WO2003040359A1 - Preparation d'un embryon somatique au moyen d'un ovocyte de lapin - Google Patents

Preparation d'un embryon somatique au moyen d'un ovocyte de lapin Download PDF

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Publication number
WO2003040359A1
WO2003040359A1 PCT/CN2001/001537 CN0101537W WO03040359A1 WO 2003040359 A1 WO2003040359 A1 WO 2003040359A1 CN 0101537 W CN0101537 W CN 0101537W WO 03040359 A1 WO03040359 A1 WO 03040359A1
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cells
cell
oocytes
somatic
nuclear transfer
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PCT/CN2001/001537
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English (en)
French (fr)
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Huizhen Sheng
Ying Chen
Qingzhang Yang
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Shanghai Second Medical University
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Priority to KR10-2004-7006900A priority Critical patent/KR20040065214A/ko
Priority to PCT/CN2001/001537 priority patent/WO2003040359A1/zh
Priority to HU0401692A priority patent/HUP0401692A3/hu
Priority to US10/494,075 priority patent/US20050066379A1/en
Priority to CA002466203A priority patent/CA2466203A1/en
Priority to EP01274634A priority patent/EP1443107A4/en
Priority to JP2003542606A priority patent/JP2005508185A/ja
Priority to CNA018237665A priority patent/CN1558949A/zh
Publication of WO2003040359A1 publication Critical patent/WO2003040359A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8777—Rabbit embryos
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/107—Rabbit

Definitions

  • the invention more particularly relates to the preparation of a human somatic embryo by transplanting a human cell nucleus into an enucleated oocyte, preferably a rabbit oocyte, and most preferably a New Zealand white rabbit enucleated oocyte. And its derived cells.
  • Nuclear transfer technology involves transferring a nuclear donor cell or nucleus into an enucleated oocyte, and the resulting nuclear transfer unit can develop into a somatic embryo or further into a normal individual.
  • Briggs and King obtained nuclear transfer frogs by injecting nuclei from the intestinal epithelial cells of the black spotted frog.
  • nuclear transfer technology was applied to mammals.
  • Various embryonic cells such as embryo blastomeres, inner cell mass cells, and late embryonic cells, have been used for nuclear transfer with some success (Collas et al, Mol. Reprod. Dev., 38: 264-2671994;, Keefer et al, Biology of Reproduction, 50: 935-939 1994; Sims et al, PNAS, 90: 6143-6147 1993).
  • Xenogeneic nuclear transfer refers to the transfer of cells or nuclei of one animal into the oocytes of another animal enucleated.
  • the resulting nuclear transfer unit can develop into a zygote embryo and implant it into the uterus.
  • xenogeneic nuclear transfer has the potential to be used for therapeutic cloning.
  • the therapeutic cloning method requires a large number of oocytes to become recipient cells. Because human oocytes are scarcely sourced, they are far from meeting experimental and clinical needs. Therefore, it is necessary to seek other sources of oocytes.
  • bovine oocytes can be used for heterologous nuclear transfer.
  • In vivo egg flushing technology can be used to flush mature oocytes directly from cattle, but the technology is complicated to operate, the oocyte yield is low (8-10), and a cattle herd must be maintained on a daily basis. high.
  • the natural estrous cycle of cattle is 21 days, and artificial hormone-stimulating ovulation is completed within 5 days. 8 to 10 oocytes can be obtained from each artificial hormone-stimulating ovulation.
  • cattle eggs are obtained from healthy living cattle.
  • Blast cells are expensive.
  • Bovine oocytes can also be obtained from slaughterhouses, however, cattle obtained from slaughterhouses
  • the ovaries often come from old, weak, sick, and disabled cattle, and are often contaminated with pathogens (such as bovine tuberculosis, foot-and-mouth disease, etc.), and the quality of oocytes is very unstable.
  • pathogens such as bovine tuberculosis, foot-and-mouth disease, etc.
  • the quality of oocytes is very unstable.
  • the cattle in slaughterhouses originate from many places, so the oocytes obtained are often genetically unclear.
  • the ovaries extracted oocytes to be cultured 18-24 hours prior to use in vitro maturation.
  • the late stage development of mature oocytes in vitro is much worse than mature oocytes in vivo.
  • a nuclear transfer unit can be obtained by transplanting human cells (such as adult somatic cells) or their nuclei into animal enucleated oocytes, and such nuclear transfer units can develop into human somatic embryos after culture .
  • human somatic nuclei can be restarted after implantation into enucleated oocytes and develop into human somatic embryos.
  • This result also proves the feasibility of interspecific nuclear transfer, that is, the transfer of animal or human cells (for example, adult somatic cells) or nuclei into rabbits, such as enucleated oocytes of rabbits.
  • the nuclear transfer unit thus prepared can develop into a somatic embryo encoded by a nuclear donor under appropriate culture conditions.
  • the object of the present invention is to provide an improved method for preparing somatic embryos using heterogeneous nuclear transfer technology.
  • Another object of the present invention is to prepare a somatic embryo by transplanting the nucleus of an animal or human cell into an enucleated oocyte of a different species.
  • the object of the present invention is particularly to transplant the nuclei of human cells into enucleated oocytes of different species of animals.
  • Another special object of the present invention is to transplant human nuclei into animal enucleated oocytes, preferably rabbits enucleated oocytes.
  • Yet another object of the present invention is to transplant the nucleus of a human cell, such as a human adult cell, into a human enucleated oocyte.
  • FIG. 1 Fibroblasts from adult foreskin
  • Figure 6 Somatic embryo hatching embryos obtained by subzonal injection method
  • Figure 7 Somatic embryos obtained by intracytoplasmic injection method (4-cell stage)
  • Figure 8 Somatic embryos obtained by intracytoplasmic injection method (mulberry) Embryo)
  • nuclear transfer or nuclear transfer (Nuclear Transfer) may be used instead.
  • nuclear transfer embryos somatic embryos or recombinant embryos may be used interchangeably.
  • nuclear transfer embryonic stem cells NT-ES cells
  • S-ES cells somatic cell-derived embryonic stem cells
  • Nuclear transfer is the transfer of a nuclear donor cell or cell nucleus into an enucleated oocyte.
  • the formed nuclear transfer unit can further develop into a somatic embryo, such as a embryo, or a normal individual.
  • the nuclear donor used for nuclear transfer can be human or other animals, for example, primates, ungulates, amphibians, rodents, etc.
  • the oocytes used for nuclear transfer can be human, or It can be other animals, for example, primates, ungulates, amphibians, rodents, etc.
  • Allogeneic nuclear transfer refers to the transfer of somatic cells or nuclei of an animal into oocytes enucleated by the same animal.
  • the resulting nuclear transfer unit can develop into a somatic embryo or further into a normal individual.
  • Xenogeneic nuclear transfer refers to the transfer of somatic cells or nuclei of an animal into oocytes enucleated by another animal.
  • the formed nuclear transfer unit can develop into a somatic embryo or further into a normal individual.
  • a nuclear transfer unit is an embryo formed by combining one cell (nuclear donor cell) and another cell (enucleated oocyte).
  • Nuclear donor cells and enucleated oocytes used to prepare nuclear transfer units can be from the same species or from different species.
  • Somatic embryos refer to embryos at various stages of development from nuclear transfer units, including different stages such as 2-cell, 4-cell, 8-cell, mulberry embryo, palatine embryo, and hatched palate embryo.
  • Rabbits are low cost, can be caged, fed with standard pellet feed, easy to manage and disease control, and under strict breeding conditions, they can be bred to SPF (Specific Pathogen Free) rabbits.
  • SPF Specific Pathogen Free
  • the natural estrous cycle of rabbits is 7-9 days.
  • Artificial hormones promote ovulation in 4 days. About 30 oocytes can be obtained from every rabbit that artificially stimulates ovulation.
  • the present invention finds that a human cell, particularly a nuclear transfer unit formed by a human fibroblast and an enucleated rabbit oocyte, can develop into a somatic embryo.
  • Human cell nuclear approval The fact that rabbit oocytes are effectively reprogrammed (reprogram), human cells can be expected to be transplanted into oocytes of other species such as other ungulates and other animals and develop into somatic embryos. For example, oocytes derived from other primates, amphibians, rodents, and the like.
  • human cells or nuclei can also be transferred into human oocytes and developed into somatic embryos, such as embryos.
  • the present invention relates to transplanting animal or human nuclei or cells into enucleated oocytes of the same or different species as the nuclear donor by injection or fusion to produce embryonic stem cells that can be used to obtain A nuclear transfer unit for embryonic stem-like cells or other types of stem cells.
  • the present invention may involve transplanting a human cell nucleus or a human cell into another species, such as another rodent or non-rodent enucleated oocyte, by injection or fusion to form a nuclear transfer unit. After the nuclear transfer unit is cultured, it can develop into somatic embryos such as mulberry embryos, pupal embryos, and hatched pupal embryos. Cells derived from these somatic embryos can be formed into embryonic stem cells, embryonic stem cell-like cells, or other types of stem cells after in vitro culture. Clinical treatment.
  • the therapeutic cloning route is as follows: Take the patient's somatic cells, develop them into somatic embryos after nuclear transfer, and generate ES cells. The ES cells are induced to differentiate into the cells required by the patient in vitro. The cells and tissues obtained in this way will be basically consistent with the patient's autogenous type. They are autologous in cell or tissue transplantation treatment and will not be rejected by the patient's immune system. Therapeutic cloning can solve the problem of patient immune rejection caused by allogeneic cell or tissue transplantation.
  • xenogeneic oocytes used for human somatic cell nuclear transfer should meet the following requirements: First, they must not carry any pathogens that can be transmitted to humans, including bacteria, viruses, and mycoplasma Etc .; Secondly, they must effectively support the reprogramming of human nuclei, and Cell embryos develop in vitro to hatch embryos or hatching embryos; moreover, they should have a clear genetic background, stable quality, and be available in large quantities.
  • rabbit oocytes are convenient, economical, stable in quality, and have a clear genetic background. There is no need to increase in vitro maturation steps in nuclear transfer operations, and the oocytes develop well in the later stages, making them more suitable for therapeutic use. clone.
  • the present invention provides a new method for preparing human somatic embryos using nuclear transfer technology. That is, by reprogramming human cells or nuclei into rabbit oocytes and developing them into human somatic embryos. Such embryos can be used to produce human embryonic stem cells, embryonic stem cell-like cells, or other types of stem cells. As mentioned above, before this, people only used large ungulates, including cattle and sheep, as oocyte donors. Compared with cattle and sheep, the use of rabbits to provide oocytes has the advantages of low cost, convenience, and large numbers.
  • rabbits can provide a large number of low-cost, oocyte-free, oocytes that are contaminated with pathogens and can efficiently activate human nuclei, in the future research and application of therapeutic cloning, rabbits are more likely to develop human eggs than cattle and sheep. Replacement of mother cells.
  • the human oocytes are preferably rabbit oocytes, and most preferably rabbit enucleated oocytes to prepare human cell embryos.
  • somatic embryos are prepared by a nuclear transfer procedure as follows: (i) obtaining the required human or animal cells for use as a nuclear donor.
  • a human or animal packet or nucleus is transferred to the perinuclear space or cytoplasm of an enucleated oocyte by injection, for example, and is electrically or chemically activated to form a nuclear transfer unit.
  • the obtained nuclear transfer unit is developed to a certain stage in vitro or in vivo, such as a pupal embryo or a hatched pupal embryo.
  • the cells used as nuclear donors are derived from human cells, preferably fibroblasts.
  • Human somatic cells can be obtained by a known method.
  • Human cells useful in the present invention include (by way of example): epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), nucleated cells Red packets, macrophages, monocytes, fibroblasts, Cardiomyocytes and other muscle cells.
  • human cells used for nuclear transfer can be obtained from different organs, such as skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney, urethra, and other urinary organs. They are only examples of suitable donor cells. Suitable donor cells, ie cells useful in the present invention (including all somatic cells) can be obtained from almost any tissue and organ of the body.
  • Oocytes used for nuclear transfer can be obtained from animals including mammals and amphibians. Suitable mammalian cell sources of oocytes include sheep, cattle, pigs, horses, rabbits, guinea pigs, mice, hamsters, rats, primates, and the like. In a preferred embodiment of the invention, the oocytes are obtained from a rabbit family, most preferably rabbits.
  • Methods for isolating oocytes are techniques well known in the art. This method basically isolates oocytes from the ovaries or reproductive tract of mammals or amphibians such as rabbits.
  • oocytes obtained from New Zealand white rabbits should be injected into humans Enucleation of chorionic gonadotropin (HCG) at 15-23 hours.
  • HCG chorionic gonadotropin
  • oocytes Prior to enucleation, oocytes are preferably moved to hyaluronidase-containing M2 culture medium (Sigma) before cell mound removal. This can be achieved by repeated suction or simple vortexing with a very fine inner diameter straw. Oocytes containing polar bodies were then screened. It is determined by the existence of polar bodies that the oocytes are in the second stage of cell division, and the oocytes are used for nuclear transfer and enucleation.
  • Immature oocytes taken from the ovary usually require in vitro maturation. This process usually brings the oocytes to metaphase I to I.
  • enucleation preferably does not exceed 20 hours after HCG injection, and more preferably 16-18 hours after HCG injection.
  • Enucleation can be achieved by micropipette removal of polar bodies and surrounding cytoplasm. Oocytes that have been successfully enucleated are then screened. This screening can be done by staining with 1 ⁇ g Hoechs t 33342 (Sigma) dye per milliliter and quickly observing the oocytes under UV light.
  • Nuclear transfer units can be prepared according to methods known in the art. For example, subzonal injection and intracytoplasmic injection.
  • Subzonal injection after enucleation, animal or human single cells heterologous to enucleated oocytes are transferred to the periovular space of enucleated oocytes.
  • the nuclear transfer unit composed of human or animal cells and oocytes of rabbits uses an electrical pulse of 90-120V for about 60 microseconds in a 0.5-wake chamber 16-20 hours after HCG injection 1-2 Perform electric fusion in the electric fusion solution (such as mannitol fusion solution, sucrose fusion solution, etc.) one or more times. After fusion, the resulting nuclear transfer unit is placed in a suitable medium such as RD, M199, DMEM, etc. until activated. Typical activation will be completed in a short period of time thereafter, generally no more than 24 hours, and preferably about 4-9 hours thereafter.
  • Electrofusion methods to fuse cells use electrical pulses sufficient to cause the plasma membrane to temporarily disintegrate Rush to achieve electrical fusion. Because the membrane rapidly reforms, the breakdown of the cytoplasmic membrane is very short. In essence, if two adjacent membranes are induced to disintegrate and re-form into a lipid bilayer that blends with each other, a small channel (pore) between the two cells will open. Due to the thermodynamic instability of this small channel, it will expand until two cells fuse into one. See
  • Nuclear transfer units can be activated by known methods.
  • the method includes, for example, cultivating a nuclear transfer unit at a sub-physiological temperature, in effect stimulating the nuclear transfer unit with cold or low temperature.
  • the nuclear transfer unit can be most conveniently operated by culturing it at room temperature, which is a low temperature relative to the normal physiological temperature conditions of the embryo.
  • the activation can be completed in sequence:
  • a kinase inhibitor such as a serine-threonine kinase inhibitor, such as 6-dimethyl-amino-purine, astrosporin, 2- Aminopurine and sphingosine.
  • phosphorylation of cellular proteins can be inhibited by introducing a tablet enzyme such as phosphatase 2 A and phosphatase 2B into an oocyte.
  • Intracytoplasmic injection Other methods can also be used for nuclear transfer, for example, direct injection of donor cell nuclei into oocyte cytoplasm instead of electroporation fusion (Collas and Barnes, Mol. Peprod. Dev., 38 : 264-267 1994). The method becomes The work enabled the human nucleus to initiate the development of human embryos in the rabbit oocyte cytoplasmic environment.
  • the activated nuclear transfer unit can be cultured in a suitable in vitro medium until a somatic embryo is produced.
  • the nucleus transfer unit for human cells / nucleated rabbit oocytes formed in RD culture medium has the highest embryogenic rate.
  • a live nuclear transfer unit can be transferred to 50 ul of micro-drop culture medium, such as RD, M199, DMEM, and coated with paraffin oil.
  • the culture environment is, for example, 38 ° C, 5% C0 2 .
  • the embryos are obtained about 6-7 days after the oocytes start to activate.
  • the somatic embryos obtained exhibit similar appearance and cell growth characteristics to embryos of nuclear donor species rather than oocyte donor species.
  • somatic embryos obtained by inserting human donor nuclei into enucleated rabbit oocytes develop similarly to human embryos rather than rabbits.
  • Human nucleus / rabbit oocyte cytoplasm-derived somatic embryos formed pupal embryos on days 6-7 of the culture, which is the same as humans, unlike rabbits (pupal embryos formed on days 3-4 of culture).
  • Culture media known in the art that can be used for rabbit embryo culture and maintenance such as DMEM + 15% FCS; M199 + 15% FCS; RD + 15% FCS and the like.
  • these media can be combined with a variety of cell types, such as granulocytes, fallopian tube cells, uterine cells, and ST0 cells, to form a co-culture system.
  • the somatic embryos produced in the present invention can be used to establish human embryonic stem cells, embryonic stem cell-like cells, or other types of stem cells, which can be applied to the treatment of multiple diseases, and may provide a brand-new means for the treatment of a large number of genetic diseases.
  • xenogeneic nuclear transfer technology can save endangered species such as Because pandas are scarce in number, it is impossible to obtain a large number of oocytes. They use their somatic cells as donor cells and transfer them to oocytes of other species. The pupal embryo was cultured in vitro, and then transferred to the womb of the surrogate mother to give birth to the giant panda.
  • the sterile foreskin tissue removed from the operation was cut into pieces, rinsed with phosphate buffered saline (PBS), and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the tissue was broken.
  • the block was digested with 0.05 Trypsin / 0.02% EDTA (Gibco) at 37 ° C for 30 minutes, and the suspended cell solution was centrifuged at 1,000 rpm for 5 minutes. The supernatant was discarded, and the cell culture medium (90% DMEM, Gibco; 10% FBS) was discarded.
  • 'Oocytes of degranulated cells are placed in M2 medium containing 7.5 g / ml CB (Sigma) and immersed for 10 minutes at room temperature. Nuclei are formed in the perinuclear space of enucleated enucleates The transplantation unit and nuclear transfer unit were equilibrated in an electrofusion buffer (0.3M Glucose, Sigma; 0.1 mM gCl 2 , Sigma; 0.05raM CaCl 2 , Sigma;) for 15 minutes and then subjected to electrical stimulation (HV 120V, 60 us, 1 time).
  • electrofusion buffer 0.3M Glucose, Sigma; 0.1 mM gCl 2 , Sigma; 0.05raM CaCl 2 , Sigma;
  • the nuclear transfer unit was cultured in RD culture medium (42.5% DMEM, Gibco; 42.5% RPMI-1640, Gibco; 15% FBS, Hyclone), and cultured for 6-7 days to obtain somatic embryos (Figure 3- 6).
  • Intracellular microenvelope injection-oocytes with degranulated cells are placed in M2 medium containing 7.5 g / ml CB (Sigma), and immersed at room temperature for 10 minutes. Then, the nuclear transfer in RD cells were cultured (42 5% DMEM, Gibco; . 42.5% RPMI- 1640, Gibco; 15 FBS, Hyclone) were cultured 6-7 days of culture, somatic Nang embryos can be obtained (FIG. 7 -10).
  • the somatic pupal embryo obtained by the above method was drawn into a thin glass needle with a diameter of 3 mm with a glass tube with a diameter of 3 mm.
  • Small cell clumps transferred to feeder layer, using ES cell culture medium (79% DMEM, Gibco; 20% FBS, Hyclone; 1% non-es sent ia 1 amino acid stock, Gibco ; 0. ImM ⁇ -merca toethanol,
  • embryonic fibroblasts were derived from rat fetuses aged 14-16 days. After aseptic removal of the head, liver, heart, and esophagus, the mouse embryos were minced and pre-warmed in 0.05% trypsin / 0.02 EDTA (GIBCO) 37. C digestion for 30 minutes. Take the suspension cell solution, centrifuge at 1000 rpm for 5 minutes and 4 ⁇ , and resuspend the cell pellet with culture medium (90% DMEM, Gibco; 10% FBS, Hyclone 50 IU penici 11 in-streptomycin, Gibco; 1% non-essential amino acid stock, Gibco; 0.
  • culture medium 90% DMEM, Gibco; 10% FBS, Hyclone 50 IU penici 11 in-streptomycin, Gibco; 1% non-essential amino acid stock, Gibco; 0.
  • the cell pellet was resuspended in fixative, left at room temperature for 15 minutes, and centrifuged at 1 000 rpm for 8 minutes. The cell pellet was dropped on a glass slide and GIEMSA stained to see the karyotype. The experimental results are shown in Figure 13.

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Description

利用兔卵母细胞制备体细胞胚胎
Figure imgf000002_0001
(简称体细胞胚胎）及其衍生细胞。 本发明更特别涉及通过将人 细胞核移植入一种去核动物卵母细胞 , 优选地为一种兔科动物卵 母细胞， 最优选地为新西兰大白兔去核卵母细胞以制备人类体细 胞胚胎及其衍生细胞。
发明背景
核移植技术是将核供体细胞或细胞核移入去核的卵母细胞 中， 所形成的核移植单元可以发育成体细胞胚胎， 也可以进一步 发育成正常个体。 50年代末首先在两栖动物上取得成功。 Briggs 和 King 采用黑斑蛙肠上皮细胞核注入卵中得到了核移植蛙。 到 80 年代后核移植技术应用到哺乳动物上来。 将各种胚胎细胞如 胚胎卵裂球、 内细胞团细胞、 晚期胚胎细胞用于核移植， 并取得 了一定的成功 （Collas et al , Mol. Reprod .Dev. , 38: 264- 2671994; , Keefer et al, Biology of Reproduction, 50: 935-939 1994; Sims et al, PNAS, 90: 6143-6147 1993)。
1997 年英国的 Wilmut Ian , et al. (Nature 1997, 385 ， 810- -813)采用 6 岁成年綿羊乳腺细胞核制备了第一只成体细胞 核移植羊。 1998 年美国成功完成了小鼠体细胞的连续核移植 (T. wakayama , et al. Nature 394 369—372)。 同一年小鼠 胚胎干细胞核移植也取得成功（Teruhiko , et al. PNAS 1998, 96 , 14984—14989) 0 成体细胞核移植的成功不仅是技术上的进 步, 更是观念上的革命。 事实说明， 只要在合适的条件下， 高度 特化的成体细 包核与胚胎细胞核一样也能重新编程， 形成新个 体。
异种核移植是指将一种动物的细胞或细胞核移入另一种动物 去核的卵母细胞中， 所形成的核移植单元可以发育成嚢胚， 并植 入子宫。
1999年 Tanja Dominko , et a l. (Biology of Reproduct ion , 60 ( 6 ), 1496) 将牛、 羊、 猪、 猴和大鼠的体细胞核注入牛 去核卵母细胞， 形成体细胞胚胎， 各种体细胞胚胎均有一定水平 的发育。 该实验从理论上说明一种动物的细胞核可以在另一种动 物的卵母细胞的细胞质中活化， 与其构成一体， 在其支持和营养 下发育成体细胞胚胎。 同一种卵母细胞质可以支持多种动物的细 胞核的发育， 这说明了多种动物卵母细胞质成分间的保守性。
对于人类医学而言， 异种核移植有可能用于治疗性克隆。 治 疗性克隆方法需要大量的卵母细胞做受核细胞。 由于人卵母细胞 来源稀少， 远不能满足实验和临床的需要。 因而寻求其他卵母 细胞的来源就非常必要了。
如上文所述， 牛卵母细胞可用于异种核移植。 应用体内冲卵 技术， 可以直接从牛体内冲出成熟的卵母细胞 , 但是该技术操作 复杂、 卵母细胞产量低（8- 10 个）， 且还要日常维持一个牛群， 这样的话成本很高。 （牛的自然发情周期为 21天， 在 5天内完成 人工激素促排卵， 从每 1 只人工激素促排卵的牛可以得到 8 - 10 个卵母细胞。）， 所以， 从健康活体牛获取牛卵母细胞成本昂贵。
牛卵母细胞也可以从屠宰场获得 , 然而， 由屠宰场获得的牛 卵巢常常来自那些老、 弱、 病、 残的牛， 并常有病原体污染 （例 如牛结核、 口蹄疫等）， 卵母细胞质量很不稳定。 再者， 屠宰场 的牛来源于许多产地， 因此得到的卵母细胞往往遗传背景不清。 另外， 卵巢中抽出的卵母细胞需在体外培养 18- 24小时成熟后才 能使用。 而且， 体外成熟的卵母细胞的后期发育远不如体内成熟 的卵母细胞。
发明简述
本发明人通过研究后发现， 可以通过将人细胞 （例如成人体 细胞） 或其细胞核移植入动物去核卵母细胞获得核转移单元, 这 种核转移单元培养后可发育成为人的体细胞胚胎。 这一结果首先 证明人类体细胞核在植入去核卵母细胞后可以被重新启动, 并发 育成为人的体细胞胚胎。 这一结果还证明了种间核移植的可行 性， 即：将动物或人细胞 （例如， 成人体细胞） 或细胞核移植入 兔科动物， 如家兔的去核卵母细胞。 这样制备的核转移单元， 在 合适的培养条件下可以发育成核供体编码的体细胞胚胎。
因此， 本发明目的在于提供利用异种核移植技术制备体细胞 胚胎的改进方法。
本发明的又一目的在于通过将动物或人细胞的细胞核移植入 不同种的去核卵母细胞制备体细胞胚胎。
本发明目的特别在于将人细胞的细胞核移植入不同种动物的 去核卵母细胞。
本发明的另一个特别目的在于将人细胞核移植入动物去核卵 母细胞， 优选为兔科动物去核卵母细胞。
本发明的又一目的在于将人细胞例如， 人成体细胞的细胞核 移植入人去核卵母细胞。 对于本发明上述以及其它目的之优点和特征将在下文描述， 的详细叙述而更易于理解。
附图说明
图 1来源成人包皮的成纤维细胞
图 1家兔卵母细胞
图 3用透明带下注射方法获得的体细胞胚胎 4细胞阶段） 图 4用透明带下注射方法获得的体细胞胚胎 桑葚胚） 图 5用透明带下注射方法获得的体细胞胚胎 嚢胚）
图 6用透明带下注射方法获得的体细胞胚胎 孵化嚢胚） 图 7用胞质内注射方法获得的体细胞胚胎 （ 4细胞阶段） 图 8用胞质内注射方法获得的体细胞胚胎 （桑葚胚）
图 9用胞质内注射方法获得的体细胞胚胎 （嚢胚）
图 10用胞质内注射方法获得的体细胞胚胎 （孵化嚢胚） 图 11 兔卵母细胞可以有效地启动人体细胞， 并支持体细胞 胚胎的早期发育
图 12起源于人类体细胞的胚胎干细月包（ So歸 t ic cel l der ived embryonic s tem ee l I s, S-E S细胞） 克隆
图 13 S- E S细胞第 26代染色体核型
发明详述
本发明中， 核转移或核移植 （ Nuc lear Transfer)可替换使 用。
本发明中， 核移植胚胎或体细胞胚胎或重组胚胎可替换使用。 本发明中， 核移植胚胎干细胞 （NT-ES细胞） 或体细胞起源的 胚胎干细胞 （ S-ES细胞） 可替换使用。 核移植是将核供体细胞或细胞核移入去核的卵母细胞中， 所 形成的核移植单元可以进一步发育成为体细胞胚胎， 例如嚢胚， 或进一步发育成正常个体。 核移植所用的核供体可以是人的， 也 可以是其他动物的， 例如， 灵长类动物、 有蹄类、 两栖动物、 啮 齿动物等； 核移植所用的卵母细胞可以是人的， 也可以是其他动 物的， 例如， 灵长类动物、 有蹄类、 两栖动物、 啮齿动物等。
同种核移植是指将一种动物的体细胞或细胞核移入同一种动 物去核的卵母细胞中， 所形成的核移植单元可以发育成为体细胞 胚胎或进一步发育成正常个体
异种核移植是指将一种动物的体细胞或细胞核移入另一种动 物去核的卵母细胞中， 所形成的核移植单元可以发育成为体细胞 胚胎或进一步发育成正常个体。
核移植单元是指由一个细胞（核供体细胞）和另一个细胞（去 核卵母细胞） 结合所形成的胚胎。 用于制备核移植单元的核供体 细胞和去核卵母细胞可以来自同一物种， 也可来自不同物种。
体细胞胚胎指从核转移单元发育来的各阶段胚胎， 包括 2 细 胞、 4细胞、 8细胞、 桑葚胚、 嚢胚和孵化嚢胚等不同阶段。
我们在异种核移植中采用合适的小型动物做卵母细胞供体， 如家兔。 家兔成本很低， 可以笼养、 喂标准的颗粒饲料、 易于管 理和疾病控制， 在严格的飼养条件下， 可以育成 SPF (无特定病 原体动物） 级兔。 兔的自然发情周期为 7-9 天， 4 天内完成人工 激素促排卵， 从每 1只人工激素促排卵的家兔中通常可以得到 30 个左右卵母细胞。
本发明发现人体细胞，特别是人成纤维细胞与去核兔卵母细胞 形成的核移植单元， 可发育成为体细胞胚胎。 基于人体细胞核可 被兔卵母细胞有效地重编程 ( reprogram ) 的事实， 可以预期人 体细胞可移植入其它物种例如其它有蹄动物以及其它动物的卵母 细^ ^并发育成体细胞胚胎。 例如， 来源于其它灵长类动物、 两栖 动物、 啮齿动物等的卵母细胞。 而且， 使用类似的方法， 也可以 将人细胞或细胞核转移到人卵母细胞中并发育成体细胞胚胎， 例 如嚢胚。
因此， 在最广泛的实施例中， 本发明涉及通过注射或融合， 将动物或人细胞核或细胞移植入与核供体相同或不同种动物的去 核卵母细胞， 产生可以用于获得胚胎干细胞， 胚胎干细胞样细胞 或其他类型干细胞的核移植单元。 例如， 本发明可以涉及通过注 射或融合将人体细胞核或人体细胞移植入其它物种，例如另一种 啮齿动物或非啮齿动物的去核卵母细胞中， 形成核移植单元。 该 核移植单元经培养后可以发育成桑葚胚、 嚢胚、 孵化嚢胚等体细 胞胚胎 , 这些体细胞胚胎所衍生的细胞经体外培养可以形成胚胎 干细胞、 胚胎干细胞样细胞或其他类型干细胞用于临床治疗。
治疗性克隆路线具体如下： 即取病人的体细胞， 通过核移植 后发育成体细胞胚胎， 并产生 ES细胞。 该 ES细胞在体外诱导分 化成病人所需的细胞， 这样获得的细胞和组织将与病人自体基因 型基本一致， 在细胞或组织移植治疗中属自体移植， 不会被病人 的免疫系统所排斥。 治疗性克隆可以解决同种异体细胞或组织移 植引起的病人免疫排斥的问题。
由于治疗性克隆所产生的干细胞将用于临床， 因此用于人类 体细胞核移植的异种卵母细胞应该符合下列要求： 首先， 它们必 须不携带任何可以传染给人的病原体， 包括细菌、 病毒、 支原体 等； 其次， 它们必须有效地支持人体细胞核的重编程， 并支持体 细胞胚胎体外发育至嚢胚或孵化嚢胚； 再者， 它们应该遗传背景 清晰、 质量稳定、 并可大量提供。
与牛和羊相比， 兔的卵母细胞来源方便、 经济、 质量稳定、 遗传背景清晰， 在核移植操作中无需增加体外成熟步骤， 且卵母 细胞的后期发育好， 更适合应用于治疗性克隆。
在一具体实施方案中， 本发明提供采用核移植技术制备人类 体细胞胚胎的新方法。 即通过将人体细胞或细胞核转入家兔卵母 细胞中使其重新编程 （reprogram ) 并发育成人类体细胞胚胎。 这种胚胎可被用于产生人类胚胎干细胞， 胚胎干细胞样细胞或其 他类型干细月包。 如上所述， 在此以前， 人们只用过大型有蹄类动 物， 包括牛和羊做卵母细胞供体。 和牛和羊相比， 利用家兔提供 卵母细胞具有低成本， 方便， 可大量提供等优点。 最重要的是， 若考虑将从体细胞胚胎获得的干细胞用于人时， 有可能通过 SPF 级 (无特定病原体动物） 管理获得无病原体污染的家兔和家兔卵 母细^^ 本发明发现， 家兔的卵母细胞可以非常有效地重新编程 人类体细胞核， 使其发育成嚢胚。 用家兔的卵母细胞重新编程的 人类体细胞发育成嚢胚的成功率在 10%左右。 已接近大规模为临 床服务的标准。
由于家兔可以提供大量的， 低成本的， 无病原体污染而又能 高效启动人体细胞核的卵母细胞， 在未来治疗性克隆的研究和应 用中， 家兔比牛和羊更有可能发展为人卵母细胞的代用品。 物卵母细胞， 优选为兔科动物卵母细胞， 最优选为家兔去核卵母 细胞中， 制备人体细胞胚胎。
通常， 体细胞胚胎通过以下步骤的核转移程序制备： ( i ) 获得所需的用作细胞核供体的人或动物细胞。
( ϋ ) 从合适的卵母细胞供体例如哺乳动物和兔科动物
(例如兔）获得卵母细胞
( iii ) 将上述卵母细胞去核；
( iv ) 将人或动物细 包或细胞核通过例如注射转移到去核 卵母细胞卵周隙或细胞质中， 并经过电激活或化学 激活形成核移植单元。
( V ) 所得到的核移植单元在体外或体内培养发育到一定 阶段， 例如嚢胚或孵化嚢胚。
核转移技术
核转移技术或核移植技术在文献中已有描述, 本发明背景引 用的一些参考文献也有描述。 特别参见， Wil匪 t Ian, et al . Nature , 385: 810— 813 ( 1997 ) , Campbell, et al. Biology of Reproduction , 43 ： 181 ( 1995 )； Collas , et al. Mol. Peprod. Dev. , 38: 264-267 ( 1994 ); Keefer, et al. Biology of Reproduction, 50： 935-939 ( 1994 ); Sims , et al.PNAS, 90： 6143-6147 ( 1993 ); 专利号 W0 94/26884; W0 94/24274; 和 W0 90/03432, 此处全文引入作为参考文献。
核供体细胞
在本发明中， 用作核供体的细胞是来源于人体细胞、 优选为 成纤维细胞。
通过公知的方法可以获得人的体细胞。 可用于本发明的人细 胞包括 （通过举例的方式）： 上皮细胞、 神经细胞、 表皮细胞、 角质化细胞、 造血细胞、 黑素细胞、 软骨细胞、 淋巴细胞 （B 和 T 淋巴细胞）、 有核红细包、 巨噬细胞、 单核细胞、 成纤维细胞、 心肌细胞和其它肌细胞等。 而且用于核转移的人细胞可以从不同 器官获得， 例如， 皮肤、 肺、 胰、 肝、 胃、 肠、 心脏、 生殖器官、 膀胱、 肾、 尿道和其它泌尿器官等。 它们仅是适合供体细胞的例 子。 合适的供体细胞， 即本发明中有用的细胞（包括所有体细胞） 可以从躯体的几乎任何组织和器官获得。
卵母细胞
用于核转移的卵母细胞可以获自动物包括哺乳动物和两栖动 物。 卵母细胞的合适哺乳动物细胞源包括绵羊、 牛、 猪、 马、 兔、 豚鼠、 小鼠、 仓鼠、 大鼠、 灵长类动物等。 在本发明的优选实施 例中， 卵母细胞获自兔科动物， 最优选为家兔。
现已发现当自然发情的母兔发情至第 到 24 小时， 可以从 其生殖道收集成熟的分裂中期 I I 期的卵母细胞， 而经人工激素 处理的母兔在注射人绒膜促性腺激素（ HCG )或类似激素后的 14-24 小时， 最优选是 15-18 小时也可收集到成熟的分裂中期 I I 期的 卵母细胞。
分离卵母细胞的方法为本领域公知的技术。 该方法基本上是 从哺乳动物或两栖动物（例如兔）的卵巢或生殖道分离卵母细胞。
曾报导核转移过程中卵母细胞的成熟程度是核移植方法是否 成功的关键（参见， 例如， Prather et a l , Different iat ion, 48: 1-8 , 1991 )。 通常， 成功的哺乳动物胚胎克隆方法使用分裂 中期 I I 期的卵母细胞作为卵母细胞， 因为认为处于该期的卵母 细胞可以被有效活化并能够以类似接受精子的方式对待导入的细 胞核。
去核
现已发现从新西兰大白家兔获得成熟的卵母细胞应在注射人 绒膜促性腺激素 （HCG ) 后第 15-23 小时去核。 去核之前卵母细 胞最好在细胞丘除去之前移至含透明质酸酶的 M2 培养液（S igma) 中。 这可以通过极细内径吸管反复吹吸或简单地旋涡震荡实现。 然后筛选分离到含极体的卵母细胞。 由存在极体确定卵母细胞处 于细胞分裂中期 I I期， 该卵母细胞用于核转移， 去核。
对于从卵巢采取的未成熟卵母细胞通常需要体外成熟。 这一 过程通常使卵母细胞到达分裂中期 I I期。
对新西兰大白家兔而言，去核优选地不超过 HCG注射后的第 20 小时， 更优选地在注射 HCG后 16-18小时。
去核可以通过显微操作用微吸管除去极体和周围的胞浆实 现。 然后筛选已被成功去核的卵母细胞。 这种筛选可以通过用每 毫升含 1微克 Hoechs t 33342 ( S igma ) 染料染色， 然后紫外线 下迅速观察卵母细胞来完成。
核移植单元制备
可以按照本领域已知方法制备核移植单元。 例如， 透明带下 注射和细胞质内注射。
透明带下注射； 去核后将与去核卵母细胞异源的动物或人单 细胞转移到去核卵母细胞的卵周隙。 优选地， 人或动物细胞和家 兔的卵母细胞构成的核移植单元在 HCG 注射后 16-20 小时后在 0. 5醒的小室中使用 90- 120V的电脉冲约 60微秒 1-2次或多次 在电融合液 （如甘露醇融合液， 蔗糖融合液等） 中进行电融合。 融合后， 产生的核移植单元置于合适培养基例如 RD、 M199、 DMEM 等培养基直到活化。 典型的活化将在其后较短的时间完成， 一般 不超过 24小时， 优选的约为之后 4- 9小时。
电融合方法融合细胞： 用足以使得细胞质膜短暂崩解的电脉 冲实现电融合。 因为膜迅速重新形成， 细胞质膜的崩解非常短暂。 实质上， 如果诱导两个邻近膜崩解并重新形成互相掺合的脂质双 分子层， 两个细胞之间的小通道（孔）将打开。 由于该小通道的 热力学不稳定性， 它将扩大直到两个细胞融合成一个。 参见
Prather et a l 的美国专利 4997384 (此处全文引入作为参考） 进一步讨论了该方法。 可以使用很多电融合介质， 包括例如， 蔗 糖、 甘露醇、 山梨醇和磷酸盐緩冲液。 也可以使用仙台病毒作为 融合剂实现融合。
可以用已知方法活化核移植单元。 该方法包括， 例如， 在亚 生理温度培养核移植单元， 实际上使用冷或者低温刺激核移植单 元。 通过室温培养核移植单元可最为方便地操作， 其相对于胚正 常的生理温度条件而言属于低温。
美国专利 No. 5496720 ( Susko-Parr i sh et a l ) 的目的即为提 供合适的卵母细胞活化的方法。
另外， 也可以顺次完成活化：
( i ) 增加卵母细胞中二价阳离子的水平， 且
( ϋ ) 降低卵母细胞中细胞蛋白质的磷酸化。
增加细月包内二价阳离子可以通过 （例 ^口， 添加激酶抑制剂， 例如丝氨酸-苏氨酸激酶抑制剂， 例如， 6-二甲基-氨基-嘌呤、 星形孢菌素、 2-氨基嘌呤和鞘氨醇。） 浓度来实现。
另外， 通过向卵母细胞导入碑酸酶例如磷酸酶 2 Α和磷酸酶 2B 能抑制细胞蛋白质的磷酸化。
细胞质内注射： 还可选用其他方法做核移植， 例如， 直接将 供体细胞核注射到卵母细胞胞质中， 而不是电穿孔融合 （Co l las 和 Barnes , Mo l . Peprod. Dev. , 38： 264-267 1994 )。 该方法成 功的使人体细胞核在兔卵母细胞质的环境中启动了人胚胎的发育 过程。
核移植单元培养
活化的核移植单元可以在合适的体外培养基中培养直到产生 体细胞胚胎。 在本发明的优选实施方案中， 优选实施例中， 对于 人细胞 /去核兔卵母细胞形成的核移植单元置于 RD培养液（详见 实施例） 中培养时嚢胚率最高。
例如， 活^ ^的核移植单元可以转移到 RD 、 M199、 DMEM 等多 种培养液 50 ul 的微滴培养基中上覆石腊油， 培养环境条件为， 例如， 38 °C , 5% C02。
根据我们的经验， 对于人细胞 /去核兔卵母细胞形成的核移 植单元， 在卵母细胞开始活化后约 6-7天得到嚢胚。 获得的体细 胞胚胎呈现与核供体物种而不是卵母细胞供体物种的胚胎相似的 外观和细胞生长特性。 例如， 人供体细胞核置入去核兔卵母细胞 获得的体细胞胚胎, 发育过程类似人的胚胎而不是兔。 人细胞核 /兔卵母细胞质衍生的体细胞胚胎在培养的第 6-7 天形成嚢胚， 这和人一样， 与兔不同 （在培养的第 3- 4天形成嚢胚）。
本领域已知的可以用于兔胚胎培养和维持的培养基，例如 DMEM +15%FCS; M199+15%FCS; RD+15%FCS 等。 另外这些培养基可以和 多种细胞类型， 例如粒细胞、 输卵管细胞、 子宫细胞和 ST0细胞 等， 结合构成共培养体系
在本发明中产生的体细胞胚胎可建立人胚胎干细胞， 胚胎干 细胞样细胞或其他类型干细胞， 其可应用在^艮多疾病的治疗中， 将可能为一大批遗传病的治疗提供全新的手段。
在实践应用上 , 异种核移植技术可以挽救濒危灭绝的物种如 大熊猫， 由于雌性数量稀少， 不可能获得大量的卵母细胞， 采用 它们的体细胞做供核细胞， 移入其他种的卵母细胞中。 在体外培 养成嚢胚， 再移入代孕妈妈子宫中生下大熊猫。
提供下列实施例以更清楚地描述本发明。 实施例 1 供体细胞的制备
在征得包皮环切术的病人同意后， 取手术切下的无菌包皮组 织， 剪成碎块， 用磷酸盐緩冲液 (PBS)冲洗， lOOOrpm 离心 5 分 钟后， 弃上清， 组织碎块用 0.05 Trypsin/0.02%EDTA (Gibco) 在 37°C条件下消化 30分钟， 取悬浮细胞液 lOOOrpm离心 5分钟 后， 弃上清， 细胞沉淀用培养液 （ 90% DMEM , Gibco ; 10% FBS , Hyclone; 50IU/ml penicillin-streptomycin , Gibco ) 重悬种于培养 i内， 37°C, 5% C02培养。 每 3天换液， 细胞长满 后传代， 传到第 7— 20代范围内用作细胞核供体细胞（图 1)。 卵母细胞的制备
新西兰大白兔 5-6 月龄， 体重 5-6 斤， 雌性， PMSG (上海第 一生化制药公司 ） 100 IU/只肌肉注射， 72 小时后耳缘静脉注 射 HCG (天津市华孚高新生物技术公司） 100IU/只 , HCG 注射 后第 I4- 16 小时， 取其输卵管， 用 M2 培养液（ Sigma) 液体沖 出带颗粒细胞的卵母细胞， 放在 300IU/ml的透明质酸酶（Sigma) 中， 在解剖镜下观察， 待颗粒细胞散开后， 立即用细玻璃针吸出 卵母细胞（图 2), 在 M2培养液中洗 3遍。 核转移程序
透明带下注射.' 去颗粒细胞的卵母细胞放在含 7.5 g/ml CB ( Sigma ) 的 M2 培养液中，室温浸泡 10 分钟后用有斜角的去核 胞的卵周隙中形成核移植单元，核移植单元在电融合缓冲液（0.3M Glucose, Sigma; 0. ImM gCl2, Sigma; 0.05raM CaCl2, Sigma; ) 中平衡 15分钟后施以电刺激（HV 120V, 60 us, 1 次）。 随后， 核移植单元在 RD培养液 （42.5% DMEM , Gibco ； 42.5% RPMI- 1640, Gibco ； 15%FBS, Hyclone )中培养， 培养 6-7 天， 即 可获得体细胞嚢胚（图 3-6)。
细月包质内注射-. 去颗粒细胞的卵母细胞放在含 7.5 g/ml CB ( Sigma ) 的 M2 培养液中，室温浸泡 10 分钟后用有斜角的去核 胞的细胞质内。 随后， 核移植单元在 RD培养液 （42.5% DMEM , Gibco ； 42.5% RPMI- 1640, Gibco ； 15 FBS, Hyclone)中培 养， 培养 6-7天， 即可获得体细胞嚢胚（图 7-10)。
Figure imgf000015_0001
从体细胞胚胎中建立人 S- ES细胞系
由上述方法获得的体细胞嚢胚，用直径 3mm 的玻璃管拉成直 径略 j、于嚢胚透明带的玻璃细针，上下轻轻吹吸嚢胚，使其脱掉透 明带，分散成多个小细胞团块，移入饲养层上， 用 ES 细胞培养液 (79%DMEM , Gibco ； 20% FBS , Hyclone； 1% non-es sent ia 1 amino acid stock, Gibco ； 0. ImM β-merca toethanol,
Gibco ； lOng/ml LIF, R & D; lOng/ml bFGF , R & I>, lOuM Forskolin , Sigma) 37°C, 5% C02条件下培养。 每 2 天换一半液 体.
培养第 2-- 4天后， 观察到细胞团贴壁生长 .7—20天可见集 落生成 （图 12 ) .用机械和酶消化方法将集落打散，传代到含新 鲜饲养层的培养板上。 传代几次后冻存 S- ES细胞， 传代 20代以 上进行 S- ES细胞的形态和生化特征的鉴定。 成纤维细胞饲喂层
胚胎成纤维细胞的原代培养物来源于 14-16天龄的鼠胎。 无 菌除去头、 肝、 心脏和食道后切碎鼠胚胎， 在预热的 0.05%trypsin/0.02 EDTA (GIBCO ) 中 37。C消化 30 分钟。 取悬 浮细胞液，离心 lOOOrpm 5分 4†,细胞沉淀用培养液重悬（ 90% DMEM, Gibco ； 10% FBS , Hyclone 50 IU penici 11 in- streptomycin, Gibco ； 1% non-essential amino acid stock, Gibco ; 0. ImM β-mercaptoethanol, Gibco )接种在组织培养皿中， 在 37 °C, 5% C02条件下培养。 传代 3 次后用 10mM Mitomycin C ( Sigma) 处理 3-4 小时后， 传代于 96孔和 4孔板上。 成纤维饲养层细胞 在潮湿并含 5% (02的大气中 37°C生长和维持。 这些具有均一的 饲喂细胞单层的培养板用于培养 S- ES细胞系。 实施例 3
S-E S第 26代染色体核型鉴定 10ug/ml 秋水仙素加入细胞中 ， 在 37 °C作用 4 小 时， 1 OOOrpm离心 8分钟， 去除上清； 用 37 °C预热的 0. 05MKCL 重悬细胞沉淀， 并在 37 °C中孵育 30分钟， l OOOrpm 离心 8分钟， 去除上清； 用固定液（甲醇： 冰醋酸 = 3： 1 ) 重悬细胞沉淀， 在室温中放置 15 分钟， l OOOrpm 离心 8分钟， 去除上清； 再用 固定液重悬细胞沉淀， 在室温中放置 15 分钟， l OOOrpm 离心 8 分钟。 取细胞沉淀滴在玻片上， GIEMSA 染色观看核型。 实验结 果如图 13所示.
虽然以上通过具体的实施方案对本发明进行了具体描述， 但 本领域技术人员可以理解， 在不背离本发明的原则和精神的情况 下可以对本发明作出各种改变和改进。 因此， 所有这些改变和改 进都应包括在本说明书和所附的权利要求书的范围之内。' ·

Claims

权 利 要 求
1.一种制备体细胞起源的胚胎的方法， 该方法包括将一种核供体 动物细胞或其细胞核移植入一种与核供体动物相同或不同种的 动物的去核卵母细胞形成核转移单元， 并在适宜条件下培养形 成的体细胞胚胎。
2.根据权利要求 1的方法，其中所说的核供体动物细胞为人细胞； 优选为胎儿或成人的体细胞;更优选为成人成纤维细胞.
3.根据权利要求 1的方法， 其中所说的卵母细胞得自哺乳动物或 两栖动物。
4.根据权利要求 3的方法， 其中所说的卵母细胞得自人。
5.根据权利要求 3 的方法, 其中所说的卵母细胞得自兔科动物， 优选家兔。
6.根据权利要求 3 - 5 中任一项的方法， 其中所说的卵母细胞处 于分裂中期， 优选处于分裂中期 I I期。
7.根据权利要求 1的方法， 其中所获得的核转移单元通过室温培 养或使用活化剂而活化。
8.根据权利要求 7的方法， 其中所说的活化剂选自甘露醇电融合 液， 葡萄糖电融合液， 山梨醇电融合液和磷酸盐緩冲液， 优选 为葡萄糖电融合液。
9.根据权利要求 1的方法， 其中活化的核移植单元在 RD培养液， M199培养液， DMEM培养液微滴中培养， 优选为 RD培养液， 使 核转移单元发育为体细胞胚胎， 包括 2 - 4 细胞， 8 -细胞， 桑 葚胚， 囊胚， 孵化囊胚等阶段。
10.根据权利要求 1的方法，其中活化的核移植单元在 RD培养液， M199培养液， DMEM培养液中培养， 也可和多种细胞类型， 例 如粒细胞、 输卵管细胞、 子宫细胞和 ST0 细胞等， 结合构成 共培养体系， 使核移植单元发育为体细胞胚胎， 包括 2 - 4细 胞， 8 -细胞， 桑葚胚， 嚢胚， 孵化嚢胚等阶段。
11.根据权利要求 1 - 10 中任一项所述的方法获得的体细胞胚 胎。
12.根据权利要求 11的体细胞胚胎， 它是通过将人体细胞或细胞 核置入兔科动物的去核卵母细胞获得。
13.根据权利要求 11的体细胞胚胎， 它是通过将成人成纤维细胞 或其细胞核移植入家兔去核卵母细胞而获得。
14.根据权利要求 11 - 13中任一项的体细胞胚胎用于制备人胚胎 干细胞， 胚胎干细胞样细胞或其他类型干细胞。
15.根据权利要求 11 - 13中任一项的体细胞胚胎及其衍生细胞用 于各种治疗和商业目的.
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