CN111944745B - 一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法 - Google Patents
一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法 Download PDFInfo
- Publication number
- CN111944745B CN111944745B CN202010858331.4A CN202010858331A CN111944745B CN 111944745 B CN111944745 B CN 111944745B CN 202010858331 A CN202010858331 A CN 202010858331A CN 111944745 B CN111944745 B CN 111944745B
- Authority
- CN
- China
- Prior art keywords
- bovine
- oocyte
- serum
- vitro maturation
- culture solution
- 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
- 210000000287 oocyte Anatomy 0.000 title claims abstract description 91
- 241000283690 Bos taurus Species 0.000 title claims abstract description 71
- 238000000338 in vitro Methods 0.000 title claims abstract description 68
- 230000035800 maturation Effects 0.000 title claims abstract description 63
- 210000002966 serum Anatomy 0.000 title claims abstract description 22
- 238000012136 culture method Methods 0.000 title claims abstract description 10
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 12
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims abstract description 10
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims abstract description 8
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 8
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 claims abstract description 7
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 claims abstract description 7
- 229960005309 estradiol Drugs 0.000 claims abstract description 7
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000004380 Cholic acid Substances 0.000 claims abstract description 6
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 6
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims abstract description 6
- 229960002471 cholic acid Drugs 0.000 claims abstract description 6
- 235000019416 cholic acid Nutrition 0.000 claims abstract description 6
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960000304 folic acid Drugs 0.000 claims abstract description 6
- 235000019152 folic acid Nutrition 0.000 claims abstract description 6
- 239000011724 folic acid Substances 0.000 claims abstract description 6
- 229920006395 saturated elastomer Polymers 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 7
- 239000000654 additive Substances 0.000 claims description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims 2
- 235000013878 L-cysteine Nutrition 0.000 claims 1
- 239000004201 L-cysteine Substances 0.000 claims 1
- 210000002257 embryonic structure Anatomy 0.000 abstract description 16
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 abstract description 5
- 238000004113 cell culture Methods 0.000 abstract description 5
- 230000013020 embryo development Effects 0.000 abstract description 3
- 238000010374 somatic cell nuclear transfer Methods 0.000 abstract description 3
- NPOAOTPXWNWTSH-UHFFFAOYSA-N 3-hydroxy-3-methylglutaric acid Chemical compound OC(=O)CC(O)(C)CC(O)=O NPOAOTPXWNWTSH-UHFFFAOYSA-N 0.000 abstract 1
- ZWQVYZXPYSYPJD-RYUDHWBXSA-N Glu-Gly-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ZWQVYZXPYSYPJD-RYUDHWBXSA-N 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 51
- 239000002609 medium Substances 0.000 description 14
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 13
- 210000001161 mammalian embryo Anatomy 0.000 description 13
- 239000002953 phosphate buffered saline Substances 0.000 description 13
- 239000007788 liquid Substances 0.000 description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 210000001672 ovary Anatomy 0.000 description 6
- 230000000392 somatic effect Effects 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 239000007640 basal medium Substances 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 230000001605 fetal effect Effects 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108010003272 Hyaluronate lyase Proteins 0.000 description 4
- 102000001974 Hyaluronidases Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000007664 blowing Methods 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- 229960002773 hyaluronidase Drugs 0.000 description 4
- 239000011777 magnesium Substances 0.000 description 4
- 210000004508 polar body Anatomy 0.000 description 4
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 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 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 239000005662 Paraffin oil Substances 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000000546 chi-square test Methods 0.000 description 3
- 210000001771 cumulus cell Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000004720 fertilization Effects 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 3
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 210000000582 semen Anatomy 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 description 2
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 229930186147 Cephalosporin Natural products 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229940124587 cephalosporin Drugs 0.000 description 2
- 150000001780 cephalosporins Chemical class 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- VVIUBCNYACGLLV-UHFFFAOYSA-N hypotaurine Chemical compound [NH3+]CCS([O-])=O VVIUBCNYACGLLV-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229940054269 sodium pyruvate Drugs 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- LJRDOKAZOAKLDU-UDXJMMFXSA-N (2s,3s,4r,5r,6r)-5-amino-2-(aminomethyl)-6-[(2r,3s,4r,5s)-5-[(1r,2r,3s,5r,6s)-3,5-diamino-2-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-hydroxycyclohexyl]oxy-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]oxyoxane-3,4-diol;sulfuric ac Chemical compound OS(O)(=O)=O.N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO LJRDOKAZOAKLDU-UDXJMMFXSA-N 0.000 description 1
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- 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 1
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-dimethylaminopyridine Substances CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 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
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- BVIAOQMSVZHOJM-UHFFFAOYSA-N N(6),N(6)-dimethyladenine Chemical compound CN(C)C1=NC=NC2=C1N=CN2 BVIAOQMSVZHOJM-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- GBOGMAARMMDZGR-UHFFFAOYSA-N UNPD149280 Natural products N1C(=O)C23OC(=O)C=CC(O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 GBOGMAARMMDZGR-UHFFFAOYSA-N 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 1
- GBOGMAARMMDZGR-JREHFAHYSA-N cytochalasin B Natural products C[C@H]1CCC[C@@H](O)C=CC(=O)O[C@@]23[C@H](C=CC1)[C@H](O)C(=C)[C@@H](C)[C@@H]2[C@H](Cc4ccccc4)NC3=O GBOGMAARMMDZGR-JREHFAHYSA-N 0.000 description 1
- GBOGMAARMMDZGR-TYHYBEHESA-N cytochalasin B Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@@H]3/C=C/C[C@H](C)CCC[C@@H](O)/C=C/C(=O)O[C@@]23C(=O)N1)=C)C)C1=CC=CC=C1 GBOGMAARMMDZGR-TYHYBEHESA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- HQQQHMUTGRTSKR-SNHSCUNWSA-L disodium;[(2r)-2-hydroxy-3-[(z)-octadec-9-enoyl]oxypropyl] phosphate Chemical compound [Na+].[Na+].CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP([O-])([O-])=O HQQQHMUTGRTSKR-SNHSCUNWSA-L 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 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 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 229960001639 penicillamine Drugs 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001540 sodium lactate Substances 0.000 description 1
- 229940005581 sodium lactate Drugs 0.000 description 1
- 235000011088 sodium lactate Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 210000004340 zona pellucida Anatomy 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
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
-
- 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
- C12N2500/38—Vitamins
-
- 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
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/21—Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
-
- 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
- C12N2501/30—Hormones
-
- 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
Abstract
本发明公开了一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法,属于细胞培养技术领域。本发明公开的一种无血清的牛卵母细胞体外成熟培养液,包括无Hepes的M199基础培养液、无脂肪酸BSA、HMG、17β‑雌二醇、EGF、L‑cysteine、bFGF、100xGlutamax、叶酸、胆酸、CXCL12。本发明公开的牛卵母细胞体外成熟培养液在不使用血清的情况下培养牛卵母细胞,提高了牛卵母细胞的成熟率,并有效提高体外受精胚胎和体细胞核移植胚胎的早期胚胎发育率。
Description
技术领域
本发明涉及细胞培养技术领域,更具体的说是涉及一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法。
背景技术
卵母细胞体外成熟培养是体外受精、体细胞核移植、动物胚胎体外生产和转基因克隆等胚胎工程技术的基础和关键环节。卵母细胞体外成熟不仅受到卵巢周期、动物品种、卵泡大小、激素、血清等多种因素影响,而且与卵母细胞体外成熟的程序和方法及培养液成分密切相关,特别是血清会导致体外生产胚胎的巨大胎儿综合征。虽然近几年卵母细胞体外成熟技术取得较大的进步,但是关于无血清培养体系的研究还并没有相关报道。
因此,提供一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养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,本发明提供了一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法,提高牛卵母细胞体外培养的成熟率,避免使用血清减少巨胎症。
为了实现上述目的,本发明采用如下技术方案:
一种无血清的牛卵母细胞体外成熟培养液,包括无Hepes的M199基础培养液,所述培养液还包括以下添加剂:无脂肪酸BSA 2-8mg/ml、HMG 0.05-0.1IU/ml、17β-雌二醇0.1-2μg/ml、EGF 1-100ng/ml、L-cysteine 0.1-1mM、bFGF 1-100ng/ml、Glutamax(100x)0.5-2μL/ml、叶酸1-100μM、胆酸1-10μg/ml、CXCL121-100ng/ml。
进一步,一种无血清的牛卵母细胞体外成熟培养液,包括无Hepes的M199基础培养液,所述培养液还包括以下添加剂:无脂肪酸BSA 6mg/ml、HMG0.075IU/ml、17β-雌二醇1μg/ml、EGF 60ng/ml、L-cysteine 0.57mM、bFGF 40ng/ml、Glutamax(100x)1.6μL/ml、叶酸50μM、胆酸2μg/ml、CXCL12 50ng/ml。
进一步,一种牛卵母细胞体外成熟培养方法,利用上述无血清的牛卵母细胞体外成熟培养液培养牛卵母细胞,具体步骤如下:将牛卵母细胞放入预热的无血清的牛卵母细胞体外成熟培养液中,置于38.5℃、5%CO2饱和湿度的培养箱中成熟培养18-22小时,获得成熟的牛卵母细胞。
进一步,所述牛卵母细胞为GV期卵母细胞。
进一步,所述无血清的牛卵母细胞体外成熟培养液预热1-3小时。
进一步,所述无血清的牛卵母细胞体外成熟培养液置于38.5℃、5%CO2饱和湿度的培养箱中预热。
进一步,每500μl无血清的牛卵母细胞体外成熟培养液中放入40-50枚牛卵母细胞。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法,本发明公开的牛卵母细胞体外成熟培养液在不使用血清的情况下培养牛卵母细胞,提高了牛卵母细胞的成熟率,并有效提高体外受精胚胎和体细胞核移植胚胎的早期胚胎发育率。
具体实施方式
下面将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。
无Hepes的M199基础培养液购自Thermo公司,特级胎牛血清(FBS)购自Gibco公司,FGF、EGF、CXCL12均购自R&D公司,其他未标明的产品均购自sigma公司。
采卵液(PBS液)
26.168mg CaCl2·2H2O、37.218mg MgSO4·7H2O、0.0203g KCl、0.0200g KH2PO4、0.0036g丙酮酸钠、0.805gNaCl、0.1153gNa2HPO4、D-Glucose 0.1g、肝素钠6mg、胎牛血清3mL,去离子水定容到100mL,1mMNaOH调pH到7.0-7.2。
SOF溶液制备:将称量的0.6294g的NaCl、0.0534g的KCl、0.162g的KH2PO4、0.0172g的CaCl2·2H2O、0.00996g的MgCl2·6H2O、0.2106g的NaHCO3、0.0033g的丙酮酸钠,及28.24L的乳酸钠加入98mL水中,1mMNaOH调pH到7.2~7.4,去离子水定容到100mL。
SOFaa溶液:以SOF溶液作为基础培养液,添加体积分数1%的必需氨基酸、体积分数1%的非必需氨基酸、6mg/mL无脂肪酸牛血清白蛋白、12g/mL庆大霉素和12μg/mL头孢霉素。
mSOFaa溶液:以SOFaa溶液作为基础培养液,预热时添加体积分数为1%的ITS-G、55μMβ-巯基乙醇、2.5mM亚牛磺酸、0.4μg/mL 1-油酰基-SN-甘油-3-磷酸钠盐、0.1mM红景天苷、10ng/mL的孕酮、100μM维生素E、1mM L-肉碱、40ng/mL FGF、10ng/mL皮质酮、20ng/mLEGF、25ng/mL IGF、20ng/mL LIF、2mg/mL透明质酸。
电融合液
电融合液包括:0.3mol/L甘露醇、0.05mol/L氯化钙、0.1mol/L硫酸镁、0.27mol/L组氨酸及1mg/mL的BSA。
受精液:以不含肌醇的SOF溶液作为基础培养液,添加8mg/mL无脂肪酸牛血清白蛋白,12μg/mL庆大霉素、12μg/mL头孢霉素、63μg/mL L-精氨酸、6μg/mL L-天冬氨酸、50μM L-肉碱、2mM肾上腺素、20mM青霉胺、10mM亚牛磺酸、10μg/mL肝素。
实施例1
无血清的牛卵母细胞体外成熟培养液,包括无Hepes的M199基础培养液,以及6mg/mL的无脂肪酸BSA、0.075IU/ml的HMG、1μg/ml的17β-雌二醇、60ng/ml的EGF、0.57mM的L-cysteine、40ng/ml的bFGF、1.6μL/ml的Glutamax(100x)、50μM的叶酸、2μg/mL的胆酸和50ng/mL的CXCL12。
对比例1
牛卵母细胞有血清体外成熟培养液,包括无Hepes的M199基础培养液以及10%体积分数的FBS、0.075IU/ml的HMG、1μg/ml的17β-雌二醇、10ng/ml的EGF、1%体积分数的ITSG、10ng/ml的bFGF、50ng/mL的CXCL12。
对比例2
牛卵母细胞无血清体外成熟培养液,包括无Hepes的M199基础培养液以及6mg/mL无脂肪酸的BSA、0.075IU/ml的HMG、1μg/ml的17β-雌二醇、10ng/ml的EGF、1%体积分数的ITSG、10ng/ml的bFGF、50ng/mL的CXCL12。
实施例2牛卵母细胞的成熟培养
牛卵巢采自陕西省西安市定点屠宰场,将卵巢置于保温瓶内含100IU/ml青霉素和100μg/ml链霉素的20~25℃的生理盐水中,5h以内运回试验室。卵巢运回后,用灭菌剪刀剪除卵巢表面的结缔组织、脂肪和附着的输卵管,在无菌生理盐水中清洗三次,用装有21G针头的10mL注射器抽取卵巢表面2~8mm卵泡中的卵母细胞,放入6cm皿中,在实体显微镜下收集卵丘-卵母细胞复合体(cumulus-oocyte complexes,COCs)。收集后在PBS中清洗三遍。选择卵母细胞形态正常的COCs用于体外成熟培养。
处理组:将选择的COCs在无血清的牛卵母细胞体外成熟培养液(实施例1的体外成熟培养液,作为处理组)中洗两遍,然后移入装有3mL实施例1的体外成熟培养液的3cm平皿中(提前在培养箱平衡一小时),在38.5℃、5%CO2、饱和湿度条件下培养18~22h。将培养成熟的COCs用PBS液清洗3次,放入含0.1%透明质酸酶的无Ca2+、Mg2+PBS中消化1~2min,并用1000mL移液枪反复吹打,以除去卵母细胞外扩散的卵丘细胞。吹打干净后,在PBS中洗3次,然后在实体视显微镜下,以异物针拨动卵母细胞,挑选有极体的卵母细胞待用。
对照组1:将选择的COCs在牛卵母细胞有血清体外成熟培养液(对比例1的体外成熟培养液,作为对照组1)中洗两遍,然后移入装有3mL的对比例1的体外成熟培养液的3cm平皿中(提前在培养箱平衡一小时),在38.5℃、5%CO2、饱和湿度条件下培养18~22h。将培养成熟的COCs用PBS液清洗3次,放入含0.1%透明质酸酶的无Ca2+、Mg2+PBS中消化1~2min,并用1000mL移液枪反复吹打,以除去卵母细胞外扩散的卵丘细胞。吹打干净后,在PBS中洗3次,然后在实体视显微镜下,以异物针拨动卵母细胞,挑选有极体的卵母细胞待用。
对照组2:将选择的COCs在牛卵母细胞无血清体外成熟培养液(对比例2的体外成熟培养液,作为对照组2)中洗两遍,然后移入装有3mL的对比例2的体外成熟培养液的3cm平皿中(提前在培养箱平衡一小时),在38.5℃、5%CO2、饱和湿度条件下培养18~22h。将培养成熟的COCs用PBS液清洗3次,放入含0.1%透明质酸酶的无Ca2+、Mg2+PBS中消化1~2min,并用1000mL移液枪反复吹打,以除去卵母细胞外扩散的卵丘细胞。吹打干净后,在PBS中洗3次,然后在实体视显微镜下,以异物针拨动卵母细胞,挑选有极体的卵母细胞待用。
观察并记录各组卵母细胞体外成熟情况,结果见表1。
表1卵母细胞体外成熟情况
注:a、b上标不同表示差异显著,显著性使用卡方检验来检测。
由表1可以看出,与对照组1、对照组2的有血清和无血清体外成熟培养液体系相比,采用本发明公开的无血清的牛卵母细胞体外成熟培养液,牛卵母细胞的成熟率明显提高。
实施例3牛体细胞克隆胚胎的制备
(1)牛胎儿成纤维细胞的培养
从液氮中取一管荷斯坦奶牛的2~5代的牛胎儿成纤维细胞(采集自杨凌科元克隆股份有限公司牛场)于39℃解冻,加0.8mL的DMEM/F12细胞培养液,离心,弃上清,加细胞培养液重悬,取3mL细胞悬浮液接种于直径6cm的培养皿中,置于CO2培养箱中38.5℃条件下培养。
待牛胎儿成纤维细胞达到80%汇合时,吸弃培养液,用无Ca2+、Mg2+的PBS冲洗细胞,加入胰蛋白酶与EDTA混合消化液,消化细胞。在倒置显微镜下观察细胞,待大多数细胞回缩、变圆、细胞间隙扩大时,用含10%胎牛血清的DMEM/F12细胞培养液终止消化,用移液器吹打后,离心收集,悬浮,按1:3的比例接种于24孔板,放入CO2培养箱中培养。进行体细胞克隆胚制作的前两天,将培养液更换成含0.5%胎牛血清的DMEM/F12。
(2)卵母细胞的成熟培养
同实施例2。
(3)牛体细胞克隆胚的构建
去核:
去核前处理组和对照组1、对照组2的卵母细胞分别先在含7.5μg/mL细胞松弛素B、4mg/mL不含脂肪酸BSA的含有Hepes的M199中孵育15min。在显微操作仪下,用内径为20μm的去核管吸取第一极体及其周围的部分突出卵胞质。
注核和电融合:
注核时,挑选直径为15~20μm的牛胎儿成纤维细胞分别注入去核的处理组和对照组1、对照组2的卵母细胞透明带下。注核后的重组胚采用微电极的方法进行融合。融合前,重组胚在电融合液中预平衡3min,电融合在150倍的显微操作仪下进行。用于融合操作的两根“Z”字形微电极顶端直径为15μm,后端连于显微操作仪上,使重组胚的供体、受体细胞膜接触面与两电极的连线垂直,融合参数:电压32V、脉冲时长20μs、2次脉冲间隔10ms。融合后半小时在显微镜下观察融合情况,将融合好的胚胎分别放回卵母细胞体外成熟培养液(对应的实施例1、对比例1、对比例2)中培养3h。
(4)牛体细胞克隆重组胚的激活
在电融合之后3h,将处理组和对照组1、对照组2的牛体细胞克隆重组胚通过离子霉素(Ionomycin)联合6-DMAP的激活处理进行激活:首先在含2~5μmol/L离子霉素的SOFaa溶液中室温孵育4min,然后在含1~2mmol/L6-DMAP的SOFaa溶液中,在38.5℃、5%CO2、饱和湿度条件下培养4h。
(5)牛体细胞克隆胚胎的体外培养
四孔板每孔加入500μL的mSOFaa溶液,mSOFaa溶液上还覆盖有500μL石蜡油,石蜡油预先在CO2培养箱中平衡至少2h,处理组和对照组1、对照组2的牛体细胞克隆重组胚在进行激活处理后,转移到上述四孔板中。每孔放置35~40枚牛体细胞克隆重组胚,在38.5℃、5%CO2、7%O2及饱和湿度条件下培养,记录发育情况,结果见表2。
表2克隆胚胎的发育情况
注:a、b、c上标不同表示差异显著,显著性使用卡方检验来检测。AB级囊胚为可移植囊胚,C级为不推荐移植囊胚,囊胚ABC分级是根据国际胚胎移植协会的囊胚质量评分标准来评定。
由表2可以看出,与对照组1、对照组2的有血清和无血清体外成熟培养液体系相比,采用本发明公开的无血清的牛卵母细胞体外成熟培养液,在核移植后获得的胚胎的可移植胚胎比率显著提高。
实施例4牛体外受精胚胎的制备方法
(1)卵母细胞的成熟培养
同实施例2。
(2)精子的解冻,纯化,受精
将冻精(内蒙古赛科星公司)从液氮罐取出后,放置于37℃水浴解冻,离心管中做Percoll分层液。将2mL 90%Percoll液置于离心管底部,把2mL45%Percoll液小心加到90%Percoll液面上,再将解冻后的精子置于45%Percoll液面上,1500rpm离心20min,小心吸取底部约200μL精液,分别加入含有处理组和对照组1、对照组2卵母细胞的受精液中,放回培养箱内使精卵进行结合8-12h。
(3)受精胚胎的培养
受精完毕后,用口吸管或者枪头吹去卵子周围的残存卵丘细胞、精子。如果无法去除干净,可以在透明质酸酶溶液中进行去除。后移入mSOFaa溶液(液面上还覆盖有500μL石蜡油,并预先在CO2培养箱中平衡至少2h)中培养,观察并记录发育情况,结果见表3。
表3体外受精胚胎的发育情况
注:a、b、c上标不同表示差异显著,显著性使用卡方检验来检测。AB级囊胚为可移植囊胚,C级为不推荐移植囊胚,囊胚ABC分级是根据国际胚胎移植协会的囊胚质量评分标准来评定。
由表3可以看出,与对照组1、对照组2的有血清和无血清体外成熟培养液体系相比,采用本发明公开的无血清的牛卵母细胞体外成熟培养液,在受精后获得的胚胎的可移植胚胎比率显著提高。
本发明公开的无血清的牛卵母细胞体外成熟培养液包括添加于基础培养液的BSA、EGF、L-cysteine、bFGF、叶酸、胆酸和CXCL12等组分;将牛卵母细胞放入预热的牛卵母细胞体外成熟培养液中,然后置于CO2体积浓度为5%的环境内成熟培养18~22小时,即获得成熟的卵母细胞。实验证实,本发明的体外成熟培养液及体外成熟培养方法,能够提高牛卵母细胞体外培养的成熟效率。
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 (6)
1.一种无血清的牛卵母细胞体外成熟培养液,包括无Hepes的M199基础培养液,其特征在于,所述培养液还包括以下添加剂:无脂肪酸BSA 6mg/ml、HMG 0.075 IU/ml、17β-雌二醇1µg/ml、EGF 60ng/ml、L-cysteine 0.57mM、bFGF 40ng/ml、100xGlutamax 1.6µL/ml、叶酸50µM、胆酸 2µg/ml、CXCL12 50ng/ml。
2.一种牛卵母细胞体外成熟培养方法,其特征在于,利用权利要求1所述的一种无血清的牛卵母细胞体外成熟培养液培养牛卵母细胞,具体步骤如下:将牛卵母细胞放入预热的无血清的牛卵母细胞体外成熟培养液中,置于38.5℃、5% CO2饱和湿度的培养箱中成熟培养20-22小时,获得成熟的牛卵母细胞。
3.根据权利要求2所述的一种牛卵母细胞体外成熟培养方法,其特征在于,所述牛卵母细胞为GV期卵母细胞。
4.根据权利要求3所述的一种牛卵母细胞体外成熟培养方法,其特征在于,所述无血清的牛卵母细胞体外成熟培养液预热1-3小时。
5.根据权利要求4所述的一种牛卵母细胞体外成熟培养方法,其特征在于,所述无血清的牛卵母细胞体外成熟培养液置于38.5℃、5% CO2饱和湿度的培养箱中预热。
6.根据权利要求5所述的一种牛卵母细胞体外成熟培养方法,其特征在于,每500 µl无血清的牛卵母细胞体外成熟培养液中放入40-50枚牛卵母细胞。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2020105750245 | 2020-06-22 | ||
CN202010575024 | 2020-06-22 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111944745A CN111944745A (zh) | 2020-11-17 |
CN111944745B true CN111944745B (zh) | 2023-12-19 |
Family
ID=73359713
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010858331.4A Active CN111944745B (zh) | 2020-06-22 | 2020-08-24 | 一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN111944745B (zh) |
WO (1) | WO2021258423A1 (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114934011A (zh) * | 2022-05-07 | 2022-08-23 | 南京优而生物科技发展有限公司 | 一种高质量培养卵母细胞的体外培养方法 |
CN115684401A (zh) * | 2022-10-27 | 2023-02-03 | 黑龙江八一农垦大学 | 一种利用血液生化指标预测奶牛酮病脂肪肝综合征的系统及应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101225373A (zh) * | 2008-01-23 | 2008-07-23 | 北京锦绣大地农业股份有限公司 | 一种牛卵母细胞体外成熟无血清培养基 |
CN108103011A (zh) * | 2018-02-09 | 2018-06-01 | 西北农林科技大学 | 一种牛卵母细胞体外成熟培养液及培养方法 |
CN110951678A (zh) * | 2019-12-26 | 2020-04-03 | 华中农业大学 | 一种促进猪卵母细胞体外成熟的培养液 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110066763A (zh) * | 2019-05-21 | 2019-07-30 | 天津博裕力牧科技有限公司 | 促进牛体外胚胎培养受精卵发育的方法 |
CN111254109B (zh) * | 2020-02-06 | 2022-07-08 | 天津力牧生物科技有限公司 | 牛卵母细胞的体外受精和胚胎培养的方法和运输培养液 |
-
2020
- 2020-07-08 WO PCT/CN2020/100828 patent/WO2021258423A1/zh active Application Filing
- 2020-08-24 CN CN202010858331.4A patent/CN111944745B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101225373A (zh) * | 2008-01-23 | 2008-07-23 | 北京锦绣大地农业股份有限公司 | 一种牛卵母细胞体外成熟无血清培养基 |
CN108103011A (zh) * | 2018-02-09 | 2018-06-01 | 西北农林科技大学 | 一种牛卵母细胞体外成熟培养液及培养方法 |
CN110951678A (zh) * | 2019-12-26 | 2020-04-03 | 华中农业大学 | 一种促进猪卵母细胞体外成熟的培养液 |
Also Published As
Publication number | Publication date |
---|---|
WO2021258423A1 (zh) | 2021-12-30 |
CN111944745A (zh) | 2020-11-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6905714B2 (ja) | 始原生殖細胞を機能的に成熟した卵母細胞へと分化させる培養方法 | |
CN108103011B (zh) | 一种牛卵母细胞体外成熟培养液及培养方法 | |
AU2002333022B2 (en) | Methods of derivation and propagation of undifferentiated human embryonic stem (HES) cells on feeder-free matrices and human feeder layers | |
CN111944745B (zh) | 一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法 | |
CN110951678B (zh) | 一种促进猪卵母细胞体外成熟的培养液 | |
US11098282B1 (en) | Serum-free culture medium for in vitro maturation of bovine oocytes and a method for culture of oocytes | |
WO2001042421A2 (en) | Long-term cell culture compositions and genetically modified animals derived therefrom | |
AU2004309300B2 (en) | Embryonic stem cell line and method for preparing the same | |
KR100417566B1 (ko) | 체세포 핵치환 복제수정란의 대량생산방법 | |
CN108707578B (zh) | 一种猪单胚胎体外培养方法 | |
EP1293561B1 (en) | Monkey-origin embryonic stem cells | |
CN107043743B (zh) | 犬卵母细胞的体外成熟方法 | |
CN110577928A (zh) | 一种卵母细胞体外成熟培养液及其应用 | |
CN110305836B (zh) | 一种牛体外受精的受精液及其使用方法 | |
US20200399587A1 (en) | Culture medium for improving development of bovine in vitro fertilized embryos and cloned embryos | |
CN114410573A (zh) | 一种卵母细胞体外成熟培养液添加剂及其应用 | |
WO2021227225A1 (zh) | 一种提高牛体外受精胚和克隆胚的发育质量的培养液 | |
WO2006083133A2 (en) | Human embryonic stem cell created from an oocyte and a somatic cell derived from non-identical individuals and a method for preparing the same | |
CN113186152B (zh) | Dna甲基转移酶抑制剂在提高动物圆形精子注射胚胎发育效率中的应用 | |
CN113943698B (zh) | 一种卵母细胞体外成熟培养的方法及其应用 | |
Gao et al. | Generation of large pig and bovine blastocysts by culturing in human induced pluripotent stem cell medium | |
CN116286617A (zh) | 一种利用mgp重组蛋白提高绵羊卵母细胞质量的方法 | |
CN114369567A (zh) | 牛扩展多能性胚胎干细胞的建系方法及培养液 | |
CN116555170A (zh) | 一种成年鸡卵原干细胞的体外分离培养方法及其应用 | |
Tripathy | ADVANCE TECHNOLOGIES TO TREAT INFERTILITY |
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 |