CN117467599B - 一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法 - Google Patents
一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法 Download PDFInfo
- Publication number
- CN117467599B CN117467599B CN202311814122.XA CN202311814122A CN117467599B CN 117467599 B CN117467599 B CN 117467599B CN 202311814122 A CN202311814122 A CN 202311814122A CN 117467599 B CN117467599 B CN 117467599B
- Authority
- CN
- China
- Prior art keywords
- chicken
- cells
- reprogramming
- mass
- chickens
- 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
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 56
- 230000008672 reprogramming Effects 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000002710 gonadal effect Effects 0.000 title claims abstract description 22
- 239000000126 substance Substances 0.000 title claims abstract description 19
- 210000001082 somatic cell Anatomy 0.000 title claims abstract description 17
- 239000000411 inducer Substances 0.000 title claims abstract description 15
- 210000001778 pluripotent stem cell Anatomy 0.000 title claims abstract description 14
- 235000013330 chicken meat Nutrition 0.000 title abstract description 46
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 claims abstract description 20
- NFLGAXVYCFJBMK-RKDXNWHRSA-N (+)-isomenthone Natural products CC(C)[C@H]1CC[C@@H](C)CC1=O NFLGAXVYCFJBMK-RKDXNWHRSA-N 0.000 claims abstract description 10
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 claims abstract description 10
- NFLGAXVYCFJBMK-UHFFFAOYSA-N Menthone Chemical compound CC(C)C1CCC(C)CC1=O NFLGAXVYCFJBMK-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229940000635 beta-alanine Drugs 0.000 claims abstract description 10
- 229930007503 menthone Natural products 0.000 claims abstract description 10
- VZQDDSYKVYARDW-UHFFFAOYSA-N n-(9,10-dioxophenanthren-2-yl)-2,2-dimethylpropanamide Chemical compound C1=CC=C2C3=CC=C(NC(=O)C(C)(C)C)C=C3C(=O)C(=O)C2=C1 VZQDDSYKVYARDW-UHFFFAOYSA-N 0.000 claims abstract description 10
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 claims abstract description 7
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 claims abstract description 7
- 229940016667 resveratrol Drugs 0.000 claims abstract description 7
- 235000021283 resveratrol Nutrition 0.000 claims abstract description 7
- 229940118537 p53 inhibitor Drugs 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims description 45
- 210000004602 germ cell Anatomy 0.000 claims description 12
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 12
- 150000001413 amino acids Chemical class 0.000 claims description 11
- 239000003112 inhibitor Substances 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 7
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 claims description 6
- 108010088751 Albumins Proteins 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 6
- 229920000669 heparin Polymers 0.000 claims description 6
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims description 6
- 229960001008 heparin sodium Drugs 0.000 claims description 6
- 229940054269 sodium pyruvate Drugs 0.000 claims description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- IYOZTVGMEWJPKR-VOMCLLRMSA-N 4-[(1R)-1-aminoethyl]-N-pyridin-4-yl-1-cyclohexanecarboxamide Chemical compound C1CC([C@H](N)C)CCC1C(=O)NC1=CC=NC=C1 IYOZTVGMEWJPKR-VOMCLLRMSA-N 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
- 230000001502 supplementing effect Effects 0.000 claims description 2
- 102100027211 Albumin Human genes 0.000 claims 2
- 239000007640 basal medium Substances 0.000 claims 2
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 10
- 238000011160 research Methods 0.000 abstract description 6
- 238000010362 genome editing Methods 0.000 abstract description 5
- IYOZTVGMEWJPKR-IJLUTSLNSA-N Y-27632 Chemical compound C1C[C@@H]([C@H](N)C)CC[C@@H]1C(=O)NC1=CC=NC=C1 IYOZTVGMEWJPKR-IJLUTSLNSA-N 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 3
- 210000002894 multi-fate stem cell Anatomy 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- -1 small molecule compounds Chemical class 0.000 description 9
- 239000000725 suspension Substances 0.000 description 8
- 241000282414 Homo sapiens Species 0.000 description 7
- 102000009027 Albumins Human genes 0.000 description 4
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 description 4
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 108010023082 activin A Proteins 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 230000000392 somatic effect Effects 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 238000010802 RNA extraction kit Methods 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- UJHBVMHOBZBWMX-UHFFFAOYSA-N ferrostatin-1 Chemical compound NC1=CC(C(=O)OCC)=CC=C1NC1CCCCC1 UJHBVMHOBZBWMX-UHFFFAOYSA-N 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 230000012447 hatching Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 description 1
- 101000642514 Homo sapiens Transcription factor SOX-4 Proteins 0.000 description 1
- 102100020677 Krueppel-like factor 4 Human genes 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102100036693 Transcription factor SOX-4 Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- KLGQSVMIPOVQAX-UHFFFAOYSA-N XAV939 Chemical compound N=1C=2CCSCC=2C(O)=NC=1C1=CC=C(C(F)(F)F)C=C1 KLGQSVMIPOVQAX-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 229940050528 albumin Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000009385 viral infection Effects 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
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- 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
- C12N2500/30—Organic components
- C12N2500/35—Polyols, e.g. glycerin, inositol
-
- 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
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- 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
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
-
- 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
- C12N2501/70—Enzymes
- C12N2501/73—Hydrolases (EC 3.)
-
- 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
- C12N2501/90—Polysaccharides
- C12N2501/91—Heparin
-
- 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
- C12N2501/998—Proteins not provided for elsewhere
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/24—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the genital tract, from non-germinal gonad cells
Abstract
本发明公开了一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法。属于多能干细胞制备技术领域。该化学诱导剂包括Y‑27632、Resveratrol、SF1670、CHIR‑99021、Menthone、β‑Alanine和p53抑制剂。本发明利用小分子化合物重编程鸡性腺体细胞,以期在短时间内获得大量鸡iPSC,为其进行离体保种、基因编辑研究等奠定良好基础。
Description
技术领域
本发明涉及多能干细胞制备技术领域,更具体的说是涉及一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法。
背景技术
诱导多能干细胞(Induced pluripotent stem cells,iPSC)是日本科学家TAKAHASHI在2006年将表达四种多能性转录因子OCT4、SOX4、KLF4和c-MYC (统称为OSKM)的逆转录病毒载体导入小鼠成纤维细胞,使成纤维细胞转化为具有多向分化潜能的多能性细胞。iPSC具有无限增殖和自我更新的能力,在适当条件下可被诱导分化为多种细胞组织,开启了生命科学的新时代。该技术的诞生,不仅给人类带来了新的、大量的干细胞来源,也为珍稀动物保种、转基因动物的生产等研究开辟了新思路。2013年,第三代基因编辑技术CRISPR问世后,开启了基因编辑技术井喷式发展的时代。利用iPSC进行相对高效安全的基因组编辑,在基础研究和临床应用上有巨大的优势。尽管小鼠和人等多个物种中iPSC的重编程非常成熟,但家禽体细胞重编程研究进程仍较为缓慢。
目前获得鸡iPSC的方法主要利用OSKM四因子及其它转录因子的不同组合在慢病毒过表达载体进行重编程,但效率较低,细胞形态从第6天开始发生变化,到15天时开始形成与多能干细胞形态类似的iPSC克隆,限制了其应用。
近年来,小分子化合物重编程的方法已经成为操控细胞命运的重要手段,利用小分子化合物可以实现细胞重编程、分化和转分化。由于OSKM因子会带来病毒感染及其它外源性细胞成分,非常不安全,需要寻求更安全、高效的重编程方法。近年来研究表明小分子化合物重编程可以避免OSKM因子带来的问题,同时,小分子化合物靶点相对清晰、作用相对可控,对体细胞重编程机制的研究起了很大的推动作用。目前已经在小鼠、人和猪等物种上实现了小分子化合物重编程体细胞为多能干细胞或胚胎干细胞。虽然小分子化合物在体细胞重编程中具有独特优势,但从上千甚至上万的小分子化合物中筛选出合适的重编程组合,绝非易事。目前还没有使用小分子化合物诱导鸡体细胞重编程为iPSC的报道。不同起始细胞和目标细胞决定了转分化过程中所需要的小分子化合物不同,因此在小分子化合物的选择上要经过详尽的调研和筛选。
综上,如何提供一种化学重编程鸡性腺体细胞为多能干细胞的方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法。
发明人前期挖掘到影响鸡原始生殖细胞自我更新的核心细胞群和新的信号通路5个,通过添加小分子化合物、抑制剂等调控这5个信号通路,显著提高了鸡原始生殖细胞体外建系效率。因此,发明人结合鸡原始生殖细胞这5个信号通路,开展鸡iPSC的化学诱导体系筛选,从众多的小分子化合物中筛选出合适的诱导组合。
本发明利用小分子化合物重编程鸡性腺体细胞,以期在短时间内获得大量鸡iPSC,为其进行离体保种、基因编辑研究等奠定良好基础。
为了实现上述目的,本发明采用如下技术方案:
一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂,包括如下组分:Y-27632、Resveratrol、SF1670、CHIR-99021、Menthone、β-Alanine和p53抑制剂;
且上述物质的质量浓度比为(5~30):(30~100):(0.5~5):(15~36):(20~100):(20~100):(10~100)。
所取得的有益效果:可以将鸡性腺体细胞重编程为表达多能性蛋白的iPSC。
进一步的,一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂,包括如下组分:Y-27632、Resveratrol、SF1670、CHIR-99021、Menthone、β-Alanine和p53抑制剂;
且上述物质的质量浓度比为20:30:1:18:40:40:20。
一种重编程鸡性腺体细胞为鸡多能干细胞的方法,使用上述的化学诱导剂。
进一步的,在鸡原始生殖细胞培养液中加入所述化学诱导剂,诱导培养鸡性腺体细胞。
进一步的,培养过程中所述化学诱导剂各成分的终质量浓度如下:
10 µM Y-27632、15 µM Resveratrol、0.5 µM SF1670、9 µM CHIR-99021、20 µMMenthone、20 µM β-Alanine和10 µM p53抑制剂。
进一步的,培养过程中所述鸡原始生殖细胞培养液包括如下组分:在基础培养基DMEM中添加如下质量浓度的组分:0.1~2%双抗、2~4% B27补充液、2~8 mM GlutaMAX、0.5~3%非必需氨基酸、0.05~0.2 mM β-巯基乙醇、0.5~2 mM丙酮酸钠、0.5~2%氨基酸溶液、0.2~1%鸡血清、1~30%白蛋白、10~100mg/mL肝素钠、20~40 µg/mL human Activin A和2~40 µg/mLrhFGF。
进一步的,培养过程中所述鸡原始生殖细胞培养液包括如下组分:在基础培养基DMEM中添加如下质量浓度的组分:1%双抗、2% B27补充液、4 mM GlutaMAX、1%非必需氨基酸、0.1 mM β-巯基乙醇、1 mM丙酮酸钠、1%氨基酸溶液、0.4%鸡血清、20%白蛋白、50mg/mL肝素钠、25 µg/mL human Activin A和25 µg/mL rhFGF。
进一步的,重编程培养时间为2~3天。
经由上述的技术方案可知,与现有技术相比,本发明取得的有益效果为:本发明的小分子化合物在2~3天可以将鸡性腺体细胞重编程为表达多能性蛋白的iPSC。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明实施例1中F组细胞培养1天、3天和4天的细胞形态;
图2附图为本发明实施例1中鸡iPSC的RT-PCR鉴定结果;
图3附图为本发明实施例1中F组细胞培养3天分别表达SSEA1、OCT4蛋白的情况。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明所需药剂为常规实验药剂,采购自市售渠道;未提及的实验方法为常规实验方法,在此不再一一赘述。
实施例1
1.材料与方法
1.1材料和试剂
种蛋来源于广东省农业科学院动物科学研究所原种鸡场,在38.5℃、相对湿度60%孵化至所需日龄。
小分子化合物CHIR-99021、Y-27632、Menthone、β-Alanine、Ferrostatin-1、XAV-939和SF1670购自selleck公司。
Human Activin A购自PeproTech公司。
p53抑制剂、Resveratrol、β-巯基乙醇购自sigma公司。
DMEM、胎牛血清、PBS溶液、双抗、非必需氨基酸、GlutaMAXTM、丙酮酸钠、B27补充液、氨基酸溶液、白蛋白、肝素钠和胰酶购自Gibco公司。
rhFGF购自R&D systems公司。
RNA抽提试剂盒、cDNA反转试剂盒和PCR扩增试剂盒购自TAKALA公司。
抗体OCT4和SSEA1购自Abcam公司。
1.2鸡性腺体细胞的分离
准备孵育至4~18天的种蛋若干,灭菌处理过的剪刀、普通镊子等,提前开紫外照射细胞房30min。用酒精棉球擦拭种蛋尖端,普通镊子敲开一个口子,用镊子小心将胚胎取出,放置于装有PBS溶液的培养皿中,除去胚胎表面的蛋黄颗粒。将清洗好的胚胎转移至新的PBS溶液中,用镊子分离性腺转移至装有300µl PBS溶液的1.5ml离心管中,加入100µl的0.25% Trypsin/EDTA溶液放置于37℃培养箱中消化25min。加入400µl完全培养基中和,之后1000g常温离心5min,弃掉上清;用300µl的完全培养基重悬细胞沉淀,转移至48孔板进行培养,培养箱条件37℃、5% CO2。细胞每天换液1次,2~4天传代。
1.3试验分组
将分离的鸡性腺体细胞随机分组:
A组为对照组,全程用鸡原始生殖细胞培养液:
鸡原始生殖细胞培养液组成:在基础培养基DMEM中添加如下质量浓度的组分:
1%双抗、2% B27补充液、4 mM GlutaMAX、1%非必需氨基酸、0.1 mM β-巯基乙醇、1mM丙酮酸钠、1%氨基酸溶液、0.4%鸡血清、20%白蛋白、50mg/mL肝素钠、25 µg/mL humanActivin A和25 µg/mL rhFGF。
B组全程在A组基础上添加10 µM XAV-939、5 µM Y-27632、3 µM Resveratrol。
C组全程在B组基础上添加0.5 µM SF1670、3 µM CHIR-99021、5 µM Menthone。
D组全程在C组基础上添加10 µM β-Alanine、2 µM Ferrostatin-1、1 µM p53抑制剂。
E组全程在D组基础上调整小分子化合物的添加量,添加量为D组的2倍:20 µMXAV-939、10 µM Y-27632、6 µM Resveratrol、1 µM SF1670、6 µM CHIR-99021、10 µMMenthone、20 µM β-Alanine、4 µM Ferrostatin-1、2 µM p53抑制剂。
F组调整部分小分子化合物及其添加量:10 µM Y-27632、15 µM Resveratrol、0.5µM SF1670、9 µM CHIR-99021、20 µM Menthone、20 µM β-Alanine和10 µM p53抑制剂。
1.4 RT-PCR鉴定
收集1×105个细胞,用RNA抽提试剂盒提取细胞总RNA。采用反转录试剂盒cDNA合成第一链cDNA,以合成的cDNA为模板进行普通PCR实验。
引物为:
Pou5f3-F:5'-GGCTCAATGAGGCAGAGAAC-'3,SEQ ID NO:1;
Pou5f3-R:5'-GGACTGGGCTTCACACATTT-'3,SEQ ID NO:2;
NANOG-F:5'-AGCAGACCTCTCCTTGACCA-'3,SEQ ID NO:3;
NANOG-R:5'-TTCCTTGTCCCACTCTCACC-'3,SEQ ID NO:4。
PCR程序为:
94℃ 5 min,之后30个循环(94℃ 30 s, 57℃ 45 s, 72℃ 30 s),最后72℃延伸5 min。
PCR产物用1%琼脂糖凝胶进行电泳。
1.5鸡iPSC的免疫荧光鉴定
细胞离心去掉上清后,转移到24孔板中用4%多聚甲醛固定10 min。用PBS洗涤3次、每次5 min(以下PBS洗涤均如此),再用0.5% Triton X-100通透5 min。PBS洗涤,然后用含1%(v/v)BSA的PBS封闭液固定细胞45 min。PBS洗涤,在4℃的湿盒内与一抗OCT4(1:50)、SSEA1(1:50)孵育过夜。PBS洗涤,孵育二抗(1:400)。PBS洗涤后,用DAPI复染细胞核,晾干后滴加DAPI染液,室温避光孵育10min。最后通过共聚焦显微镜进行分析。
2.结果与分析
A~D组细胞培养7天,细胞有增殖,但形态基本一致,大部分处于贴壁状态。E组细胞培养4天形态开始发生变化,小部分细胞由贴壁变为悬浮,并变圆而亮,7天后大部分细胞都是悬浮状态。F组细胞培养2~3天形态开始发生变化,部分细胞由贴壁变为悬浮,并变圆而亮;而且细胞增殖较快,第三天24孔板一个孔的细胞量由最初的1.3×104到3天后增殖为2.1×105,其中悬浮细胞量即iPSC为6.9×104,此时1个孔的细胞传到3个孔;4天后几乎所有细胞都是悬浮状态,每个孔细胞量为7.8×105,此时1个孔的细胞传到3个孔(图1)。
选取D组培养4天的悬浮细胞和F组培养0~5天的悬浮细胞进行RT-PCR鉴定,试验结果表明D组细胞培养4天不表达iPSC标记基因Pou5f3和NANOG,而F组在2~3天即开始表达(图2)。
免疫荧光鉴定结果表明(图3),F组培养3天的悬浮细胞均表达iPSC标志蛋白SSEA1和OCT4。上述结果表明F组的鸡性腺体细胞在培养2~3天后重编程为鸡iPSC。
表1 鸡性腺体细胞在不同培养基中连续培养7天诱导的iPSC数量*
* 24孔板一个孔的悬浮细胞量,不包括贴壁细胞(未诱导成功的细胞)。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (7)
1.一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂,其特征在于,包括如下组分:Y-27632、Resveratrol、SF1670、CHIR-99021、Menthone、β-Alanine和p53抑制剂;
且上述物质的摩尔浓度比为20:30:1:18:40:40:20。
2.一种重编程鸡性腺体细胞为鸡多能干细胞的方法,其特征在于,使用权利要求1所述的化学诱导剂。
3.如权利要求2所述的方法,其特征在于,在鸡原始生殖细胞培养液中加入所述化学诱导剂,诱导培养鸡性腺体细胞。
4.如权利要求3所述的方法,其特征在于,培养过程中所述化学诱导剂各成分的终摩尔浓度如下:
10μMY-27632、15μM Resveratrol、0.5μM SF1670、9μM CHIR-99021、20μM Menthone、20μMβ-Alanine和10μM p53抑制剂。
5.如权利要求3所述的方法,其特征在于,培养过程中所述鸡原始生殖细胞培养液包括如下组分:在基础培养基DMEM中添加如下组分:质量浓度为0.1~2%的双抗、质量浓度为2~4%的B27补充液、2~8mM GlutaMAX、质量浓度为0.5~3%的非必需氨基酸、0.05~0.2mMβ-巯基乙醇、0.5~2mM丙酮酸钠、质量浓度为0.5~2%的氨基酸溶液、质量浓度为0.2~1%的鸡血清、质量浓度为1~30%的白蛋白、10~100mg/mL肝素钠、20~40μg/mLhumanActivinA和2~40μg/mLrhFGF。
6.如权利要求3所述的方法,其特征在于,培养过程中所述鸡原始生殖细胞培养液包括如下组分:在基础培养基DMEM中添加如下组分:质量浓度为1%的双抗、质量浓度为2%的B27补充液、4mM GlutaMAX、质量浓度为1%的非必需氨基酸、0.1mMβ-巯基乙醇、1mM丙酮酸钠、质量浓度为1%的氨基酸溶液、质量浓度为0.4%的鸡血清、质量浓度为20%的白蛋白、50mg/mL肝素钠、25μg/mL humanActivinA和25μg/mL rhFGF。
7.如权利要求3所述的方法,其特征在于,重编程培养时间为2~3天。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311814122.XA CN117467599B (zh) | 2023-12-27 | 2023-12-27 | 一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311814122.XA CN117467599B (zh) | 2023-12-27 | 2023-12-27 | 一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117467599A CN117467599A (zh) | 2024-01-30 |
CN117467599B true CN117467599B (zh) | 2024-03-29 |
Family
ID=89633314
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311814122.XA Active CN117467599B (zh) | 2023-12-27 | 2023-12-27 | 一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117467599B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102093981A (zh) * | 2010-12-17 | 2011-06-15 | 深圳市北科生物科技有限公司 | 一种高效诱导人类体细胞重编程为多潜能干细胞的方法 |
CN106987555A (zh) * | 2016-01-20 | 2017-07-28 | 中国科学院上海生命科学研究院 | 高效诱导人多能干细胞向心肌细胞分化的小分子化合物组合物 |
CN111534481A (zh) * | 2020-05-18 | 2020-08-14 | 扬州大学 | 一种提高鸡胚成纤维细胞体外诱导重编程为iPS细胞效率的方法 |
CN115747140A (zh) * | 2022-11-25 | 2023-03-07 | 扬州大学 | 一种诱导鸡胚胎干细胞分化生成原始生殖细胞的方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9752124B2 (en) * | 2009-02-03 | 2017-09-05 | Koninklijke Nederlandse Akademie Van Wetenschappen | Culture medium for epithelial stem cells and organoids comprising the stem cells |
CN105688968A (zh) * | 2016-01-20 | 2016-06-22 | 陕西科技大学 | 一种钒酸铋/氮化硼复合光催化材料及其制备方法 |
-
2023
- 2023-12-27 CN CN202311814122.XA patent/CN117467599B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102093981A (zh) * | 2010-12-17 | 2011-06-15 | 深圳市北科生物科技有限公司 | 一种高效诱导人类体细胞重编程为多潜能干细胞的方法 |
CN106987555A (zh) * | 2016-01-20 | 2017-07-28 | 中国科学院上海生命科学研究院 | 高效诱导人多能干细胞向心肌细胞分化的小分子化合物组合物 |
CN111534481A (zh) * | 2020-05-18 | 2020-08-14 | 扬州大学 | 一种提高鸡胚成纤维细胞体外诱导重编程为iPS细胞效率的方法 |
CN115747140A (zh) * | 2022-11-25 | 2023-03-07 | 扬州大学 | 一种诱导鸡胚胎干细胞分化生成原始生殖细胞的方法 |
Non-Patent Citations (2)
Title |
---|
Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis;Xiao-Xu Qiu等;Journal of the American Heart Association;20171002;1-22 * |
诱导多能干细胞与细胞重编程技术的研究与未来;赵 星 等;中国组织工程研究;20131203;第17卷(第49期);8608-8614 * |
Also Published As
Publication number | Publication date |
---|---|
CN117467599A (zh) | 2024-01-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5902092B2 (ja) | 心筋細胞の生成 | |
Xie et al. | Sertoli cell-mediated differentiation of male germ cell-like cells from human umbilical cord Wharton’s jelly-derived mesenchymal stem cells in an in vitro co-culture system | |
JP2006519015A (ja) | 幹細胞を単離または生成するためのマーカーとしてのIslet1の使用 | |
JP2004057198A (ja) | 多能性体細胞 | |
CN110713973B (zh) | 一种诱导多能干细胞分化为间充质干细胞的培养基组合及方法 | |
WO2001053465A9 (en) | Human embryoid body-derived cells | |
US11366115B2 (en) | Isolation of human lung progenitors derived from pluripotent stem cells | |
EP3699275A1 (en) | Organ organoid and method for producing same | |
US20040234972A1 (en) | Method for identifying and purifying smooth muscle progenitor cells | |
KR101562366B1 (ko) | 메타크릴레이트화된 젤라틴을 포함하는, 심근세포분화 유도용 세포 지지체 | |
JP2005520516A (ja) | 幹細胞の特定細胞系統への分化を誘発する方法 | |
CN103555661A (zh) | 一种无血清、无饲养层的多能干细胞培养方法 | |
Dai et al. | Maintenance and neuronal differentiation of chicken induced pluripotent stem-like cells | |
JP4576545B2 (ja) | 羊膜由来因子による胚性幹細胞の培養方法 | |
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 | |
JP2018183137A (ja) | 多能性幹細胞から樹状分岐した集合管を伴う腎臓構造を作製する方法 | |
CN117467599B (zh) | 一种重编程鸡性腺体细胞为鸡多能干细胞的化学诱导剂及重编程方法 | |
CN117487743B (zh) | 一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂及诱导方法 | |
WO2021201175A1 (ja) | 下垂体ホルモン産生細胞及びその前駆細胞の分離法 | |
CN109852587B (zh) | 一种人科凯恩氏综合征特异性成体干细胞的制备方法 | |
JP2008206510A (ja) | 腸管幹/前駆細胞の取得方法 | |
Mujtaba et al. | Isolation of lineage-restricted neural precursors from cultured ES cells | |
JP6516280B2 (ja) | iPS細胞の樹立方法および幹細胞の長期維持方法 | |
US20240139256A1 (en) | Methods for the production of cardiac fibroblasts | |
CN111718890B (zh) | 一种将成纤维细胞转分化为腺上皮细胞的方法及其培养体系和应用 |
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 |