CN117431203B - 一种增强体外培养肝细胞凝血因子表达的培养添加物及其应用 - Google Patents
一种增强体外培养肝细胞凝血因子表达的培养添加物及其应用 Download PDFInfo
- Publication number
- CN117431203B CN117431203B CN202311758235.2A CN202311758235A CN117431203B CN 117431203 B CN117431203 B CN 117431203B CN 202311758235 A CN202311758235 A CN 202311758235A CN 117431203 B CN117431203 B CN 117431203B
- Authority
- CN
- China
- Prior art keywords
- culture
- vitro
- hepatocyte
- expression
- coagulation factor
- 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
- 239000003114 blood coagulation factor Substances 0.000 title claims abstract description 50
- 102000015081 Blood Coagulation Factors Human genes 0.000 title claims abstract description 48
- 108010039209 Blood Coagulation Factors Proteins 0.000 title claims abstract description 48
- 210000003494 hepatocyte Anatomy 0.000 title claims abstract description 45
- 238000000338 in vitro Methods 0.000 title claims abstract description 30
- 230000002708 enhancing effect Effects 0.000 title claims abstract description 15
- 239000000654 additive Substances 0.000 title abstract description 31
- 230000000996 additive effect Effects 0.000 title abstract description 29
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 claims abstract description 10
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229940093265 berberine Drugs 0.000 claims abstract description 10
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 claims abstract description 8
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 5
- 239000013589 supplement Substances 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 6
- GMRQFYUYWCNGIN-ZVUFCXRFSA-N 1,25-dihydroxy vitamin D3 Chemical compound C1([C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=CC=C1C[C@@H](O)C[C@H](O)C1=C GMRQFYUYWCNGIN-ZVUFCXRFSA-N 0.000 claims description 5
- 229930003448 Vitamin K Natural products 0.000 claims description 5
- 235000019168 vitamin K Nutrition 0.000 claims description 5
- 239000011712 vitamin K Substances 0.000 claims description 5
- 150000003721 vitamin K derivatives Chemical class 0.000 claims description 5
- 229940046010 vitamin k Drugs 0.000 claims description 5
- 229960002882 calcipotriol Drugs 0.000 claims description 4
- LWQQLNNNIPYSNX-UROSTWAQSA-N calcipotriol Chemical compound C1([C@H](O)/C=C/[C@@H](C)[C@@H]2[C@]3(CCCC(/[C@@H]3CC2)=C\C=C\2C([C@@H](O)C[C@H](O)C/2)=C)C)CC1 LWQQLNNNIPYSNX-UROSTWAQSA-N 0.000 claims description 4
- 229960005084 calcitriol Drugs 0.000 claims description 4
- 235000020964 calcitriol Nutrition 0.000 claims description 4
- 239000011612 calcitriol Substances 0.000 claims description 4
- GMRQFYUYWCNGIN-NKMMMXOESA-N calcitriol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C GMRQFYUYWCNGIN-NKMMMXOESA-N 0.000 claims description 4
- 241000124008 Mammalia Species 0.000 claims description 3
- 150000003180 prostaglandins Chemical class 0.000 claims description 2
- 229940104691 vitamin k 1 10 mg Drugs 0.000 claims description 2
- 210000005229 liver cell Anatomy 0.000 abstract description 25
- 229940019700 blood coagulation factors Drugs 0.000 abstract description 16
- 230000023555 blood coagulation Effects 0.000 abstract description 4
- 229960002986 dinoprostone Drugs 0.000 abstract description 4
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 abstract description 4
- ABSPRNADVQNDOU-UHFFFAOYSA-N Menaquinone 1 Natural products C1=CC=C2C(=O)C(CC=C(C)C)=C(C)C(=O)C2=C1 ABSPRNADVQNDOU-UHFFFAOYSA-N 0.000 abstract description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 abstract description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 abstract description 3
- 239000003112 inhibitor Substances 0.000 abstract description 3
- 230000037361 pathway Effects 0.000 abstract description 3
- 235000019175 phylloquinone Nutrition 0.000 abstract description 3
- 239000011772 phylloquinone Substances 0.000 abstract description 3
- MBWXNTAXLNYFJB-NKFFZRIASA-N phylloquinone Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CCC[C@H](C)CCC[C@H](C)CCCC(C)C)=C(C)C(=O)C2=C1 MBWXNTAXLNYFJB-NKFFZRIASA-N 0.000 abstract description 3
- 229960001898 phytomenadione Drugs 0.000 abstract description 3
- 239000000018 receptor agonist Substances 0.000 abstract description 3
- 229940044601 receptor agonist Drugs 0.000 abstract description 3
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 abstract description 3
- 102000009310 vitamin D receptors Human genes 0.000 abstract description 3
- 108050000156 vitamin D receptors Proteins 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 12
- 230000000694 effects Effects 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 238000012795 verification Methods 0.000 description 6
- 238000011529 RT qPCR Methods 0.000 description 5
- 102000007469 Actins Human genes 0.000 description 4
- 108010085238 Actins Proteins 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 210000001778 pluripotent stem cell Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 102100023804 Coagulation factor VII Human genes 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 108010076282 Factor IX Proteins 0.000 description 2
- 108010014172 Factor V Proteins 0.000 description 2
- 108010023321 Factor VII Proteins 0.000 description 2
- 108010054218 Factor VIII Proteins 0.000 description 2
- 102000001690 Factor VIII Human genes 0.000 description 2
- 108010014173 Factor X Proteins 0.000 description 2
- 108010074864 Factor XI Proteins 0.000 description 2
- 108010080865 Factor XII Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 108010000499 Thromboplastin Proteins 0.000 description 2
- 102000002262 Thromboplastin Human genes 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229960000987 paricalcitol Drugs 0.000 description 2
- BPKAHTKRCLCHEA-UBFJEZKGSA-N paricalcitol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](\C=C\[C@H](C)C(C)(C)O)C)=C\C=C1C[C@@H](O)C[C@H](O)C1 BPKAHTKRCLCHEA-UBFJEZKGSA-N 0.000 description 2
- 238000003672 processing method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 102100037529 Coagulation factor V Human genes 0.000 description 1
- 102100029117 Coagulation factor X Human genes 0.000 description 1
- 102100030563 Coagulation factor XI Human genes 0.000 description 1
- 102100030556 Coagulation factor XII Human genes 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 102000000429 Factor XII Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 229940105772 coagulation factor vii Drugs 0.000 description 1
- 229940105756 coagulation factor x Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229940012413 factor vii Drugs 0.000 description 1
- 229960000301 factor viii Drugs 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000008717 functional decline Effects 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000011880 melting curve analysis Methods 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
-
- 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
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/02—Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
-
- 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
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2501/999—Small molecules not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及肝细胞培养技术领域，具体公开一种增强体外培养肝细胞凝血因子表达的培养添加物，所述培养添加物包含：维生素D受体激动剂、转化生长因子beta通路抑制剂、小檗碱、前列腺素E2和维生素K1。凝血功能减退和丢失是肝细胞体外培养的主要问题之一，在培养基中添加本发明提供的培养添加物可明显提高体外培养的肝细胞中凝血因子的表达水平，利于体外培养的肝细胞的应用。
Description
技术领域
本发明涉及肝细胞培养技术领域，具体涉及一种增强体外培养肝细胞凝血因子表达的培养添加物及其应用。
背景技术
体外培养技术是进行肝细胞研究的重要手段，但是，肝细胞是一种功能复杂多样的细胞，具有代谢功能、解毒和生物转化功能、凝血功能、分泌功能、免疫防御功能等。在体外培养时，由于脱离体内肝细胞所处的微环境，致使肝细胞的功能特性出现减退甚至丧失。其中在凝血功能方面，除凝血因子Ⅲ（组织因子）（由血管外组织产生，在血管破损时启动凝血过程）和凝血因子Ⅳ（钙离子）外，其他十种凝血因子均由肝细胞合成分泌，因此，体外培养的肝细胞均会出现不同程度的凝血功能减退，表现为只表达几种凝血因子或表达水平很低，与正常肝细胞的凝血因子相比差距很大，因此，将其用于体外研究或应用存在明显缺陷。
发明内容
本发明的第一个目的是提供一种增强体外培养肝细胞凝血因子表达的培养添加物，在正常肝细胞培养基中添加该培养添加物可有效提高肝细胞中多种凝血因子的表达水平，解决了体外培养的肝细胞凝血因子表达减弱或丢失的问题。
作为一个具体的实施例，本发明提供了一种增强体外培养肝细胞凝血因子表达的培养添加物，其特征在于，所述培养添加物包含：维生素D受体激动剂、转化生长因子beta通路抑制剂、小檗碱、前列腺素E2和维生素K1。
进一步，本发明所述的培养添加物，其特征在于，所述维生素D受体激动剂包括1,25(OH)2D3、骨化三醇、帕立骨化醇、艾地骨化醇和卡泊三醇中的至少一种；其中：
所述1,25(OH)2D3的浓度为10-8M~10-6M；所述骨化三醇的浓度为5~50μM、所述帕立骨化醇的浓度为10-7M~10-5M、所述艾地骨化醇的浓度为0.05~5μM、所述卡泊三醇的浓度为1~10μM；
所述转化生长因子beta通路抑制剂包括LY2157299、LY2109761、LY364947、K02288和A-83-01中的至少一种；所述LY2157299的浓度为10~1000μM、所述LY2109761的浓度为10~500μM、所述LY364947的浓度为1~500μM、所述K02288的浓度为10~1000μM、所述A-83-01的浓度为10~500μM；
所述小檗碱的浓度为1~10mmol/L；
所述前列腺素E2的浓度为0.5~10mmol/L；
所述维生素K1的浓度为10~500mg/L。
作为更进一步技术方案，本发明提供的培养添加物由以下成分组成：
1,25(OH)2D310-7M、K02288 100μM、小檗碱 5mmol/L、前列腺素E2 5mmol/L、维生素K1 200mg/L。
本发明的第二个目的是提供一种增强体外培养肝细胞凝血因子表达的培养添加物在体外肝细胞培养中的应用。所述肝细胞为各种来源获得的具有肝细胞特征的细胞，具体的可以应用在原代肝细胞，也可以应用在连续传代的肝细胞如永生化的肝细胞和肝癌细胞系，以及其他来源的肝细胞，如干细胞分化的肝细胞或转分化来源的肝细胞等。
本发明所述肝细胞来源于哺乳动物，不局限于人类，其他种属的肝细胞与人肝细胞具有类似特征的哺乳动物肝细胞均可适用，因此，本发明所述的培养添加物也适用于其他种属如鼠、兔、绵羊或猪的肝细胞的体外培养。
在使用时，将本发明提供的培养添加物以1:100的比例加入到肝细胞的培养基中，可明显提高肝细胞中多种凝血因子的表达水平，满足了科学研究和应用开发对体外培养肝细胞的要求。
综上所述，本发明所提供的一种增强体外培养肝细胞凝血因子表达的培养添加物具有如下优点：（1）可明显增强体外培养肝细胞中凝血因子的表达；（2）无动物源性成分；（3）所有组分均为商业上易于获得的，制备简单，使用方便，价格低廉。
附图说明
图1. qPCR 检测采用本发明的培养添加物前后原代肝细胞中凝血因子表达情况图；
图2. qPCR 检测采用本发明的培养添加物前后永生化肝细胞中凝血因子表达情况图；
图3. qPCR 检测采用本发明的培养添加物前后干细胞分化的肝细胞中凝血因子表达情况图。
实施方式
下面结合附图和实施例进一步说明本发明，这些附图和实施例仅作为说明本发明的例子，不限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、
一种增强体外培养肝细胞凝血因子表达培养添加物，由以下组分组成：骨化三醇5μM、LY2157299 10μM、小檗碱 1mmol/L、前列腺素E2 0.5mmol/L、维生素K1 10mg/L。
实施例2、
一种增强体外培养肝细胞凝血因子表达培养添加物，由以下组分组成：卡泊三醇10μM、 A-83-01 500μM、小檗碱 10mmol/L、前列腺素E2 10mmol/L、维生素K1 500mg/L。
实施例3、
一种增强体外培养肝细胞凝血因子表达培养添加物，由以下组分组成：1,25(OH)2D310-7M、K02288 100μM、小檗碱 5mmol/L、前列腺素E2 5mmol/L、维生素K1 200mg/L。
效果验证
1、本发明的培养添加物对体外培养的原代肝细胞凝血因子表达的影响
原代肝细胞的获取为现有技术，参照文献描述(Bader A, Hansen T, KirchnerG, et al. Primary porcine enterocyte and hepatocyte cultures to study drugoxidation reactions. Pharmacol. 2000 Jan;129(2):331-42.)采用胶原酶灌注技术进行分离。从猪肝组织中经过胶原酶灌注消化法分离获得原代猪肝细胞，经离心洗涤后取1*106个肝细胞接种于2瓶T25细胞培养瓶中。接种时两瓶使用相同的肝细胞培养基：DMEM/F12基础培养基，添加10%胎牛血清、ITS（购自西格玛公司（Sigma公司），货号I3146）、肝细胞生长因子（20ng/ml）、胰岛素（160IU/L）和地塞米松（10-8M），接种完成后向其中一瓶中添加1%（v/v）的实施例1的培养添加物，此瓶细胞标记为“有添加物组”，另一瓶标记为“无添加物组”。分别培养14天后，提取两组细胞的总RNA，进行各种凝血因子的表达检测。
具体的，应用总RNA提取试剂盒（购自北京天根生化科技有限公司）提取两组细胞的总RNA，然后应用逆转录试剂盒（购自北京全式金生物技术股份有限公司）将1μg总RNA逆转录为cDNA。应用实时定量PCR 试剂盒-SYBR® Green Realtime PCR Master Mix (购自东洋纺（上海）生物科技有限公司)，在ABI 7900荧光定量PCR仪上进行各种凝血因子表达水平的检测，以管家基因β-actin作为内参。所有引物由上海生工生物工程有限公司合成，引物序列参照引物银行（PrimerBank）网站，具体检测的凝血因子及引物银行（PrimerBank）网站的引物ID号见表1。扩增条件为：初始变性温度95℃ 1min，循环变性温度95℃ 15s，退火和延伸温度60℃ 60s（收集信号），共40个循环，然后进行融解曲线分析。结果分析采用2-ΔCt的方法。
表1. PCR 扩增引物信息
| 凝血因子名称 | PrimerBank引物ID号 |
| 凝血因子Ⅰ (FⅠ) | 70906432c1 |
| 凝血因子Ⅱ (FⅡ) | 169808403c3 |
| 凝血因子Ⅴ (FⅤ) | 119395710c3 |
| 凝血因子Ⅶ (FⅦ) | 116805320c1 |
| 凝血因子Ⅷ (FⅧ) | 192448441c1 |
| 凝血因子Ⅸ (FⅨ) | 183979970c2 |
| 凝血因子Ⅹ (FⅩ) | 89142731c3 |
| 凝血因子Ⅺ (FⅪ) | 116805318c3 |
| 凝血因子Ⅻ (FⅫ) | 145275212c1 |
| β-actin | 4501885a1 |
qPCR程序结束后，检查融解曲线，确定没有二聚体或非特异扩增产物干扰后，进行结果处理，将各凝血因子的Ct值减去β-actin的Ct值，得到ΔCt，计算2-ΔCt，即各凝血因子表达水平相对于β-actin表达水平的倍数，结果如附图1所示，各凝血因子的表达水平各不相同，以凝血因子1和2表达较强，而凝血因子Ⅴ、Ⅶ和Ⅷ的表达较低。但和无添加物组相比，有添加物组的各凝血因子表达水平均有所上调，特别是凝血因子Ⅴ、Ⅶ和Ⅷ均提高到可容易检测到的水平。
2、本发明的培养添加物对体外培养的永生化肝细胞凝血因子表达的影响
采用文献描述方法进行小鼠原代肝细胞的分离和永生化（Song XG, Bian PF, YuSL, et al. Expression of hepatitis B virus 1.3-fold genome plasmid in an SV40T-antigen-immortalized mouse hepatic cell line. World J Gastroenterol. 2013Nov 28;19(44):8020-7.）经筛选获得永生化小鼠肝细胞，使用效果验证1中描述的肝细胞培养基进行培养。使用第10代的细胞进行本实验，将细胞分别接种于2个T25瓶中，一瓶使用肝细胞培养基（无添加物组），另一瓶使用添加了1%(v/v)实施例2的培养添加物的肝细胞培养基（有添加物组）。分别培养3天后，提取两组细胞的总RNA，进行各种凝血因子的表达检测。检测的基因和使用的引物同效果验证1。
总RNA的提取、逆转录、荧光定量PCR过程和结果处理方法同效果验证1。
结果如附图2所示，各凝血因子的表达水平各不相同，以凝血因子1和2表达较强，而凝血因子Ⅴ、Ⅶ和Ⅷ的表达非常低。但和无添加物组相比，有添加物组各凝血因子的表达水平均明显上调，凝血因子Ⅴ、Ⅶ和Ⅷ均提高到可检测水平。
3、本发明的培养添加物对体外培养的干细胞分化的肝细胞凝血因子表达的影响
采用文献描述方法将人多能干细胞细胞向肝细胞进行分化（Chen Y‐F, Tseng C‐Y, Wang H‐W, et al. Rapid generation of mature hepatocyte‐like cells fromhuman induced pluripotent stem cells by an efficient three‐step protocol.Hepatology. 2012;55(4):1193‐1203.）。将所得到的人多能干细胞源的肝细胞同样接种在2个T25瓶中，一瓶使用肝细胞培养基（无添加物组），另一瓶使用添加了1%(v/v)实施例3的培养添加物的肝细胞培养基（有添加物组）。分别培养3天后，提取两组细胞的总RNA，进行各种凝血因子的表达检测。检测的基因和使用的引物同效果验证1。
总RNA的提取、逆转录、荧光定量PCR过程和结果处理方法同效果验证1。
结果如附图3所示，无添加物组，人源多能干细胞源肝细胞表达各凝血因子的表达水平也以凝血因子1和2表达较强，而凝血因子Ⅴ、Ⅶ和Ⅷ的表达低于检测限度，而有添加物组各凝血因子的表达水平均明显上调，说明使用本发明的培养添加物可有效改善该细胞的凝血因子表达。
以上所述仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是在本发明的构思下，利用本发明说明书及附图内容所作的等效变换，或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。
Claims (7)
1.一种增强体外培养肝细胞凝血因子表达的培养添加物，其特征在于，所述培养添加物由以下组分组成：骨化三醇5μM、LY2157299 10μM、小檗碱1mmol/L、前列腺素E20.5mmol/L、维生素K1 10mg/L。
2.一种增强体外培养肝细胞凝血因子表达的培养添加物，其特征在于，所述培养添加物由以下组分组成：卡泊三醇10μM、A-83-01 500μM、小檗碱10mmol/L、前列腺素E210mmol/L、维生素K1 500mg/L。
3.一种增强体外培养肝细胞凝血因子表达的培养添加物，其特征在于，所述培养添加物由以下组分组成：1,25(OH)2D3 10-7M、K02288 100μM、小檗碱5mmol/L、前列腺素E25mmol/L、维生素K1 200mg/L。
4.根据权利要求1～权利要求3所述的培养添加物在增强体外培养肝细胞的凝血因子表达上的应用。
5.根据权利要求4所述的应用，其中本发明的培养添加物以1：100的比例添加到培养基中。
6.根据权利要求4所述的应用，其特征在于，所述肝细胞来源于哺乳动物。
7.根据权利要求6所述的应用，其特征在于，所述哺乳动物为人、鼠或猪。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311758235.2A CN117431203B (zh) | 2023-12-20 | 2023-12-20 | 一种增强体外培养肝细胞凝血因子表达的培养添加物及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311758235.2A CN117431203B (zh) | 2023-12-20 | 2023-12-20 | 一种增强体外培养肝细胞凝血因子表达的培养添加物及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117431203A CN117431203A (zh) | 2024-01-23 |
| CN117431203B true CN117431203B (zh) | 2024-03-22 |
Family
ID=89548357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311758235.2A Active CN117431203B (zh) | 2023-12-20 | 2023-12-20 | 一种增强体外培养肝细胞凝血因子表达的培养添加物及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117431203B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330099A (zh) * | 2017-03-22 | 2018-07-27 | 上海赛立维生物科技有限公司 | 个性化肝细胞的培养和扩增方法及其应用 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020172792A1 (en) * | 2019-02-26 | 2020-09-03 | Peking University | Compositions and methods for long term culture of hepatocytes |
| JPWO2021125176A1 (zh) * | 2019-12-16 | 2021-06-24 |
-
2023
- 2023-12-20 CN CN202311758235.2A patent/CN117431203B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330099A (zh) * | 2017-03-22 | 2018-07-27 | 上海赛立维生物科技有限公司 | 个性化肝细胞的培养和扩增方法及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117431203A (zh) | 2024-01-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Suthen et al. | Hypoxia‐driven immunosuppression by Treg and type‐2 conventional dendritic cells in HCC | |
| CN109749986B (zh) | 一种由人多能干细胞分化获得胰腺前体细胞及胰岛β细胞的方法 | |
| Jansen et al. | Functional differences between mesenchymal stem cell populations are reflected by their transcriptome | |
| Li et al. | Hepatoblast-like progenitor cells derived from embryonic stem cells can repopulate livers of mice | |
| Peters et al. | Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells | |
| Garvin et al. | The in vitro growth, heterotransplantation, and differentiation of a human rhabdomyosarcoma cell line. | |
| CN110804586B (zh) | 一种临床级人诱导多能干细胞源间充质干细胞的制备方法及试剂盒 | |
| Govarthanan et al. | Glycogen synthase kinase 3β inhibitor-CHIR 99021 augments the differentiation potential of mesenchymal stem cells | |
| TW201138790A (en) | Hepatic progenitor cells and uses thereof | |
| CN117431203B (zh) | 一种增强体外培养肝细胞凝血因子表达的培养添加物及其应用 | |
| EP1385936A2 (en) | Method for the isolation of stem cells by immuno-labeling with hla/mhc gene product marker | |
| CN109913461B (zh) | 根尖牙乳头干细胞外泌体piRNA生物标志物及其筛选应用 | |
| Devito et al. | Comparison of human isogeneic Wharton’s jelly MSCs and iPSC-derived MSCs reveals differentiation-dependent metabolic responses to IFNG stimulation | |
| Gomes-Alves et al. | In vitro expansion of human cardiac progenitor cells: exploring'omics tools for characterization of cell-based allogeneic products | |
| KR101981450B1 (ko) | 인간 제대 조직-유래된 세포의 검출 | |
| Leung et al. | mAb C19 targets a novel surface marker for the isolation of human cardiac progenitor cells from human heart tissue and differentiated hESCs | |
| Sivalingam et al. | Improved erythroid differentiation of multiple human pluripotent stem cell lines in microcarrier culture by modulation of Wnt/β-Catenin signaling | |
| US20050054096A1 (en) | Pluripotent cells from monocytes, and methods of making and using pluripotent cells | |
| CN117802031A (zh) | 肝细胞的体外扩增培养方法及应用 | |
| Kuci et al. | Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells | |
| Harkness et al. | Molecular characterisation of stromal populations derived from human embryonic stem cells: Similarities to immortalised bone marrow derived stromal stem cells | |
| JPWO2002103033A1 (ja) | ｄｌｋを用いた未分化肝細胞の検出および分離方法 | |
| Hasan et al. | In vitro differentiation of human umbilical cord blood mesenchymal stem cells into functioning hepatocytes | |
| KR102018783B1 (ko) | 간세포의 약물대사 기능 향상방법 | |
| CN113106160A (zh) | 评估肝谱系细胞成熟度的标志物、双组学试剂盒及构建方法 |
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 |