CN112852711B - 刀鲚性腺体细胞系的建立及其应用 - Google Patents
刀鲚性腺体细胞系的建立及其应用 Download PDFInfo
- Publication number
- CN112852711B CN112852711B CN202110172872.6A CN202110172872A CN112852711B CN 112852711 B CN112852711 B CN 112852711B CN 202110172872 A CN202110172872 A CN 202110172872A CN 112852711 B CN112852711 B CN 112852711B
- Authority
- CN
- China
- Prior art keywords
- cells
- coilia ectenes
- gonadal
- cell line
- stem cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241001460967 Coilia nasus Species 0.000 title claims abstract description 80
- 210000001082 somatic cell Anatomy 0.000 title claims abstract description 35
- 210000002149 gonad Anatomy 0.000 title claims abstract description 33
- 210000004027 cell Anatomy 0.000 claims abstract description 134
- 210000000130 stem cell Anatomy 0.000 claims abstract description 56
- 230000002710 gonadal effect Effects 0.000 claims abstract description 52
- 238000000034 method Methods 0.000 claims abstract description 19
- 241000251468 Actinopterygii Species 0.000 claims abstract description 18
- 230000004069 differentiation Effects 0.000 claims abstract description 10
- 238000007747 plating Methods 0.000 claims abstract description 5
- 241000276569 Oryzias latipes Species 0.000 claims description 34
- 239000002609 medium Substances 0.000 claims description 19
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 18
- 241000283073 Equus caballus Species 0.000 claims description 14
- 210000001161 mammalian embryo Anatomy 0.000 claims description 13
- 239000000284 extract Substances 0.000 claims description 12
- 241000723298 Dicentrarchus labrax Species 0.000 claims description 11
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 11
- 244000304217 Brassica oleracea var. gongylodes Species 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 9
- 239000007640 basal medium Substances 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 8
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 8
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 7
- 229940024606 amino acid Drugs 0.000 claims description 7
- 150000001413 amino acids Chemical class 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 239000012091 fetal bovine serum Substances 0.000 claims description 7
- 229960002989 glutamic acid Drugs 0.000 claims description 7
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 6
- 210000002257 embryonic structure Anatomy 0.000 claims description 6
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 6
- 229960001471 sodium selenite Drugs 0.000 claims description 6
- 239000011781 sodium selenite Substances 0.000 claims description 6
- 235000015921 sodium selenite Nutrition 0.000 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 5
- 230000001939 inductive effect Effects 0.000 claims description 4
- -1 hydroxyethylpiperazine ethylsulfanilic acid Chemical compound 0.000 claims description 3
- 229940054269 sodium pyruvate Drugs 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 abstract description 16
- 239000001963 growth medium Substances 0.000 description 26
- 239000008055 phosphate buffer solution Substances 0.000 description 20
- 230000021595 spermatogenesis Effects 0.000 description 19
- 239000013592 cell lysate Substances 0.000 description 15
- 235000019688 fish Nutrition 0.000 description 15
- 210000004602 germ cell Anatomy 0.000 description 15
- 230000006698 induction Effects 0.000 description 15
- 239000006228 supernatant Substances 0.000 description 12
- 230000029087 digestion Effects 0.000 description 10
- 210000003495 flagella Anatomy 0.000 description 10
- 108010019160 Pancreatin Proteins 0.000 description 9
- 239000012228 culture supernatant Substances 0.000 description 9
- 229940055695 pancreatin Drugs 0.000 description 9
- 108010010803 Gelatin Proteins 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 7
- 229920000159 gelatin Polymers 0.000 description 7
- 239000008273 gelatin Substances 0.000 description 7
- 235000019322 gelatine Nutrition 0.000 description 7
- 235000011852 gelatine desserts Nutrition 0.000 description 7
- 238000003757 reverse transcription PCR Methods 0.000 description 7
- 241000972773 Aulopiformes Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000002771 cell marker Substances 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 6
- 238000000227 grinding Methods 0.000 description 6
- 235000019515 salmon Nutrition 0.000 description 6
- 239000002356 single layer Substances 0.000 description 6
- 101100408379 Drosophila melanogaster piwi gene Proteins 0.000 description 5
- 238000003501 co-culture Methods 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 108010054624 red fluorescent protein Proteins 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000000920 spermatogeneic effect Effects 0.000 description 5
- 210000001550 testis Anatomy 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 101150090523 DAZL gene Proteins 0.000 description 4
- 101100263436 Drosophila melanogaster vas gene Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 4
- 101100049051 Penaeus vannamei vasa gene Proteins 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000004660 morphological change Effects 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 230000000392 somatic effect Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 101150105763 FSHR gene Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 201000010208 Seminoma Diseases 0.000 description 3
- 210000001789 adipocyte Anatomy 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical compound [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 description 2
- 101100278180 Danio rerio dnd gene Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 101100096236 Xenopus laevis sox9-b gene Proteins 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007844 bleaching agent Substances 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 108091092328 cellular RNA Proteins 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 229940076788 pyruvate Drugs 0.000 description 2
- 210000004336 spermatogonium Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000825055 Coilia Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241001514662 Leptospermum Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001327682 Oncorhynchus mykiss irideus Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 101100096235 Xenopus laevis sox9-a gene Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 230000006543 gametophyte development Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229940082569 selenite Drugs 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000024642 stem cell division Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0683—Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells
-
- 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
- C12N5/0608—Germ cells
- C12N5/061—Sperm cells, spermatogonia
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/24—Genital tract cells, non-germinal cells from gonads
- C12N2502/246—Cells of the male genital tract, non-germinal testis cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Reproductive Health (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及生物技术领域,具体为刀鲚性腺体细胞系的建立及应用。步骤包括:(1)分离和消化刀鲚的性腺组织,取细胞铺板培养;(2)细胞贴壁后进行传代,连续培养45‑60代。所获得的刀鲚性腺体细胞系能够稳定传代和增殖,并且能够诱导鱼类精原干细胞在体外分化产生精子。
Description
技术领域
本发明涉及生物技术领域,具体为刀鲚性腺体细胞系的建立及其应用。
背景技术
性腺对脊椎动物的繁殖至关重要,性腺由生殖细胞、支持细胞和间质细胞组成,支持细胞和间质细胞统称为体细胞。生殖细胞最终发育产生配子,支持细胞包裹着生殖细胞,促进生殖细胞的生长、发育和配子产生,支持细胞紧靠间质细胞。几种细胞之间相互作用,维持性腺的正常发育和生理机能。
精原干细胞(Spermatogonial stem cells,SSCs)是精子发生的基础,对遗传信息的传递至关重要。鱼类和其他两性生殖动物一样,其SSCs具有高度的自我更新和分化能力,为精子产生提供了稳定的生殖细胞来源。精子发生是一个复杂的过程,经历了干细胞更新和分化的有丝分裂期、减数分裂期和精子发生后的减数分裂期。在体外完全重现精子发生非常复杂且不稳定,一方面需要获得能够稳定培养的正常精原干细胞系,另一方面需要模拟精子发生的体内微环境,高效诱导SSCs发生减数分裂产生精母细胞,以及后续精子细胞形变产生精子。
精子发生受到各种内源和外源信号的共同调控。内源因子包括几种重要的生殖细胞标记分子,如dnd、piwi等,它们的缺失都会导致不育;外源信号包括各类激素、生长因子等,它们通过调节支持细胞的活性、结构以及性类固醇产生,从而调节生殖细胞的发育和分化。另外,支持细胞上存在着一些分子,能够与生殖细胞上的受体相互作用,在生殖细胞的自我更新和分化过程中必不可少。因此,性腺体细胞与生殖细胞之间存在着紧密联系,二者在鱼类性腺的有效发育和配子发生过程中缺一不可。有研究表明,将青鳉精原干细胞与虹鳟性腺体细胞混合培养,可诱导青鳉精原干细胞体外精子发生,但由于混合后细胞成分复杂,一定程度上限制了鱼类精子发生机制的相关研究。
基于性腺体细胞在精子发生过程中的重要作用,获得稳定培养的性腺体细胞系对探究鱼类精子发生过程至关重要。探究鱼类的精子发生,不仅有利于干细胞生物学的基础研究,对濒危珍稀鱼种的保护,缩短性成熟周期长的经济鱼类的育种周期也有重要价值。
发明内容
本发明旨在提供一种建立刀鲚性腺体细胞系的方法。
本发明还提供了上述刀鲚性腺体细胞系的应用。
技术方案为,一种建立刀鲚性腺体细胞系的方法,包括以下步骤:
(1)分离和消化刀鲚的性腺组织,取细胞铺板培养;
(2)细胞贴壁后进行传代,连续培养45-60代。
优选的,在步骤(2)中,连续培养50代获得刀鲚性腺体细胞系。
优选的,所述的刀鲚为长江刀鲚。
以含有羟乙基哌嗪乙硫磺酸的DMEM培养基为基础培养基,并含有胎牛血清、青鳉鱼胚胎提取物、海鲈鱼血清、碱性成纤维细胞生长因子、L-谷氨酸、非必要氨基酸;培养条件为:27-30℃培养。
青鳉鱼胚胎提取物的制备方法为:收取发育至5-8天的青鳉鱼胚胎进行研磨,收集研磨物,液氮中冻融2-4次后继续研磨,将研磨物在10000-15000g条件下,1-4℃离心15-40min,收集上清;用pH=7.0-7.4的PBS对胚胎提取物进行溶解并定容。优选的,收取发育至7天的青鳉鱼胚胎进行提取。
海鲈鱼血清的制备方法为:抽取海鲈鱼血(从海鲈鱼尾静脉抽血),室温静置1-4h后,转移至4℃放置8-12h,然后4000-8000g、1-4℃离心20-40min,收集上清。
优选的,每升基础培养基中添加:120-180mL胎牛血清、以青鳉鱼胚胎个数计200-600个青鳉鱼胚胎提取物、5-20mL海鲈鱼血清、2-8μg碱性成纤维细胞生长因子、0.5-2UPenicillin-streptomycin双抗,0.01-0.03M L-谷氨酸,以及非必要氨基酸(各0.005-0.02mM)。更为优选的,每升基础培养基中添加:150mL胎牛血清、以青鳉鱼胚胎个数计400个青鳉鱼胚胎提取物、2mL海鲈鱼血清、5μg碱性成纤维细胞生长因子、1U Penicillin-streptomycin双抗、0.02M L-谷氨酸,以及非必要氨基酸(各0.01mM)。pH=7.5-7.8,优选为7.7。
培养基中还含有β-巯基乙醇和亚硒酸钠。每升基础培养基中β-巯基乙醇的添加量为0.001-0.005μM,亚硒酸钠的添加量为0.01-0.05mM。优选的,每升基础培养基中β-巯基乙醇的添加量为0.002μM,亚硒酸钠的添加量为0.02mM。
通过本发明方法可以利用刀鲚性腺细胞建立起一种能在体外连续传代的刀鲚性腺体细胞系。在体外持续培养时间已超过11个月,细胞长势良好,保持高增殖活性。
该细胞系经鉴定为性腺体细胞,而非生殖细胞,有47条染色体。
所培养的刀鲚性腺体细胞可诱导鱼类的精原干细胞体外分化产生精子。例如马口鱼精原干细胞和青鳉鱼精原干细胞。
通过将刀鲚性腺体细胞与鱼类的精原干细胞共同培养,精原干细胞发生形态变化,细胞逐渐拉长,可以实现精原干细胞体外精子发生。优选的,刀鲚性腺体细胞与鱼类的精原干细胞的比例为5:1-1:5,更优选为2:1-1:2。在本发明的一个优选方式中,比例为1:1。
所培养的刀鲚性腺体细胞的细胞裂解液可以诱导鱼类的精原干细胞体外分化产生精子。例如马口鱼精原干细胞和青鳉鱼精原干细胞。
将培养的刀鲚性腺体细胞消化后重悬于培养基中,在-70至-85℃冻融2-4次,优选为在-80℃冻融3次。所述的培养基以含有羟乙基哌嗪乙硫磺酸的DMEM培养基为基础培养基,含有胎牛血清、海鲈鱼血清、L-谷氨酸、非必要氨基酸。
优选的,用于重悬细胞的培养基与用于培养刀鲚性腺体细胞的培养基相比,不含青鳉鱼胚胎提取物和碱性成纤维细胞生长因子。
将上述刀鲚性腺体细胞裂解液作为培养基用于培养鱼类的精原干细胞。以刀鲚性腺体细胞计,细胞裂解液中的细胞数量为1×106-1×109/mL,优选为5×106-1×108/mL。
综上所述,本方法培养获得了一种能够稳定传代和增殖的刀鲚性腺体细胞系,能够诱导鱼类精原干细胞在体外分化产生精子,对于经济鱼类的繁殖生产研究具有重要意义。
附图说明
图1为建立的刀鲚性腺体细胞系图片。
图2为RT-PCR鉴定刀鲚性腺细胞的结果,显示表达体细胞标记,不表达生殖细胞标记。
图3为刀鲚性腺体细胞的核型分析结果,具有47条染色体,为二倍体核型。
图4为长江刀鲚性腺体细胞诱导马口鱼精原干细胞体外精子发生。
图5为长江刀鲚性腺体细胞诱导青鳉精原干细胞体外精子发生。
图6为长江刀鲚性腺体细胞裂解液诱导马口鱼精原干细胞体外精子发生。
图7为长江刀鲚性腺体细胞培养上清诱导马口鱼精原干细胞体外精子发生。
图8为长江刀鲚性腺体细胞裂解液诱导青鳉精原干细胞体外精子发生。
具体实施方式
实验材料
培养基的配制:每1L灭菌ddH2O中加入13.37g DMEM和4.77g Hepes,NaOH调节pH值至7.7,获得基础培养基DMEM。每1L基础培养基DMEM中依次加入以下成分:150mL胎牛血清(FBS),1mL青鳉胚胎提取物(自制,收取发育至7天的青鳉鱼胚胎,进行研磨,收集研磨物,液氮中冻融3次后继续研磨,将研磨物转移至1.5mL离心管中,12000g,4℃离心30min,收集上清,按照每400颗胚胎提取1mL的比例,用pH=7.4的PBS对胚胎提取物进行定容,分装保存在-20℃备用),2mL海鲈鱼血清(自制,取体重500g左右的海鲈鱼,麻醉后用5mL注射器,尾静脉抽血,约每0.1mL血液置于1mL离心管内,室温静置2h后,转移至4℃过夜,5000g,4℃离心30min,收集上清,分装保存在-20℃备用),50μL碱性成纤维细胞生长因子(bFGF,0.1mg/mL),10mL L-谷氨酸(L-Glu,100×,2mM),10mL非必须氨基酸(non-essential amino acid,100×,1mM),10mL丙酮酸钠(Na-pyruvate,100×,1mM),10mL双抗(Penicillin-streptomycin,100×,100U ug/mL),4mLβ-巯基乙醇(β-Mercaptoethanol,50mM),1mL亚硒酸钠(Na-selenite,1000×,2μM)。配制好的培养基过滤后4℃保存。除非特别说明,在后续实验中均使用该培养基。
实验中所用器具均灭菌后使用。
实施例1刀鲚性腺体细胞培养
1.刀鲚性腺细胞的分离与消化
取长江刀鲚,用含0.5%漂白粉的PBS浸泡鱼体1min,用含1%双抗、pH=7.4的PBS清洗3次后进行解剖。剪刀剪开鱼腹,镊子小心夹出性腺,置于含有1%双抗PBS的培养皿内,用镊子小心剥离性腺表面的脂肪组织,含1%双抗的PBS清洗3次后,将性腺转移至装有500μL胰酶的1.5mL EP管内,用剪刀适当剪碎组织,置于冰上消化。1h后加入1mL培养基终止消化,用移液枪轻轻吹散组织块,将细胞悬液转移至铺过0.1%明胶的24孔板内,置于28℃培养。
2.刀鲚性腺体细胞的培养与传代
第二天显微镜下观察细胞贴壁情况,并确认细胞未被污染,继续置于28℃培养。铺板后第3天,更换一半培养基,后续根据细胞生长状况进行换液,直至细胞铺满单层。待细胞铺满单层后,按照1:2的比例进行传代。首先,吸出孔内培养基,用500μL PBS洗细胞3次,200μL胰酶消化30s,吸出消化液,加入2mL培养基重悬细胞,吸出1mL至新的孔内,继续置于28℃培养。待细胞铺满单层后,继续按照1:2的比例进行传代。连续培养50代后,表明细胞系已基本建成,可用于后续的鉴定及其他实验。
显微镜下观察发现,性腺细胞悬液在接种后第1天有少量细胞贴壁,细胞多呈长形,此后每天更换一半培养基,约一周后细胞聚集生长,出现致密的细胞团,胰酶消化后,加入新的培养基重悬细胞,轻轻吹打细胞团使其分散均匀,细胞再次贴壁后,同样每天更换一半培养基,约一周后细胞铺满单层,按1:2的比例传代(图1,P0为刚刚贴壁的细胞状态,P38为第38代的细胞,P78为第78代细胞)。目前,该株性腺细胞在体外已持续培养超过11个月,细胞长势良好,依然保持高增殖活性。
实施例2刀鲚性腺体细胞的鉴定
1.RT-PCR鉴定培养的刀鲚性腺体细胞类型
为鉴定所培养的刀鲚性腺细胞类型,提取细胞RNA反转为cDNA,进行RT-PCR检测。
用Primer Premier 6设计刀鲚性腺体细胞标记基因sox9、fshr和clu引物及生殖细胞标记基因vasa、dazl和piwi引物(引物序列信息见表1),提取细胞RNA并反转为cDNA,采用RT-PCR鉴定刀鲚性腺细胞类型。
表1 RT-PCR引物序列信息
基因名 | 引物序列(F:上游,R:下游) |
sox9b | F:TGGACCCCTACCTGAAGATG;R:AGTCCAGTCGTAGCCCTTGA |
fshr | F:GTGGTGCTGGTGTTGCTGCTTA;R:TGGACGAGTGAGTAGATAGTGCCTTC |
clu | F:TCTCTGCTCTGTGTCTTATC;R:AACTTCTTGTGGTCCTCTC |
piwi | F:CGACATCCACCAGCACAGA;R:AACGCCACGCATCTCCTT |
vasa | F:CGCCATCTTCAATCAGTTCCA;R:AGTGTCTGCCTCTCCTCCT |
dazl | F:CTCGAGATGGATATCAACAAGCC;R:CAGCACAGTCAACATAGTC |
取四个孔的细胞,胰酶消化后,用PBS重悬细胞至1.5mL EP管内,3500g离心5min,PBS洗一次,小心吸去上清,按照以下步骤提取细胞RNA。首先,加入500μL Trizol至装有细胞沉淀的EP管内,上下颠倒混匀,在冰上静置5min;加入200μL氯仿,剧烈震荡15s,冰上静置5min,4℃12000rpm离心20min;吸取约400μl的上清至一个新的1.5mL EP管中,加入等体积的酚:氯仿,涡旋30s,4℃12000rpm离心20min;吸取约300μL的上清至一个新的1.5mL EP管中,加入0.7倍体积(约210μL)的异丙醇,上下颠倒混均后室温孵育15min,4℃12000rpm离心15min;弃上清,加入75%的乙醇洗涤沉淀两次,4℃7500rpm离心5min;弃上清,室温静置3min以使乙醇完全挥发。加入20μL DEPC水反复吹打溶解沉淀,获得RNA可在-80℃长期保存。按照Takara反转说明书合成cDNA,以此为模板进行RT-PCR检测,反应体系及程序如下:上下游引物各1μL、模板2μL、dNTPs 1μL,Buffer 2μL,加入ddH2O定容至20μL;95℃预变性5min;95℃变性20s,58℃退火20s,72℃延伸1-2min,42个循环,72℃延伸10min。
结果显示,刀鲚性腺细胞表达体细胞标记分子clu、fshr、sox9b,不表达生殖细胞标记分子vasa、dazl、piwi,如图2所示。由此可以判定培养的刀鲚性腺细胞为性腺体细胞,而非生殖细胞。
2.刀鲚性腺细胞的核型分析
将细胞接种至包被过明胶的6孔板内扩大培养,每孔加2mL培养基,待细胞长至80%汇合度,加入1mg/mL的秋水仙素使终浓度为20μg/mL,28℃处理4h。除去培养基,pH=7.4的PBS洗2次,胰酶消化后,用1mL PBS重悬并转移至1.5mL EP管内,3500g离心5min;pH=7.4的PBS洗1次,弃上清,加入40mM KCl进行低渗处理,枪头轻轻吹打重悬细胞,室温孵育30min;加入0.2mL新鲜固定液(甲醇:乙酸=3:1),倒置混匀,3500g离心5min,弃部分上清,约留0.3mL重悬细胞;滴加1mL固定液,室温固定10min,3500g离心5min;弃上清,滴加1mL固定液二次固定30min,3500g离心5min;用0.2-0.5mL固定液重悬细胞。预冷载玻片,吸取20μL左右细胞悬液,在距离玻片上方5cm处滴片,使染色体从细胞内摔出,室温风干;5%Gimesa(1/15M磷酸盐缓冲液,pH6.8)室温染色10min,流水冲洗后,室温风干;显微镜下观察,细胞核和染色体呈暗红色,细胞质呈淡蓝色,100倍油镜下计数染色体。
结果如图3所示。刀鲚性腺体细胞的核型分析显示,该细胞系具有47条染色体。
实施例3刀鲚性腺体细胞诱导马口鱼精原干细胞
1.马口鱼精原干细胞培养
A.马口鱼精巢细胞的分离与消化
取马口鱼幼鱼,用含0.5%漂白粉的PBS浸泡鱼体1min,用含1%双抗的PBS清洗3次后进行解剖。剪刀剪开鱼腹,镊子小心夹出精巢,置于含有1%双抗PBS的培养皿内,用镊子小心剥离精巢表面的脂肪组织,含1%双抗的PBS清洗3次后,将精巢转移至装有500μL胰酶的1.5mL EP管内,用剪刀适当剪碎组织,置于冰上消化。1h后加入1mL培养基终止消化,用移液枪轻轻吹散组织块,将细胞悬液转移至铺过0.1%明胶的24孔板内,置于28℃培养。
培养基的配制:每1L灭菌ddH2O中加入13.37g DMEM和4.77g Hepes,NaOH调节pH值至7.7,获得基础培养基DMEM。每1L基础培养基DMEM中依次加入以下成分:150mL胎牛血清(FBS),2mL青鳉胚胎提取物,2mL海鲈鱼血清,100μL碱性成纤维细胞生长因子(bFGF,0.1mg/mL),10mL L-谷氨酸(L-Glu,100×,2mM),10mL非必须氨基酸(non-essential amino acid,100×,1mM),10mL丙酮酸钠(Na-pyruvate,100×,1mM),10mL双抗(Penicillin-streptomycin,100×,100U ug/mL),4mLβ-巯基乙醇(β-Mercaptoethanol,50mM),1mL亚硒酸钠(Na-selenite,1000×,2μM)。配制好的培养基过滤后4℃保存。
在精原干细胞建系阶段(细胞培养至50代前),采用上述培养基,在精原干细胞建系成功后,在培养基中bFGF的添加量为50μL,青鳉胚胎提取物的添加量为1mL。用此培养基进行后续的转染和诱导分化。
2.马口鱼精巢细胞的培养与传代
第二天显微镜下观察细胞贴壁情况,并确认细胞未被污染,继续置于28℃培养。铺板后第3天,更换一半培养基,后续根据细胞生长状况和培养基颜色变化进行换液,直至细胞铺满单层。待细胞铺满单层后,按照1:2的比例进行传代。首先,吸出孔内培养基,用500μLpH=7.4PBS洗细胞3次,200μL胰酶消化5s,吸出消化液,加入2mL培养基重悬细胞,吸出1mL至新的孔内,继续置于28℃培养。待细胞铺满单层后,继续按照1:2的比例进行传代。连续培养50代后,表明细胞系已基本建成,可用于后续的鉴定及其他实验。
显微镜下观察发现,马口鱼幼鱼精巢细胞悬液中含有大量脂肪,细胞量较少,培养3d后未见明显的细胞贴壁,大量脂肪细胞漂浮在培养基上层,小心吸出培养基上层的脂肪细胞,注意不要吸出全部培养基,补充新鲜培养基,一周后,脂肪细胞基本去除干净,可见培养板底部有个别贴壁的细胞,细胞偏圆形或者不规则形状,生长缓慢,每隔1d更换800μL培养基,约一周后细胞开始缓慢增殖,约两周后形成较大的细胞集落,中间细胞由于没有足够生长空间导致堆积生长,此时,吸出培养基,PBS洗一次,用胰酶消化5s后,加入新的培养基重悬,将细胞集落轻轻吹开,使细胞均匀分布在孔内。约一周后,细胞铺满单层,此时,按1:2的比例传代。
碱性磷酸酶染色后细胞呈深紫色,说明培养的细胞具有较强的碱性磷酸酶活性;结合RT-PCR鉴定,细胞表达生殖细胞标记分子vasa、piwi、dnd和dazl,干细胞标记分子nanog和gfrα1,不表达体细胞标记分子dmrt。说明所建立的细胞系为马口鱼精原干细胞。
将pCV-pr质粒转染进马口鱼精原干细胞,构建获得稳定表达红色荧光蛋白的马口鱼精原干细胞系。
将长江刀鲚性腺体细胞与稳定表达红色荧光蛋白的马口鱼精原干细胞按照1:1的比例接种至24孔板共培养,检测长江刀鲚性腺体细胞的诱导能力。培养条件为:置于28℃培养,每隔一天更换一半培养基。
细胞贴壁后,显微镜下观察发现,在共培养后第一天,马口鱼精原干细胞发生形态变化,细胞拉长,在共培养的第四天,形成精子样的细胞,在第八天产生了具有头和尾的精子(图4)。结果显示刀鲚性腺体细胞能够诱导马口鱼精原干细胞体外精子发生。
实施例4诱导青鳉精原干细胞
青鳉精原干细胞系SG3是已经建立好的细胞系,可参考“Establishment of anormal medakafish spermatogonial cell line capable of sperm production invitro”。青鳉鱼精原干细胞系的培养同马口鱼精原干细胞。
将pCV-pr质粒转染进青鳉精原干细胞系SG3,构建获得了稳定表达红色荧光蛋白的prSG3细胞系。将该细胞系与长江刀鲚性腺体细胞共培养,探究长江刀鲚性腺体细胞是否具备广泛的诱导精原干细胞精子发生的能力。显微镜下观察发现,在共培养的第二天,青鳉精原干细胞发生形态变化,细胞逐渐拉长,在共培养的十天内,发生形态变化产生鞭毛的细胞越来越多(图5)。
实施例5刀鲚性腺体细胞裂解液的制备
将稳定传代的刀鲚性腺体细胞接种至24孔板,待细胞铺满单层,按照以下步骤收取并裂解细胞:吸去24孔板内的培养基,PBS洗三次后,加入胰酶消化5s,用不含碱性磷酸酶生长因子(bFGF)和青鳉鱼胚胎提取物(MEE)的培养基重悬细胞,每4孔细胞(约2×107个细胞)用1mL培养基重悬。将细胞悬液在-80℃冻融3次后,用0.22μm滤器过滤后使用。
同时,收集刀鲚性腺体细胞的培养上清作为对照,0.22μm滤器过滤后使用。
实施例6刀鲚性腺体细胞裂解液诱导马口鱼精原干细胞
将5×105稳定表达红色荧光蛋白的马口鱼精原干细胞接种至包被过明胶的24孔板内,加入1mL上述制备的刀鲚性腺体细胞裂解液,细胞置于28℃培养,每隔24h在显微镜下观察细胞分化情况并拍照,连续观察14d,每隔2-3d更换500μL刀鲚性腺体细胞裂解液。
结果显示,用刀鲚性腺体细胞裂解液诱导马口鱼精原干细胞,1-2d后,显微镜下可见大量聚集的带着长长鞭毛的精子细胞团,同时也可见单个具有鞭毛的精子细胞,随着诱导时间的延长,具有鞭毛的精子细胞越来越多,在诱导5-8d后,精子细胞头部进一步收缩至3-4μm,形成精子。如图6所示。
作为对照,按照上述步骤将马口鱼精原干细胞接种至包被明胶的24孔板内,加入1mL刀鲚性腺体细胞培养上清,细胞置于28℃培养,每隔24h在显微镜下观察细胞分化情况并拍照,连续观察14d,每隔2-3d更换500μL刀鲚性腺体细胞培养上清。
结果显示,刀鲚性腺体细胞培养上清诱导马口鱼精原干细胞后,少量马口鱼精原干细胞在诱导后第4-8d形态拉长,带有一条微短的鞭毛,产生接近圆形的精子细胞,其后并未见此类精子细胞进一步形变,诱导过程中未见有带着长长鞭毛的精子。如图7所示。
刀鲚性腺体细胞培养上清的诱导效率远低于细胞裂解液的诱导效率,诱导精子发生的有效成分在刀鲚性腺体细胞中,而非刀鲚性腺体细胞培养上清中。
实施例7刀鲚性腺体细胞裂解液诱导青鳉精原干细胞
将5×105稳定表达红色荧光蛋白的青鳉精原干细胞接种至包被过明胶的24孔板内,加入1mL上述制备的刀鲚性腺体细胞裂解液,细胞置于28℃培养,每隔24h在显微镜下观察细胞分化情况并拍照,连续观察14d,每隔2-3d更换500μL刀鲚性腺体细胞裂解液。
结果显示,用刀鲚性腺体细胞裂解液诱导青鳉精原干细胞,1-2d后,青鳉精原干细胞逐渐形成偏圆形或偏长形的精子细胞,鞭毛长短不一,第3d时,可见聚集的带有鞭毛的精子细胞团,第6-8d后,带有鞭毛的精子细胞越来越多,同时鞭毛逐渐拉长,精细胞头部也有一定程度收缩。如图8所示。
作为对照,按照上述步骤将青鳉精原干细胞接种至包被明胶的24孔板内,加入1mL刀鲚性腺体细胞培养上清,细胞置于28℃培养,每隔24h在显微镜下观察细胞分化情况并拍照,连续观察14d,每隔2-3d更换500μL刀鲚性腺体细胞培养上清。结果显示,未能形成精子细胞。
Claims (2)
1.刀鲚性腺体细胞系的建立方法,其特征在于:
(1)分离和消化刀鲚的性腺,取细胞铺板培养;所述的刀鲚为长江刀鲚;
(2)细胞贴壁后进行传代,连续培养45-60代;
以含有羟乙基哌嗪乙硫磺酸的DMEM培养基为基础培养基,每升基础培养基中添加:150mL胎牛血清、以青鳉鱼胚胎个数计400个青鳉鱼胚胎提取物、2 mL海鲈鱼血清、5 μg碱性成纤维细胞生长因子、1 U Penicillin-streptomycin双抗,并含有0.02 mM L-谷氨酸、0.01mM非必要氨基酸、0.2 mM β-巯基乙醇、0.002 μM亚硒酸钠和0.01mM丙酮酸钠;培养条件为:27-30℃培养。
2.权利要求1所述的刀鲚性腺体细胞系的建立方法在诱导鱼类精原干细胞分化为精子方面的应用,其特征在于,所述的鱼类精原干细胞为马口鱼精原干细胞或青鳉鱼精原干细胞。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110172872.6A CN112852711B (zh) | 2021-02-08 | 2021-02-08 | 刀鲚性腺体细胞系的建立及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110172872.6A CN112852711B (zh) | 2021-02-08 | 2021-02-08 | 刀鲚性腺体细胞系的建立及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112852711A CN112852711A (zh) | 2021-05-28 |
CN112852711B true CN112852711B (zh) | 2024-05-17 |
Family
ID=75989160
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110172872.6A Active CN112852711B (zh) | 2021-02-08 | 2021-02-08 | 刀鲚性腺体细胞系的建立及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112852711B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114736847B (zh) * | 2022-05-18 | 2023-08-22 | 中国水产科学研究院长江水产研究所 | 一种快速获取大鲵原代性腺细胞的方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104152402A (zh) * | 2014-08-26 | 2014-11-19 | 中国水产科学研究院黄海水产研究所 | 一种半滑舌鳎伪雄鱼性腺细胞系的构建与应用方法 |
CN104762252A (zh) * | 2015-04-27 | 2015-07-08 | 上海海洋大学 | 一种草金鱼腹鳍细胞系的构建方法 |
CN109295046A (zh) * | 2018-05-15 | 2019-02-01 | 中山大学 | 一种抗赤点石斑鱼神经坏死病毒青鳉单倍体胚胎干细胞的制备方法及应用 |
CN110684724A (zh) * | 2019-09-18 | 2020-01-14 | 中山大学 | 一种3d培养中华乌塘鳢精巢细胞产生精子的方法和应用 |
-
2021
- 2021-02-08 CN CN202110172872.6A patent/CN112852711B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104152402A (zh) * | 2014-08-26 | 2014-11-19 | 中国水产科学研究院黄海水产研究所 | 一种半滑舌鳎伪雄鱼性腺细胞系的构建与应用方法 |
CN104762252A (zh) * | 2015-04-27 | 2015-07-08 | 上海海洋大学 | 一种草金鱼腹鳍细胞系的构建方法 |
CN109295046A (zh) * | 2018-05-15 | 2019-02-01 | 中山大学 | 一种抗赤点石斑鱼神经坏死病毒青鳉单倍体胚胎干细胞的制备方法及应用 |
CN110684724A (zh) * | 2019-09-18 | 2020-01-14 | 中山大学 | 一种3d培养中华乌塘鳢精巢细胞产生精子的方法和应用 |
Non-Patent Citations (2)
Title |
---|
沈关心、周汝霖主编.《现代免疫学实验技术》.湖北科学技术出版社,1998,第368页右栏第5段. * |
齐文闯.南方鲇(Silurus meridionalis Chen)性腺体细胞系的建立、鉴定及应用.《中国优秀硕士论文全文数据库 农业科技辑》.2014,(第10期),第3页第2段,第15-16页第1.5-1.7节,第31页第2段,第32页第2-5段. * |
Also Published As
Publication number | Publication date |
---|---|
CN112852711A (zh) | 2021-05-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113025565B (zh) | 马口鱼精原干细胞系的建立及其诱导分化方法 | |
US11773368B2 (en) | Culture method for differentiating primordial germ cells into functionally mature oocytes | |
AU2005205516B2 (en) | Novel culture systems for ex vivo development | |
Han et al. | Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period | |
KR101829488B1 (ko) | 인간 다분화능 세포 배양을 위한 단순화된 기본 배지 | |
Kautz et al. | Expression of genes involved in the embryo–maternal interaction in the early-pregnant canine uterus | |
US20030070186A1 (en) | Method of cloning animals | |
JP2008017840A (ja) | 胚性幹細胞の成長 | |
US6107543A (en) | Culture of totipotent embryonic inner cells mass cells and production of bovine animals | |
CN108384749B (zh) | 鸡性腺原始生殖细胞快速分离与建系的方法 | |
CA2417345A1 (en) | Method of cloning porcine animals | |
EP1487968A1 (en) | Methods of inducing differentiation of stem cells into a specific cell lineage | |
KR100569168B1 (ko) | 조류 정원줄기세포의 배양방법 및 이에 의해 수득한 조류정원줄기세포 | |
CN112852711B (zh) | 刀鲚性腺体细胞系的建立及其应用 | |
KR20050084889A (ko) | 다능성 줄기 세포 배양용 조성물 및 이의 용도 | |
AU699869B2 (en) | EG Cells of Ungulates | |
Parsons et al. | Defining conditions for sustaining epiblast pluripotence enables direct induction of clinically-suitable human myocardial grafts from biologics-free human embryonic stem cells | |
KR20060105041A (ko) | 배아 줄기 세포주 및 이의 제조방법 | |
CN117007813A (zh) | Foxo3基因及其靶向抑制剂在鸡原始生殖细胞增殖及迁移定植中的应用 | |
CN114276984B (zh) | 雌性生殖干细胞转分化至功能精子的方法及应用 | |
KR20020092900A (ko) | 조류 배반엽 세포주 | |
Honda et al. | Large-scale production of growing oocytes in vitro from neonatal mouse ovaries | |
KR102004958B1 (ko) | 고대 멸종 생물 사체 또는 화석으로부터 살아있는 세포를 분리 및 배양하는 방법 | |
Suemori et al. | Growth and differentiation of cynomolgus monkey ES cells | |
KR100662706B1 (ko) | 양수세포를 배양한 후 수거한 배양액을 이용한 인간배아 줄기세포의 배양방법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |