WO2016206086A1 - 一种猪卵母细胞体外成熟培养液及其制备方法和培养方法 - Google Patents

一种猪卵母细胞体外成熟培养液及其制备方法和培养方法 Download PDF

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WO2016206086A1
WO2016206086A1 PCT/CN2015/082485 CN2015082485W WO2016206086A1 WO 2016206086 A1 WO2016206086 A1 WO 2016206086A1 CN 2015082485 W CN2015082485 W CN 2015082485W WO 2016206086 A1 WO2016206086 A1 WO 2016206086A1
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porcine
culture
culture solution
vitro maturation
oocytes
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PCT/CN2015/082485
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French (fr)
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蔡志明
牟丽莎
冯万有
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深圳市第二人民医院
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Priority to PCT/CN2015/082485 priority Critical patent/WO2016206086A1/zh
Priority to CN201580000472.XA priority patent/CN105518124A/zh
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  • the invention relates to the technical field of cell culture, in particular to an in vitro maturation culture liquid for pig oocytes and a preparation method thereof, and a method for culturing pig oocytes using the culture liquid.
  • embryonic technology has greatly promoted the process of animal husbandry and medicine, such as variety improvement, breeding of transgenic disease-resistant varieties, preparation of human disease models, production of animal protein bioreactors, and exploration of developmental mechanisms.
  • somatic cell cloning and disease model preparation of pigs require a large number of oocytes with developmental potential, but the current in vitro maturation rate of pig oocytes is not ideal.
  • pig oocytes are cultured in the following in vitro culture base fluids, such as the culture fluid developed by North Carolina State University (North Carolina State University (NCSU23/NCSU37), PZM series (PZM-3/PZM-4/PZM- 5) Culture medium and TCM-199.
  • TCM-199 is recognized as the most successful culture base liquid, which can basically meet the developmental metabolism and nutrient requirements of pig oocytes in vitro.
  • these in vitro cultured pig oocytes indicate that they are mature. The rate is very low, we also need to improve the in vitro culture to improve the maturation rate of pig oocytes in vitro.
  • the inventors have studied to significantly increase the maturation rate of porcine oocytes in vitro by adding hormones and nutrients on the basis of TCM-199 base fluid.
  • the broth of the present invention can be stably stabilized at 80% after being cultured for 42-44 hours after oocyte collection, and is not required for the entire mature culture process.
  • the culture solution is changed and the operation is simple.
  • the present invention provides a pig oocyte in vitro maturation medium, a preparation method thereof, and a method for culturing a pig oocyte using the culture solution.
  • the present invention provides a porcine oocyte in vitro maturation medium comprising TCM-199 basal medium and added porcine follicular fluid, fetal calf serum, eCG, hCG, EGF and IGF-1.
  • 10% porcine follicular fluid and 10% fetal bovine serum are contained by volume, and 10-15 IU/mL eCG, 10-15 IU/ml hCG, Less than 100 ng/mL of EGF and less than 40 ng/mL of IGF-1.
  • glutamine and gentamicin are also included.
  • porcine oocytes according to the first aspect, which comprises porcine follicular fluid, fetal bovine serum, eCG, hCG, EGF and IGF- 1 is added to the TCM-199 basal medium, and optionally, glutamine and gentamicin are added to prepare the above culture solution.
  • the above culture solution comprises the following components:
  • the present invention provides a method for in vitro maturation of a porcine oocyte, which comprises culturing a porcine oocyte in the culture solution described in the first aspect.
  • the culture solution is placed in an incubator at 5% CO 2 saturated humidity for at least 6 hours and then used to culture the porcine oocytes.
  • the aforementioned porcine oocytes are picked from 4-7 mm follicles.
  • the above porcine oocytes are cultured for 42-44 hours in an incubator at 38.5 ° C, 5% CO 2 saturated humidity.
  • the in vitro maturation medium of the porcine oocyte of the invention can significantly increase the in vitro culture of the porcine oocyte Maturity rate.
  • the experiment confirmed that after the oocyte collection, the polar body rate can be stabilized at 80% after 42-44h mature culture, and the development rate of the parthenogenetic embryo can reach 85%.
  • the culture medium does not need to be replaced during the whole mature culture process. ,easy to use.
  • Figure 1 is a diagram showing the in vitro maturation effect of eCG and hCG hormone and EGF and IGF-1 on porcine oocytes using TCM-199 base fluid in one embodiment of the present invention.
  • the present inventors have made a remarkable ability by adding porcine follicular fluid (PFF), fetal bovine serum (FBS), eCG, hCG, EGF and IGF-1 on the basis of the currently used TCM-199 basal medium.
  • PFF porcine follicular fluid
  • FBS fetal bovine serum
  • eCG eCG
  • hCG hCG
  • EGF eGF-1
  • IGF-1 IGF-1
  • the culture broth of one embodiment of the present invention comprises TCM-199 basal medium and added porcine follicular fluid, fetal calf serum, eCG, hCG, EGF and IGF-1.
  • TCM-199 basal medium is a classic cell culture system, which is safe and reliable as a mother liquor.
  • TCM-199 basic medium is commercially available, such as Gibco or Sigma.
  • Porcine follicular fluid can be obtained by centrifugation when collected from in vitro oocytes. Its role is to provide the necessary factors for oocyte development.
  • Fetal bovine serum is one of the components in various in vitro culture fluids, and its effectiveness and safety as one of the components of the culture medium for in vitro culture has been demonstrated.
  • eCG also known as pregnant horse serum gonadotropin, namely PMSG (Pregnant Mare Serum Gonadotropin), also known as horse chorionic gonadotropin. It is a hormone found in the serum of pregnant mares. It is also known to be produced in pregnant animals ( ⁇ , zebra, etc.), so it is currently commonly referred to as the equine animal chorionic gonadotropin (eCG).
  • PMSG Pregnant Mare Serum Gonadotropin
  • eCG equine animal chorionic gonadotropin
  • hCG a human chorionic gonadotropin
  • EGF epidermal growth factor
  • epidermal growth factor is a small peptide composed of 53 amino acid residues. It is a member of the large EGF-like family. It is a multifunctional growth factor that affects a variety of tissue cells in vitro and in vivo. Has a strong mitogenic effect.
  • IGF-1 insulin-like growth factor 1
  • Csomatomedin C growth regulator
  • PFF, FBS, eCG, hCG, EGF and IGF-1 act synergistically in TCM-199 basal medium to jointly improve the maturation rate of porcine oocytes in vitro.
  • 10% porcine follicular fluid and 10% fetal bovine serum are contained by volume, and 10-15 IU/mL eCG, 10-15 IU/ml hCG, 50 ng are contained. /mL of EGF and 10 ng/mL of IGF-1. IU stands for International Unit.
  • the EGF content can achieve the desired effect below 100 ng / mL, and the effect thereof increases with the increase of EGF content in this range, but the EGF content exceeds 100 ng / mL and does not show further improvement. Therefore, from the economic and practical point of view, the content of EGF is below 100 ng/mL; the content of IGF-1 is below 40 ng/mL, and the effect is in accordance with the content of IGF-1. The increase has an increasing trend. However, the IGF-1 content exceeding 40 ng/mL does not show any further improvement. Therefore, from the economical and practical point of view, the content of IGF-1 is selected to be 40 ng/mL or less.
  • glutamine may be added to ensure that the energy required for the porcine oocytes is sufficient.
  • gentamicin in order to impart antibacterial properties to the culture solution, gentamicin may be added, which inhibits exogenous contamination such as bacteria without affecting the normal growth and maturation of the pig oocyte.
  • IVM solution The formula of the in vitro culture (IVM solution) used in the present embodiment is as follows:
  • Equilibrium of culture medium Add 1 mL of the above IVM solution to a 60 cm glass culture dish to cover the bottom, and then cover 500 ⁇ L of mineral oil on the surface of the liquid, and equilibrate at least in an incubator with 5% CO 2 saturated humidity. 6 hours.
  • Collect oocytes use a 10 mL syringe to provide negative pressure to extract the eggs, select 4-7 mm follicles, and insert the needles in parallel. During the collection process, it is necessary to avoid the insertion of the medulla and extract excessive tissue fragments, which makes it difficult to lick eggs and delay the time of licking eggs.
  • picking oocytes use a simple sputum egg device, one end of the silicone tube is placed close to the ovum needle, the other end is tightly packed with a 10 mL syringe, and the thumb and forefinger are tightly gripped with the silicone tube. The granule cell layer is selected and dense, and the cytoplasm is uniform. Pork oocytes.
  • Mature culture of in vitro oocytes 30-50 of the above selected pig oocytes are placed in a medium, and cultured in an incubator at 38.5 ° C and 5% CO 2 saturated humidity for 42-44 hours. Then follow up.
  • the inventors have confirmed that the glutamine content is in the range of 0.05-0.20 mg/mL, the gentamicin content is in the range of 0.02-0.10 mg/mL, the eCG content is in the range of 10-15 IU/mL, and the hCG content is in the 10- In the range of 15 IU/mL, the effect obtained was not significantly different from the above examples.
  • the polar body rate can reach 80%, and the development rate of parthenogenetic embryos can reach 85%.
  • the inventors investigated the effect of each component in the IVM liquid formulation of Example 1 on the polar body rate.
  • Table 1 shows the effect of using different base fluids on the porcine oocyte polar body excretion rate.
  • the MII egg indicates the oocyte during the first polar body exclusion period, that is, the oocyte entering the second metaphase of the second subdivision; the different letters in the column indicate significant difference (P ⁇ 0.05).
  • Table 2 shows the effect of adding hormones on the porcine oocyte polar body excretion rate using TCM-199 base fluid, wherein various hormones were added in an amount of 12 IU/mL.
  • the culture solution of the present invention contains eCG and hCG as essential components.
  • the inventors also confirmed that the various hormones in Table 2 were in the range of 10-15 IU/mL, and there was no significant difference in the results of the groups in each group, indicating that the addition amount can be controlled at 10-15 IU/mL.
  • Table 3 shows the effect of adding EGF and IGF-1 on the developmental rate of early parthenogenetic embryos in pigs after adding the above-mentioned eCG and hCG hormones using TCM-199 base solution, wherein the addition amount of EGF is 50 ng/mL, IGF- The amount of 1 added was 10 ng/mL.
  • the culture solution of the present invention contains EGF and IGF-1 as essential components.
  • Figure 1 shows the in vitro maturation effect of porcine oocytes supplemented with hormones and EGF and IGF-1 using TCM-199 base fluid.
  • Figure 1A shows a 4x optical magnification of the porcine cumulus oocyte complex (COC);
  • Figure 1B shows a 10x optical magnification of COC;
  • Figure 1C shows a 10x optical magnification of COC for 22h;
  • Figure 1D shows COC culture 10x optical magnification of 44h.

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Abstract

提供了一种猪卵母细胞体外成熟培养液及其制备方法和培养方法，该培养液包括TCM-199基础培养基和添加的猪卵泡液、胎牛血清、eCG、hCG、EGF和IGF-1。

Description

一种猪卵母细胞体外成熟培养液及其制备方法和培养方法 技术领域
本发明涉及细胞培养技术领域，尤其涉及一种猪卵母细胞体外成熟培养液及其制备方法，以及使用该培养液培养猪卵母细胞的方法。
背景技术
卵母细胞体外培养成熟是胚胎生物技术的基础和关键环节。目前，胚胎技术已极大地推畜牧业和医学的进程，如品种改良、转基因抗病品种培育、人类疾病模型的制备、动物生物反应器药用蛋白的生产及发育机理的探究等。
猪的体细胞克隆及疾病模型制备等都需要大量具有发育潜能的卵母细胞，但目前的猪卵母细胞体外成熟率不够理想。
目前，猪的卵母细胞培养有以下几种体外培养基础液，如北卡罗来纳州立大学研制的培养液(North Carolina State University(NCSU23/NCSU37)、PZM系列(PZM-3/PZM-4/PZM-5)培养液和TCM-199。其中TCM-199是公认的最成功的培养基础液，基本能满足猪卵母细胞体外发育代谢和营养需要。然而这些经体外培养猪卵母细胞表明，其成熟率很低，我们还需要对体外培养液进行改进，以提高猪卵母细胞体外培养的成熟率。
发明内容
发明人经研究，在TCM-199基础液基础上，通过添加激素和营养物质，显著提高猪卵母细胞体外培养的成熟率。与现有报道的猪卵母细胞体外成熟培养液相比，本发明的培养液在卵母细胞采集之后，经42-44h成熟培养后极体率可稳定在80％，整个成熟培养过程中无需更换培养液，操作简单。
基于此，本发明提供一种猪卵母细胞体外成熟培养液及其制备方法，以及使用该培养液培养猪卵母细胞的方法。
根据本发明的第一方面，本发明提供一种猪卵母细胞体外成熟培养液，包括TCM-199基础培养基和添加的猪卵泡液、胎牛血清、eCG、hCG、EGF和IGF-1。
在本发明的一个优选实施方案中，按体积百分含量计，含有10％的猪卵泡液和10％的胎牛血清，并含有10-15IU/mL的eCG、10-15IU/ml的hCG、低于100ng/mL的EGF和低于40ng/mL的IGF-1。
在本发明的一个优选实施方案中，还包括谷氨酰胺和庆大霉素。
在本发明的一个优选实施方案中，包括如下组分：
Figure PCTCN2015082485-appb-000001
根据本发明的第二方面，本发明提供一种制备第一方面所述的猪卵母细胞体外成熟培养液的方法，该方法将猪卵泡液、胎牛血清、eCG、hCG、EGF和IGF-1添加到TCM-199基础培养基中，任选地，加入谷氨酰胺和庆大霉素，制得上述培养液。
在本发明的一个优选实施方案中，上述培养液包括如下组分：
Figure PCTCN2015082485-appb-000002
根据本发明的第三方面，本发明提供一种猪卵母细胞体外成熟培养方法，该方法包括将猪卵母细胞置于第一方面所述的培养液中培养。
在本发明的一个优选实施方案中，上述培养液置于5％CO2饱和湿度的培养箱至少平衡6小时再用于培养所述猪卵母细胞。
在本发明的一个优选实施方案中，上述猪卵母细胞从4-7mm的卵泡挑取。
在本发明的一个优选实施方案中，上述猪卵母细胞在38.5℃、5％CO2饱和湿度的培养箱中培养42-44小时。
本发明的猪卵母细胞体外成熟培养液能够显著地提高猪卵母细胞体外培养 的成熟率。实验证实，本发明的培养液在卵母细胞采集之后，经42-44h成熟培养后极体率可稳定在80％，孤雌胚胎发育率可达85％，整个成熟培养过程中无需更换培养液，操作简单。
附图说明
图1为本发明一个实施例中使用TCM-199基础液添加eCG和hCG激素以及EGF和IGF-1对猪卵母细胞体外成熟效果。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
提高猪卵母细胞体外培养的成熟率，对于获取大量具有发育潜能的卵母细胞具有重要意义，从而为猪的体细胞克隆及疾病模型制备准备材料。然而，目前的猪卵母细胞体外培养使用的培养液还不能使猪卵母细胞体外培养的成熟率达到理想的水平。
本发明人在目前常用的TCM-199基础培养基的基础上，通过添加猪卵泡液(PFF)、胎牛血清(FBS)、eCG、hCG、EGF和IGF-1，制得了一种能够显著地提高猪卵母细胞体外培养的成熟率的培养液。
本发明一个实施方案的培养液，包括TCM-199基础培养基和添加的猪卵泡液、胎牛血清、eCG、hCG、EGF和IGF-1。其中，TCM-199基础培养基是一种经典的细胞体外培养系统，用其作为母液安全、可靠。TCM-199基础培养基可以从商业渠道购得，如Gibco公司或Sigma公司。
猪卵泡液(PFF)，可以获取自体外卵母细胞收集时离心所得。其作用是提供卵母细胞发育所需的必须因子。
胎牛血清(FBS)是多种体外培养液中的成分之一，已经证明其作为体外培养用的培养液成分之一的有效性和安全性。
eCG，又称孕马血清促性腺激素，即PMSG(Pregnant Mare Serum Gonadotropin)，也称为马绒毛膜促性腺激素。它是在怀孕母马血清中发现的一种激素。并已知在妊娠马属动物(驴、斑马等)都有产生，所以目前通常称为马属动物绒毛膜促性腺激素(eCG)。
hCG，即人绒毛膜促性腺激素，是由胎盘的滋养层细胞分泌的一种糖蛋白，它是由α和β二聚体的糖蛋白组成。
EGF，即表皮生长因子，是一种小肽，由53个氨基酸残基组成，是类EGF大家族的一个成员，是一种多功能的生长因子，在体内体外都对多种组织细胞 有强烈的促分裂作用。
IGF-1，即胰岛素样生长因子1，也被称为生长调节素Csomatomedin C，是一个由人类基因IGF1编码的蛋白质。
本发明中，PFF、FBS、eCG、hCG、EGF和IGF-1在TCM-199基础培养基中协同作用，共同提高猪卵母细胞体外培养的成熟率。
本发明的一个优选实施方案中，按体积百分含量计，含有10％的猪卵泡液和10％的胎牛血清，并含有10-15IU/mL的eCG、10-15IU/ml的hCG、50ng/mL的EGF和10ng/mL的IGF-1。IU表示国际单位。
发明人已经证实，EGF的含量在100ng/mL以下均能取得预期效果，并且在此范围内其效果随着EGF含量的增加有增加的趋势，然而EGF含量超出100ng/mL并没有显示效果进一步提高，因此从经济和实用的角度考虑，选择EGF的含量在100ng/mL以下；IGF-1的含量在40ng/mL以下均能取得预期效果，并且在此范围内其效果随着IGF-1含量的增加有增加的趋势，然而IGF-1含量超出40ng/mL并没有显示效果进一步提高，因此从经济和实用的角度考虑，选择IGF-1的含量在40ng/mL以下。
本发明一个优选的实施方案中，为了给猪卵母细胞提供额外的能量底物，可以添加谷氨酰胺，保证猪卵母细胞所需的能量足够。
本发明一个优选的实施方案中，为了赋予培养液抗菌性能，可以添加庆大霉素，它抑制细菌等外源性污染的同时不影响猪卵母细胞的正常生长和成熟。
因此，本发明的一个更优选实施方案中，包括如下组分：
Figure PCTCN2015082485-appb-000003
以下结合发明人的研究实施例，对本发明进行进一步详细说明。应当理解，以下实施例并不构成对本发明保护范围的限制。
实施例1
本实施例使用的平衡卵母细胞体外培养液(in vitro mature，IVM液)的配方如下：
Figure PCTCN2015082485-appb-000004
培养液的平衡：在60cm玻璃培养皿中加入1mL上述的IVM液，使其铺满底部，再于液体表面覆盖500μL矿物油(mineral oil)，置于5％CO2饱和湿度的培养箱至少平衡6小时。
采集卵母细胞：采用10mL注射器提供负压抽取卵子，选取4-7mm的卵泡，平行进针。采集过程中，须避免插入髓质部，抽取过多组织碎片，造成捡卵困难，拖延捡卵时间。在挑取卵母细胞时，采用简易的捡卵装置，硅胶管一端套紧捡卵针，另一端套紧10mL注射器，拇指和食指握紧硅胶管，选择颗粒细胞层多且致密，胞质均匀的猪卵母细胞。
体外卵母细胞的成熟培养：将上述挑选出来的猪卵母细胞，平均30-50个放入一个培养基中，在38.5℃、5％CO2饱和湿度的培养箱中培养42-44小时，然后进行后续操作。
结果：经成熟培养42-44小时后，极体率达到80％，孤雌胚胎发育率达到85％。
发明人已经证实，谷氨酰胺含量在0.05-0.20mg/mL范围内，庆大霉素含量在0.02-0.10mg/mL范围内，eCG含量在10-15IU/mL范围内，hCG含量在10-15IU/mL范围内，取得的效果与上述实施例没有显著差别。经成熟培养42-44小时后，极体率均能达到80％，孤雌胚胎发育率均能达到85％。
试验例1
发明人对实施例1的IVM液配方中各组分对于极体率的影响进行了研究。
表1示出了使用不同基础液对猪卵母细胞极体排出率的影响。
表1
Figure PCTCN2015082485-appb-000005
注：MII卵表示第一极体排除时期的卵母细胞，也就是进入减数第二次分裂中期的卵母细胞；列内不同字母表示差异显著(P<0.05)。
表1所示的结果显示：TCM-199相比NCSU-37和PZM-3对于猪卵母细胞，具有更高的极体排出率，这一点与目前对这几种培养液的认识相符。
表2示出了使用TCM-199基础液添加激素对猪卵母细胞极体排出率的影响，其中各种激素均按照12IU/mL的量添加。
表2
Figure PCTCN2015082485-appb-000006
注：列内不同字母表示差异显著(P<0.05)。
表2所示的结果显示：相比添加FSH+hCG和FSH+LH的实验组，添加eCG+hCG的实验组能够显著地提高猪卵母细胞极体排出率。因此，本发明的培养液以eCG和hCG作为必要成分。
发明人还证实，表2中的各种激素在10-15IU/mL的范围内，每组实验的组内结果没有显著性差异，说明其添加量可以控制在10-15IU/mL。
表3示出了在使用TCM-199基础液添加上述eCG和hCG激素后，又添加EGF及IGF-1对猪早期孤雌胚胎发育率的影响，其中EGF的添加量为50ng/mL，IGF-1的添加量为10ng/mL。
表3
Figure PCTCN2015082485-appb-000007
注：列内不同字母表示差异显著(P<0.05)。
表3所示的结果显示：添加EGF能够提高猪早期孤雌胚胎发育率，在EGF的基础上再添加IGF-1，能够进一步提高猪早期孤雌胚胎发育率。因此，本发明的培养液以EGF和IGF-1作为必要成分。
图1示出了使用TCM-199基础液添加激素和EGF及IGF-1对猪卵母细胞体外成熟效果。图1A显示猪卵丘卵母细胞复合体(COC)的4倍光学放大结果；图1B显示COC的10倍光学放大结果；图1C显示COC培养22h的10倍光学放大结果；图1D显示COC培养44h的10倍光学放大结果。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种猪卵母细胞体外成熟培养液，其特征在于，包括TCM-199基础培养基和添加的猪卵泡液、胎牛血清、eCG、hCG、EGF和IGF-1。
  2. 根据权利要求1所述的猪卵母细胞体外成熟培养液，其特征在于，按体积百分含量计，含有10％的猪卵泡液和10％的胎牛血清，并含有10-15IU/mL的eCG、10-15IU/ml的hCG、低于100ng/mL的EGF和低于40ng/mL的IGF-1。
  3. 根据权利要求1或2所述的猪卵母细胞体外成熟培养液，其特征在于，还包括谷氨酰胺和庆大霉素。
  4. 根据权利要求3所述的猪卵母细胞体外成熟培养液，其特征在于，包括如下组分：
    Figure PCTCN2015082485-appb-100001
  5. 一种制备权利要求1-4任一项所述的猪卵母细胞体外成熟培养液的方法，其特征在于，所述方法将猪卵泡液、胎牛血清、eCG、hCG、EGF和IGF-1添加到TCM-199基础培养基中，任选地，加入谷氨酰胺和庆大霉素，制得所述培养液。
  6. 根据权利要求5所述的制备猪卵母细胞体外成熟培养液的方法，其特征在于，所述培养液包括如下组分：
    Figure PCTCN2015082485-appb-100002
    Figure PCTCN2015082485-appb-100003
  7. 一种猪卵母细胞体外成熟培养方法，其特征在于，所述方法包括将猪卵母细胞置于权利要求1-4任一项所述的培养液中培养。
  8. 根据权利要求7所述的猪卵母细胞体外成熟培养方法，其特征在于，所述培养液置于5％CO2饱和湿度的培养箱至少平衡6小时再用于培养所述猪卵母细胞。
  9. 根据权利要求7所述的猪卵母细胞体外成熟培养方法，其特征在于，所述猪卵母细胞从4-7mm的卵泡挑取。
  10. 根据权利要求7所述的猪卵母细胞体外成熟培养方法，其特征在于，所述猪卵母细胞在38.5℃、5％CO2饱和湿度的培养箱中培养42-44小时。
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