WO2021031614A1 - 一种高效抗氧化的人卵母细胞冷冻保护剂 - Google Patents

一种高效抗氧化的人卵母细胞冷冻保护剂 Download PDF

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WO2021031614A1
WO2021031614A1 PCT/CN2020/088708 CN2020088708W WO2021031614A1 WO 2021031614 A1 WO2021031614 A1 WO 2021031614A1 CN 2020088708 W CN2020088708 W CN 2020088708W WO 2021031614 A1 WO2021031614 A1 WO 2021031614A1
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melatonin
freezing
human
liquid
mol
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PCT/CN2020/088708
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English (en)
French (fr)
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章志国
丁丁
穆耀琴
邹慧娟
邹薇薇
陈蓓丽
周平
魏兆莲
曹云霞
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安徽医科大学第一附属医院
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

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  • the invention relates to a highly effective anti-oxidant cryoprotectant for human oocytes, which belongs to the field of reproductive medicine engineering.
  • freezing eggs can solve many religious, legal and ethical issues on the one hand, and on the other hand, for women who have damaged or lost ovarian function due to reproductive system diseases, such as For patients with early-onset ovarian failure, endometriosis, and malignant tumors undergoing chemotherapy or radiation therapy, egg freezing is an effective way to preserve the fertility of such women [1] .
  • human egg freezing technology has made great breakthroughs in recent years, and the survival rate has been greatly improved, human egg freezing has not been carried out routinely in the field of assisted reproduction technology like embryo and sperm freezing [2] .
  • the root cause is that the accumulated oxygen free radicals during the freezing process make the egg's oxidation-antioxidant system unbalanced, causing oxidative stress in the oocytes [3] .
  • This oxidative stress causes structural changes such as loosening of the cytoplasm of the egg, granulation, and increased vesicle rate, as well as a decrease in the membrane potential of the mitochondria and an increase in intracellular calcium levels [4-5] .
  • oxidative damage is one of the most important freezing damages. This damage not only causes irreversible damage to the structure and function of human oocytes, but also greatly reduces the recovery rate and development potential [5-6] . Oxidative damage is mainly manifested as an increase in the level of oxygen free radicals and calcium ions in the cell, which in turn causes the damage of mitochondrial function, and ultimately leads to serious damage or loss of cell function [7-8] .
  • the purpose of the present invention is to provide a cryoprotectant for human oocytes with high-efficiency antioxidant properties, which is added with the endogenous high-efficiency antioxidant melatonin on the basis of commonly used cryoprotectants for human oocytes (melatonin), not only inhibits the formation of vacuoles in the cytoplasm of human oocytes after freezing and thawing, but also reduces the level of oxygen free radicals in the cells, protects the function of mitochondria, and effectively protects the quality of human eggs after freezing and thawing .
  • the present invention provides a high-efficiency anti-oxidant cryoprotectant for human oocytes, which includes egg freezing liquid and egg thawing liquid.
  • the egg freezing liquid includes a balance liquid and a freezing liquid, and its composition is:
  • Freezing solution 40% buffer (V/V)+20% human serum substitute (V/V)+20%PROH(V/V)+20%EG(V/V)+0.65mol/L trehalose+ 10 -9 mol/L melatonin (melatonin);
  • the egg thawing liquid includes resuscitation liquid, diluent-1, diluent-2 and cleaning liquid, and its composition is:
  • Resuscitation solution 80% buffer solution (V/V) + 20% human serum substitute (V/V) + 1.0 mol/L trehalose + 10 -9 mol/L melatonin (melatonin),
  • Diluent-1 80% buffer (V/V) + 20% human serum substitute (V/V) + 0.5 mol/L trehalose + 10 -9 mol/L melatonin (melatonin),
  • Diluent-2 80% buffer (V/V) + 20% human serum substitute (V/V) + 0.2 mol/L trehalose + 10 -9 mol/L melatonin (melatonin),
  • Washing solution 80% buffer (V/V) + 20% human serum substitute (V/V) + 10 -9 mol/L melatonin (melatonin).
  • the buffer is selected from HTF 1024 (SAGE, USA), HTF1023 (SAGE, USA), GMOPS (Vitrolife, Sweden) or DPBS (SAGE, USA).
  • the human serum substitute is selected from SPS (SAGE, USA), SSS (Irovine Scientific, USA) or HAS (Vitrolife, Sweden), more preferably SSS (Irovine Scientific, USA).
  • Buffer covers HTF 1024 (SAGE, USA), HTF1023 (SAGE, USA), GMOPS (Vitrolife, Sweden) and DPBS (SAGE, USA), etc.; human serum substitute covers SPS (SAGE, USA), SSS (Irovine Scientific, USA) and HSA (Vitrolife, Sweden); PROH (1,2-Propanediol, MERCK, USA); EG (Ethylene glycol, MERCK, USA); Trehalose (Trehalose, ⁇ -D-glucopyranosyl ⁇ -D-glucopyranoside, C 12 H 22 O 11 ⁇ 99.0, FLUKA, USA); Melatonin (SIGMA). On the other hand, V/V represents the volume ratio.
  • the preparation method is to prepare a mixture according to the volume ratio of buffer, human serum substitute, PROH, and EG at 4:2:2:2.
  • the mixed solution of this ratio can be used to prepare a solution containing 0.65 mol/L of trehalose and 10-9 mol/L of melatonin.
  • cryoprotectants propylene glycol and ethylene glycol, an impermeable trehalose protective agent, and melatonin are used as antioxidants to develop the present invention. So far, there is still no human egg cryoprotectant in the reproductive world that contains antioxidants.
  • the existing cryoprotectants for human eggs only contain non-permeable protective agents and osmotic resistance agents, which cannot prevent human eggs from freezing and thawing. The oxidative damage suffered.
  • the present invention adds melatonin with high-efficiency antioxidant properties to the cryoprotectant of human oocytes, which not only inhibits the formation of intracytoplasmic vacuoles in human oocytes after freezing and thawing, but also reduces the level of intracellular oxygen free radicals As well as the level of calcium ions, it has the function of protecting mitochondria, preventing the oxidative damage of human oocytes during the freezing and thawing process, and effectively protecting the quality of human eggs after freezing and thawing.
  • Figure 1 The effect of different concentrations of melatonin on the structure of human oocytes after freezing and thawing (arrows indicate cytoplasmic vacuoles).
  • Figure 2 The effect of different concentrations of melatonin on the level of ROS in human oocytes after freezing and thawing;
  • A Fluorescence image of human oocytes stained with DCFH kit (green fluorescence intensity from strong to weak is 0> 10 -5 >10 -7 >10 -9 );
  • B the effect of melatonin on the level of ROS in human oocytes. *Represents significant difference between groups (P ⁇ 0.05).
  • Figure 3 The effect of different concentrations of melatonin on the mitochondrial membrane potential of human oocytes after freezing and thawing.
  • A Fluorescence image of human oocytes stained with JC-1 kit (from top to bottom the first row shows red fluorescence, the second row shows green fluorescence, and the third row shows yellow fluorescence);
  • B Melatonin Effect on the mitochondrial membrane potential of human oocytes. *Represents significant difference between groups (P ⁇ 0.05).
  • Figure 4 The effect of different concentrations of melatonin on the calcium ion concentration in human oocytes after freezing and thawing.
  • A Fluorescence image of human oocytes stained with Fluo-4AM kit (the intensity of green fluorescence is 0>10 -5 >10 -7 >10 -9 from strong to weak);
  • B Melatonin to human The influence of calcium ions in oocytes. *Represents significant difference between groups (P ⁇ 0.05).
  • the cryoprotectant for human oocytes of the present invention includes egg freezing solution and egg thawing solution.
  • the egg freezing solution includes a balance solution and a freezing solution, and its composition is:
  • the egg thawing liquid includes resuscitation liquid, diluent-1, diluent-2 and cleaning liquid, and its composition is:
  • Freezing fluid 40%HTF 1024(V/V)+20%SSS(V/V)+20%PROH(V/V)+20%EG(V/V)+0.65mol/L trehalose+ 10 -9 mol/L melatonin (melatonin),
  • the egg thawing liquid includes resuscitation liquid, diluent-1, diluent-2 and cleaning liquid, and its composition is:
  • Resuscitation fluid 80% HTF 1024 (V/V) + 20% SSS (V/V) + 1.0 mol/L trehalose + 10 -9 mol/L melatonin (melatonin),
  • Diluent-1 (DM-1): 80% HTF 1024 (V/V) + 20% SSS (V/V) + 0.5 mol/L trehalose + 10 -9 mol/L melatonin (melatonin),
  • Diluent-2 (DM-2): 80% HTF 1024 (V/V) + 20% SSS (V/V) + 0.2 mol/L trehalose + 10 -9 mol/L melatonin (melatonin),
  • Washing solution 80% HTF 1024 (V/V) + 20% SSS (V/V) + 10 -9 mol/L melatonin (melatonin).
  • MI/GV immature human oocytes
  • TM resuscitation solution
  • the DCFH kit was used to determine the level of ROS in living human oocytes. After freezing and thawing, the human oocytes were cultured in vitro for 2-2.5 hours, and then washed in pre-warmed 1X PBS. Then stain with 10 ⁇ mol/L DCFHDA solution, and treat for 20 minutes at 37°C and 5% CO 2 in the dark. After washing three times in 1X PBS, the human oocytes were transferred to a glass dish, and the fluorescence was photographed immediately under a Zeiss inverted confocal microscope. The intensity of green fluorescence represents the level of ROS in the cell, and the picture is analyzed by a confocal microscope image processing system.
  • JC-1 Assay Kit use the JC-1 Assay Kit to evaluate the mitochondrial membrane potential.
  • Human oocytes were cultured in vitro for 2-2.5 hours. The frozen human oocytes were washed with preheated 0.1% PVA/PBS, and stained with 8 ⁇ mol/L JC-1 solution in 5% CO 2 at 37°C, 5 Incubate for 30 minutes in the dark in a %CO 2 environment. In order to eliminate the influence of autofluorescence on the experiment, a human oocyte was selected as a negative control in each experiment and treated with 1XBuffer. The human oocytes were washed again, and fluorescence imaging was performed immediately under a Zeiss inverted confocal microscope. The mitochondrial membrane potential is expressed by the ratio of red fluorescence to green fluorescence, and the picture is analyzed by a confocal microscope image processing system.
  • Melatonin untreated group (melatonin concentration is 0): In this case, a total of 53 human oocytes were collected. After freezing and thawing, 51 human eggs survived, with a survival rate of 96.23%;
  • the concentration of 10 -9 mol/L melatonin treatment group a total of 34 human oocytes were collected. After freezing and thawing, 34 human oocytes survived, with a survival rate of 100%;
  • Treatment group with a concentration of 10 -7 mol/L melatonin A total of 38 human oocytes were collected. After freezing and thawing, 38 human oocytes survived, with a survival rate of 100%;
  • the melatonin treatment group with a concentration of 10 -5 mol/L A total of 38 human oocytes were collected. After freezing and thawing, 36 human oocytes survived, with a survival rate of 94.74%.
  • FIG. 1 There are differences in cell morphology between groups with different concentrations of melatonin. Among them, the human oocytes in the melatonin-untreated group had no abnormal morphology, and the cytoplasm was heterogeneous and darkened. The cytoplasm contained vacuoles (as shown by the arrow) and granular dark areas.
  • the first polar body was smooth , Intact and round, with normal zona pellucida and perivitelline space; human oocytes treated with a concentration of 10 -9 mol/L melatonin had no abnormal morphology, and the cytoplasm was clear and homogeneous, without vacuoles or debris , No granular dark areas, the first polar body is smooth, complete, round, zona pellucida and perivitelline space are normal; human oocytes treated with a concentration of 10 -7 mol/L melatonin have no abnormal morphology, The cytoplasm is relatively clear and homogeneous, with vacuoles (as indicated by the arrow), no fragments, no granular dark areas, the first polar body is smooth, complete, and round, and the zona pellucida and the perivitelline space are normal; the concentration is 10
  • the human oocytes in the -5 mol/L melatonin treatment group had no abnormal morphology, and the cytoplasm
  • the levels of intracellular oxygen free radicals are different between the melatonin groups of different concentrations.
  • the intracellular oxygen free radical level of the 10 -9 mol/L melatonin treatment group was the lowest and was significantly lower than the 10 -5 mol/L melatonin treatment group and the melatonin untreated group, which was statistically significant Significance (P ⁇ 0.05);
  • the level of intracellular oxygen free radicals in the 10 -7 mol/L melatonin treatment group was significantly lower than the 10 -5 mol/L melatonin treatment group and the melatonin untreated group, and there was Significant statistical significance (P ⁇ 0.05);

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Abstract

本发明公开了一种高效抗氧化的人卵母细胞冷冻保护剂，包括卵子冷冻液和卵子解冻液，所述卵子冷冻液包括平衡液和冷冻液，所述卵子解冻液包括复苏液、稀释液-1、稀释液-2和清洗液。本发明联合应用了丙二醇和乙二醇两种渗透性冷冻保护剂、海藻糖非渗透性保护剂以及褪黑素(melatonin)抗氧化剂。本发明在人卵母细胞冷冻保护剂中添加具备高效抗氧化特性的褪黑素，不仅抑制了冻融后人卵母细胞胞浆内空泡的形成，而且降低了细胞内的氧自由基水平以及钙离子水平，具备保护线粒体功能，防止了人卵子在冻融过程中所遭受的氧化损伤，进而有效地保护了冻融后的人卵子质量。

Description

一种高效抗氧化的人卵母细胞冷冻保护剂 技术领域
本发明涉及一种高效抗氧化的人类卵母细胞冷冻保护剂，属于生殖医学工程领域。
背景技术
2006年1月，我国首例、全球第二例“三冻(冻卵、冻精、冻胚胎，再解冻移植入母体子宫内)”试管婴儿在北京大学第三医院诞生。目前，中国ART(辅助生殖技术)衍生技术的应用范围和技术水准已处于世界前列。随着全球性不孕人口上升，女性育龄的推迟、癌症患者的增加，女性生育能力保存的需求也日益增加。卵子冷冻技术也已逐步应用于临床，与冷冻胚胎相比较，冷冻卵子一方面可使许多宗教、法律及伦理问题得以解决，另一方面对于因生殖系统疾病导致卵巢功能损伤甚至散失的妇女，如早发性卵巢衰竭、子宫内膜异位症及接受化学治疗或放射线治疗恶性肿瘤的患者，卵子冷冻是保存此类妇女生育能力的有效途径 [1]。
尽管人类卵子冷冻技术近年来取得了较大的突破，成活率大大提高，但人卵子冷冻至今仍未能像胚胎及精子冷冻在辅助生殖技术领域常规开展 [2]。根本原因是冷冻过程中累积的氧自由基使卵子的氧化-抗氧化系统失衡，造成卵母细胞的氧化应激 [3]。这种氧化应激使卵子的胞浆松散、颗粒化、含泡率升高等结构改变，以及线粒体的膜电位降低、胞内钙离子水平升高 [4-5]，这种细胞结构和功能的改变会造成冻融后的人卵子质量下降，导致人卵子冷冻技术进展较为缓慢。因此开发一种更加高效安全的人类卵子冷冻保护剂是解决人类卵子冷冻效果低下的唯一路径。
目前，没有一种人卵子冷冻保护剂具备高效的抗氧化特性，现有的人卵子冷冻保护剂仅包含非渗透性保护剂和/或渗透性保护剂。冷冻保护剂的广泛使用旨在防止人卵子遭受冷冻损伤。其中，氧化损伤是最主要的冷冻损伤之一，这种损伤不仅造成了人卵母细胞结构和功能的不可逆损伤，而且使其复苏率和发育潜能大大降低 [5-6]。氧化损伤主要表现为细胞内氧自由基水平以及钙离子水平的升高，进而引发线粒体功能的损伤，最终导致细胞功能的严重损伤或丧失 [7-8]。
迄今为止，生殖界仍没有一种人类卵子冷冻保护剂具备高效地抗氧化特性，现 有的人卵子冷冻保护剂仅包含非渗透性保护剂和渗透性抵抗剂，现有的保护剂无法防止人卵子在冻融过程中所遭受的氧化损伤，这种损伤不仅造成了人卵母细胞结构和功能的不可逆损伤，而且使其复苏率和发育潜能大大降低，严重阻碍了在辅助生殖技术领域常规开展。再者，虽然有报道采用褪黑素在其他实验动物如小鼠卵细胞培养方面具备一定应用，但其研究仅限于某一方面或者其所要研究的方面而非卵母细胞冷冻损伤的防护研究；并且人卵母细胞样本相对于实验动物样本更加难以获取，这就给人卵母细胞的研究造成了较大困难，即使能够获取足够量的人卵母细胞样本，也会因为种属间的差异，如卵母细胞的直径大小，卵胞膜渗透性及弹性等参数的完全不同导致动物样本的实验结果与人的结果存在本质差异，即动物的研究结果不能直接用于人类样本。另外，迄今为止，没有关于褪黑素应用于人卵母细胞冷冻方面的研究报道。
发明内容
本发明的目的在于提供了一种具备高效抗氧化特性的人类卵母细胞冷冻保护剂，该保护剂在常用的人类卵母细胞冷冻保护剂的基础上添加内源性的高效抗氧化剂褪黑素(melatonin)，不仅抑制了冻融后人卵母细胞胞浆内空泡的形成，而且降低了细胞内的氧自由基水平、保护线粒体的功能，进而有效地保护了冻融后的人卵子质量。
本发明所提供的一种高效抗氧化的人卵母细胞冷冻保护剂，包括卵子冷冻液和卵子解冻液，所述卵子冷冻液包括平衡液和冷冻液，其组成为：
平衡液：60％缓冲液(V/V)+20％人血清替代物(V/V)+10％PROH(V/V)+10％EG(V/V)+10 -9mol/L褪黑素(melatonin)，
冷冻液：40％缓冲液(V/V)+20％人血清替代物(V/V)+20％PROH(V/V)+20％EG(V/V)+0.65mol/L海藻糖+10 -9mol/L褪黑素(melatonin)；
所述卵子解冻液包括复苏液、稀释液-1、稀释液-2和清洗液，其组成为：
复苏液：80％缓冲液(V/V)+20％人血清替代物(V/V)+1.0mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
稀释液-1：80％缓冲液(V/V)+20％人血清替代物(V/V)+0.5mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
稀释液-2：80％缓冲液(V/V)+20％人血清替代物(V/V)+0.2mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
清洗液：80％缓冲液(V/V)+20％人血清替代物(V/V)+10 -9mol/L褪黑素(melatonin)。
所述缓冲液选自HTF 1024(SAGE,USA)、HTF1023(SAGE,USA)、GMOPS(Vitrolife,Sweden)或DPBS(SAGE,USA)。
所述人血清替代物选自SPS(SAGE,USA)、SSS(IrovineScientific，USA)或HAS(Vitrolife,Sweden)，更优选SSS(IrovineScientific，USA)。
缓冲液涵盖HTF 1024(SAGE,USA)、HTF1023(SAGE,USA)、GMOPS(Vitrolife,Sweden)及DPBS(SAGE,USA)等；人血清替代物涵盖SPS(SAGE,USA)、SSS(Irovine Scientific，USA)及HSA(Vitrolife,Sweden)；PROH(1，2-Propanediol，MERCK，USA)；EG(Ethylene glycol，MERCK，USA)；海藻糖(Trehalose，α-D-glucopyranosylα-D-glucopyranoside，C 12H 22O 11≥99.0，FLUKA，USA)；褪黑素(SIGMA)。另V/V代表体积比。
以冷冻液(VM)为例(其他溶液的配制同此)，其配制方法为，按缓冲液、人血清替代物、PROH、EG的体积比4：2：2：2配成混合液，以该配比的混合液来配制含海藻糖0.65mol/L和褪黑素10-9mol/L的溶液即可。
本发明联合应用了丙二醇和乙二醇两种渗透性冷冻保护剂、海藻糖非渗透性保护剂以及褪黑素(melatonin)作为抗氧化剂开发了本发明。迄今为止，生殖界仍没有一款人类卵子冷冻保护剂含有抗氧化物质成分，现有的人卵子冷冻保护剂仅包含非渗透性保护剂和渗透性抵抗剂，无法防止人卵子在冻融过程中所遭受的氧化损伤。本发明在人卵母细胞冷冻保护剂中添加具备高效抗氧化特性的褪黑素，不仅抑制了冻融后人卵母细胞胞浆内空泡的形成，而且降低了细胞内的氧自由基水平以及钙离子水平，具备保护线粒体功能，防止了人卵母细胞在冻融过程中所遭受的氧化损伤，进而有效地保护了冻融后的人卵子质量。
附图说明
图1：不同浓度的褪黑素对冻融后人卵母细胞结构的影响(箭头所示为卵子胞浆空泡)。
图2：不同浓度的褪黑素对冻融后人卵母细胞内ROS水平的影响；(A)用DCFH试剂盒染色人卵母细胞的荧光图(绿色荧光强度由强到弱依次为0＞10 -5＞10 -7＞10 -9)；(B)褪黑素对人卵母细胞内ROS水平的影响。*代表组间差异有显着性(P<0.05)。
图3：不同浓度的褪黑素对冻融后人卵母细胞线粒体膜电位的影响。(A)用JC-1试剂盒染色人卵母细胞的荧光图(从上到下第一排显红色荧光，第二排显绿色荧光，第三排显黄色荧光)；(B)褪黑素对人卵母细胞线粒体膜电位的影响。*代表组间差异有显着性(P<0.05)。
图4：不同浓度的褪黑素对冻融后人卵母细胞内钙离子浓度的影响。(A)用Fluo-4AM试剂盒染色人卵母细胞的荧光图(绿色荧光强度由强到弱依次为0＞10 -5＞10 -7＞10 -9)；(B)褪黑素对人卵母细胞内钙离子的影响。*代表组间差异有显着性(P<0.05)。
具体实施方式
下述实施例是对于本发明内容的进一步说明以作为对本发明技术内容的阐释，但本发明的实质内容并不仅限于下述实施例所述，本领域的普通技术人员可以且应当知晓任何基于本发明实质精神的简单变化或替换均应属于本发明所要求的保护范围。
实施例1
本发明的人类卵母细胞冷冻保护剂，包括卵子冷冻液和卵子解冻液，所述卵子冷冻液包括平衡液和冷冻液，其组成为：
所述卵子解冻液包括复苏液、稀释液-1、稀释液-2和清洗液，其组成为：
平衡液(EM)：60％HTF 1024(V/V)+20％SSS(V/V)+10％PROH(V/V)+10％EG(V/V)+10 -9mol/L褪黑素(melatonin)，
冷冻液(VM)：40％HTF 1024(V/V)+20％SSS(V/V)+20％PROH(V/V)+20％EG(V/V)+0.65mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
所述卵子解冻液包括复苏液、稀释液-1、稀释液-2和清洗液，其组成为：
复苏液(TM)：80％HTF 1024(V/V)+20％SSS(V/V)+1.0mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
稀释液-1(DM-1)：80％HTF 1024(V/V)+20％SSS(V/V)+0.5mol/L海藻糖 +10 -9mol/L褪黑素(melatonin)，
稀释液-2(DM-2)：80％HTF 1024(V/V)+20％SSS(V/V)+0.2mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
清洗液(WM)：80％HTF 1024(V/V)+20％SSS(V/V)+10 -9mol/L褪黑素(melatonin)。
对比例
仅调整褪黑素(melatonin)浓度分别为0、10 -7mol/L、10 -5mol/L，其他同实施例1。
人卵母细胞冻融技术的实施如下：
由安徽医科大学第一附属医院生殖医学中心从受试者为来我们中心寻求IVF/ICSI治疗的不孕症患者，年龄均小于35岁。收集废弃的未成熟人卵母细胞(MI/GV)于体外培养24小时，随后体外成熟的人卵母细胞行冷冻-解冻过程。
冷冻过程:
将人卵母细胞移至平衡液(EM)，放置15min后,转移至冷冻液(VM)液中1min，随后将人卵母细胞转移至特定的载体上并迅速投入液氮,最后再将含有人卵母细胞的载体移入液氮罐中-196℃保存。
解冻过程:
冷冻2周后，将含人卵母细胞的载体从液氮罐中取出，迅速将载体插入复苏液(TM)中，人卵母细胞在TM液中自动脱落，快速将人卵母细胞转移至稀释液-1(DM-1)，室温下放置3min后依次转至稀释液-2(DM-2)和清洗液，各3min，最后，人卵母细胞被移入配子培养液，在37℃、5％CO 2及饱和湿度条件下培养。
(2)冻融后的人卵子结构与功能检测的实施如下：
形态学检测：
冻融后的人卵母细胞体外培养2-2.5小时后，在倒置电子显微镜下观察形态并拍照记录。
细胞内ROS水平检测：
使用DCFH试剂盒测定活人卵母细胞中的ROS水平。冻融后的人卵母细胞体外培养2-2.5小时后，在预热的1X PBS中洗涤。然后用10μmol/L DCFHDA溶液染色，在37℃、5％CO 2的环境下避光处理20分钟。在1X PBS中洗涤三次后将人卵 母细胞转移至玻璃皿中，立即在Zeiss倒置共聚焦显微镜下进行荧光拍摄。绿色荧光的强度代表细胞内ROS的水平，图片用共聚焦显微镜图像处理系统分析。
线粒体膜电位检测：
根据制造商的说明，使用JC-1测定试剂盒评估线粒体膜电位。人卵母细胞体外培养2-2.5小时，用预热的0.1％PVA/PBS洗涤冻融的人卵母细胞，并用5％CO 2中的8μmol/L JC-1溶液染色，在37℃、5％CO 2的环境下避光孵育30分钟。为了消除自发荧光对实验的影响，在每个实验中选择一个人卵母细胞作为阴性对照，并用1XBuffer缓冲液处理。再次洗涤人卵母细胞，立即在Zeiss倒置共聚焦显微镜下进行荧光拍摄。线粒体膜电位用红色荧光与绿色荧光的比率表示，图片用共聚焦显微镜图像处理系统分析。
细胞内钙离子水平检测：
根据制造商的说明，使用Fluo4 AM试剂盒评估细胞内钙浓度。人卵母细胞体外培养2-2.5小时，将冻融的人卵母细胞在预热的1X PBS中洗涤三次，并用5μmol/L Fluo4 AM溶液染色，在37℃、5％CO 2的环境下避光处理30分钟。用1X PBS处理阴性对照组人卵母细胞，再次洗涤所有的人卵母细胞，并避光孵育15-30分钟，立即在Zeiss倒置共聚焦显微镜下进行荧光拍摄。绿色荧光的强度代表细胞内钙离子的浓度。荧光强度越弱，钙离子浓度越低，图片用共聚焦显微镜图像处理系统分析。
目前，本发明相关实验已在安徽医科大学第一附属医院生殖医学中心完成。实验研究及结果如下：
褪黑素未处理组(褪黑素浓度为0)：本例共收集人卵母细胞53枚，人卵子冻融后，存活51枚，存活率96.23％；
浓度为10 -9mol/L褪黑素处理组：共收集人卵母细胞34枚，人卵子冻融后，存活34枚，存活率100％；
浓度为10 -7mol/L褪黑素处理组：共收集人卵母细胞38枚，人卵子冻融后，存活38枚，存活率100％；
浓度为10 -5mol/L褪黑素处理组：共收集人卵母细胞38枚，人卵子冻融后，存活36枚，存活率94.74％。
如图1所示：不同浓度的褪黑素组之间细胞形态有差异。其中，褪黑素未处理组的人卵母细胞无异常形态出现，胞浆不均质、发黑，胞浆内含有空泡(如箭头所 示)及颗粒状暗区，第一极体光滑、完整，呈圆形，透明带及卵周间隙正常；浓度为10 -9mol/L褪黑素处理组的人卵母细胞无异常形态出现，胞质清亮、均质，无空泡、碎片，无颗粒状暗区，第一极体光滑、完整，呈圆形，透明带及卵周间隙正常；浓度为10 -7mol/L褪黑素处理组的人卵母细胞无异常形态出现，胞质较为清亮、均质，有空泡(如箭头所示)，无碎片，无颗粒状暗区，第一极体光滑、完整，呈圆形，透明带及卵周间隙正常；浓度为10 -5mol/L褪黑素处理组的人卵母细胞无异常形态出现,胞浆不均质、较黑，胞浆内含有空泡(如箭头所示)及局部颗粒状暗区，第一极体光滑、完整，呈圆形，透明带，卵周间隙大。
如图2所示：不同浓度的褪黑素组之间细胞内氧自由基水平有差异。其中，浓度为10 -9mol/L褪黑素处理组细胞内氧自由基水平最低且明显低于10 -5mol/L褪黑素处理组与褪黑素未处理组，有显著性统计学意义(P<0.05)；浓度为10 -7mol/L褪黑素处理组细胞内氧自由基水平明显低于10 -5mol/L褪黑素处理组与褪黑素未处理组，且有显著性统计学意义(P<0.05)；
如图3所示：不同浓度的褪黑素组之间线粒体膜电位水平有差异。其中，浓度为10 -9mol/L褪黑素处理组的线粒体膜电位水平最高且明显高于褪黑素未处理组，有显著性统计学意义(P<0.05)；浓度为10 -7mol/L褪黑素处理组线粒体膜电位水平明显高于褪黑素未处理组，且有显著性统计学意义(P<0.05)；
如图4所示：不同浓度褪黑素组之间细胞内钙离子水平有差异。其中，浓度为10 -9mol/L褪黑素处理细胞内钙离子水平最低且明显低于其他各组，有显著性统计学意义(P<0.05)；浓度为10 -7mol/L褪黑素处理组细胞内钙离子水平明显低于褪黑素未处理组，且有显著性统计学意义(P<0.05)。
以上结果表明，在人卵母细胞冷冻保护剂中添加具备高效抗氧化特性的褪黑素且浓度10 -9mol/L，不仅抑制了冻融后人卵母细胞胞浆内空泡的形成，而且降低了细胞内的氧自由基水平以及钙离子水平、具备保护线粒体的功能，进而能够有效地保护冻融后的人卵子质量。
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Claims (3)

  1. 一种高效抗氧化的人卵母细胞冷冻保护剂，包括卵子冷冻液和卵子解冻液，其特征在于，所述卵子冷冻液包括平衡液和冷冻液，其组成为：
    平衡液：60％缓冲液(V/V)+20％人血清替代物(V/V)+10％PROH(V/V)+10％EG(V/V)+10 -9mol/L褪黑素(melatonin)，
    冷冻液：40％缓冲液(V/V)+20％人血清替代物(V/V)+20％PROH(V/V)+20％EG(V/V)+0.65mol/L海藻糖+10 -9mol/L褪黑素(melatonin)；
    所述卵子解冻液包括复苏液、稀释液-1、稀释液-2和清洗液，其组成为：
    复苏液：80％缓冲液(V/V)+20％人血清替代物(V/V)+1.0mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
    稀释液-1：80％缓冲液(V/V)+20％人血清替代物(V/V)+0.5mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
    稀释液-2：80％缓冲液(V/V)+20％人血清替代物(V/V)+0.2mol/L海藻糖+10 -9mol/L褪黑素(melatonin)，
    清洗液：80％缓冲液(V/V)+20％人血清替代物(V/V)+10 -9mol/L褪黑素(melatonin)。
  2. 如权利要求1所述高效抗氧化的人卵母细胞冷冻保护剂，其特征在于，所述缓冲液选自HTF1024(SAGE,USA)、HTF1023(SAGE,USA)、GMOPS(Vitrolife,Sweden)或DPBS(SAGE,USA)。
  3. 如权利要求1所述高效抗氧化的人卵母细胞冷冻保护剂，其特征在于，所述人血清替代物选自SPS(SAGE,USA)、SSS(IrovineScientific，USA)或HSA(Vitrolife,Sweden)。
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