CN105130930A - In-vitro maturation medium of bovine oocytes and culturing method - Google Patents
In-vitro maturation medium of bovine oocytes and culturing method Download PDFInfo
- Publication number
- CN105130930A CN105130930A CN201510631508.6A CN201510631508A CN105130930A CN 105130930 A CN105130930 A CN 105130930A CN 201510631508 A CN201510631508 A CN 201510631508A CN 105130930 A CN105130930 A CN 105130930A
- Authority
- CN
- China
- Prior art keywords
- vitro
- fertilization
- compound
- bovine oocyte
- nutrient 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.)
- Granted
Links
- 210000000287 oocyte Anatomy 0.000 title claims abstract description 78
- 238000000338 in vitro Methods 0.000 title claims abstract description 72
- 230000035800 maturation Effects 0.000 title claims abstract description 54
- 241000283690 Bos taurus Species 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000012258 culturing Methods 0.000 title abstract description 5
- 150000001875 compounds Chemical class 0.000 claims abstract description 71
- 235000002956 Gynostemma pentaphyllum Nutrition 0.000 claims abstract description 17
- 210000002966 serum Anatomy 0.000 claims abstract description 10
- 229930182555 Penicillin Natural products 0.000 claims abstract description 8
- 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 claims abstract description 8
- 229940049954 penicillin Drugs 0.000 claims abstract description 8
- 239000002904 solvent Substances 0.000 claims abstract description 5
- 240000006509 Gynostemma pentaphyllum Species 0.000 claims abstract description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 98
- 235000015097 nutrients Nutrition 0.000 claims description 64
- ZQSIJRDFPHDXIC-UHFFFAOYSA-N daidzein Chemical compound C1=CC(O)=CC=C1C1=COC2=CC(O)=CC=C2C1=O ZQSIJRDFPHDXIC-UHFFFAOYSA-N 0.000 claims description 58
- 230000004720 fertilization Effects 0.000 claims description 51
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 46
- 235000007240 daidzein Nutrition 0.000 claims description 29
- 210000001161 mammalian embryo Anatomy 0.000 claims description 26
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 19
- 238000010828 elution Methods 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 16
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000000741 silica gel Substances 0.000 claims description 9
- 229910002027 silica gel Inorganic materials 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
- 239000003463 adsorbent Substances 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.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](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.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](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 claims description 7
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- 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 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 229960002897 heparin Drugs 0.000 claims description 4
- 229920000669 heparin Polymers 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000012259 ether extract Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000010829 isocratic elution Methods 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003921 oil Substances 0.000 claims description 2
- 238000011161 development Methods 0.000 abstract description 4
- 210000002257 embryonic structure Anatomy 0.000 abstract description 4
- 150000003505 terpenes Chemical class 0.000 abstract description 3
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 abstract 3
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 abstract 3
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 abstract 3
- 239000002609 medium Substances 0.000 abstract 3
- 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 abstract 2
- 210000000805 cytoplasm Anatomy 0.000 abstract 1
- 229960005322 streptomycin Drugs 0.000 abstract 1
- 241001065361 Gynostemma Species 0.000 description 13
- 229930187479 gypenoside Natural products 0.000 description 7
- ZRBFCAALKKNCJG-UHFFFAOYSA-N gypenoside-XVII Natural products C1CC(C2(CCC3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC(C(C(O)C1O)O)OC1COC1OC(CO)C(O)C(O)C1O ZRBFCAALKKNCJG-UHFFFAOYSA-N 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 210000000625 blastula Anatomy 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 210000002459 blastocyst Anatomy 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- 229930014626 natural product Natural products 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000001471 micro-filtration Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000000967 suction filtration Methods 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- 102000015779 HDL Lipoproteins Human genes 0.000 description 2
- 108010010234 HDL Lipoproteins Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 102000007330 LDL Lipoproteins Human genes 0.000 description 2
- 108010007622 LDL Lipoproteins Proteins 0.000 description 2
- -1 Methoxyl group Chemical group 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000008143 early embryonic development Effects 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical group [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001896 rotating frame Overhauser effect spectroscopy Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 240000008027 Akebia quinata Species 0.000 description 1
- 235000007756 Akebia quinata Nutrition 0.000 description 1
- 241000303854 Aralia leschenaultii Species 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 206010062129 Tongue neoplasm Diseases 0.000 description 1
- 206010044302 Tracheitis Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- OBOXTJCIIVUZEN-UHFFFAOYSA-N [C].[O] Chemical group [C].[O] OBOXTJCIIVUZEN-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- VILAVOFMIJHSJA-UHFFFAOYSA-N dicarbon monoxide Chemical compound [C]=C=O VILAVOFMIJHSJA-UHFFFAOYSA-N 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- GZYPWOGIYAIIPV-JBDTYSNRSA-N ginsenoside Rb1 Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GZYPWOGIYAIIPV-JBDTYSNRSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- HZVOZRGWRWCICA-UHFFFAOYSA-N methanediyl Chemical compound [CH2] HZVOZRGWRWCICA-UHFFFAOYSA-N 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 150000002773 monoterpene derivatives Chemical group 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 238000010449 nuclear transplantation Methods 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 210000004508 polar body Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 201000006134 tongue cancer Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
- C07D303/02—Compounds containing oxirane rings
- C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
- C07D303/32—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by aldehydo- or ketonic radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D301/00—Preparation of oxiranes
- C07D301/32—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
-
- 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
Landscapes
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Gynecology & Obstetrics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Reproductive Health (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an in-vitro maturation medium of bovine oocytes and a culturing method. The in-vitro maturation medium of the bovine oocytes is prepared from compounds (I) 50-70 micrograms/mL, isoflavoues aglycone 0.4-0.6 microgram per milliliter, penicillin 90-110 IU/mL, streptomycin 90-110 micrograms per milliliter, 8%-20% of blood serum and one or several of solvent of M199, DMEM, F12 and MEM, and new miscellaneous terpenoids separated from dry leaves of gynostemma pentaphylla are adopted as the compounds (I). According to the in-vitro maturation medium of the bovine oocytes and the culturing method, the compounds (I) and the isoflavoues aglycone are combined to be applied to in-vitro maturation culture of the bovine oocytes, compared with a method of using the compounds (I) or the isoflavoues aglycone singly, the maturity of cytoplasm of the oocytes can be significantly promoted, and the problem that at present, the in-vitro development rate of embryos is low after the in-vitro matured bovine oocytes are fertilized is solved.
Description
Technical field
The invention belongs to field of cell culture, be specifically related to a kind of natural product that separation and purification goes out from gynostemma pentaphylla, the bovine oocyte in vitro maturation culture solution containing this natural product and cultural method.
Background technology
Gynostemma pentaphylla (Gynostemmapentaphyllum (Thunb.) Maki-no, GP) belongs to the perennial draft of overgrowing of Curcurbitaceae, has another name called Rhizome or herb of Fiveleaf Gynostemma, Pentapanax leschenaultii etc.Yin Qiye is pleasantly sweet to be called as " Herb Gynostemmae Pentaphylli " in Japan.Cool in nature, bitter, micro-sweet, return lung, spleen, kidney channel.Have and replenish qi to invigorate the spleen, preventing phlegm from forming and stopping coughing, clearing heat and detoxicating effect, can be used for that treatment body void is weak, nephrasthenia emission, leukopenia, hyperlipidaemia, viral hepatitis, chronic gastroenteritis, chronic tracheitis.This plant primary growth is in subtropics, and about there are 16 kinds and 3 mutation in the whole world, and there are 15 kinds and 3 mutation in China, extensively the distribution Qinling Mountains and the Changjiang river areas to the south.
Gynostemma pentaphylla main chemical compositions is gypenoside, is distributed in vegetable nutritorium and comprises assimilation tissue and phloem parenchyma cells, and its content is difference to some extent because the position of plant is different, and generally in gynostemma pentaphyllum leaf, content is the highest.Be divided into from obtaining more than 140 kind of gypenoside from gynostemma pentaphylla at present, wherein gypenoside 3,4,8,12 respectively with ginsenoside Rb
1, Rb
3, Rd, F
2structure is identical, is synonym.In addition, gynostemma pentaphylla is also containing number of chemical compositions such as flavonoid, carbohydrate, terpenes.
Modern pharmacological research shows, gynostemma pentaphylla is the plant that a kind of chemical composition is clearer and more definite, has the features such as drug effect is good, toxic side effect is little.Gypenoside is cultivated cancer cells to cancer of the stomach, cervical cancer, tongue cancer etc. and is all had significant cytotoxicity.Gypenoside have significantly reduce cholesterol (TCH), triglyceride (TG), low-density lipoprotein (LDL), high density lipoprotein increasing (HDL), protection blood vessel; lipid is stoped to deposit at vessel wall, the effect of arteriosclerosis.Gynostemma pentaphylla can regulate oxygen to decompose the refuse of rear generation in vivo---and lipid acid, reaches the object of reducing blood-fat.Gypenoside has and obviously reduces blood viscosity, adjustment blood pressure function, can prevent microthrombusis and increase the endurance of myocardial cell to anoxic simultaneously, plays the effect of protection cardiac muscle.Gypenoside can improve senescent fibroblast multiplication capacity, and multiplication effect is time and concentration dependent, delaying cell aging.Gynostemma pentaphylla has immunoregulation effect, and its active major embodiment is: strengthen non-specific immunity, humoral immunization, cellular immunization; Affect lymphocyte transformation and interleukin II secretion; Strengthen the function of natural killer cell (NK).In addition, gynostemma pentaphylla also can be made into the functional foodstuffs such as health tea, beverage, foodstuff additive, has wide DEVELOPMENT PROSPECT.
Along with the modern times breeding development of biotechnology and the gradual perfection of embryo IVC technology, the technology production embryo in vitro such as in vitro fertilization, body-cell neucleus transplanting are utilized to present wide application prospect.Livestock embryo produced in vitro especially body-cell neucleus transplanting is the effective way of the outstanding population quantity of rapid expansion.The mature oocyte obtaining sufficient amount is the precondition of embryo IVC.Although obtain Preliminary Applications aborning from living animal oocyte collection, this method has complicated operation, injures greatly dam, needs precision instrument and obtain the deficiencies such as oocyte number is few.For large-scale by vitro fertilization or body-cell neucleus transplanting production embryo in vitro, from the ovary that slaughterhouse gathers, extracting ovocyte and carrying out vitro culture is obtain effective, the most most economical approach of mature oocyte.
Current research shows, the ovocyte of maturation in vitro carry out in vitro fertilization after, its fertilization, the spilting of an egg and early stage embryo development rate are lower, and blastocyst rate is lower than 50%, the developmental rate of nuclear transfer embryo is lower, limits embryo IVC technology applying in practice.Extracorporeal embryo development rate low major cause is caused to be that the defect of oocyte in vitro maturation and culture systems causes caused by the non-fully matured of kytoplasm.Oocyte maturation comprises Nuclear maturity and cytoplasmic maturation, only has the two equal maturation just to have fertility and developmental potentiality.The mark of Nuclear maturity is the discharge of first polar body, and the not yet unification of the judging criterion of cytoplasmic maturation.All events of first three cell cycle after oocyte fertilization have been responsible for by rRNA, mRNA of being stored in ripening process in tenuigenin and protein, and therefore cytoplasmic maturation seems particularly important to the impact of developmental potentiality after oocyte fertilization.Same to the receptor oocytes for nuclear transplantation, can the ripe quality of kytoplasm directly will affect reconstructed embryo and maintain normal division and later stage growth.Therefore, In vitro maturation condition is improved significant to raising embryo production in vitro efficiency.
Summary of the invention
The object of the present invention is to provide a kind of natural product that separation and purification goes out from gynostemma pentaphylla, the bovine oocyte in vitro maturation culture solution containing this natural product and cultural method.
Above-mentioned purpose is achieved by the following technical solution:
There is the compound (I) of following structural formula,
The preparation method of described compound (I), comprise following operation steps: the dry leave of gynostemma pentaphylla is pulverized by (a), extract with 80 ~ 90% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract D101 type macroporous adsorbent resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, use 80% ethanol elution, 6 column volumes again, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract; C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,25:1,12:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, in the preparation method of described compound (I), in step (a), the ethanol of thermal backflow is 85% ethanol.
Further, in the preparation method of described compound (I), described in step (b), macroporous adsorbent resin is D101 type macroporous adsorbent resin.
A kind of bovine oocyte in vitro maturation culture solution, described bovine oocyte in vitro maturation culture solution is composed as follows: described compound (I) 50 ~ 70 μ g/mL, daidzein 0.4 ~ 0.6 μ g/mL, penicillin 90 ~ 110IU/mL, Streptomycin sulphate 90 ~ 110 μ g/mL, serum volumn concentration 8% ~ 20%, solvent is cellar culture liquid; Described cellar culture liquid is one or more in M199, DMEM, F12, MEM.
Further, described nutrient solution is composed as follows: described compound (I) 60 μ g/mL, daidzein 0.5 μ g/mL, penicillin 100IU/mL, Streptomycin sulphate 100 μ g/mL, serum volumn concentration 10%, solvent is M199 nutrient solution.
Utilize described bovine oocyte in vitro maturation culture solution to carry out bovine oocyte vitro culture and method in vitro fertilization, described method comprises: (1) preparation bovine oocyte in vitro maturation culture solution; (2) bovine oocyte in vitro maturation culture solution is formed on disposable plastic culture dish the cultivation droplet that volume is 50 ~ 100 μ L, to put in 38 ~ 39 DEG C of environment preheating 1 ~ 3 hour, bovine oocyte puts into the cultivation droplet after preheating after rinsing, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 20 ~ 24 hours; (3) on disposable plastic culture dish, form with fertilization nutrient solution the fertilization droplet that volume is 50 ~ 100 μ L, to put in 38 ~ 39 DEG C of environment preheating 1 ~ 3 hour, the essence of freezing be stored in liquid nitrogen container is thawed, then the bovine oocyte after cultivating with step (2) together puts into fertilization droplet, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 15 ~ 18 hours, be fertilized, obtain the body early embryo of after fertilization; (4) on disposable plastic culture dish, form with embryo in-vitro culture solution the cultivation droplet that volume is 50 ~ 100 μ L, to put in 38 ~ 39 DEG C of environment preheating 1 ~ 3 hour, after the body early embryo of after fertilization is rinsed, put into and cultivate droplet, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate.
Further, in described bovine oocyte vitro culture and method in vitro fertilization, the described fertilization nutrient solution of step (3) is improve liquid containing the BO liquid of 5 ~ 10mg/mL heparin or Tyrode ' s.
Further, in described bovine oocyte vitro culture and method in vitro fertilization, step (4) described embryo in-vitro culture solution is the SOF nutrient solution containing volume percent 10% serum.
Described compound (I) is preparing the application in bovine oocyte in vitro maturation culture solution.
Beneficial effect: the assorted terpenoid (I) that the invention provides a kind of novel structure, the bovine oocyte in vitro maturation culture solution containing this compound, and based on the bovine oocyte in vitro maturation cultural method of this nutrient solution; The present invention by compound (I) and daidzein Combination application in bovine oocyte vitro culture, compared to being used alone compound (I) or daidzein, significantly can promote the Cytoplasmic maturation of ovocyte, solve the problem that vitro Development of Embryos rate after the oocyte fertilization of current ox maturation in vitro is low.This may to have provide protection to bovine oocyte relevant with compound (I) and daidzein coupling.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula;
Fig. 2 is that the theoretical ECD value of compound (I) compares with experiment ECD value.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Embodiment 1: compound (I) is separated preparation and structural identification
Reagent source: ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Preparation method: the dry leave (10kg) of gynostemma pentaphylla is pulverized by (a), (25L × 3 time) are extracted with 85% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (3L), use sherwood oil (3L × 3 time), ethyl acetate (3L × 3 time) and water saturated propyl carbinol (3L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (431g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract D101 type macroporous adsorbent resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, use 80% ethanol elution, 6 column volumes again, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract (157g); C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, successively with volume ratio be 75:1 (8 column volumes), the methylene chloride-methanol gradient elution of 45:1 (8 column volumes), 25:1 (8 column volumes), 12:1 (8 column volumes) and 1:1 (5 column volumes) obtains 5 components; D component 4 (49g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 20:1 (8 column volumes), the methylene chloride-methanol gradient elution of 12:1 (10 column volumes) and 5:1 (8 column volumes) obtains 3 components; E in () step (d), component 2 (22g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10-12 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I) (838mg).
Structural identification: HR-ESIMS shows [M+Na]
+for m/z377.1212, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
17h
22o
8, degree of unsaturation is 7.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 600MHz): H-2 (6.37, s), H-5 (3.52, d, J=15.8), H-5 (2.49, d, J=15.8), H-7 (2.73, d, J=8.0), H-8 (3.68, m), H-9 (2.39, m), H-9 (1.71, t, J=13.0), H-11 (3.69, s), and H-15 (1.27, s), H-16 (1.36, s), and 1-OH (11.88, s), 4-OH (9.58, s), 6-OH (4.09, s), and 7-OH (5.77, s), 8-OH (4.40, s), 3-OCH
3(3.87, s); Carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 125Hz): 158.5 (C, 1-C), 99.0 (CH, 2-C), 153.1 (C, 3-C), 138.2 (C, 4-C), 29.4 (CH
2, 5-C), 61.3 (C, 6-C), 69.8 (CH, 7-C), 68.7 (CH, 8-C), 43.1 (CH
2, 9-C), 61.6 (C, 10-C), 63.2 (CH, 11-C), 197.4 (C, 12-C), 114.2 (C, 13-C), 120.5 (C, 14-C), 21.1 (CH
3, 15-C), 16.8 (CH
3, 16-C), 56.1 (CH
3, 3-OCH
3); Carbon atom mark is see Fig. 1.Infrared spectra shows that this compound contains oh group (3440cm
-1); In addition this compound has uv-absorbing in 365 and 287nm, illustrates containing many conjugated structures.1DNMR spectrum and hsqc spectrum show 17 carbon signals, are respectively three methyl (comprising a methoxyl group), two methylene radical, four methynes (comprising an alkene carbon) and eight quaternary carbons.In addition, olefinic proton signals δ H6.37 (1H, a s, and six olefinic carbon signals δ C158.5 (C, 1-C) H-2), 99.0 (CH, 2-C), 153.1 (C, 3-C), 138.2 (C, 4-C), 114.2 (C, 13-C) He 120.5 (C, 14-C), show that this compound contains one five benzene ring structure replaced.In HMBC spectrum, hydroxyl proton (δ H11.88, s, 1-OH) and C-1, C-2, the dependency of C-13 and δ C197.4 (C, 12-C) shows that carbonyl carbon (C-12) is connected with the C-13 of phenyl ring, and this compound also demonstrates this conclusion in the uv-absorbing of 365 and 287nm.In HMBC spectrum, hydroxyl proton δ H9.58 (1H, s, 4-OH) and δ C138.2 (C, 4-C), 153.1 (C, 3-C) and 120.5 (C, 14-C); Methoxyl group proton δ H3.87 (s, 3-OCH
3) and C-3; And the dependency of H-2 and C-3 shows that methoxyl group and hydroxyl lay respectively on C-3 and C-4 position.Remaining 10 carbon atoms (removing seven carbon of phenyl ring and methoxyl group) define a ten-ring monoterpene structure.In addition, two show existence 10,11-epoxide groups containing oxygen carbon signal δ C61.6 (C, 10-C) and 63.2 (CH, 11-C).In HMBC spectrum, H-11 (δ H3.69, s) and C-12 and C-10; H
3the above-mentioned inference of the relevance verification of-16 (δ H1.36, s) and C-10 and C-16 (δ C16.8).In ROESY spectrum, H-5b and H-8 and H
3dependency in-16 shows, H-8 and H
3-16 are positioned at homonymy, are beta comfiguration, then C-8-OH is α configuration.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and ROESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as shown in Figure 1, steric configuration is determined further by ECD test, theoretical value and experimental value basically identical (Fig. 2).
Embodiment 2: preparation is containing the oocyte in vitro maturation culture solution of compound (I)
Take daidzein 10mg, be dissolved in the DMSO of 1mL, be made into mother liquor and be stored in 4 DEG C of refrigerators.Accurate Weigh Compound (I) 3mg, the M199 nutrient solution (Gibco company) of 20mL is first used to dissolve, then in the M199 nutrient solution being dissolved with compound (I), the above-mentioned daidzein mother liquor of 2.5 μ L, 5mL calf serum, the penicillin of 5000IU, the Streptomycin sulphate of 5000 μ g is added, gentle agitation is even, add M199 nutrient solution again to 50mL volume, again stirring and evenly mixing.After microfiltration membrane (MilliporeCorporation, Bedford.Mass.USA) the suction filtration sterilization of 0.22 μM, store at 4 DEG C, for subsequent use.
Embodiment 3: the nutrient solution containing compound (I) is used for milk cow In vitro maturation
Milk bovine oocyte respectively the contrast nutrient solution (M199 nutrient solution) not containing compound (I) and daidzein, only containing compound (I) 60 μ g/mL M199 nutrient solution, only prepare containing the M199 nutrient solution of daidzein 0.5 μ g/mL and embodiment 2 contain maturation culture in the bovine oocyte in vitro maturation culture solution of compound (I) and daidzein, then carry out in vitro fertilization.Concrete grammar is as follows:
A, maturation culture: bovine oocyte in vitro maturation culture solution forms the cultivation droplet that volume is about 80 ~ 100 μ L on disposable plastic culture dish, to put in 38 ~ 39 DEG C of environment preheating 2 hours, bovine oocyte puts into the cultivation droplet after preheating after rinsing, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 24 hours;
B, in vitro fertilization: on disposable plastic culture dish, to form with fertilization nutrient solution the fertilization droplet that volume is 80 ~ 100 μ L, to put in 38 ~ 39 DEG C of environment preheating 2 hours, the essence of freezing be stored in liquid nitrogen container is thawed, then the bovine oocyte after cultivating with steps A together puts into fertilization droplet, 38 ~ 39 DEG C, cultivate 16 hours in the environment of 5%CO2, be fertilized, obtain the body early embryo of after fertilization; Fertilization nutrient solution is the BO liquid containing 8mg/mL heparin;
C, the cultivation droplet making volume 50 ~ 100 μ L with embryo in-vitro culture solution on disposable plastic culture dish to put in 38 ~ 39 DEG C of environment preheating 1 ~ 3 hour.After the body early embryo embryo in-vitro culture solution of after fertilization is rinsed three times, put into and cultivate droplet, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 8 days; Embryo in-vitro culture solution is the SOF nutrient solution containing volume percent 10% serum.In cultivate after the 48th hour and the 8th day respectively statistics embryos cleave rate and blastula stage developmental rate.
Result shows: the 2nd day of cultivation, division rate after not containing the oocyte fertilization in the contrast nutrient solution of compound (I) is 67.2%, is 83.5% in embodiment 2 preparation containing the division rate after the oocyte fertilization in compound (I) oocyte in vitro maturation, improves 16.3% than control group.The 8th day that cultivates, developmental rate blastula stage after not containing the oocyte fertilization in the contrast nutrient solution of compound (I) is 19.8%, is 60.1% in embodiment 2 preparation containing developmental rate blastula stage after the oocyte fertilization in the oocyte in vitro maturation of compound (I), improves 40.3% than control group.(see table 1)
Table 1
| Group | Cleavage rates (%) | Blastocyst rate (%) |
| Not containing compound (I) and daidzein nutrient solution group | 67.2 | 19.8 |
| Only containing the nutrient solution group of compound (I) | 67.8 | 20.2 |
| Only containing the nutrient solution group of daidzein | 66.9 | 20.8 |
| Embodiment 2 nutrient solution group | 83.5* | 60.1* |
Note: * represent with not containing compound (I) and daidzein nutrient solution group, only to compare significant difference (P < 0.05) containing the nutrient solution group of compound (I) and the nutrient solution group that to contain daidzein.
Result confirms, effectively can improve the early embryonic development rate of after fertilization with the bovine oocyte in vitro maturation culture solution In-vitro maturation milk bovine oocyte containing compound (I) prepared by embodiment 2.
Embodiment 4: the nutrient solution containing compound (I) is used for ox In vitro maturation
Ox ovocyte is maturation culture in the oocyte in vitro maturation not containing the contrast nutrient solution of compound (I) and the compound (I) containing embodiment 2 preparation respectively, and then carry out in vitro fertilization, method is with embodiment 3.
Result shows, the 2nd day that cultivates, division rate after not containing the oocyte fertilization in the contrast nutrient solution of compound (I) is 67.6%, is 83.1% in embodiment 2 preparation containing the division rate after the oocyte fertilization in compound (I) oocyte in vitro maturation, improves 15.5% than control group.The 8th day that cultivates, developmental rate blastula stage after not containing the oocyte fertilization in the contrast nutrient solution of compound (I) is 19.6%, is 60.5% in embodiment 2 preparation containing developmental rate blastula stage after the oocyte fertilization in the oocyte in vitro maturation of compound (I), improves 40.9% (see table 2) than control group.
Table 2
| Group | Cleavage rates (%) | Blastocyst rate (%) |
| Not containing compound (I) and daidzein nutrient solution group | 67.6 | 19.6 |
| Only containing the nutrient solution group of compound (I) | 67.9 | 20.4 |
| Only containing the nutrient solution group of daidzein | 67.3 | 20.3 |
| Embodiment 2 nutrient solution group | 83.1* | 60.5* |
Note: * represent with not containing compound (I) and daidzein nutrient solution group, only to compare significant difference (P < 0.05) containing the nutrient solution group of compound (I) and the nutrient solution group that to contain daidzein.
Result confirms, effectively can improve the early embryonic development rate of after fertilization with the bovine oocyte in vitro maturation culture solution In-vitro maturation ox ovocyte containing compound (I) prepared by embodiment 2.
Embodiment 5: the nutrient solution containing compound (I) is used for milk cow In vitro maturation and bovine embryo is cultivated outward
Concrete grammar is as follows:
A, maturation culture: bovine oocyte in vitro maturation culture solution forms the cultivation droplet that volume is about 80 ~ 100 μ L on disposable plastic culture dish, to put in 38 ~ 39 DEG C of environment preheating 2 hours, bovine oocyte puts into the cultivation droplet after preheating after rinsing, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 24 hours; With the M199 nutrient solution (control group 1) not containing compound (I) and daidzein, only containing compound (I) 60 μ g/mL M199 nutrient solution (control group 2), only contain the M199 nutrient solution (control group 3) of daidzein 0.5 μ g/mL in contrast.
Bovine oocyte in vitro maturation culture solution is prepared: take daidzein 10mg, be dissolved in the DMSO of 1mL, be made into mother liquor and be stored in 4 DEG C of refrigerators.Accurate Weigh Compound (I) 3mg, the M199 nutrient solution (Gibco company) of 20mL is first used to dissolve, then in the M199 nutrient solution being dissolved with compound (I), the above-mentioned daidzein mother liquor of 2.5 μ L, 5mL calf serum, the penicillin of 5000IU, the Streptomycin sulphate of 5000 μ g is added, gentle agitation is even, add M199 nutrient solution again to 50mL volume, again stirring and evenly mixing.After microfiltration membrane (MilliporeCorporation, Bedford.Mass.USA) the suction filtration sterilization of 0.22 μM, store at 4 DEG C, for subsequent use.
B, in vitro fertilization: on disposable plastic culture dish, to form with fertilization nutrient solution the fertilization droplet that volume is 80 ~ 100 μ L, to put in 38 ~ 39 DEG C of environment preheating 2 hours, the essence of freezing be stored in liquid nitrogen container is thawed, then the bovine oocyte after cultivating with steps A together puts into fertilization droplet, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 16 hours, be fertilized, obtain the body early embryo of after fertilization; Fertilization nutrient solution is the BO liquid containing 8mg/mL heparin;
C, on disposable plastic culture dish, to make volume 50 ~ 100 μ L with ox embryo in vitro culturing liquid cultivate droplet and to put in 38 ~ 39 DEG C of environment preheating 1 ~ 3 hour.After the body early embryo embryo in-vitro culture solution of after fertilization is rinsed three times, put into and cultivate droplet, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 8 days, with the SOF nutrient solution (control group 1) not containing compound (I), only containing compound (I) 60 μ g/mL SOF nutrient solution (control group 2), only containing daidzein 0.5 μ g/mL SOF nutrient solution (control group 3) in contrast.
Ox embryo in vitro culturing liquid is prepared: accurately Weigh Compound (I) 3mg, first dissolve with the SOF nutrient solution of 20mL, then in nutrient solution, add the penicillin of 400mg bovine serum albumin and 5000IU, the Streptomycin sulphate of 5000 μ g, gentle agitation is even, add SOF nutrient solution again to 50mL volume, again stirring and evenly mixing.After microfiltration membrane (MilliporeCorporation, Bedford.Mass.USA) the suction filtration sterilization of 0.22 μM, store at 4 DEG C, for subsequent use.In cultivate after the 48th hour and the 8th day respectively statistics embryos cleave rate and blastula stage developmental rate, the results are shown in Table 3.
Table 3
| Group | Cleavage rates (%) | Blastocyst rate (%) |
| Not containing compound (I) and daidzein nutrient solution group (control group 1) | 67.1 | 19.7 |
| Only containing the nutrient solution group (control group 2) of compound (I) | 67.7 | 20.1 |
| Only containing the nutrient solution group (control group 3) of daidzein | 66.8 | 20.7 |
| Embodiment 2 nutrient solution group | 83.4* | 60.0* |
Note: * represent with not containing compound (I) and daidzein nutrient solution group, only to compare significant difference (P < 0.05) containing the nutrient solution group of compound (I) and the nutrient solution group that to contain daidzein.
Claims (10)
1. there is the compound (I) of following structural formula,
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: the dry leave of gynostemma pentaphylla is pulverized by (a), extract with 80 ~ 90% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract D101 type macroporous adsorbent resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, use 80% ethanol elution, 6 column volumes again, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract; C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,25:1,12:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. preparation method according to claim 2, is characterized in that: in step (a), the ethanol of thermal backflow is 85% ethanol.
4. preparation method according to claim 2, is characterized in that: macroporous adsorbent resin described in step (b) is D101 type macroporous adsorbent resin.
5. a bovine oocyte in vitro maturation culture solution, it is characterized in that described bovine oocyte in vitro maturation culture solution is composed as follows: described compound (I) 50 ~ 70 μ g/mL, daidzein 0.4 ~ 0.6 μ g/mL, penicillin 90 ~ 110IU/mL, Streptomycin sulphate 90 ~ 110 μ g/mL, serum volumn concentration 8% ~ 20%, solvent is cellar culture liquid; Described cellar culture liquid is one or more in M199, DMEM, F12, MEM.
6. bovine oocyte in vitro maturation culture solution according to claim 5, it is characterized in that described nutrient solution is composed as follows: described compound (I) 60 μ g/mL, daidzein 0.5 μ g/mL, penicillin 100IU/mL, Streptomycin sulphate 100 μ g/mL, serum volumn concentration 10%, solvent is M199 nutrient solution.
7. utilize bovine oocyte in vitro maturation culture solution described in claim 5 or 6 to carry out bovine oocyte vitro culture and method in vitro fertilization, it is characterized in that described method comprises: (1) preparation bovine oocyte in vitro maturation culture solution; (2) bovine oocyte in vitro maturation culture solution is formed on disposable plastic culture dish the cultivation droplet that volume is 50 ~ 100 μ L, to put in 38 ~ 39 DEG C of environment preheating 1 ~ 3 hour, bovine oocyte puts into the cultivation droplet after preheating after rinsing, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 20 ~ 24 hours; (3) on disposable plastic culture dish, form with fertilization nutrient solution the fertilization droplet that volume is 50 ~ 100 μ L, to put in 38 ~ 39 DEG C of environment preheating 1 ~ 3 hour, the essence of freezing be stored in liquid nitrogen container is thawed, then the bovine oocyte after cultivating with step (2) together puts into fertilization droplet, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate 15 ~ 18 hours, be fertilized, obtain the body early embryo of after fertilization; (4) on disposable plastic culture dish, form with embryo in-vitro culture solution the cultivation droplet that volume is 50 ~ 100 μ L, to put in 38 ~ 39 DEG C of environment preheating 1 ~ 3 hour, after the body early embryo of after fertilization is rinsed, put into and cultivate droplet, at 38 ~ 39 DEG C, 5%CO
2environment in cultivate.
8. bovine oocyte vitro culture according to claim 7 and method in vitro fertilization, is characterized in that the described fertilization nutrient solution of step (3) is the BO liquid or Tyrode ' the s improvement liquid that contain 5 ~ 10mg/mL heparin.
9. bovine oocyte vitro culture according to claim 7 and method in vitro fertilization, is characterized in that step (4) described embryo in-vitro culture solution is the SOF nutrient solution containing volume percent 10% serum.
10. compound according to claim 1 (I) is preparing the application in bovine oocyte in vitro maturation culture solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510631508.6A CN105130930B (en) | 2015-09-29 | 2015-09-29 | A kind of bovine oocyte in vitro maturation culture solution and cultural method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510631508.6A CN105130930B (en) | 2015-09-29 | 2015-09-29 | A kind of bovine oocyte in vitro maturation culture solution and cultural method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105130930A true CN105130930A (en) | 2015-12-09 |
| CN105130930B CN105130930B (en) | 2016-05-18 |
Family
ID=54716562
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510631508.6A Expired - Fee Related CN105130930B (en) | 2015-09-29 | 2015-09-29 | A kind of bovine oocyte in vitro maturation culture solution and cultural method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105130930B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861427A (en) * | 2016-05-20 | 2016-08-17 | 内蒙古大学 | New application of glial cell line-derived neurotrophic factor (GDNF) |
| CN105997980A (en) * | 2016-06-03 | 2016-10-12 | 武晓丹 | Application of meroterpenoid compound in preparation of drugs for treating bone destructive disease |
| CN106083766A (en) * | 2016-06-03 | 2016-11-09 | 武晓丹 | A kind of miscellaneous note compounds and preparation method thereof |
| CN113444683A (en) * | 2021-07-12 | 2021-09-28 | 华南农业大学 | Additive for improving oocyte in-vitro maturation quality, culture method and application |
-
2015
- 2015-09-29 CN CN201510631508.6A patent/CN105130930B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| XIA YIN: "Highly Oxygenated Meroterpenoids from Fruiting Bodies of the Mushroom Tricholoma terreum", 《JOURNAL OF NATURAL PRODUCTS》, vol. 76, no. 7, 9 July 2013 (2013-07-09), pages 1365 - 1368 * |
| 张岭,王茵: "染料木黄酮对哺乳动物卵母细胞成熟的影响", 《中国药理学与毒理学杂志》, vol. 27, no. 1, 30 November 2013 (2013-11-30), pages 133 - 134 * |
| 徐翠凤，罗嘉梁，王碧兰，郁青，沈兆邦: "绞股蓝化学成分分析", 《林产化工通讯》, no. 2, 31 December 1994 (1994-12-31), pages 3 - 7 * |
| 李群峰: "绞股蓝的化学成分与药理作用研究进展", 《光明中医》, vol. 24, no. 12, 31 December 2009 (2009-12-31), pages 2396 - 2398 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861427A (en) * | 2016-05-20 | 2016-08-17 | 内蒙古大学 | New application of glial cell line-derived neurotrophic factor (GDNF) |
| CN105997980A (en) * | 2016-06-03 | 2016-10-12 | 武晓丹 | Application of meroterpenoid compound in preparation of drugs for treating bone destructive disease |
| CN106083766A (en) * | 2016-06-03 | 2016-11-09 | 武晓丹 | A kind of miscellaneous note compounds and preparation method thereof |
| CN113444683A (en) * | 2021-07-12 | 2021-09-28 | 华南农业大学 | Additive for improving oocyte in-vitro maturation quality, culture method and application |
| CN113444683B (en) * | 2021-07-12 | 2022-11-04 | 华南农业大学 | Additive for improving oocyte in-vitro maturation quality, culture method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105130930B (en) | 2016-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105130930A (en) | In-vitro maturation medium of bovine oocytes and culturing method | |
| Lee et al. | Influence of inoculum density and aeration volume on biomass and bioactive compound production in bulb-type bubble bioreactor cultures of Eleutherococcus koreanum Nakai | |
| CN101760496A (en) | Biological dehydrogenation preparation method of steroid drug intermediate | |
| Shohael et al. | Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides | |
| CN105198893A (en) | Diterpenoid compounds for treating stomach cancer | |
| CN105294665A (en) | Novel diterpene compound for neuroprotection | |
| CN108689851B (en) | Tiglic alkane type diterpene compound and preparation method and application thereof | |
| CN101113430A (en) | Method for gaining panax japonicus secondary metabolite by using panax japonicus cell culture and biological transformation technique | |
| CN103845420B (en) | The yellow extract of a kind of tower and application thereof | |
| CN108277197B (en) | Method for increasing mogroside V content in suspension cells of momordica grosvenori | |
| CN105524063A (en) | Novel terpene indole alkaloid compound, and preparation method and medical application thereof | |
| Ernst | Pimpinella anisum L.(Anise): Cell culture, somatic embryogenesis, and the production of anise oil | |
| CN103122334A (en) | Primary culture and purification method of intestinal epithelial cells of carassius auratus gibelio | |
| CN105237499A (en) | Natural product and method using same to prepare CIK cell with high cytotoxic activity | |
| CN105884720A (en) | Buspirone hydrochloride pharmaceutical composition and medical application thereof | |
| CN105622629A (en) | Kaurane type diterpenoid compound for treating prostatic cancer | |
| CN105586314A (en) | Method for inducing directional differentiation of stem cells | |
| CN105534968A (en) | Application of diterpenoid compound to preparation of drug for treating prostate cancer | |
| CN105566130A (en) | Novel enamine compound and preparation method and medical application thereof | |
| CN108347915A (en) | A kind of frozen stock solution and preparation method thereof for adult stem cell long-term preservation | |
| Yoshikawa | Production of ginsenosides in ginseng hairy root cultures | |
| CN110776409B (en) | Method for extracting pterocarpus indicus and application of extract in antitumor drugs | |
| CN110204589B (en) | Effective component of feather cockscomb seed, extraction method and application thereof in preparing neuroprotective medicament | |
| CN104829567B (en) | Compounds, containing its pharmaceutical composition and its preparation method and application | |
| CN113387997A (en) | Triterpene saponin compound with function of improving glycolipid metabolic disturbance and preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160406 Address after: 325000 Kunlun Road, Lucheng District, Zhejiang, No. 72, No. Applicant after: Li Qunxing Address before: Tong Xiang, Gulou District of Nanjing city of Jiangsu Province, China Medicine University No. 24 210009 Applicant before: Yang Tingwen |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information |
Inventor after: Ma Fengzhao Inventor before: Yang Tingwen |
|
| CB03 | Change of inventor or designer information | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20170317 Address after: Nansha District Fengze road Guangzhou city Guangdong province 511400 No. 106 (self building No. 1) X1301-F1378 Patentee after: Guangzhou Yi Yi Biotechnology Co., Ltd. Address before: 325000 Kunlun Road, Lucheng District, Zhejiang, No. 72, No. Patentee before: Li Qunxing |
|
| TR01 | Transfer of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160518 Termination date: 20190929 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |