WO2016119085A1 - 对甲氧基桂皮酸乙酯及其衍生物在维持干细胞自我更新和多潜能性中的应用 - Google Patents

对甲氧基桂皮酸乙酯及其衍生物在维持干细胞自我更新和多潜能性中的应用 Download PDF

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WO2016119085A1
WO2016119085A1 PCT/CN2015/000311 CN2015000311W WO2016119085A1 WO 2016119085 A1 WO2016119085 A1 WO 2016119085A1 CN 2015000311 W CN2015000311 W CN 2015000311W WO 2016119085 A1 WO2016119085 A1 WO 2016119085A1
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ethyl
och
methoxycinnamate
stem cells
trans
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PCT/CN2015/000311
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English (en)
French (fr)
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鲍永利
李玉新
秦洪双
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东北师范大学
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Priority to US15/546,504 priority Critical patent/US10723998B2/en
Publication of WO2016119085A1 publication Critical patent/WO2016119085A1/zh
Priority to AU2017100034A priority patent/AU2017100034A4/en

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    • 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
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the invention belongs to the technical field of biomedicine and relates to the application of a small molecule compound, namely ethyl p-methoxycinnamate and its derivatives, in maintaining self-renewal and pluripotency of stem cells.
  • the invention further relates to the use of such small molecule compounds for the preparation of medicaments and cosmetic skin care products for the treatment of cell-deficient or damaging diseases, as well as for the in vitro expansion of stem cells and the preparation of induced pluripotent stem cell iPS.
  • Stem cells are a class of primitive cells with self-renewal capacity and multi-directional differentiation potential, including embryonic stem cells, adult stem cells, and induced pluripotent stem cell iPS. Stem cells have very important theoretical research significance and clinical application value.
  • the use of embryonic stem cells to establish in vitro differentiation systems for a variety of cell types allows one to study mammalian development at the cellular level; to compare embryonic stem cells and gene transcription and expression of stem cells and differentiated cells at different developmental stages, to determine embryos The molecular mechanism of development and cell differentiation, as well as the discovery of new genes; stem cell research provides new research tools for the pharmacology, pharmacodynamics, toxicology and pharmacokinetics of new drugs, overcoming the above experiments can not be directly carried out in the human body.
  • stem cells Difficulties, while improving the effectiveness and safety of drug applications; stem cells also have important applications in regenerative medicine.
  • the application of stem cells in regenerative medicine involves all tissues and organs of the human body, as well as most medical problems faced by humans, such as Cardiovascular disease, diabetes, Parkinson's disease, cancer, Alzheimer's disease, autoimmune diseases, severe burns, Spinal cord injury, hereditary diseases, osteoporosis, aging, etc.
  • stem cell transplantation has become an important means of treating diseases such as cell transplantation and organ transplantation.
  • Cell transplantation therapy is the purpose of healing by transplanting healthy stem cells into a patient, repairing or replacing damaged cells or tissues.
  • Organ transplantation treatment is to use stem cells as "seeds", which are induced to differentiate in vitro to form the required tissues and organs, thereby replacing the diseased tissues and organs.
  • stem cells for the treatment of stem cells in clinical practice, there should be sufficient sources. For example, adult blood transfusion cells usually require 1-4 ⁇ 10 6 /kg body weight, and the amount of stem cells in each cord blood is only about 1 ⁇ 10 7 . Therefore the current treatment is limited to children.
  • growth factors and biologically active proteins can be used as regulating drugs for stem cells
  • growth factors and biologically active proteins can act as macromolecular active substances, which can intervene in the physiological process of crisscrossing in vivo, exhibit extremely complicated multi-regulation functions, and are expensive and difficult to be used. Has medicinal and clinical therapeutic value. Therefore, the potential of small molecule compounds as stem cell regulating drugs has received increasing attention.
  • Small molecule compounds have their unique advantages as drugs or medium additives: small molecule compounds are easy to administer and easy to withdraw after physiological function recovery; small molecule compounds are easy to synthesize and industrialize, and have good medicinal prospects; small Molecular compounds, especially natural small molecule compounds, undergo a biological metabolic process and have good biocompatibility, especially with less toxic side effects. So looking for stem cells The small molecule compound regulator has become a research hotspot of stem cell drugs.
  • the present invention provides the use of ethyl p-methoxycinnamate and its derivatives for the preparation of a self-renewing and pluripotent drug, medium or modulator for maintaining stem cells.
  • R 1 is 4-OCH 3
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 4-OCH 3
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH 2 CH(CH 3 )CH 3 ;
  • R 1 is 4-OCH 3
  • R 2 is H
  • R 3 is COOCH 2 CH 3
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 4-OCH 3
  • R 2 is H
  • R 3 is COCH 3
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 4-OCH 3
  • R 2 is H
  • R 3 is CN
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 4-OCH 3
  • R 2 is CH 3
  • R 3 is H
  • R 4 is -CH 2 CH(CH 2 CH 3 )CH 2 CH 2 CH 2 CH 3 ;
  • R 1 is 3,4-OCH 3
  • R 2 is H
  • R 3 is COCH 2 CH 3
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 3,4-OCH 3
  • R 2 is H
  • R 3 is CN
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 2,4-OCH 3
  • R 2 is H
  • R 3 is CN
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 3,4,5-OCH 3 , R 2 is CH 3 , R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 2-OCH 2 CH 3 , R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 4-OCH 2 CH 3 , R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 4-OCH 2 CH 2 CH 2 CH 3 , R 2 is H, R 3 is CN, and R 4 is -CH 2 CH 3 ;
  • R 1 is 3-COOCH 3 , R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 2-CH 3 -5-COOCH 3
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 4-OCOCH 3
  • R 2 is H
  • R 3 is COCH 2 CH 3
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 3-OCH 3 -4-OCH 2 COOCH 2 CH 3 , R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 2-OH, R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 3,4-OH, R 2 is H, R 3 is H, and R 4 is -CH 2 CHC(CH 3 )CH 3 ;
  • R 1 is 3,4-OH, R 2 is H, R 3 is H, and R 4 is -CH 2 CH 2 C 6 H 5 ;
  • R 1 is 3,4-OH
  • R 2 is H
  • R 3 is H
  • R 4 is
  • R 1 is 3,4-OH
  • R 2 is H
  • R 3 is H
  • R 4 is
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH 3 ;
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is -CH(CH 3 )CH 3 ;
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH 2 CH 2 CH 3 ;
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH(CH 3 )CH 3 ;
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is -CH(CH 3 )CH 2 CH 3 ;
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH 2 CH(CH 3 )CH 3 ;
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH 2 C 6 H 5 ;
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH(CH 3 )C 6 H 5 ;
  • R 1 is H
  • R 2 is H
  • R 3 is CN
  • R 4 is -CH 2 CH 3 ;
  • R 1 is H
  • R 2 is CH 3
  • R 3 is H
  • R 4 is -CH 2 CH 3 ;
  • R 1 is H
  • R 2 is C 6 H 5
  • R 3 is CN
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 2,6-Cl
  • R 2 is H
  • R 3 is H
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 4-Br, R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 2-CF 3 , R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 2-OH-4-Br, R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 4-Cl, R 2 is H, R 3 is COOCH 2 CH 3 , and R 4 is -CH 2 CH 3 ;
  • R 1 is 4-Cl, R 2 is H, R 3 is OCH 2 CH 3 , and R 4 is -CH 2 CH 3 ;
  • R 1 is 3,4-F
  • R 2 is H
  • R 3 is OCH 2 CH 3
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 2-Cl-4-F, R 2 is H, R 3 is CN, and R 4 is -CH 2 CH 3 ;
  • R 1 is 3-Cl-4-OCH 3
  • R 2 is H
  • R 3 is CN
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 2-Cl-4-OCH 3
  • R 2 is H
  • R 3 is CN
  • R 4 is -CH 2 CH 3 ;
  • R 1 is 4-NH 2 , R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 3-CN, R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 4-NO 2 , R 2 is H, R 3 is H, and R 4 is -CH 2 CH 3 ;
  • R 1 is 4-(2,4-FC 6 H 4 ), R 2 is CH 3 , R 3 is H, and R 4 is —CH 2 CH 3 ;
  • R 1 is 4-OCH 3
  • R 2 is H
  • R 3 is H
  • R 1 is 4-OCH 3
  • R 2 is H
  • R 3 is H
  • R 1 , R 2 , R 3 form a heterocyclic structure
  • R 4 is -CH 2 CH 3 ;
  • R 1 , R 2 , R 3 form a heterocyclic structure
  • R 4 is -CH 2 CH 3 ;
  • R 1 , R 2 , R 3 form a heterocyclic structure
  • R 4 is -CH 2 CH 3 ;
  • R 1 , R 2 , R 3 form a heterocyclic structure
  • R 4 is -CH 2 CH 3 ;
  • R 1 is -N-CH 3 (CH 3 ), R 2 is H, and R 3 and R 4 form a heterocyclic structure
  • the ethyl p-methoxycinnamate and its derivatives according to the present invention include: ethyl p-methoxycinnamate, isoamyl methoxycinnamate, and diethyl 2-benzylbenzylidene malonate.
  • Ethyl ester ethyl 2-(4-methoxybenzylidene)acetate, ethyl 2-cyano-3-(4-hydroxyethylphenyl)acrylate, octyl 4-methoxycinnamate, 2- (3,4-dimethoxybenzylidene) diethyl malonate, ethyl 2-cyano-3-(3,4-dimethoxyphenyl)-2-butenoate, trans Ethyl 2-cyano-3-(2,4-dimethoxyphenyl)-2-propenoate, trans 3-(3,4,5-trimethoxyphenyl)-2-butenoic acid ethyl ester, Ethyl 2-hydroxyethyl cinnamate, ethyl trans-4-ethoxycinnamate, ethyl 3-(4-butoxyphenyl)-2-cyano-acrylate, 3-(trans-3 Methyl-hydroxyethyl-3-oxo-1-prop
  • the stem cells of the present invention are adult stem cells, embryonic stem cells or induced pluripotent stem cells iPS.
  • the ethyl p-methoxycinnamate and its derivatives maintain the self-renewal ability and pluripotency state of stem cells by promoting the expression of the Oct4 gene.
  • the pharmaceutical composition of the present invention comprises a therapeutically effective amount of ethyl p-methoxycinnamate and a derivative thereof, and the carrier is a solvent, a dispersing agent, a coating, an antibacterial and an antifungal agent. , isotonic agent and absorption delaying agent.
  • the medicament of the present invention can be diluted or directly used by a user before use.
  • the formulation can be prepared by conventional processing methods well known to those skilled in the art.
  • the medicament of the present invention can be prepared into a pharmaceutical dosage form suitable for different administration routes as needed, and a more preferred dosage form is a capsule or a tablet.
  • the capsules may comprise any standard pharmaceutically acceptable substance such as gelatin or cellulose.
  • Tablets can be prepared by compressing the pharmaceutical composition with a solid support and a lubricant in a conventional manner.
  • the solid support comprises starch and glaze.
  • compositions of the invention may also be administered parenterally.
  • Dosage forms for parenteral administration include aqueous solutions of the pharmaceutical compositions of the invention, isotonic saline solutions or 5% sugar solutions, and formulations formed with other pharmaceutically acceptable excipients known in the art.
  • the p-methoxycinnamic acid ethyl ester and the derivative thereof according to the present invention have a self-renewal ability and a pluripotent state pharmaceutical preparation dosage in the range of 0.01 to 100 mg/kg.
  • ethyl p-methoxycinnamate and its derivatives in the preparation of stem cells in vitro is specifically used for expanding stem cells in vitro and preparing inducible pluripotent stem cells iPS.
  • the present invention also provides the use of ethyl p-methoxycinnamate and a derivative thereof for the preparation of a medicament for treating a cell loss or an injurious disease, the damage disease being a disease of a cell, a tissue or an organ, specifically It is a nervous system disease, an immune system disease, a blood system disease, a cardiovascular disease or a skin disease.
  • the invention also provides the use of ethyl p-methoxycinnamate and its derivatives in the preparation of cosmetic skin care products.
  • the ethyl p-methoxycinnamate and its derivatives of the present invention can be suspended in a pharmaceutically acceptable carrier (such as a physiological solution) by oral or intravenous infusion, or by subcutaneous, intramuscular, intrathoracic, intraperitoneal, rectal Internal, intravaginal, intranasal, intragastric, intragastric, intrapulmonary injection or infusion, etc. Route administration.
  • a pharmaceutically acceptable carrier such as a physiological solution
  • the ethyl p-methoxycinnamate and its derivatives of the invention can be suspended in a cosmetic skin care product, and the effect of activating skin stem cells can be achieved by smearing.
  • the ethyl p-methoxycinnamate and its derivatives of the present invention can be directly added to a cell culture solution for expanding stem cells in vitro.
  • the beneficial effects of the present invention are as follows: by studying small molecule compounds, it is found that ethyl p-methoxycinnamate and its derivatives can promote the expression of Oct4 gene, thereby maintaining the self-renewal ability and pluripotent state of stem cells, and can be used as Regulatory drugs or media additives that maintain stem cell self-renewal and pluripotency can provide a large number of stem cells for clinical transplantation, providing new insights and new ideas for the treatment of cell loss or injury diseases.
  • Figure 1 is the effect of ethyl p-methoxycinnamate on the activity of Oct4 promoter
  • Figure 2 is the effect of ethyl p-methoxycinnamate on the expression of Oct4 mRNA
  • Figure 3 is the effect of ethyl p-methoxycinnamate on the expression of Oct4 protein
  • Figure 4 is a graph showing the effect of ethyl p-methoxycinnamate on the formation of stem cell microspheres in suspension culture
  • Figure 5 is a graph showing the effect of ethyl p-methoxycinnamate on the formation of adherent cultured stem cell microspheres
  • Figure 6 is a graph showing the effect of a derivative of p-methoxycinnamate on the expression of Oct4;
  • a for chlorogenic acid B for ethyl 2-cyano-3,3-diphenylacrylate, and C for ⁇ -2 - ethyl carboxylate
  • D represents ethyl cinnamate
  • E represents 3-(4-methoxyphenyl)-acrylic acid-4-[3-(4-methoxyphenyl)-acryloyloxy]-butyl-2 - alkynyl ester
  • F stands for 3-(4-methoxybenzene)-acrylic acid -3-[3-(4-Methoxyphenyl)-acryloyloxymethyl]-phenylmethyl ester
  • G represents 3-propylacrylic acid-3-cincosyloxypropyl ester
  • H represents 4-acetic acid Diethyl benzylidene malonate.
  • Transfection was performed according to the instructions using Engreen's liposome transfection reagent EntransterTM-D.
  • the target plasmid pGL3-Oct4P-Basic was transfected into P19 cells, and the total amount of plasmid used for transfection was 3 ⁇ g/well.
  • the plasmid was added to 50 ⁇ L of serum-free DMEM medium and mixed well. Then, 9 ⁇ L of the transfection reagent was added to another 50 ⁇ L of serum-free DMEM medium, mixed, and allowed to stand for 5 min.
  • the DMEM medium containing the transfection reagent was added to the DMEM medium containing the plasmid, and immediately squirted 10 times or more, and allowed to stand at room temperature for 30 minutes.
  • 900 ⁇ L of DMEM medium containing 10% fetal calf serum was added to the mixture, and gently mixed. Discard the old cell culture medium in the 6-well plate and replace with the above mixture.
  • the cells were resuspended in DMEM medium containing 10% fetal bovine serum. The above cells were seeded at a density of 8 x 103 cells/well in 96-well cell plates.
  • the medium was aspirated, DMEM medium containing 3% fetal bovine serum was added, and the drug to be screened was added to the medium to a final concentration of 5 ⁇ g/mL, and DMSO was used as a control. After continuing to culture for 12-24 hours, the cells were lysed and assayed for luciferase activity.
  • Luciferase activity of transfected cell lysates was detected using Luciferase Reporter Assay System and FluoStar Optima. Take 45 ⁇ L of cell lysate, add 5 ⁇ L of Assay Cocktail reagent, mix and add 100 ⁇ L of Luciferin reaction solution, and measure the fluorescence count of 2 s using FluoStar Optima instrument. This count reflects the expression of the plasmid reporter gene expression firefly luciferase. Activity level.
  • pCMV- ⁇ -gal expresses the level of activity of ⁇ -galactosidase.
  • the ratio of the activity of firefly luciferase to the activity of ⁇ -galactosidase ( ⁇ -gal) eliminates the difference in transfection efficiency of the sample and can objectively reflect the difference in promoter activity between samples.
  • the relative activity of luciferase in different samples was calculated by the following formula:
  • P19 cells were seeded at a density of 2 ⁇ 10 5 /well in 6-well cell culture plates. After 24 h, the cells were replaced with DMEM medium containing 3% serum. Methyl p-methoxycinnamate was added to the experimental group to a final concentration of 5 ⁇ g/mL, and the control group was treated with DMSO. After 12 h, the 6-well plates overgrown with monolayers were removed from the cell culture incubator and the cells were washed 3 times with pre-chilled PBS. Add 1 mL of pre-cooled Trizol solution, transfer vigorously to EP tube, and let stand at room temperature for 5 min.
  • Reverse transcription was performed using TaKaRa's Reverse Transcription System kit, and the reaction system was as follows:
  • RNA template 0.1% DEPC water and Oligo(dT)15primer
  • a clean EP tube water bath at 70 ° C for 2 min. Centrifuge at 1000 rpm for 1 min. The other components of the reverse transcription were added to the EP tube and a water bath at 42 ° C for 1 h. The reaction was stopped at 70 ° C for 8 min, and finally 0.1% DEPC water was added to 100 ⁇ L.
  • Upstream primer 5'-CTCGAACCACATCCTTCTCT-3'; downstream primer: 5'-TCGTGCGTGACATTAAGGAG-3.
  • the PCR product was added to the 10 ⁇ gel loading buffer in proportion, mixed, and carefully added to the sample wells with a micropipette. After the sample is loaded, close the electrophoresis tank cover and turn on the power to keep the voltage at 100-140V. When the bromophenol blue band moved about 2 cm from the gel front, electrophoresis was stopped and observed and photographed using a gel imaging system.
  • lysate 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM was added to each tube pellet.
  • NaF, 0.5% NP-40, 2 ⁇ g/mL Aprotinin, 1 mM PMSF) incubated on ice for 30 min, vortexed every 5 min for sufficient cell lysis. After centrifugation at 12000 rpm for 10 min, the supernatant was transferred to a new EP tube, and 1/3 volume of 4 ⁇ protein loading buffer was added, mixed, boiled for 10 min, centrifuged or centrifuged, or placed at 80 ° C for free storage. .
  • the transmembrane buffer was prepared and pre-cooled at 4 °C.
  • the PVDF membrane was immersed in methanol for 40 s, taken out, and immersed in a transfer buffer for use. Place the transfer buffer in the square plate and install the transfer device under the liquid surface.
  • the membrane transfer device was placed in an electrophoresis tank, and the membrane buffer was poured. After the membrane was transferred at 100 V for 2 hours, the PVDF membrane was taken out, and the membrane was labeled with the front and back surfaces and the electrophoresis direction. Wash the PVDF membrane with TBST (0.2% Tween20), wash once every 5 minutes, wash a total of 2 After that, it was blocked with 5% skim milk powder for 2 h at room temperature.
  • the secondary antibody HRP-goat anti-mouse (0.8 mg/mL) was diluted 1:2000 with TBST (0.2% Tween 20), and then placed in a hybridization bag together with the PVDF membrane, and incubated for 40 min at room temperature on a shaker.
  • the PVDF membrane was taken out, washed rapidly with TBST (0.2% Tween 20) for 2 times, and then washed 5 times 2 times, 15 minutes 3 times.
  • the PVDF film was subjected to ECL color development.
  • Example 4 p-Methoxycinnamate and its derivatives promote the formation of stem cell microspheres in suspension culture
  • Example 5 p-Methoxy cinnamate promotes the formation of stem cell microspheres in adherent culture
  • mouse teratoma cells P19 were seeded in a 6-well cell plate at a density of 1 ⁇ 10 4 cells/well, and 2 mL was supplemented with DMEM medium containing 10% fetal calf serum. Gently shake the cell culture plate to evenly distribute the cells in the cell culture plate, and then incubate them in a cell culture incubator. After 24 h, the old medium was discarded, and 3% fetal bovine serum DMEM medium containing p-methoxycinnamate was added to give a final concentration of p-methoxycinnamate ethyl 5 ⁇ g/mL.
  • the old medium was discarded every 24 hours, and a new DMEM medium containing 3% fetal bovine serum was added, and the final concentration of the ethyl p-methoxycinnamate was also 5 ⁇ g/mL. After continuous culture for 22 days, the cells were photographed.
  • the plasmid was added to 25 ⁇ L of serum-free DMEM medium and mixed well. Then 3 ⁇ L of the transfection reagent was added to an additional 25 ⁇ L of serum-free DMEM medium, mixed, and allowed to stand for 5 min. Thereafter, the DMEM medium containing the transfection reagent was added to the DMEM medium containing the plasmid, and immediately blown 10 times or more, and allowed to stand at room temperature for 30 minutes. 250 ⁇ L of DMEM medium containing 10% fetal calf serum was added to the mixture, and gently mixed. Discard the old cell culture medium in the 24-well plate and replace with the above mixture.
  • Luciferase activity of transfected cell lysates was detected using Luciferase Reporter Assay System and FluoStar Optima. Take 45 ⁇ L of cell lysate, add 5 ⁇ L of Assay Cocktail reagent, mix and add 100 ⁇ L of Luciferin reaction solution, and measure the fluorescence count of 2 s using FluoStar Optima instrument. This count reflects the expression of the plasmid reporter gene expression firefly luciferase. Activity level. Another 20 ⁇ L of cell lysate was added, 37.5 ⁇ L ⁇ -gal Buffer was added, 12.5 ⁇ L ONPG (6 mg/mL) was added, and the mixture was allowed to stand at 37 ° C for 30 min.
  • the absorbance at 450 nm was measured by a microplate reader. This value reflects the internal reference.
  • the plasmid pCMV- ⁇ -gal expresses the level of activity of ⁇ -galactosidase.
  • the ratio of the activity of firefly luciferase to the activity of ⁇ -galactosidase ( ⁇ -gal) eliminates the difference in transfection efficiency of the sample and can objectively reflect the difference in promoter activity between samples.
  • the relative activity of luciferase in different samples was calculated by the following formula:
  • A represents chlorogenic acid
  • B represents ethyl 2-cyano-3,3-diphenylacrylate
  • C represents ethyl phthalate-2-carboxylate
  • D represents ethyl cinnamate
  • E represents 3-(4).
  • F represents 3-(4-methoxyphenyl)-acrylic acid-3- [3-(4-Methoxyphenyl)-acryloyloxymethyl]-phenylmethyl ester
  • G represents 3-propylacrylic acid-3-cincosyloxypropyl ester
  • H represents 4-acetoxybenzoic acid Diethyl methylmalonate.

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Abstract

本发明公开了对甲氧基桂皮酸乙酯及其衍生物在制备维持干细胞自我更新和多潜能性药物、培养基、调节剂或美容护肤品中的应用，所述的干细胞包括成体干细胞、胚胎干细胞和诱导性多潜能干细胞iPS。本发明还公开了对甲氧基桂皮酸乙酯及其衍生物在体外扩增干细胞及制备诱导性多潜能干细胞iPS中的应用，以及对甲氧基桂皮酸乙酯及其衍生物在制备治疗细胞缺失或损伤性疾病的药物中的应用。

Description

对甲氧基桂皮酸乙酯及其衍生物在维持干细胞自我更新和多潜能性中的应用 技术领域
本发明属于生物医药技术领域，涉及一类小分子化合物即对甲氧基桂皮酸乙酯及其衍生物在维持干细胞自我更新和多潜能性中的应用。本发明还涉及此类小分子化合物在制备治疗细胞缺失或损伤性疾病的药物及美容护肤品中的用途，以及在体外扩增干细胞及制备诱导性多潜能干细胞iPS中的用途。
背景技术
干细胞是一类具有自我更新能力和多向分化潜能的原始细胞，包括胚胎干细胞、成体干细胞和诱导性多潜能干细胞iPS。干细胞具有非常重要的理论研究意义和临床应用价值。利用胚胎干细胞建立起多种细胞类型的体外分化系统，使得人们能够在细胞分子水平上研究哺乳动物的发育过程；比较胚胎干细胞以及不同发育阶段的干细胞和分化细胞的基因转录和表达，能够确定胚胎发育及细胞分化的分子机制，同时能够发现新的基因；干细胞的研究为新药的药理、药效、毒理及药代等提供了新的研究手段，克服了以上实验不能直接在人体内进行的困难，同时提高了药物应用的效果和安全性；干细胞在再生医学中也有着重要的应用，干细胞在再生医学中的应用涉及人体所有的组织和器官，也涉及人类面临的大多数医学难题，如心血管疾病、糖尿病、帕金森病、癌症、早老性痴呆、自身免疫性疾病、严重烧伤、 脊髓损伤、遗传性疾病、骨质疏松、衰老等。
在再生医学中，干细胞移植治疗已经成为治疗疾病的重要手段，如细胞移植、器官移植等。细胞移植治疗是把健康的干细胞移植到患者体内，修复或替换受损细胞或组织，从而达到治愈的目的。器官移植治疗是以干细胞为“种子”，在体外经过定向的诱导分化，形成需要的组织和器官，从而替换病变的组织和器官。干细胞应用于临床治疗，首先应该有充足的来源，例如成人输用血液干细胞一般要求1-4×106个/kg体重，而每份脐血中干细胞的含量仅在1×107个左右，因此目前的治疗仅限于儿童。临床应用表明输入的量越大，得以成功移植归巢的越多，长期生根繁殖的机率也更大。但是哺乳动物的干细胞数量极少，且倾向于自发的分化，所以很难在体外进行长期、大量地培养。若能设法在体外分离培养过程中提高干细胞的获得率，无疑将为干细胞的临床应用开辟广阔的前景。
虽然生长因子和生物活性蛋白可以作为干细胞的调节药物，但是生长因子和生物活性蛋白作为大分子活性物质，可以介入体内纵横交错的生理过程，呈现极其复杂的多重调控功能，且价格昂贵，很难具有药用和临床治疗价值。因此，小分子化合物作为干细胞调节药物的潜力越来越受到人们的重视。小分子化合物作为药物或培养基添加剂有其独特的优势：小分子化合物易于给药，在生理功能恢复后又易于撤出；小分子化合物易于人工合成及工业化生产，具有良好的药用前景；小分子化合物尤其是天然小分子化合物经过了生物代谢过程，具有良好的生物适应性，尤其具有较小的毒副作用。因此，寻找干细胞 的小分子化合物调节剂成为了干细胞药物的研究热点。
发明内容
本发明的目的在于提供一类具有维持干细胞自我更新和多潜能性作用的小分子化合物即对甲氧基桂皮酸乙酯及其衍生物。
本发明具体通过以下技术方案实现：
本发明提供了对甲氧基桂皮酸乙酯及其衍生物在制备维持干细胞自我更新和多潜能性药物、培养基或调节剂中的应用。
所述的对甲氧基桂皮酸乙酯及其衍生物的结构通式如下所示：
Figure PCTCN2015000311-appb-000001
其中R1为4-OCH3，R2为H，R3为H，R4为-CH2CH3；
或R1为4-OCH3，R2为H，R3为H，R4为-CH2CH2CH(CH3)CH3；
或R1为4-OCH3，R2为H，R3为COOCH2CH3，R4为-CH2CH3；
或R1为4-OCH3，R2为H，R3为COCH3，R4为-CH2CH3；
或R1为4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
或R1为4-OCH3，R2为CH3，R3为H，R4为-CH2CH(CH2CH3)CH2CH2CH2CH3；
或R1为3，4-OCH3，R2为H，R3为COCH2CH3，R4为-CH2CH3；
或R1为3，4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
或R1为2，4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
或R1为3，4，5-OCH3，R2为CH3，R3为H，R4为-CH2CH3；
或R1为2-OCH2CH3，R2为H，R3为H，R4为-CH2CH3；
或R1为4-OCH2CH3，R2为H，R3为H，R4为-CH2CH3；
或R1为4-OCH2CH2CH2CH3，R2为H，R3为CN，R4为-CH2CH3；
或R1为3-COOCH3，R2为H，R3为H，R4为-CH2CH3；
或R1为2-CH3-5-COOCH3，R2为H，R3为H，R4为-CH2CH3；
或R1为4-OCOCH3，R2为H，R3为COCH2CH3，R4为-CH2CH3；
或R1为3-OCH3-4-OCH2COOCH2CH3，R2为H，R3为H，R4为-CH2CH3；
或R1为2-OH，R2为H，R3为H，R4为-CH2CH3；
或R1为3，4-OH，R2为H，R3为H，R4为-CH2CHC(CH3)CH3；
或R1为3，4-OH，R2为H，R3为H，R4为-CH2CH2C6H5；
或R1为3，4-OH，R2为H，R3为H，R4为
Figure PCTCN2015000311-appb-000002
或R1为3，4-OH，R2为H，R3为H，R4为
Figure PCTCN2015000311-appb-000003
或R1为H，R2为H，R3为H，R4为-CH2CH3；
或R1为H，R2为H，R3为H，R4为-CH(CH3)CH3；
或R1为H，R2为H，R3为H，R4为-CH2CH2CH2CH3；
或R1为H，R2为H，R3为H，R4为-CH2CH(CH3)CH3；
或R1为H，R2为H，R3为H，R4为-CH(CH3)CH2CH3；
或R1为H，R2为H，R3为H，R4为-CH2CH2CH(CH3)CH3；
或R1为H，R2为H，R3为H，R4为-CH2CH2CH2OOCCH＝CHC6H5；
或R1为H，R2为H，R3为H，R4为-CH2CH2C6H5；
或R1为H，R2为H，R3为H，R4为-CH2CH(CH3)C6H5；
或R1为H，R2为H，R3为CN，R4为-CH2CH3；
或R1为H，R2为CH3，R3为H，R4为-CH2CH3；
或R1为H，R2为C6H5，R3为CN，R4为-CH2CH3；
或R1为2，6-Cl，R2为H，R3为H，R4为-CH2CH3；
或R1为4-Br，R2为H，R3为H，R4为-CH2CH3；
或R1为2-CF3，R2为H，R3为H，R4为-CH2CH3；
或R1为2-OH-4-Br，R2为H，R3为H，R4为-CH2CH3；
或R1为4-Cl，R2为H，R3为COOCH2CH3，R4为-CH2CH3；
或R1为4-Cl，R2为H，R3为OCH2CH3，R4为-CH2CH3；
或R1为3，4-F，R2为H，R3为OCH2CH3，R4为-CH2CH3；
或R1为2-Cl-4-F，R2为H，R3为CN，R4为-CH2CH3；
或R1为3-Cl-4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
或R1为2-Cl-4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
或R1为4-NH2，R2为H，R3为H，R4为-CH2CH3；
或R1为3-CN，R2为H，R3为H，R4为-CH2CH3；
或R1为4-NO2，R2为H，R3为H，R4为-CH2CH3；
或R1为4-(2，4-F-C6H4)，R2为CH3，R3为H，R4为-CH2CH3；
或R1为4-OCH3，R2为H，R3为H，R4为-CH2-C6H4-CH2-OOC-CH＝CH-C6H4-O-CH3；
或R1为4-OCH3，R2为H，R3为H，R4为-CH2-C-CH2-OOC-CH ＝CH-C6H4-O-CH3；
或R1，R2，R3形成杂环结构
Figure PCTCN2015000311-appb-000004
R4为-CH2CH3；
或R1，R2，R3形成杂环结构
Figure PCTCN2015000311-appb-000005
R4为-CH2CH3；
或R1，R2，R3形成杂环结构
Figure PCTCN2015000311-appb-000006
R4为-CH2CH3；
或R1，R2，R3形成杂环结构
Figure PCTCN2015000311-appb-000007
R4为-CH2CH3；
或R1为-N-CH3(CH3)，R2为H，R3与R4形成杂环结构
Figure PCTCN2015000311-appb-000008
本发明所述的对甲氧基桂皮酸乙酯及其衍生物包括：对甲氧基桂皮酸乙酯、甲氧基桂皮酸异戊酯、4-甲氧基亚苄基丙二酸二乙酯、2-(4-甲氧基苯亚甲基)乙酰乙酸乙酯、2-氰基-3-(4-羟乙基苯)丙烯酸乙酯、4-甲氧基桂皮酸辛酯、2-(3，4-二甲氧基苯亚甲基)丙二酸二乙酯、2-氰基-3-(3，4-二甲氧基苯)-2-丁烯酸乙酯、反式2-氰基-3-(2，4-二甲氧基苯)-2-丙烯酸乙酯、反式3-(3，4，5-三甲氧基苯)-2-丁烯酸乙酯、2-羟乙基桂皮酸乙酯、反式-4-乙氧基桂皮酸乙酯、3-(4-丁氧基苯)-2-氰基-丙烯酸乙酯、3-(反式-3-羟乙基-3-氧-1-丙烯基)苯甲酸甲酯、3-(反式-3-乙氧基-3-氧-1-丙烯基)-4-甲基苯甲酸甲酯、4-乙酸基苯亚甲基丙二酸二乙酯、3-(3-甲氧基-4-甲氧基羰基甲氧基苯)-丙烯酸乙酯、反式-2-羟基桂皮酸乙酯、咖啡酸-1，1-二甲基丙烯酸酯、反式-咖啡酸-1-苯基乙酯、新绿原酸、绿原酸、桂皮酸乙酯、桂皮酸异丙酯、桂皮酸丁酯、桂皮酸异丁酯、桂皮酸-3-甲基丙酯、桂皮酸异戊酯、3-丙基丙烯酸-3-桂皮酰氧基丙酯、桂皮酸 苯乙酯、3-苯丙基桂皮酸、反式-2-氰基桂皮酸乙酯、反式-β-甲基桂皮酸乙酯、2-氰基-3，3-二苯基丙烯酸乙酯、2，6-二氯桂皮酸乙酯、反式-4-溴桂皮酸乙酯、反式-3-(2-三氟甲基苯)-2-丙烯酸乙酯、反式-3-(4-溴-2-羟基苯基)-2-丙烯酸乙酯、4-氯苯亚甲基丙二酸二乙酯、顺式-3-(4-氯苯基)-2-羟乙基-2-丙烯酸乙酯、顺式-3-(3，4-二氟苯基)-2-乙氧基-2-丙烯酸乙酯、3-(2-氯-4-氟苯基)-2-氰基-2-丙烯酸乙酯、3-(3-氯-4-甲氧基苯基)-2-氰基丙烯酸乙酯、3-(2-氯-4-甲氧基苯基)-2-氰基丙烯酸乙酯、4-氨基桂皮酸乙酯、3-氰基桂皮酸乙酯、4-硝基桂皮酸乙酯、3-[2，，4，-二氟(1，1，-联苯)-4-基]-2-丁烯酸乙酯、3-(4-甲氧基苯)-丙烯酸-3-[3-(4-甲氧基苯基)-丙稀酰氧甲基]-苯甲酯、3-(4-甲氧基苯)-丙烯酸-4-[3-(4-甲氧基苯)-丙烯酰氧]-丁-2-炔基酯、吲哚-2-羧酸乙酯、6-甲氧基-1-苯并呋喃-2-羧酸乙酯、6-氨基-3-甲基-1-苯并呋喃-2-羧酸乙酯、5-甲基吲哚-2-羧酸乙酯、反式-3-(4-二甲氨基苯亚甲基)二氢-2(3H)-呋喃酮。
本发明所述的干细胞为成体干细胞、胚胎干细胞或诱导性多潜能干细胞iPS。
所述的对甲氧基桂皮酸乙酯及其衍生物通过促进Oct4基因的表达来维持干细胞的自我更新能力和多潜能状态。
本发明所述的药物组合物包括治疗有效量的对甲氧基桂皮酸乙酯及其衍生物以及可药用载体，所述的载体为溶剂、分散剂、包衣、抗细菌和抗真菌剂、等张剂和吸收延迟剂。
本发明所述的药物可以由使用者在使用前经稀释或直接使用，其 配制可由通常的本领域技术人员所公知的加工方法制备。
本发明所述的药物根据需要可制备成适应于不同给药途径的药物剂型，其中较优选的剂型为胶囊剂、片剂。胶囊剂可包含任何标准的可药用物质如明胶或纤维素。片剂可按传统方法即将药物组合物与固相载体以及润滑剂压缩制得。所述固相载体包括淀粉和糖斑脱土。
本发明的药物组合物还可通过非肠道途径给药。非肠道途径给药剂型包括本发明的药物组合物的水剂、等张盐溶液或5％的糖溶液以及与其他本领域公知的可药用赋形剂形成的制剂。
本发明所述的对甲氧基桂皮酸乙酯及其衍生物在维持干细胞的自我更新能力和多潜能状态药物制剂用量范围为0.01～100mg/kg。
本发明所述的对甲氧基桂皮酸乙酯及其衍生物在体外大量制备干细胞中的应用，具体为在体外扩增干细胞及制备诱导性多潜能干细胞iPS中的应用。
本发明还提供了对甲氧基桂皮酸乙酯及其衍生物在制备治疗细胞缺失或损伤性疾病的药物中的应用，所述的损伤性疾病为细胞、组织或器官的损伤性疾病，具体为神经系统疾病、免疫系统疾病、血液系统疾病、心血管系统疾病或皮肤疾病。
本发明还提供了对甲氧基桂皮酸乙酯及其衍生物在制备美容护肤品中的应用。
本发明的对甲氧基桂皮酸乙酯及其衍生物可悬溶于可药用载体(如生理溶液)中，通过口服或静脉输液，或通过皮下、肌下、胸内、腹膜内、直肠内、阴道内、鼻内、胃内、气道内、肺内注射或输液等 途径给药。
本发明的对甲氧基桂皮酸乙酯及其衍生物可悬溶于美容护肤品中，通过涂抹方式达到激活皮肤干细胞的功效。
本发明的对甲氧基桂皮酸乙酯及其衍生物可直接添加于细胞培养液中，用于体外扩增干细胞。
本发明的有益效果为：通过对小分子化合物的研究，发现对甲氧基桂皮酸乙酯及其衍生物能促进Oct4基因的表达，从而来维持干细胞的自我更新能力和多潜能状态，可以作为维持干细胞自我更新和多潜能性的调节药物或培养基添加剂，能够为临床移植提供大量干细胞，为细胞缺失或损伤性疾病的治疗提供新视点和新思路。
附图说明
图1是对甲氧基桂皮酸乙酯对Oct4启动子活性的影响；
图2是对甲氧基桂皮酸乙酯对Oct4mRNA表达的影响；
图3是对甲氧基桂皮酸乙酯对Oct4蛋白表达的影响；
图4是对甲氧基桂皮酸乙酯对悬浮培养的干细胞微球体形成的影响；
图5是对甲氧基桂皮酸乙酯对贴壁培养的干细胞微球体形成的影响；
图6是对甲氧基桂皮酸乙酯的衍生物对Oct4表达的影响；A代表绿原酸，B代表2-氰基-3，3-二苯基丙烯酸乙酯，C代表吲哚-2-羧酸乙酯，D代表肉桂酸乙酯，E代表3-(4-甲氧基苯)-丙烯酸-4-[3-(4-甲氧基苯)-丙烯酰氧]-丁-2-炔基酯，F代表3-(4-甲氧基苯)-丙烯酸 -3-[3-(4-甲氧基苯基)-丙稀酰氧甲基]-苯甲酯，G代表3-丙基丙烯酸-3-桂皮酰氧基丙酯，H代表4-乙酸基苯亚甲基丙二酸二乙酯。
具体实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
实施例1对甲氧基桂皮酸乙酯对Oct4启动子活性的影响
按文献所述方法(Yang H M，Do H J，Oh J H，et al.Characterization of putative cis-regulatory elements that control the transcriptional activity of the human Oct4 promoter[J].Journal of cellular biochemistry，2005，96(4)：821-830.)钓取Oct4基因的启动子，并克隆入pGL3-Basic中，从而构建出pGL3-Oct4P-Basic报告质粒。在转染前一天将小鼠畸胎瘤细胞P19以2×105个/孔的密度接种于6孔细胞板中，使细胞在转染前长到80％左右。使用Engreen公司的脂质体转染试剂EntransterTM-D，按照说明书进行转染。将目的质粒pGL3-Oct4P-Basic转染P19细胞，转染所用质粒总量为3μg/孔。将质粒加入到50μL无血清的DMEM培养基中，充分混匀。然后将9μL的转染试剂加入到另外的50μL无血清DMEM培养基中，混匀，静置5min。之后将含有转染试剂的DMEM培养基加入到含有质粒的 DMEM培养基中，立即吹打10次以上，室温静置30min。在混合液中加入900μL含10％胎牛血清的DMEM培养基，轻柔混匀。弃去6孔板中旧的细胞培养基，换成上述混合液。在37℃、5％CO2的细胞培养箱中培养4-6h后，用含10％胎牛血清的DMEM培养基使细胞重新悬浮。将上述细胞以8×103个/孔的密度接种于96孔细胞板中。培养24h后，吸去培养基，加入含3％胎牛血清的DMEM培养基，在此培养基中加入待筛选药物，使其终浓度为5μg/mL，同时以DMSO做为对照。继续培养12-24h后，裂解细胞，检测荧光素酶活性。
用Luciferase Reporter Assay System和FluoStar Optima检测转染细胞裂解液的荧光素酶活性。取45μL细胞裂解液，加5μL的Assay Cocktail试剂，混匀后加入100μL Luciferin反应液，利用FluoStar Optima仪器测2s的荧光计数，该计数反映了研究质粒报告基因表达萤火虫荧光素酶(firefly luciferase)的活性水平。另取20μL细胞裂解液，加入37.5μLβ-gal Buffer，再加入12.5μL ONPG(6mg/mL)，混匀后37℃放置30min，利用酶标仪检测450nm下的吸收值，该值反映了内参质粒pCMV-β-gal表达β半乳糖苷酶的活性水平。萤火虫荧光素酶(firefly luciferase)的活性与β半乳糖苷酶(β-gal)的活性之比，剔除了样本转染效率的差异，可以比较客观地反映样本间启动子活性的差异。不同样品的荧光素酶相对活性以下述公式计算：
Figure PCTCN2015000311-appb-000009
结果如图1所示，对甲氧基桂皮酸乙酯对Oct4启动子活性的影 响达到了统计学意义上的显著性差异(*p＜0.05)。
实施例2对甲氧基桂皮酸乙酯对Oct4mRNA表达的影响
1)mRNA的提取与逆转录
将P19细胞以2×105个/孔的密度接种于6孔细胞培养板中。24h后，换成含3％血清的DMEM培养基，实验组中加入对甲氧基桂皮酸乙酯，使其终浓度为5μg/mL，对照组用DMSO处理。12h后，将长满单层细胞的6孔板从细胞培养箱中取出，用预冷的PBS洗涤细胞3次。加入1mL预冷的Trizol溶液，剧烈吹打后转移至EP管中，室温静止5min。然后加入200μL氯仿，剧烈混匀15s，室温静止2min，4℃12000rpm离心15min。吸取上层水相至另一个EP管中，加入等体积预冷的异丙醇，颠倒数次，室温静止10min沉淀RNA。12000rpm离心10min，弃上清，沉淀中加入1mL 75％乙醇(0.1％DEPC水配制)洗涤，7500rpm离心5min(此步骤重复一次)。弃上清，自然干燥(或者37℃烘干)，加入适量的0.1％DEPC水，37℃溶解30min(或者55℃溶解10min)，将溶解的RNA放在-80℃保存备用。
使用TaKaRa公司的Reverse Transcription System试剂盒进行逆转录，反应体系如下：
Figure PCTCN2015000311-appb-000010
首先将RNA模板、0.1％DEPC水和Oligo(dT)15primer加入 到干净的EP管中，70℃水浴2min。1000rpm离心1min。在EP管中加入逆转录的其它组分，42℃水浴1h。70℃8min终止反应，最后补0.1％DEPC水至100μL。
2)RT-PCR反应
上游引物：5’-CTCGAACCACATCCTTCTCT-3’；下游引物：5’-TCGTGCGTGACATTAAGGAG-3。
PCR反应体系(25μL)：
Figure PCTCN2015000311-appb-000011
PCR反应条件：
Figure PCTCN2015000311-appb-000012
3)琼脂糖凝胶电泳
称取0.4g琼脂糖，置于锥形瓶中，加入40mL 1×TAE电泳缓冲液，放入微波炉里加热至琼脂糖全部熔化，取出摇匀，重复三次。待溶液冷却至50℃左右，加入20μL花青素染料，终浓度为0.5mg/mL，充分混匀。将电泳胶溶液倒至胶槽中，静置冷却，凝固30min。待胶完全凝固后将梳子拨出，向电泳槽中倒入1×TAE电泳缓冲液至液面 恰好没过胶板上表面。PCR产物依比例加入10×凝胶上样缓冲液，混匀，用微量移液器小心加入样品孔中。加样完毕后，合上电泳槽盖，接通电源，使电压保持在100-140V。当溴酚蓝条带移动到距凝胶前沿2cm左右时，停止电泳，使用凝胶成像系统观察并拍照。
结果如图2所示，对甲氧基桂皮酸乙酯能够显著促进Oct4mRNA的表达。
实施例3对甲氧基桂皮酸乙酯对Oct4蛋白表达的影响
用Western Blot方法检测对甲氧基桂皮酸乙酯对Oct4蛋白表达的影响。将小鼠畸胎瘤细胞P19接种于6孔板中，24h后加入终浓度为5μg/mL的对甲氧基桂皮酸乙酯，继续作用24h，对照组用DMSO处理。用PBS洗涤细胞3次后，将细胞用刮刀刮起，转移至EP管中，4000rpm离心5min，弃上清，每管沉淀中加入90μL裂解液(50mM Tris-HCl pH7.5，150mM NaCl，1mM NaF，0.5％ NP-40，2μg/mL Aprotinin，1mM PMSF)，冰上作用30min，每隔5min涡旋一次，以便细胞充分裂解。12000rpm离心10min后，将上清转移至新EP管中，加入1/3体积的4×蛋白上样缓冲液，混匀，煮沸10min，瞬时离心后上样电泳或者置于一80℃冻存备用。
配制转膜缓冲液并于4℃预冷。将PVDF膜用甲醇浸泡40s，取出后浸泡于转膜缓冲液中备用。将方盘中放入转膜缓冲液，在液面下进行转膜装置的安装。并将转膜装置放入电泳槽中，倒入转膜缓冲液，100V转膜2h后取出PVDF膜，将膜标记正反面及电泳方向。用TBST(0.2％的Tween20)洗涤PVDF膜，每隔5min洗涤一次，共洗涤2 次，再用5％的脱脂奶粉室温封闭2h。之后用TBST(0.2％的Tween20)冲洗2次，每次5min(洗膜时均放于摇床上，以下同)。将Oct4的抗体用TBST(0.2％的Tween20)1∶500稀释后，与PVDF膜一同装入杂交袋中，4℃过夜。取出PVDF膜，用TBST(0.2％的Tween20)快速冲洗2遍，再洗涤5min 2次，15min 3次。将二抗HRP-山羊抗小鼠(0.8mg/mL)用TBST(0.2％的Tween20)1∶2000稀释后，与PVDF膜一同装入杂交袋中，室温摇床孵育40min。取出PVDF膜，用TBST(0.2％的Tween20)快速冲洗2遍，再洗涤5min 2次，15min3次。将PVDF膜进行ECL显色。
结果如图3所示，对甲氧基桂皮酸乙酯能够促进Oct4蛋白的表达。
实施例4对甲氧基桂皮酸乙酯及其衍生物促进悬浮培养的干细胞微球体的形成
称取5g琼脂置于三角瓶中，再加入100mL超纯水，混匀，封口，然后高压灭菌30min。在超净台中，吸取9mL预热的(50℃-60℃)无血清DMEM培养基于10mL离心管中，然后加入1mL未冷却的5％的琼脂，迅速用吹打管混匀，即得到了0.5％的琼脂。吸取1mL 0.5％的琼脂置于6孔板的一个孔中，室温冷却20-30min，即得到了0.5％的软琼脂培养基。
在超净台中，将D-Hanks轻轻加入到软琼脂培养基的表面，轻柔摇动2-3次后，弃去D-Hanks。重复清洗一次。将小鼠畸胎瘤细胞P19轻轻地接种于软琼脂培养基的表面，细胞密度为4×105个/孔。细胞 接种之后，将对甲氧基桂皮酸乙酯加入到1mL含10％胎牛血清的DMEM培养基中，混匀后，再轻轻加入到实验组中，对甲氧基桂皮酸乙酯的终浓度为5μg/mL。对照组用DMSO处理。隔天进行半量换液，方法是从孔的一侧轻轻地吸去1mL培养基(注意不能吸到细胞)，然后加入新鲜的培养基，在其中加入对甲氧基桂皮酸乙酯，使其终浓度为5μg/mL。7d后，在对照组和实验组中各加入200μL MTT(5mg/mL)，放回培养箱中继续培养4h。染色成功后，对细胞进行拍照。
结果如图4所示，与对照组相比，在实验组中，P19细胞能够形成更多更大更典型的微球体。说明对甲氧基桂皮酸乙酯具有维持干细胞自我更新和多潜能性的作用。
实施例5对甲氧基桂皮酸乙酯促进贴壁培养的干细胞微球体的形成
在超净台中，将小鼠畸胎瘤细胞P19以1×104个/孔的密度接种于6孔细胞板中，用含10％胎牛血清的DMEM培养基补足2mL。轻轻晃动细胞培养板，使细胞均匀分布于细胞培养板中，然后放入细胞培养箱中培养。24h后，弃去旧的培养基，加入含有对甲氧基桂皮酸乙酯的3％胎牛血清DMEM培养基，使对甲氧基桂皮酸乙酯的终浓度为5μg/mL。每隔24h，弃去旧的培养基，加入新的含3％胎牛血清的DMEM培养基，同样使其中对甲氧基桂皮酸乙酯的终浓度为5μg/mL。连续培养22d后，对细胞进行拍照。
结果如图5所示，对照组不能形成微球体，但是在实验组中，P19 细胞能够形成典型的微球体。再次说明对甲氧基桂皮酸乙酯具有维持干细胞自我更新和多潜能性的作用。
实施例6对甲氧基桂皮酸乙酯的衍生物能够促进Oct4的表达
运用荧光素酶报告基因法检测对甲氧基桂皮酸乙酯的衍生物对Oct4表达的影响，包括4-甲氧基亚苄基丙二酸二乙酯，3-(3-甲氧基-4-甲氧基羰基甲氧基苯)-丙烯酸乙酯，4-乙酸基苯亚甲基丙二酸二乙酯，绿原酸，桂皮酸乙酯，3-丙基丙烯酸-3-桂皮酰氧基丙酯，2-氰基-3，3-二苯基丙烯酸乙酯，2，6-二氯桂皮酸乙酯，4-氯苯亚甲基丙二酸二乙酯，4-氨基桂皮酸乙酯，3-氰基桂皮酸乙酯，4-硝基桂皮酸乙酯，3-(4-甲氧基苯)-丙烯酸3-[3-(4-甲氧基苯基)-丙稀酰氧甲基]-苯甲酯，3-(4-甲氧基苯)-丙烯酸-4-[3-(4-甲氧基苯)-丙烯酰氧]-丁-2-炔基酯，吲哚-2-羧酸乙酯，6-甲氧基-1-苯并呋喃-2-羧酸乙酯。
在转染前一天将小鼠畸胎瘤细胞P19以1×105个/孔的密度接种于24孔细胞板中，使细胞在转染前长到80％左右。使用Engreen公司的脂质体转染试剂EntransterTM-D，按照说明书进行转染，将目的质粒(pGL3-Oct4P-Basic或pGL3-Basic)与参照质粒(pCMV-β-gal)共转染P19细胞，转染所用质粒总量为1μg/孔(目的质粒：参照质粒＝2∶1)。将质粒加入到25μL无血清的DMEM培养基中，充分混匀。然后将3μL的转染试剂加入到另外的25μL无血清DMEM培养基中，混匀，静置5min。之后将含有转染试剂的DMEM培养基加入到含有质粒的DMEM培养基中，立即吹打10次以上，室温静置30min。在混合液中加入250μL含10％胎牛血清的DMEM培养基，轻柔混 匀。弃去24孔板中旧的细胞培养基，换成上述混合液。在37℃、5％CO2的细胞培养箱中培养4-6h，然后吸去旧的培养基，换成500μL含10％胎牛血清的DMEM培养基。培养24-36h后，吸去培养基，加入含3％胎牛血清的DMEM培养基，在此培养基中加入对甲氧基桂皮酸乙酯，使其终浓度为5μg/mL，同时以DMSO做为对照。继续培养12-24h后，裂解细胞，检测荧光素酶活性。
利用Luciferase Reporter Assay System和FluoStar Optima检测转染细胞裂解液的荧光素酶活性。取45μL细胞裂解液，加5μL的Assay Cocktail试剂，混匀后加入100μL Luciferin反应液，利用FluoStar Optima仪器测2s的荧光计数，该计数反映了研究质粒报告基因表达萤火虫荧光素酶(firefly luciferase)的活性水平。另取20μL细胞裂解液，加入37.5μL β-gal Buffer，再加入12.5μL ONPG(6mg/mL)，混匀后37℃放置30min，利用酶标仪检测450nm下的吸收值，该值反映了内参质粒pCMV-β-gal表达β半乳糖苷酶的活性水平。萤火虫荧光素酶(firefly luciferase)的活性与β半乳糖苷酶(β-gal)的活性之比，剔除了样本转染效率的差异，可以比较客观地反映样本间启动子活性的差异。不同样品的荧光素酶相对活性以下述公式计算：
Figure PCTCN2015000311-appb-000013
结果如图6所示，所检测的8种化合物都具有增强Oct4启动子活性的作用。其中A代表绿原酸，B代表2-氰基-3，3-二苯基丙烯酸乙酯，C代表吲哚-2-羧酸乙酯，D代表肉桂酸乙酯，E代表3-(4-甲氧 基苯)-丙烯酸-4-[3-(4-甲氧基苯)-丙烯酰氧]-丁-2-炔基酯，F代表3-(4-甲氧基苯)-丙烯酸-3-[3-(4-甲氧基苯基)-丙稀酰氧甲基]-苯甲酯，G代表3-丙基丙烯酸-3-桂皮酰氧基丙酯，H代表4-乙酸基苯亚甲基丙二酸二乙酯。鉴于这8种化合物均具有促进Oct4表达的作用，所以我们认为是它们结构中的共同部分在发挥此作用，而且据此我们可以推断其它具有这个共同结构的对甲氧基桂皮酸乙酯衍生物，即具有以下通式的化合物也应该具有相同的维持干细胞特性的活性，可以作为干细胞的调节药物。

Claims (9)

  1. 对甲氧基桂皮酸乙酯及其衍生物在制备维持干细胞自我更新和多潜能性的药物、培养基、调节剂或美容护肤品中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述的对甲氧基桂皮酸乙酯及其衍生物的结构通式如下所示：
    Figure PCTCN2015000311-appb-100001
    其中R1为4-OCH3，R2为H，R3为H，R4为-CH2CH3；
    或R1为4-OCH3，R2为H，R3为H，R4为-CH2CH2CH(CH3)CH3；
    或R1为4-OCH3，R2为H，R3为COOCH2CH3，R4为-CH2CH3；
    或R1为4-OCH3，R2为H，R3为COCH3，R4为-CH2CH3；
    或R1为4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
    或R1为4-OCH3，R2为CH3，R3为H，R4为-CH2CH(CH2CH3)CH2CH2CH2CH3；
    或R1为3，4-OCH3，R2为H，R3为COCH2CH3，R4为-CH2CH3；
    或R1为3，4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
    或R1为2，4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
    或R1为3，4，5-OCH3，R2为CH3，R3为H，R4为-CH2CH3；
    或R1为2-OCH2CH3，R2为H，R3为H，R4为-CH2CH3；
    或R1为4-OCH2CH3，R2为H，R3为H，R4为-CH2CH3；
    或R1为4-OCH2CH2CH2CH3，R2为H，R3为CN，R4为-CH2CH3；
    或R1为3-COOCH3，R2为H，R3为H，R4为-CH2CH3；
    或R1为2-CH3-5-COOCH3，R2为H，R3为H，R4为-CH2CH3；
    或R1为4-OCOCH3，R2为H，R3为COCH2CH3，R4为-CH2CH3；
    或R1为3-OCH3-4-OCH2COOCH2CH3，R2为H，R3为H，R4为-CH2CH3；
    或R1为2-OH，R2为H，R3为H，R4为-CH2CH3；
    或R1为3，4-OH，R2为H，R3为H，R4为-CH2CHC(CH3)CH3；
    或R1为3，4-OH，R2为H，R3为H，R4为-CH2CH2C6H5；
    或R1为3，4-OH，R2为H，R3为H，R4为
    Figure PCTCN2015000311-appb-100002
    或R1为3，4-OH，R2为H，R3为H，R4为
    Figure PCTCN2015000311-appb-100003
    或R1为H，R2为H，R3为H，R4为-CH2CH3；
    或R1为H，R2为H，R3为H，R4为-CH(CH3)CH3；
    或R1为H，R2为H，R3为H，R4为-CH2CH2CH2CH3；
    或R1为H，R2为H，R3为H，R4为-CH2CH(CH3)CH3；
    或R1为H，R2为H，R3为H，R4为-CH(CH3)CH2CH3；
    或R1为H，R2为H，R3为H，R4为-CH2CH2CH(CH3)CH3；
    或R1为H，R2为H，R3为H，R4为-CH2CH2CH2OOCCH＝CHC6H5；
    或R1为H，R2为H，R3为H，R4为-CH2CH2C6H5；
    或R1为H，R2为H，R3为H，R4为-CH2CH(CH3)C6H5；
    或R1为H，R2为H，R3为CN，R4为-CH2CH3；
    或R1为H，R2为CH3，R3为H，R4为-CH2CH3；
    或R1为H，R2为C6H5，R3为CN，R4为-CH2CH3；
    或R1为2，6-Cl，R2为H，R3为H，R4为-CH2CH3；
    或R1为4-Br，R2为H，R3为H，R4为-CH2CH3；
    或R1为2-CF3，R2为H，R3为H，R4为-CH2CH3；
    或R1为2-OH-4-Br，R2为H，R3为H，R4为-CH2CH3；
    或R1为4-Cl，R2为H，R3为COOCH2CH3，R4为-CH2CH3；
    或R1为4-Cl，R2为H，R3为OCH2CH3，R4为-CH2CH3；
    或R1为3，4-F，R2为H，R3为OCH2CH3，R4为-CH2CH3；
    或R1为2-Cl-4-F，R2为H，R3为CN，R4为-CH2CH3；
    或R1为3-Cl-4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
    或R1为2-Cl-4-OCH3，R2为H，R3为CN，R4为-CH2CH3；
    或R1为4-NH2，R2为H，R3为H，R4为-CH2CH3；
    或R1为3-CN，R2为H，R3为H，R4为-CH2CH3；
    或R1为4-NO2，R2为H，R3为H，R4为-CH2CH3；
    或R1为4-(2，4-F-C6H4)，R2为CH3，R3为H，R4为-CH2CH3；
    或R1为4-OCH3，R2为H，R3为H，R4为-CH2-C6H4-CH2-OOC-CH＝CH-C6H4-O-CH3；
    或R1为4-OCH3，R2为H，R3为H，R4为-CH2-C-CH2-OOC-CH＝CH-C6H4-O-CH3；
    或R1，R2，R3形成杂环结构
    Figure PCTCN2015000311-appb-100004
    R4为-CH2CH3；
    或R1，R2，R3形成杂环结构
    Figure PCTCN2015000311-appb-100005
    R4为-CH2CH3；
    或R1，R2，R3形成杂环结构
    Figure PCTCN2015000311-appb-100006
    R4为-CH2CH3；
    或R1，R2，R3形成杂环结构R4为-CH2CH3；
    或R1为-N-CH3(CH3)，R2为H，R3与R4形成杂环结构
    Figure PCTCN2015000311-appb-100008
  3. 根据权利要求2所述的应用，其特征在于：所述的对甲氧基桂皮酸乙酯及其衍生物包括：对甲氧基桂皮酸乙酯、甲氧基桂皮酸异戊酯、4-甲氧基亚苄基丙二酸二乙酯、2-(4-甲氧基苯亚甲基)乙酰乙酸乙酯、2-氰基-3-(4-羟乙基苯)丙烯酸乙酯、4-甲氧基桂皮酸辛酯、2-(3，4-二甲氧基苯亚甲基)丙二酸二乙酯、2-氰基-3-(3，4-二甲氧基苯)-2-丁烯酸乙酯、反式2-氰基-3-(2，4-二甲氧基苯)-2-丙烯酸乙酯、反式3-(3，4，5-三甲氧基苯)-2-丁烯酸乙酯、2-羟乙基桂皮酸乙酯、反式-4-乙氧基桂皮酸乙酯、3-(4-丁氧基苯)-2-氰基-丙烯酸乙酯、3-(反式-3-羟乙基-3-氧-1-丙烯基)苯甲酸甲酯、3-(反式-3-乙氧基-3-氧-1-丙烯基)-4-甲基苯甲酸甲酯、4-乙酸基苯亚甲基丙二酸二乙酯、3-(3-甲氧基-4-甲氧基羰基甲氧基苯)-丙烯酸乙酯、反式-2-羟基桂皮酸乙酯、咖啡酸-1，1-二甲基丙烯酸酯、反式-咖啡酸-1-苯基乙酯、新绿原酸、绿原酸、桂皮酸乙酯、桂皮酸异丙酯、桂皮酸丁酯、桂皮酸异丁酯、桂皮酸-3-甲基丙酯、桂皮酸异戊酯、3-丙基丙烯酸-3-桂皮酰氧基丙酯、桂皮酸苯乙酯、3-苯丙基桂皮酸、反式-2-氰基桂皮酸乙酯、反式-β-甲基桂皮酸乙酯、2-氰基-3，3-二苯基丙烯 酸乙酯、2，6-二氯桂皮酸乙酯、反式-4-溴桂皮酸乙酯、反式-3-(2-三氟甲基苯)-2-丙烯酸乙酯、反式-3-(4-溴-2-羟基苯基)-2-丙烯酸乙酯、4-氯苯亚甲基丙二酸二乙酯、顺式-3-(4-氯苯基)-2-羟乙基-2-丙烯酸乙酯、顺式-3-(3，4-二氟苯基)-2-乙氧基-2-丙烯酸乙酯、3-(2-氯-4-氟苯基)-2-氰基-2-丙烯酸乙酯、3-(3-氯-4-甲氧基苯基)-2-氰基丙烯酸乙酯、3-(2-氯-4-甲氧基苯基)-2-氰基丙烯酸乙酯、4-氨基桂皮酸乙酯、3-氰基桂皮酸乙酯、4-硝基桂皮酸乙酯、3-[2，，4，-二氟(1，1，-联苯)-4-基]-2-丁烯酸乙酯、3-(4-甲氧基苯)-丙烯酸-3-[3-(4-甲氧基苯基)-丙稀酰氧甲基]-苯甲酯、3-(4-甲氧基苯)-丙烯酸-4-[3-(4-甲氧基苯)-丙烯酰氧]-丁-2-炔基酯、吲哚-2-羧酸乙酯、6-甲氧基-1-苯并呋喃-2-羧酸乙酯、6-氨基-3-甲基-1-苯并呋喃-2-羧酸乙酯、5-甲基吲哚-2-羧酸乙酯、反式-3-(4-二甲氨基苯亚甲基)二氢-2(3H)-呋喃酮。
  4. 根据权利要求1所述的应用，其特征在于：所述的对甲氧基桂皮酸乙酯及其衍生物通过促进Oct4基因的表达来维持干细胞的自我更新能力和多潜能状态。
  5. 根据权利要求1所述的应用，其特征在于：所述的干细胞包括成体干细胞、胚胎干细胞和诱导性多潜能干细胞iPS。
  6. 根据权利要求1所述的应用，其特征在于：对甲氧基桂皮酸乙酯及其衍生物在维持干细胞的自我更新能力和多潜能状态药物制剂用量范围为0.01～100mg/kg。
  7. 权利要求1所述的对甲氧基桂皮酸乙酯及其衍生物在体外大 量制备干细胞中的应用。
  8. 根据权利要求7所述的应用，其特征在于：所述的对甲氧基桂皮酸乙酯及其衍生物在体外扩增干细胞及制备诱导性多潜能干细胞iPS中的应用。
  9. 权利要求1所述的对甲氧基桂皮酸乙酯及其衍生物在制备治疗细胞缺失或损伤性疾病的药物中的应用。
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