ................................................... 5
§1.4 卵母细胞玻璃化冷冻存在的问题 ...................................................................... 7
§1.5 研究任务 .............................................................................................................. 8
第二章 卵母细胞玻璃化冷冻的数值模拟...................................................................10
§2.1 数值模拟软件简介 ............................................................................................. 10
§2.1.1 程序的结构和计算流程...............................................................................11
§2.1.2 FLUENT 程序求解范围.............................................................................. 11
§2.1.3 FLUENT 程序求解问题的步骤.................................................................. 11
§2.1.4 FLUENT 计算方法选择.............................................................................. 12
§2.2 CFD 在本课题中的应用 .................................................................................... 12
§2.2.1 算法选择......................................................................................................12
§2.2.2 材料特性......................................................................................................13
§2.3 CFD（GAMBIT）建模步骤 ..............................................................................13
§2.4 CFD(FLUENT)模拟计算步骤 ........................................................................... 17
§2.5 四种卵母细胞玻璃化冷冻方法传热特性的比较 ............................................ 18
§2.5.1 四种冷冻方法传热特点的比较...................................................................18
§2.5.2 四种冷冻方法细胞冷冻速率的比较..........................................................19
§2.6 Cryotop 法传热性能优化的模拟计算 ............................................................... 20
§2.6.1 不同的载板厚度对 Cryotop 法细胞冷冻速率的影响 ............................... 20
§2.6.2 不同的冷冻保护液体积对 Cryotop 法细胞冷冻速率的影响 ................... 21
§2.6.3 不同的冷源温度对 Cryotop 法细胞冷冻速率的影响 .............................. 22
§2.6.4 不同的对流换热系数对 Cryotop 法细胞冷冻速率的影响 ....................... 23
VI
§2.7 本章小结 ............................................................................................................ 24
第三章 Cryotop 快速冷冻速率的测量 ........................................................................ 25
§3.1 快速测温装置 ..................................................................................................... 25
§3.2 Cryotop 在液氮中冷却时冷冻速率的测量 ....................................................... 26
§3.2.1 材料与方法..................................................................................................26
§3.2.2 结果与分析...................................................................................................27
§3.3 Cryotop 在浆态氮中冷却时降温速率的测量 ................................................... 28
§3.3.1 浆态氮的提出背景和制作原理..................................................................28
§3.3.2 材料与方法...................................................................................................29
§3.3.3 结果与分析..................................................................................................29
§3.4 纳米冷冻保护剂在 Cryotop 法中冷冻速率的测量 ......................................... 30
§3.4.1 纳米冷冻保护剂的配制...............................................................................30
§3.4.2 纳米冷冻保护剂分散稳定性表征...............................................................31
§3.4.3 纳米颗粒冷冻保护剂在 Cryotop 法中冷冻速率的测量 ........................... 32
§3.5 本章小结 ............................................................................................................ 33
第四章 Cryotop 冷冻方案中对流换热系数和冷冻保护剂玻璃化转变所需冷冻速率
阈值的讨论.....................................................................................................................35
§4.1 Cryotop 冷冻过程的模拟计算中所需对流换热系数的讨论 ........................... 35
§4.1.1 Cryotop 冷冻速率的模拟计算及结果 ........................................................ 35
§4.1.2 快速测温系统测量冷冻速率及其结果......................................................37
§4.1.3 结果分析与讨论..........................................................................................37
§4.2 冷冻保护剂实现玻璃化冷冻所需冷冻速率阈值的讨论 ................................. 38
§4.2.1 冷冻保护剂溶液玻璃化的结晶动力学模型...............................................38
§4.2.2 实验测定
)( 0
TK
............................................................................................40
§4.2.3 冷冻保护剂实现玻璃化冷冻所需冷冻速率阈值的确定..........................40
§4.2.4 结果与分析..................................................................................................41
§4.3 本章小结 ............................................................................................................ 43
第五章 猪 GV 期卵母细胞 Cryotop 法玻璃化冷冻的初步实验研究........................44
§5.1 猪卵母细胞玻璃化冷冻的简介 ........................................................................ 44
§5.2 材料与方法 ........................................................................................................ 44
§5.2.1 实验设备和材料..........................................................................................44
VII
§5.2.2 实验方法和步骤..........................................................................................45
§5.3 Cryotop 载板厚度对卵母细胞玻璃化冷冻效果的影响 ................................... 47
§5.4 纳米颗粒对卵母细胞玻璃化冷冻效果的影响 ................................................. 48
§5.5 本章小结 ............................................................................................................ 51
第六章 结论与展望.....................................................................................................52
§6.1 文章主要结论 .................................................................................................... 52
§6.2 展望及建议 ........................................................................................................ 53
参考文献.........................................................................................................................55
在读期间公开发表的论文和承担科研项目及取得成果.............................................61
致谢.................................................................................................................................62
摘要：

I摘要卵母细胞的低温保存技术是人类辅助生殖领域的重要工具，通过对健康的卵母细胞进行低温保存，将能够为需要的人提供更多的生育机会。此外，家畜胚胎体外规模化生产、核移植研究、转基因动物等研究对卵母细胞的需求日益增加，卵母细胞的低温保存技术可以为家禽胚胎工程技术的开展提供充足的材料来源，同时它也是优质家畜保种和珍稀濒危野生动物保护的一条新途径。Cryotop技术是目前诸多卵母细胞玻璃化冷冻方案中最有效的方法之一。然而，Cryotop方案中样品所能够达到的最高冷冻速率有限，要实现玻璃化必须采用高浓度的冷冻保护剂，这使得卵母细胞经Cryotop法玻璃化冷冻后的发育潜能显著低于对照组。因此提高Cryoto...

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