WO2020030091A1 - 用于治疗组织坏死或改善心脏功能的药物 - Google Patents

用于治疗组织坏死或改善心脏功能的药物 Download PDF

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Publication number
WO2020030091A1
WO2020030091A1 PCT/CN2019/099947 CN2019099947W WO2020030091A1 WO 2020030091 A1 WO2020030091 A1 WO 2020030091A1 CN 2019099947 W CN2019099947 W CN 2019099947W WO 2020030091 A1 WO2020030091 A1 WO 2020030091A1
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days
eggs
amniotic fluid
age
pharmaceutical composition
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PCT/CN2019/099947
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English (en)
French (fr)
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钱进
孙宁
高翔
崔白苹
郑煜凡
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浙江楚沅生物科技有限公司
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Priority to JP2021531168A priority Critical patent/JP2021534235A/ja
Priority to US17/267,586 priority patent/US20210308191A1/en
Priority to KR1020217006661A priority patent/KR20210044242A/ko
Priority to AU2019318661A priority patent/AU2019318661A1/en
Priority to EP19847075.9A priority patent/EP3834834A4/en
Priority to CA3109199A priority patent/CA3109199A1/en
Priority to CN201980053589.2A priority patent/CN112566646A/zh
Publication of WO2020030091A1 publication Critical patent/WO2020030091A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/57—Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells

Definitions

  • the present invention relates to a therapeutic drug for treating tissue necrosis or improving cardiac function.
  • hypoxia due to ischemia results in the local tissue and cell metabolism to stop and its functions completely lost.
  • Cells can die by changes in nuclear enrichment, nuclear fragmentation, and lysis, resulting in tissue necrosis.
  • Myocardial infarction is one of the examples of tissue necrosis caused by ischemia and hypoxia. It refers to the interruption of coronary blood on the basis of coronary artery disease, which causes the corresponding myocardium to develop severe and persistent acute ischemia, eventually leading to acute, persistent Myocardial necrosis caused by ischemia and hypoxia (coronary insufficiency).
  • Patients with acute myocardial infarction often have persistent severe sternum pain, fever, increased white blood cell count, elevated serum myocardial enzymes, and electrocardiograms that reflect a series of characteristic evolutions of acute myocardial injury, ischemia, and necrosis.
  • the occurrence of arrhythmia, shock, and heart failure is a serious type of coronary heart disease. It can be complicated by arrhythmia, shock, or heart failure and is often life-threatening.
  • the invention provides a pharmaceutical composition, the pharmaceutical composition contains:
  • Amniotic fluid and / or its extracts from non-human animals and
  • An optional pharmaceutically acceptable carrier is an optional pharmaceutically acceptable carrier
  • the amniotic fluid is derived from eggs with an embryo age of 5-12 days, preferably eggs with an embryo age of 6-11 days, more preferably eggs with an embryo age of 7-9 days, and more preferably eggs with an embryo age of 7-8 days Or eggs from avians other than chickens at a developmental stage corresponding to the developmental period of the embryonic age eggs; or embryos from rodents with a gestational age of 8-20 days, preferably 8-14 days, Or embryos from non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-20 days, preferably 8-14 days.
  • the pharmaceutical composition is a cryopreserved amniotic fluid and / or an extract thereof, or a lyophilized reagent of the amniotic fluid and / or an extract thereof.
  • the pharmaceutical composition is an infusion solution.
  • the pharmaceutical composition does not contain or contain isotonic saline, water for injection or glucose injection.
  • the eggs of the other birds are birds with an embryonic age of 5-20 days.
  • the eggs of the other birds are eggs with an embryo age of 6-15 days.
  • the amniotic fluid is derived from eggs with an embryonic age of 7 days.
  • the eggs of the other birds are duck eggs, goose eggs, or a combination thereof.
  • the invention also provides the use of amniotic fluid and / or its extract in the preparation of a medicament for treating tissue necrosis caused by ischemia and hypoxia, wherein the amniotic fluid is derived from eggs with an embryonic age of 5-12 days, and preferably with an embryonic age of 6 -11 days of eggs, more preferably 7-9 days of eggs, more preferably 7-8 days of eggs, or chickens from a development period corresponding to the development period in which the eggs of the embryo age Eggs from birds other than birds; or embryos from rodents with gestational age of 8-20 days, preferably 8-14 days, or from rodents with developmental and gestational ages of 8-20 days, preferably 8-14 days
  • the animal's developmental stage corresponds to embryos of non-human mammals other than rodents.
  • the tissue necrosis is myocardial ischemic necrosis.
  • the tissue necrosis is a myocardial infarction.
  • the eggs of the other birds are birds with an embryonic age of 5-20 days.
  • the eggs of the other birds are eggs with an embryo age of 6-15 days.
  • the amniotic fluid is derived from eggs with an embryonic age of 7 days.
  • the eggs of the other birds are duck eggs, goose eggs, or a combination thereof.
  • the invention also provides the use of amniotic fluid and / or its extract in the preparation of a medicament for improving cardiac function, wherein the amniotic fluid is derived from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more Eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or eggs from birds other than chickens whose developmental period corresponds to the developmental period in which the embryonic-aged eggs are located; Or from embryos of rodents with a gestational age of 8-20 days, preferably 8-14 days, or from developmental stages corresponding to the developmental period of rodents with a gestational age of 8-20 days, preferably 8-14 days Embryos of non-human mammals other than rodents.
  • the eggs of the other birds are birds with an embryonic age of 5-20 days.
  • the eggs of the other birds are eggs with an embryo age of 6-15 days.
  • the amniotic fluid is derived from eggs with an embryonic age of 7 days.
  • the eggs of the other birds are duck eggs, goose eggs, or a combination thereof.
  • the medicament is used to improve heart failure, especially senile heart failure or heart failure.
  • the present invention also provides a method for treating tissue necrosis caused by ischemia and hypoxia, which method comprises administering a therapeutically effective amount of amniotic fluid and / or an extract thereof to a subject in need, wherein the amniotic fluid comes from an embryonic age of 5- 12-day eggs, preferably eggs with an embryo age of 6-11 days, more preferably eggs with an embryo age of 7-9 days, more preferably eggs with an embryo age of 7-8 days, or Eggs of other birds other than chickens corresponding to the developmental period of the eggs; or embryos from rodents with a gestational age of 8-20 days, preferably 8-14 days, or from the developmental period and gestational age of 8-20 Embryos of non-human mammals other than rodents corresponding to the developmental period of the rodents, preferably 8-14 days.
  • the method includes intravenously injecting a pharmaceutical formulation containing the amniotic fluid and / or its extract, or infusion of a pharmaceutical formulation containing the amniotic fluid and / or its extract.
  • the eggs of the other birds are birds with an embryonic age of 5-20 days.
  • the eggs of the other birds are eggs with an embryo age of 6-15 days.
  • the amniotic fluid is derived from eggs with an embryonic age of 7 days.
  • the eggs of the other birds are duck eggs, goose eggs, or a combination thereof.
  • the present invention also provides a method for improving cardiac function, the method comprising administering to a subject in need thereof a therapeutically effective amount of amniotic fluid and / or an extract thereof, wherein the amniotic fluid is derived from eggs with an embryo age of 5-12 days, preferably embryos.
  • mice 6-11 days old eggs, more preferably 7-9 days old eggs, more preferably 7-8 days old eggs, or derived from a developmental period that is in phase with the developmental period in which the eggs are located
  • Eggs from other birds other than chickens or embryos from rodents with gestational age of 8-20 days, preferably 8-14 days, or from developmental stage and gestational age of 8-20 days, preferably 8-14 days
  • the developmental stages of rodents correspond to embryos of non-human mammals other than rodents.
  • the method includes intravenously injecting a pharmaceutical formulation containing the amniotic fluid and / or its extract, or infusion of a pharmaceutical formulation containing the amniotic fluid and / or its extract.
  • the eggs of the other birds are birds with an embryonic age of 5-20 days.
  • the eggs of the other birds are eggs with an embryo age of 6-15 days.
  • the amniotic fluid is derived from eggs with an embryonic age of 7 days.
  • the eggs of the other birds are duck eggs, goose eggs, or a combination thereof.
  • the method for improving cardiac function is a method for improving heart failure, especially a method for improving senile cardiac hypofunction or cardiac insufficiency.
  • FIG. 1 Ejection fraction of myocardial infarction mice. Cardiac ultrasound can measure the ejection fraction and left ventricular short axis shortening rate in mice. It can be seen from the figure that the treatment of amniotic fluid (EE) significantly increased the ejection fraction of myocardial infarction mice and significantly improved cardiac function.
  • EE amniotic fluid
  • FIG. 2 Left ventricular short-axis shortening rate in myocardial infarction mice. Cardiac ultrasound can measure the ejection fraction and left ventricular short axis shortening rate in mice. It can be seen from the figure that the treatment of amniotic fluid (EE) significantly increased the left ventricular short axis shortening rate of myocardial infarction mice and significantly improved cardiac function.
  • EE amniotic fluid
  • FIG. 3 Masson trichrome staining of myocardial infarcted mouse hearts. It can be seen from the figure that myocardial infarction mice have severe fibrosis and the left ventricular wall is significantly thinner. After treatment with amniotic fluid (EE), the left ventricular wall became thinner and fibrosis was significantly reduced.
  • EE amniotic fluid
  • FIG. 4 Immunofluorescence staining (PH3, cTnT, DAPI) of the heart of myocardial infarction mice.
  • FIG. 5 Immunofluorescence staining of hearts of myocardial infarcted mice (AuroraB, cTnT, DAPI). It can be seen from the figure that the PH3 positive and AuroraB positive cells in the heart of the mice in the treatment group increased significantly, indicating that EE treatment significantly induced heart cell regeneration in myocardial infarction mice.
  • FIG. 6 The area of cardiac fibrosis in mice with myocardial infarction was significantly reduced after treatment with amniotic fluid (EE) than in the untreated group (NS).
  • EE amniotic fluid
  • Figure 7 HPLC results of amniotic fluid of eggs at 7 days of age.
  • Figure 8 HPLC results of amniotic fluid of eggs at 11 days of age.
  • Figure 9 HPLC results of amniotic fluid of eggs at 13 days of age.
  • Figure 10 Free radical resistance of amniotic fluid of eggs of different embryo ages.
  • the abscissa indicates the embryonic age, and the ordinate indicates the clearance rate.
  • Figure 11 Growth curves of chicken embryo fibroblasts under different culture conditions. Amniotic fluid of eggs can promote cell growth.
  • Figure 12 Effect of amniotic fluid from eggs on the growth viability and migration capacity of human umbilical vein endothelial cells (HUVEC).
  • the abscissa indicates the culture medium, and the ordinate indicates the OD450 value.
  • Figure 13 The effect of amniotic fluid from duck eggs on the growth viability and migration capacity of chicken embryo fibroblasts.
  • the abscissa indicates the culture medium, and the ordinate indicates the OD450 value.
  • Figure 14 Gel column GE HiLoad 16/600 Superdex 75pg separation chromatogram.
  • Figure 15 Cell viability detection gel column GE HiLoad 16/600 Superdex 75pg fractions.
  • the abscissa indicates the culture medium, where FBS indicates fetal calf serum; DMEM is Dulbecco's Modified Eagle Medium; EE indicates amniotic fluid; "EE” indicates lyophilized amniotic fluid; S-200B indicates the fraction of the B peak; Q UNBOUND indicates the unbound fraction of the anion column ; 3-1 to 3-6 represent the third step purification of medium volume fractions 1-6, respectively.
  • Figure 16 Unbound fractions separated by cell viability detection cation exchange column GE HiPrep SP and anion exchange column HiPrep Q.
  • the abscissa indicates the culture medium, where FBS indicates fetal calf serum; DMEM is Dulbecco's Modified Eagle Medium; EE indicates amniotic fluid; "EE” indicates lyophilized amniotic fluid; Hiprep SP-UN indicates fractions that do not bind Hiprep SP column; Hiprep Q-UN Denotes the fraction not bound to the Hiprep Q column; Hiprep Q-Bound represents the fraction bound to the Hiprep Q column.
  • Figure 17 Amniotic fluid from eggs promotes the growth of primary cardiomyocytes. Among them, the abscissa indicates the culture medium, and the ordinate indicates the OD450 value.
  • Figure 18 EE improves cardiac function and reduces left ventricular remodeling in MI pigs.
  • FIG. 19 EE reduces myocardial infarct size and prolongs activity time in IR pigs.
  • Figure 20 The effect of amniotic fluid from mice on the growth viability of AC16 cells.
  • amniotic fluid and / or its extracts are collectively referred to herein as EE.
  • amniotic fluid can come from poultry eggs and non-human mammals.
  • Poultry eggs refer to the eggs of birds.
  • Preferred birds are poultry such as chicken, duck and goose.
  • the present invention uses poultry eggs with an embryo age of 5-20 days, preferably 6-15 days. It should be understood that the appropriate embryo age may not be the same for different poultry eggs. For example, when eggs are used, eggs with an embryonic age of 5-12 days are preferred, eggs with an embryonic age of 6-11 days are more preferred, eggs with an embryonic age of 7-9 days are more preferred, and embryonic age is more preferred. Eggs for 7-8 days.
  • eggs whose development period corresponds to the development period of the above-mentioned embryonic age eggs may be used.
  • duck eggs with an embryo age of 8-10 days, especially 8-9 days, may be the best.
  • Poultry eggs and amniotic fluid can be obtained by conventional methods.
  • the blunt end of an egg of the corresponding embryo age can be tapped to break the egg shell and peel the egg shell to form a mouth with a diameter of about 2 cm.
  • the amniotic membrane and the associated tissues surrounding the embryo are poured from the shell into a petri dish, and the amniotic fluid is drawn through the amniotic membrane with a syringe until the amniotic membrane is close to the embryo, thereby obtaining the amniotic fluid used in the present invention.
  • amniotic fluid can also come from non-human mammals, especially rodents, such as from mice.
  • Other non-human mammals can be common domestic animals, such as cattle, sheep, dogs, cats, pigs, and the like.
  • the amniotic fluid is derived from an embryo of a rodent having a gestational age of 8-20 days, preferably 8-14 days or 11-16 days, and more preferably 13-14 days, or from a period of development and gestational age that is An embryo of a non-human mammal corresponding to the developmental period of the rodent at 8-20 days, preferably 8-14 days or 11-16 days, more preferably 13-14 days.
  • Amniotic fluid can be obtained by conventional methods.
  • amniotic fluid used in the present invention can be obtained.
  • amniotic fluid may be centrifuged to separate impurities that may be contained, such as egg yolk, etc., to obtain pure amniotic fluid as much as possible.
  • the supernatant obtained after centrifugation is the amniotic fluid used in the present invention.
  • amniotic fluid as used herein shall mean "pure” amniotic fluid, that is, isolated from avian eggs or non-human mammalian embryos that do not contain Amniotic fluid of other components in bird eggs or non-human mammal embryos, and not contaminated with foreign substances. Pure amniotic fluid can be stored in a refrigerator below -60 ° C and thawed before use.
  • the invention uses extracts of amniotic fluid.
  • the extract does not bind to the ion exchange column between pH 5.8 and 8.0, and the molecular weight of the contained component is in the range of 500 to 1200 Daltons.
  • the neutral fraction having a molecular weight of 500 to 1200 Daltons can be separated from the amniotic fluid, thereby obtaining the extract.
  • the methods herein can be performed using gel columns and ion exchange columns well known in the art.
  • a well-known gel chromatography column (such as the various gel chromatography columns described below) can be used to separate a molecular weight of 500-1200 Daltons from amniotic fluid, and then use an ion exchange method (such as the following using The ion exchange column) separates the neutral fraction from the fraction.
  • the neutral fraction can be separated from the amniotic fluid by ion exchange (such as using an ion exchange column described below) before using a gel chromatography column (such as various gel chromatography columns described below). ) The fraction with a molecular weight in the range of 500-1200 Daltons in the neutral fraction is separated.
  • a neutral fraction having a molecular weight of 500-2000 Daltons may be separated from the amniotic fluid first, and then a fraction having a molecular weight in the range of 500-1200 Daltons may be separated therefrom.
  • the method may include the following steps:
  • a neutral fraction having a molecular weight of 500-1200 Daltons is separated from the neutral fraction having a molecular weight of 500-2000 Daltons.
  • Step (1) can be achieved by using gel chromatography and ion exchange methods. Components with a molecular weight of 500-2000 Daltons in amniotic fluid were separated by a gel chromatography column, and an uncharged (neutral) fraction was obtained by ion exchange.
  • gel chromatography columns can be used to perform gel chromatography.
  • Such gel chromatography columns include, but are not limited to, GE's SephacrylS-100, SephacrylS-200, SephacrylS-300, Sephacryl S -400, Superose 12, Superose 6, Superdex 12, and Superdex 6. It should be understood that any other gel chromatography packing with a separation range of 500-10,000 Daltons can also be used.
  • the gel chromatography column can be first equilibrated with ddH 2 O, and the flow rate can be determined according to the actual situation.
  • the flow rate may be 0.5-50 ml / min, such as 1 ml / min.
  • UV absorption is between 200-300 nm, such as 280 nm. After the UV absorption curve is stable and the baseline is returned, the equilibrium is ended. After equilibration is complete, the sample can be loaded. The loading flow rate is determined according to the actual preparation. After loading, the crude product can be eluted with degassed ddH 2 O, and fractions with molecular weights between 500 and 2000 Daltons are collected. If necessary, the gel chromatography separation can be repeated several times, and the fractions with the same peak time at each separation can be mixed.
  • anion exchange and cation exchange can be used in the method of the invention.
  • anion exchange methods are used herein.
  • Commercially available anion exchange columns can be used, including, but not limited to, DEAE Sepharose, ANX Sepharose, Q Sepharose, Capto DEAE, Capto Q, Mono Q, and Mini Q from GE. It should be understood that other brands of anion exchange packings can also be used.
  • commercially available cation exchange columns can also be used, including but not limited to CM Sepharose, SP Sepharose, CaptoS, MonoS, and MiniS.
  • the ion exchange column is first equilibrated with a buffer when performing ion exchange.
  • the buffer may be a conventional buffer in the art, for example, a phosphate buffer, especially a sodium phosphate buffer may be used.
  • the pH of the buffer can be determined based on the ion exchange column used. For example, when an anion exchange column is used, the anion exchange column can be equilibrated with a buffer having a pH of 7.5 to 8.5, preferably 7.5 to 8.0; when a cation exchange column is used, the buffer can be equilibrated with a pH of 5.8 to 7.0, preferably 5.8 to 6.5. Cation exchange column.
  • the sodium phosphate buffer contains Na 2 HPO 4 and NaH 2 PO 4 at a pH of about 5.8 or 8.0.
  • the present invention preferably uses an anion exchange column for separation.
  • the flow rate can be determined according to the actual situation.
  • the flow rate may be 0.5-50 ml / min, such as 1 ml / min.
  • the ultraviolet absorption curve at 280 nm is stable, and the equilibrium is ended after returning to the baseline.
  • step (1) gel chromatography may be performed first to separate a fraction having a molecular weight of 500-2000 Daltons, and then ion exchange may be performed to separate a neutral fraction; or, ion exchange may be performed first to separate out
  • the neutral fraction of amniotic fluid is then separated by gel chromatography to separate the active ingredients with molecular weights in the range of 500-2000 Daltons to obtain neutral fractions with molecular weights between 500-2000 Daltons. .
  • the main purpose in step (2) is to further separate the neutral fraction obtained in step (1) to obtain an active ingredient with a molecular weight in the range of 500-1200 Daltons.
  • a commercially available gel chromatography column can be used to separate fractions having a molecular weight in the range of 500-1200 Daltons.
  • Suitable gel chromatography columns include, but are not limited to, HiLoad Superdex 16/600, Superdex 75pg, Superdex Peptide, Superdex 200, and Superdex 30 from GE. It should be understood that other brands of gel chromatography packings with a separation range of 500-10,000 Daltons can also be used.
  • the gel column can be equilibrated with ddH 2 O first, and the flow rate can be determined according to the actual situation.
  • the flow rate may be 0.5-50 ml / min, such as 1 ml / min.
  • the ultraviolet absorption curve at 280 nm is stable, and the equilibrium is ended after returning to the baseline.
  • the sample can be loaded. The loading flow rate is determined according to the actual preparation.
  • the crude product can be eluted with degassed ddH 2 O, and the fractions are collected to obtain fractions with molecular weights ranging from 500 to 1200 Daltons, which are the extracts described herein.
  • the extract obtained by the above method is formulated into a solution with a pH of 5.8-8.0 and passed through a variety of ion exchange columns (including DEAE Sepharose, Q Sepharose, Mono Q, CM Sepharose, SP Sepharose, and Mono S). No active ingredients are bound to these ion exchange columns.
  • amniotic fluid and its extracts can promote cell growth (including but not limited to promoting heart cell regeneration after myocardial infarction, such as cardiomyocytes), and improve or improve heart function.
  • tissue may be various tissues of the human or animal body, including but not limited to cartilage tissue, meniscus tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascial tissue, periosteum Any one or more of tissue, eye tissue, pericardial tissue, lung tissue, synovial tissue, neural tissue, kidney tissue, bone marrow, urogenital tissue, intestinal tissue, liver tissue, pancreatic tissue, spleen tissue and adipose tissue organization.
  • the tissue necrosis due to ischemia and hypoxia is myocardial ischemic necrosis. In certain embodiments, the tissue necrosis is myocardial necrosis caused by acute, persistent ischemia and hypoxia, ie, myocardial infarction.
  • the amniotic fluid and its extracts herein can also be used to improve heart function, especially to improve heart function in patients with heart failure. In certain embodiments, the amniotic fluid and its extracts herein can be used to improve the heart function and the heart function of patients with senile heart failure or heart dysfunction.
  • cardiac dysfunction or cardiac insufficiency is due to a variety of reasons leading to a decrease in the contractile function of the myocardium, reducing the forward blood flow of the heart, and causing symptoms of blood stasis in the systemic circulation or pulmonary circulation.
  • amniotic fluid and / or its extracts described herein can be used directly for the purposes described herein and administered to a subject in need.
  • the method of administration can be parenteral, intravenous or intracardiac.
  • a therapeutically effective amount of amniotic fluid and / or its extract can be mixed with an appropriate amount of physiological saline for injection, water for injection or glucose injection and then administered by, for example, intravenous infusion or intracardiac injection .
  • the dosage and frequency of administration can be determined by the medical staff according to the specific condition, the age and sex of the patient, and so on.
  • a therapeutically effective amount refers to a dose sufficient to ameliorate or in some way reduce the symptoms associated with the disease.
  • Such a dose may be administered as a single dose or may be administered according to an effective treatment regimen.
  • the dosage may also cure the disease, but it is usually administered to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired improvement in symptoms.
  • a dose to be administered to a human it may usually be 1-200 ml / time, and it may be administered by injection daily or weekly.
  • the frequency of administration may be once every two days, every three days, every four days, every five days, or every six days, or every half month, or monthly.
  • provided herein is a method of treating tissue necrosis caused by ischemia and hypoxia, the method comprising administering to a subject in need thereof a therapeutically effective amount of the amniotic fluid and / or its extract or containing The step of the pharmaceutical composition of amniotic fluid and / or its extract.
  • a method of improving cardiac function in a subject comprising administering to a subject in need thereof an effective amount of amniotic fluid and / or extracts thereof or containing said amniotic fluid and / or Steps of extracting a pharmaceutical composition.
  • subject generally refers to animals, such as mammals, and especially humans.
  • this document provides a method for treating myocardial ischemic necrosis (especially myocardial infarction), and a method for improving heart failure (especially senile heart failure or cardiac insufficiency) in a subject, the method comprising administering treatment to the subject in need
  • the avian eggs are as defined herein or as described in any embodiment herein.
  • the poultry eggs are poultry eggs, especially poultry eggs having an embryonic age of 5-12 days, more preferably 6-11 days, more preferably 6-9, more preferably 7-8 days, especially It's an egg.
  • the amniotic fluid or extract thereof is the amniotic fluid or extract thereof described herein from a rodent from 8-20 days, preferably 8-14 or 11-16 days, more preferably 13-14 days of pregnancy.
  • amniotic fluid and / or extracts thereof as described herein for the manufacture of a medicament for the treatment of tissue necrosis caused by ischemia and hypoxia. Also provided are amniotic fluid and / or extracts thereof as described herein for use in the treatment of tissue necrosis caused by ischemia and hypoxia.
  • amniotic fluid and / or its extracts in the manufacture of a medicament for improving cardiac function. Also provided are amniotic fluid and / or extracts thereof as described herein for improving cardiac function.
  • amniotic fluid and / or extracts thereof as described herein, especially amniotic fluid and / or extracts thereof in poultry eggs, more preferably with an embryonic age of 5-12
  • the amniotic fluid and / or its extract of eggs is more preferably 6-11 days, more preferably 6-9 days, more preferably 7-8 days.
  • the amniotic fluid or an extract thereof is an amniotic fluid or an extract thereof as described herein from a rodent that is 8-20 days, preferably 8-14 days or 11-16 days, and more preferably 13-14 days pregnant.
  • the pharmaceutical composition may be cryopreserved amniotic fluid and / or an extract thereof or a lyophilized reagent thereof, such as lyophilized amniotic fluid and / or an extract thereof.
  • the pharmaceutical composition may also contain other pharmaceutically acceptable carriers or excipients, such as physiological saline for injection, water for injection or glucose injection.
  • Microcomputer automatic incubator (Zhengda TM ZF880), clean petri dish, 1.0ml syringe (Jiangxi Hongda TM ), 70% alcohol-sterilized forceps, stainless steel sieve, sterile centrifuge tube ( # SCT-50ML-25-S) and low-speed refrigerated centrifuge (Zhongjia KDC-2046).
  • amniotic membrane and the associated tissues from the shell into the petri dish, and pierce the amniotic fluid with a syringe to extract the amniotic fluid.
  • the bevel of the needle should be facing away from the embryo until the amniotic membrane is close to the embryo, and then the clear, colorless, and foreign body-free Amniotic fluid is poured into a centrifuge tube inside the ice box.
  • amniotic fluid extract collected through the test of the Midalta TM 1800 UV spectrophotometer.
  • standard operating procedures of the photometer refer to the instruction manual. Qualified ones can be used for mixing and trimming.
  • Rabbit anti-human / mouse Aurora B antibody was purchased from Sigma Aldrich, USA; rabbit anti-human / mouse phosphorylated histone H3 polyclonal antibody was purchased from Merck Millipore, Germany; rabbit anti-human / mouse cTnT polyclonal antibody was purchased from Abcam, UK; Alexa Fluor-594 labeled goat anti-rabbit IgG, Alexa Fluor-488 labeled goat anti-rabbit IgG, Alexa Fluor-594 labeled goat anti-mouse IgG, and Alexa Fluor-488 labeled goat anti-mouse IgG were purchased from American Life Technologies Company; DAPI was purchased from American Sigma Aldrich company; goat Serum working fluid was purchased from Wuhan Baoshide Biological Engineering Co., Ltd.
  • Trizol was purchased from Invitrogen Corporation; Doxorubicin Hydrochloride was purchased from Shanghai Shenggong Biological Engineering Co., Ltd.
  • the experimental animal was a male C57BL / 6J mouse and was purchased from Shanghai Slark Experimental Animal Co., Ltd.
  • Leica Dmi8 fluorescence microscope and Leica IM50 image acquisition system were purchased from Leica Company in Germany; the small animal ultrasound diagnostic instrument was purchased from Canada's VisualSonics Company.
  • Triton X-100 Triton X-100 stock solution 5ml, 1 ⁇ PBS 995ml.
  • the collagen fibers, mucus, and cartilage were blue (such as light green liquor stained green), the cytoplasm, muscle, cellulose, and glial cells were red, and the nuclei were black and blue.
  • mice received isoflurane anesthesia in an induction box, the ventilator frequency was 115 beats / min, the respiration ratio was 1: 1, and the tidal volume was 1.5 ml.
  • a 20 g indwelling needle plastic tube was intubated through the mouth and trachea, connected to a small animal ventilator, and continuously anesthetized with pure oxygen containing 2.5% isoflurane. Prepare the skin, open the chest between 3-4 ribs, expose the heart, and ligate the left anterior descending branch with a 7-0 prolene thread. You will see the apex turn white, suture the intercostal space, suture the skin, and disinfect. Switch off the anesthetic and continue aeration until the mouse wakes up.
  • mice C57BL / 6J male mice were injected with doxorubicin (5mg / kg) once every 7 days for 8 weeks, and a total of four injections would lead to heart failure in mice, which was verified by cardiac ultrasound.
  • mice were sacrificed by intraperitoneal injection of 10% chloral hydrate (200 mg / kg), and the heart was removed. Liver and kidney, OCT embedding or paraffin embedding were also taken for 1 week.
  • mice Establish a mouse myocardial infarction model by referring to the method described in (4) above.
  • the established mouse myocardial infarction model was divided into control group (NS) and chicken embryo amniotic fluid (EE) treatment group (6 animals in each group).
  • EE treatment group 100 ⁇ l of the EE prepared in Example 1 was injected through the tail vein every two days. By the 21st day of the third week, a total of 10 injections were made.
  • the control group was injected with 100 microliters of physiological saline 10 times in the same manner.
  • Left ventricular ejection fraction is a key classic indicator of left ventricular function.
  • An increase in left ventricular ejection fraction indicates that cardiac function in mice is improved after myocardial infarction.
  • the ejection fraction of the mouse was calculated by echocardiography, and the results are shown in FIG. 1. It can be seen from Figure 1 that by the third week, the treatment of EE significantly increased the left ventricular ejection fraction of myocardial infarction mice, indicating that the treatment of EE significantly improved the cardiac function of mice after myocardial infarction.
  • the left ventricular short-axis shortening rate (LVFS) of each group of mice was calculated by echocardiography, and the results are shown in FIG. 2. It can be seen from FIG. 2 that by the third week, the treatment of EE significantly improved the LVFS of myocardial infarction mice, that is, the cardiac function of mice after myocardial infarction.
  • Masson's staining is a classic method for judging myocardial infarction and fibrous tissue.
  • the mice of each group treated for 21 days were sacrificed, paraffin sections of myocardial tissue were prepared, and stained according to the above point (3), and the results are shown in FIG. 3.
  • blue is the infarcted fibrosis tissue and red is the muscle tissue.
  • myocardial infarction mice have severe fibrosis, and fibrosis is significantly reduced after EE treatment; suggesting that the treatment of EE prevents small Fibrosis after Myocardial Infarction in Rats
  • the size of the left ventricular cavity is the basis for judging the presence or absence of ventricular dilatation after myocardial infarction.
  • Ventricular dilatation is an important sign of decreased cardiac function. It can also be seen from FIG. 3 that the ventricular cavity of the myocardial infarction mice in the control group was severely enlarged, but after 3 weeks of EE treatment, the left ventricular cavity of the mice in the treatment group was not significantly enlarged.
  • PH3 staining is an indicator for judging cell regeneration in the heart.
  • the mice of each group treated for 21 days were sacrificed, frozen sections of myocardial tissue were prepared, and PH3 staining was performed according to the method described in point (1) above, and the results are shown in FIG. 4.
  • FIG. 4 cells with positive PH3 staining (green fluorescent dots, indicated by arrows) in the heart tissue of the EE-treated mice significantly increased, indicating that the treatment of EE promoted the regeneration of cells in the heart tissue.
  • AuroraB staining is an indicator for judging cell regeneration in the heart.
  • AuroraB staining was performed according to the method described in point (1) above, and the results are shown in Figure 5. From Figure 5, it can be clearly seen that AuroraB staining (green fluorescent dots, arrows) cells in the heart tissue of mice treated with EE The significant increase indicates that the treatment of EE promotes the regeneration of cells in cardiac tissue.
  • mice heart failure model Refer to the method (5) above to construct a mouse heart failure model.
  • the established mouse heart failure model was divided into a control group and a chicken embryo component extract (EE) treatment group (6 animals in each group).
  • EE treatment group 100 ⁇ l of the EE prepared in Example 1 was injected through the tail vein every two days. By the 21st day of the third week, a total of 10 injections were made.
  • the control group was injected with 100 microliters of physiological saline 10 times in the same manner.
  • Left ventricular ejection fraction is a key classic indicator of left ventricular function.
  • An increase in left ventricular ejection fraction indicates that cardiac function in mice is improved after heart failure.
  • the ejection fraction of the mice was measured by echocardiography, and the results are shown in FIG. 6. It can be seen from Figure 6 that by the third week, the treatment of EE significantly increased the left ventricular ejection fraction of mice with heart failure, indicating that the treatment of EE significantly improved the heart function of mice with heart failure. The area of left ventricular fibrosis was significantly reduced.
  • Hitachi Primaide-type high performance liquid chromatography is used to detect the amniotic fluid components of eggs of different embryo ages.
  • Test according to the instruction manual of the chromatograph. Among them, the test was first washed with 100% acetonitrile for 30 minutes, and the flow rate time was 0.8 mL / min, and then the water was equilibrated for 30 minutes, and the flow rate was 0.8 mL / min time. Take a 25 ⁇ L sample and eliminate air bubbles. Click the “Data Acquisition” button of the software that comes with the chromatograph, select “Method 2”, and click “Single Analysis Start” at the bottom of the screen. When the system waits for injection, start to inject the sample. The injection should be quick, and the valve should be switched after the injection.
  • the method 2 is as follows:
  • amniotic fluid with a gestational age of 7 days, 11 days, and 13 days is detected, and the results are shown in Figs. 7-9.
  • DPPH 1,1-diphenyl-2-picrylhydrazine radical, and its structure is as follows:
  • DPPH a stable free radical, provides an ideal and simple pharmacological model for the detection of free radical scavenging activity.
  • DPPH is used to detect the free radical resistance of chicken embryo amniotic fluid.
  • Example 1 The method described in Example 1 was used to obtain amniotic fluid of chicken embryos with embryonic ages of 6, 7, 8, 9, 9, and 11 days, and then stored in a refrigerator at 4 ° C for use after centrifugation.
  • Vitamin C was used as a positive control to determine the standard curve. Take different volumes of 0.04mg / ml Vc samples and add 0.6ml of DPPH, add absolute ethanol to make up to 1ml, mix well, use methanol as a control to zero, and measure the absorbance at 519nm wavelength. Plot the data after three replicates.
  • test group Sample solution 95% ethanol (or anhydrous ethanol) DPPH test solution total capacity Blank group 0mL 0.4mL 0.6mL 1mL Vc n ⁇ L (400-n) ⁇ L 0.6mL 1mL Sample set 0.4mL 0mL 0.6mL 1mL
  • Creatine clearance (inhibition rate) from the following companies:
  • This example tests the effect of egg amniotic fluid (EE) of Example 1 on the growth of chicken embryo fibroblasts under different culture conditions.
  • the composition of the DMEM medium used in this example is as follows: # Cat.11960077, add 1% L-glutamine ( # G0200) and 5% FBS ( # Cat.10099141)), 0.25% trypsin (Hangzhou Keyi Biological TM # CY003), PBS (BI TM # 02-024-1ACS), 0.4% trypan blue stain (BBI TM # 72-57-1) .
  • Discard the supernatant add 4 ml of DMEM medium, and resuspend the cells with a pipette tip. Inject 1 ml of the cell suspension into 10 cm cell culture dishes containing 10 ml of fresh medium containing different volume ratios of amniotic fluid. Shake the culture dish in the cross direction, at least 20 times in each direction, to make the cells uniformly distributed, and culture at 37 ° C and 5% CO 2 .
  • Figure 11 shows that after 96 hours of incubation, the number of chicken embryo fibroblasts in the experimental group with EE was significantly higher than the number of cells with the control without EE.
  • Example 2 The same method as in Example 1 was used to obtain amniotic fluid of duck eggs with an embryonic age of 8 days. Scratch experiments were used to test the effects of egg amniotic fluid on chicken embryo fibroblasts and duck egg amniotic fluid on human umbilical vein endothelial cells (HUVEC) growth viability and migration ability. Duck eggs and amniotic fluid were obtained from duck eggs with an embryonic age of 8 days and obtained by the method of Example 1. Chicken embryo fibroblasts were obtained by the method described in Example 5, and human umbilical vein endothelial cells were obtained from a commercially available route.
  • VEC human umbilical vein endothelial cells
  • composition of the DMEM medium used in this example is as follows: # Cat.11960077, add 1% L-glutamine ( # G0200) and 5% FBS ( # Cat.10099141)), 0.25% trypsin (Hangzhou Keyi Biological TM # CY003), PBS (BI TM # 02-024-1ACS), 0.4% trypan blue stain (BBI TM # 72-57-1) .
  • the width is preferably 1000-2000 ⁇ m. Wash each well 3 times with 2ml PBS and wash away the cells from the scratches. 2ml of medium containing different content of EE was added to each well, and the culture was changed every 48 hours. The timing of the scratch was 0h, and photos were taken at fixed points every 24 hours to measure the cell spacing on both sides of the scratch. Observe the cell growth status in each well; draw a chart with time (days) as the horizontal axis and the scratch distance in each well as the vertical axis; calculate the healing speed of the scratches in each well.
  • FIG. 12 shows the effect of amniotic fluid from eggs on the growth viability and migration ability of human umbilical vein endothelial cells (HUVEC). The addition of 5% (volume ratio) amniotic fluid significantly promoted the healing of HUVEC.
  • Figure 13 shows the effect of amniotic fluid from duck eggs on the growth viability and migration capacity of chicken embryo fibroblasts. The addition of amniotic fluid also showed a very significant promotion effect on the healing of chicken embryo fibroblasts.
  • HUVEC human umbilical vein endothelial cells
  • the purpose of this example is to gradually purify the biologically active compounds of chicken embryo amniotic fluid through analytical column gel column SephacrylS-200, anion exchange column HiPrep Q, desalting column HiPrep 26/10 Desalting, HiLoad 16/600 Superdex75pg.
  • Step 1 Gel column GE Sephacryl S-200
  • Step 2 Anion exchange column GE HiPrep Q
  • Sample loading Take the biologically active fraction purified in the first step, use a pump to load the sample at a flow rate of 1.5ml / min, and load 250ml, while collecting the unbound portion of the anion column with an equal volume, 2ml / tube;
  • Desalting The bound and unbound fractions in the ion column are replaced with degassed ddH 2 O by GE HiPrep 26/10 Desalting, respectively, and the desalted portion is collected;
  • Step 3 Gel column GE HiLoad 16/600 Superdex75pg
  • Test cell activity After digesting AC16 with better growth, spread it in 96-well plate, 8000 cells / well, five duplicate wells per group. The cells were adhered in a 5% CO 2 saturated humidity 37 ° C incubator for 2 hours. After 24 hours of starvation in culture with DMEM, it was replaced with 10% FBS DMEM, DMEM, and 20% (volume ratio) added medium. After 24 hours of incubation, 10 ⁇ l of CCK-8 reagent was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm on a microplate reader.
  • Step 1 Gel column GE Sephacryl S-200
  • Step 2 cation exchange column GE HiPrep SP
  • Sodium phosphate buffer A 50mM Na 2 HPO 4 + NaH 2 PO 4 , pH 5.8 equilibrated cation exchange column: flow rate 2ml / min, until the ultraviolet absorption curve at 280nm is stable, and returns to baseline;
  • Sample loading Take the fraction with molecular weight in the range of 500-2000 Dalton obtained in the first step, use the pump to load the flow rate of 1.5ml / min, the loading volume is 250ml, and collect the unbound part of the cation exchange column;
  • Step 3 Gel column GE HiLoad 16/600 Superdex75pg
  • the better-growing AC16 was digested and spread in 96-well plates at 8,000 cells / well, with five replicates in each group.
  • the cells were adhered in a 5% CO 2 saturated humidity 37 ° C incubator for 2 hours. After 24 hours of starvation in culture with DMEM, it was replaced with 10% FBS DMEM, DMEM, and 20% (volume ratio) added medium. After 24 hours of incubation, 10 ⁇ l of CCK-8 reagent was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm on a microplate reader.
  • the cell viability of the unbound region after cation exchange column GE HiPrep SP treatment is shown in FIG. 16.
  • the first step an ion exchange column, an anion exchange column HiPrep Q can be used to make the pH of each solution 5.8 and 8.0, and then load and ion exchange column, flow rate 2ml / min, until the UV absorption curve at 280nm is stable, return to baseline ;
  • Sample loading Take amniotic fluid, use the pump to load the flow rate of 1.5ml / min, the loading volume is 50ml, and collect the unbound fraction of the ion column;
  • Step 2 Gel column GE Sephacryl S-200
  • the sample is the unbound fraction in the first step, the flow rate is 1ml / min, and the loading amount is 10ml;
  • Step 3 Gel column GEHiLoad 16/600 Superdex75pg
  • Sample loading Take the fractions in the range of 500-2000 Dalton obtained in the second step, and load the sample at a flow rate of 1ml / min and a loading volume of 10ml;
  • the better-growing AC16 was digested and spread in 96-well plates at 8,000 cells / well, with five replicates in each group.
  • the cells were adhered in a 5% CO 2 saturated humidity 37 ° C incubator for 2 hours. After 24 hours of starvation in culture with DMEM, it was replaced with 10% FBS DMEM, DMEM, and 20% (volume ratio) added medium. After 24 hours of incubation, 10 ⁇ l of CCK-8 reagent was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm on a microplate reader.
  • the cell viability of the non-binding region after anion exchange column GE HiPrep Q treatment is shown in FIG. 16.
  • the ventricles of the suckling rats were washed with pre-chilled PBS, and then the heart tissue was cut in DMEM / F12. Shake at 37 ° C in a water bath and digest with 0.04% collagenase II + 0.08% trypsin. Filter and centrifuge the digested cells with a sieve, 1000 r / min, 10 min. Add 15% FBS cell culture solution to plate and incubate in a 5% CO 2 saturated humidity 37 ° C incubator.
  • Figure 18 shows that chicken EE treatment of myocardial infarction in large white pigs can increase left ventricular ejection fraction and short-axis shortening rate of myocardial infarction in large white pigs.
  • Postoperative heart function of large white pigs in the control group showed a gradual decline, while Ventricular function improved to some extent, and EF and FS were significantly higher at 2 weeks, 4 weeks, and 8 weeks than the control group ( Figure 18, A and C).
  • ⁇ EF and ⁇ FS statistics were calculated using the difference from the preoperative baseline values. It was found that the EF and FS significantly decreased compared to the preoperative decrease values at 1 week after EE treatment, and the decrease values at 2 weeks, 4 weeks, and 8 weeks in the treatment group were significant.
  • EE can significantly increase the left ventricular ejection fraction and stroke volume of ischemia-reperfusion pigs, reduce left ventricular remodeling caused by myocardial infarction, reduce pulmonary congestion in ischemia-reperfusion pigs, and increase Daily activity.
  • TTC staining results showed that the area of myocardial infarction in the EE treatment group was significantly lower than that of the control group.
  • Tissue Masson staining results showed that the left anterior wall of the large white pigs in the control group had transmural infarction and the fibrotic area was significantly higher than that of the EE treatment group ; Fluorescence staining results show that EE can increase the angiogenesis in the infarcted area of white pigs.
  • Example 1 Refer to the method described in Example 1 to obtain amniotic fluid of 13-14 days of gestational age in mice. After equilibrating the centrifuge tube collecting the amniotic fluid extract, use a Zhongjia TM KDC-2046 low-speed refrigerated centrifuge at 5 ° C and 3500 rpm for 21 minutes. (Refer to the manual for the standard operating procedure of the centrifuge). The supernatant was decanted and transferred to a clean plastic storage tank and stored in a -80 ° C refrigerator. Reserve 5ml samples for each batch for subsequent testing. All steps are performed under sterile conditions.
  • Test cell activity After digesting AC16 with better growth, spread it in 96-well plate, 8000 cells / well, five duplicate wells per group. The cells were adhered in a 5% CO 2 saturated humidity 37 ° C incubator for 2 hours. After 24 hours of starvation in DMEM, 10% FBS DMEM and DMEM were replaced with 2.5%, 5%, 10%, and 20% (volume ratio) mouse EE medium. After 24 hours of incubation, 10 ⁇ l of CCK-8 reagent was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm on a microplate reader.

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Abstract

一种用于治疗缺血和缺氧引起的组织坏死或改善心脏功能的药物，该药物含有来自非人动物的羊水和/或其提取物，和任选的药学上可接受的载体，所述羊水来自于来自胚龄为5-12天的鸡蛋、发育时期相对应的其他禽类的蛋、胎龄为8-20天啮齿类动物的胚胎或发育时期相对应的其他非人哺乳动物的胚胎。能促进人脐静脉内皮细胞、AC16细胞和鸡胚胎成纤维细胞的生长，提高心肌梗塞后的心脏功能，促进心肌梗死后心脏细胞再生。

Description

用于治疗组织坏死或改善心脏功能的药物 技术领域
本发明涉及用于治疗组织坏死或改善心脏功能的治疗药物。
背景技术
通常，由于缺血导致缺氧，结果导致局部组织、细胞新陈代谢停止，其功能完全丧失，细胞可出现核浓缩、核碎裂及核溶解等变化而死亡，结果导致组织坏死。
心肌梗塞是由于缺血缺氧导致的组织坏死的例子之一，指在冠状动脉病变的基础上，冠状动脉的血液中断，使相应的心肌出现严重而持久的急性缺血，最终导致急性、持续性缺血、缺氧(冠状动脉功能不全)所引起的心肌坏死。
发生急性心肌梗塞的病人，在临床上常有持久的胸骨后剧烈疼痛、发热、白细胞计数增高、血清心肌酶升高以及心电图反映心肌急性损伤、缺血和坏死的一系列特征性演变，并可出现心律失常、休克和心力衰竭，属冠心病的严重类型，可并发心律失常、休克或心力衰竭等合并症，常会危及生命。
发明内容
本发明提供一种药物组合物，该药物组合物含有：
非人动物的羊水和/或其提取物，和
任选的药学上可接受的载体；
其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-20天、优选8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天、优选8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
在一个或多个实施方案中，所述药物组合物为-60℃以下冷冻保存羊水和/或其提取物，或为所述羊水和/或其提取物的冻干试剂。
在一个或多个实施方案中，所述药物组合物为输注液。
在一个或多个实施方案中，所述药物组合物不含有或含有等渗的生理盐水、注射用水或葡萄糖注射液。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为5-20天的禽蛋。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为6-15天的禽蛋。
在一个或多个实施方案中，所述羊水来自胚龄为7天的鸡蛋。
在一个或多个实施方案中，所述其它禽类的蛋为鸭蛋、鹅蛋或其组合。
本发明还提供羊水和/或其提取物在制备治疗缺血和缺氧引起的组织坏死的药物中的应用，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-20天、优选8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天、优选8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
在一个或多个实施方案中，所述组织坏死是心肌缺血坏死。
在一个或多个实施方案中，所述组织坏死是心肌梗塞。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为5-20天的禽蛋。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为6-15天的禽蛋。
在一个或多个实施方案中，所述羊水来自胚龄为7天的鸡蛋。
在一个或多个实施方案中，所述其它禽类的蛋为鸭蛋、鹅蛋或其组合。
本发明还提供羊水和/或其提取物在制备改善心脏功能的药物中的应用，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-20天、优选8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天、优选8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为5-20天的禽蛋。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为6-15天的禽蛋。
在一个或多个实施方案中，所述羊水来自胚龄为7天的鸡蛋。
在一个或多个实施方案中，所述其它禽类的蛋为鸭蛋、鹅蛋或其组合。
在一个或多个实施方案中，所述药物用于改善心力衰竭，尤其是老年性心功能减退或心功能不全。
本发明还提供一种治疗缺血和缺氧引起的组织坏死的方法，所述方法包括给予需要的对象治疗有效量的羊水和/或其提取物，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自 胎龄为8-20天、优选8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天、优选8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
在一个或多个实施方案中，所述方法包括静脉注射含所述羊水和/或其提取物的药物制剂，或输注含所述羊水和/或其提取物的药物制剂。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为5-20天的禽蛋。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为6-15天的禽蛋。
在一个或多个实施方案中，所述羊水来自胚龄为7天的鸡蛋。
在一个或多个实施方案中，所述其它禽类的蛋为鸭蛋、鹅蛋或其组合。
本发明还提供一种改善心脏功能的方法，所述方法包括给予需要的对象治疗有效量的羊水和/或其提取物，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-20天、优选8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天、优选8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
在一个或多个实施方案中，所述方法包括静脉注射含所述羊水和/或其提取物的药物制剂，或输注含所述羊水和/或其提取物的药物制剂。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为5-20天的禽蛋。
在一个或多个实施方案中，所述其它禽类的蛋为胚龄为6-15天的禽蛋。
在一个或多个实施方案中，所述羊水来自胚龄为7天的鸡蛋。
在一个或多个实施方案中，所述其它禽类的蛋为鸭蛋、鹅蛋或其组合。
在一个或多个实施方案中，所述改善心脏功能的方法为改善心力衰竭的方法，尤其是改善老年性心功能减退或心功能不全的方法。
附图说明
图1：心肌梗塞小鼠的射血分数。通过心脏超声可以测算出小鼠的射血分数和左心室短轴缩短率。从图中可以看出羊水(EE)的治疗显著地提升了心肌梗塞小鼠的射血分数，心功能明显改善。
图2：心肌梗塞小鼠的左心室短轴缩短率。通过心脏超声可以测算出小鼠的射血分数和左心室短轴缩短率。从图中可以看出羊水(EE)的治疗显著地提升了心梗小鼠的左心室短轴缩短率，心功能明显改善。
图3：心肌梗塞小鼠心脏的马松三色染色。从图中可以看到心梗小鼠有严重的纤维化，左心室壁显著变薄。而羊水(EE)治疗后，左心室壁变薄不明显，纤维化显著减少。
图4：心肌梗塞小鼠心脏的免疫荧光染色(PH3，cTnT，DAPI)。
图5：心肌梗塞小鼠心脏的免疫荧光染色(AuroraB，cTnT，DAPI)。从图中可以看到治疗组小鼠心脏的PH3阳性和AuroraB阳性细胞显著增加，说明EE治疗显著地引发了心梗小鼠的心脏细胞再生。
图6：心肌梗塞小鼠心脏纤维化面积在羊水(EE)治疗后比未治疗组(NS)明显减小。
图7：胚龄为7天的鸡蛋的羊水的HPLC检测结果。
图8：胚龄为11天的鸡蛋的羊水的HPLC检测结果。
图9：胚龄为13天的鸡蛋的羊水的HPLC检测结果。
图10：不同胚龄的鸡蛋羊水的抗自由基能力。横坐标表示胚龄，纵坐标表示清除率。
图11：鸡胚胎成纤维细胞在不同培养条件下的生长曲线，鸡蛋的羊水能促进细胞生长。
图12：来自鸡蛋的羊水对人脐静脉内皮细胞(HUVEC)的生长活力和迁移能力的影响。横坐标表示培养基，纵坐标表示OD450值。
图13：来自鸭蛋的羊水对鸡胚胎成纤维细胞的生长活力和迁移能力的影响。横坐标表示培养基，纵坐标表示OD450值。
图14：凝胶柱GE HiLoad 16/600 Superdex75pg分离色谱图。
图15：细胞活力检测凝胶柱GE HiLoad 16/600 Superdex75pg分离馏分。横坐标表示培养基，其中，FBS表示胎牛血清；DMEM为Dulbecco's Modified Eagle Medium；EE表示羊水；“EE”表示冻干羊水；S-200B表示B峰的馏分；Q UNBOUND表示阴离子柱未结合馏分；3-1到3-6分别表示第三步纯化中等体积的馏分1-6。
图16：细胞活力检测阳离子交换柱GE HiPrep SP和阴离子交换柱HiPrep Q分离得到的未结合馏分。横坐标表示培养基，其中，FBS表示胎牛血清；DMEM为Dulbecco's Modified Eagle Medium；EE表示羊水；“EE”表示冻干羊水；Hiprep SP-UN表示未结合Hiprep SP柱的馏分；Hiprep Q-UN表示未结合Hiprep Q柱的馏分；Hiprep Q-Bound表示结合Hiprep Q柱的馏分。
图17：来自鸡蛋的羊水促进原代心肌细胞的生长。其中，横坐标表示培养基，纵坐标表示OD450值。
图18：EE提高心梗猪的心功能，并减轻左心室重构。
图19：EE降低IR猪的心脏梗死面积，并延长活动时间。
图20：来自小鼠的羊水对AC16细胞的生长活力的影响。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本文中，羊水和/或其提取物统称为EE，除非特别标注。本文中，羊水可来自禽蛋和非人哺乳动物。禽蛋指禽类的蛋。优选的禽类为家禽，如鸡、鸭和鹅。优选的是，本发明使用胚龄在5-20天、优选6-15天的禽蛋。应理解，不同禽蛋，合适的胚龄未必相同。例如，当使用鸡蛋时，优选使用胚龄为5-12天的鸡蛋，更优选使用胚龄为6-11天的鸡蛋，更优选使用胚龄为7-9天的鸡蛋、更优选使用胚龄为7-8天的鸡蛋。当使用其它禽类的蛋时，可使用其发育时期与上述胚龄的鸡蛋所处的发育时期相对应的蛋。例如，当使用鸭蛋时，胚龄为8-10天、尤其是8-9天的鸭蛋可能是最好的。
可采用常规的方法获得禽蛋羊水。例如，可敲击相应胚龄的蛋的钝端，使蛋壳碎裂，将蛋壳剥开形成一个直径约为2厘米的口子。然后用镊子小心撕开壳膜和卵黄膜，注意不要破坏羊膜。将包裹着胚胎的羊膜和相连组织从壳中倾倒至培养皿中，用注射器刺入羊膜抽取羊水，直至羊膜紧贴胚胎，由此即可获得用于本发明的羊水。
本文中，羊水还可来自非人哺乳动物，尤其是啮齿类动物，如来自小鼠。其它非人哺乳动物可以是常见的家畜，例如牛、羊、狗、猫、猪等。在某些实施方案中，羊水来自胎龄为8-20天、优选8-14天或11-16天、更优选13-14天的啮齿类动物的胚胎，或来自其发育时期与胎龄为8-20天、优选8-14天或11-16天、更优选13-14天的啮齿类动物所处的发育时期相对应的非人哺乳动物的胚胎。可采用常规的方法获得羊水。例如，用手术剪剪开怀孕8-20天、优选8-14天或11-16天、更优选13-14天的小鼠腹腔，小心取出并剪开子宫，用注射器刺入羊膜抽取羊水，直至羊膜紧贴胚胎，由此即可获得用于本发明的羊水。
应理解，必要时，可对羊水进行离心，以分离出可能含有的杂质，例如卵黄等，尽可能获得纯的羊水。离心后获得的上清液即为用于本发明的羊水。应理解的是，获取羊水的所有步骤都需在无菌条件下进行；另外，本文所示的“羊水”应指“纯”的羊水，即分离自禽蛋或非人哺乳动物胚胎的不含有禽蛋内或非人哺乳动物胚胎内其它成分、且也未被外源物质污染的羊水。纯的羊水可储存于-60℃以下的冰箱中，解冻后再使用。
在某些实施方案中，本发明使用羊水的提取物。优选地，该提取物在pH5.8-8.0之间不和离子交换柱子结合，且其所含成分的分子量在500-1200道尔顿范围内。可从羊水中分离出分子量为500-1200道尔顿的中性馏分，由此获得所述提取物。可采用本领域周知的凝胶柱和离子交换柱来实施本文的方法。例如，可使用周知的凝胶层析柱(如下文所述的各种凝胶层析柱)从羊水中分离出分子量为500-1200道尔顿的馏分，然后使用离子交 换方法(如使用下文所述的离子交换柱)从该馏分中分离出中性馏分。或者，也可先用离子交换方法(如使用下文所述的离子交换柱)从羊水中分离出中性馏分，然后再使用凝胶层析柱(如下文所述的各种凝胶层析柱)分离出该中性馏分中分子量在500-1200道尔顿范围内的馏分。
在某些实施方案中，可先从羊水中分离得到分子量在500-2000道尔顿的中性馏分，然后再从中分离得到分子量在500-1200道尔顿范围内的馏分。具体而言，该方法可包括以下步骤：
(1)从羊水中分离得到分子量为500-2000道尔顿的中性馏分；和
(2)从该分子量为500-2000道尔顿的中性馏分中分离得到分子量为500-1200道尔顿的中性馏分。
步骤(1)可通过使用凝胶层析和离子交换方法来实现。通过凝胶层析柱分离出羊水中分子量在500-2000道尔顿的成分，而通过离子交换可获得不带电荷(中性)的馏分。
本文中，可采用各种市售的凝胶层析柱来实施凝胶层析，这类凝胶层析柱包括但不限于GE公司的SephacrylS-100、SephacrylS-200、SephacrylS-300、Sephacryl S-400、Superose 12、Superose 6、Superdex 12和Superdex 6等。应理解，也可使用分离范围为500-10000道尔顿的其它任意凝胶层析填料。通常，在使用凝胶层析柱时，可先用ddH 2O平衡凝胶层析柱，流速可根据实际情况确定。例如，在某些实施方案中，流速可以是0.5-50ml/min，如1ml/min。通常，紫外吸收在200-300nm之间，如280nm。待紫外吸收曲线平稳，回归基线后，结束平衡。平衡结束后，可上样。上样流速根据实际制备情况确定。上样结束后，可用脱气ddH 2O洗脱粗品，收集分子量在500-2000道尔顿之间的馏分。需要时，可重复凝胶层析的分离数次，混合每一次分离时相同出峰时间的馏分。
本文中，可采用本领域周知的方法将带电荷的成分与不带电荷的成分分离。例如，可使用离子交换方法实现。阴离子交换和阳离子交换都可用于本发明方法中。在某些实施方案中，本文采用阴离子交换法。可使用市售的阴离子交换柱，包括但不限于GE公司的DEAE Sepharose、ANX Sepharose、Q Sepharose、Capto DEAE、Capto Q、Mono Q和Mini Q等。应理解，也可使用其它品牌的阴离子交换填料。或者，也可使用市售的阳离子交换柱，包括但不限于CM Sepharose、SP Sepharose、Capto S、Mono S和Mini S等。
通常，在实施离子交换时，先用缓冲液平衡离子交换柱。缓冲液可以是本领域常规的缓冲液，例如可使用磷酸盐缓冲液，尤其是磷酸钠缓冲液。缓冲液的pH可根据所使用的离子交换柱确定。例如，使用阴离子交换柱时，可使用pH为7.5～8.5、优选7.5～8.0的缓冲液平衡阴离子交换柱；使用阳离子交换柱时，可使用pH为5.8～7.0、优选5.8～6.5的缓冲液平衡阳离子交换柱。在某些实施方案中，该磷酸钠缓冲液含有Na 2HPO 4和 NaH 2PO 4，pH为约5.8或8.0。本发明优选使用阴离子交换柱进行分离。流速可根据实际情况确定。例如，在某些实施方案中，流速可以是0.5-50ml/min，如1ml/min。通常，待280nm紫外吸收曲线平稳，回归基线后，结束平衡。平衡结束后，可上样，收集流出部分(即未与柱结合的部分)。上样流速根据实际制备情况确定。
步骤(1)中，可先进行凝胶层析，分离出分子量在500-2000道尔顿的馏分，然后再进行离子交换，分离出中性馏分；或者，也可以先进行离子交换，分离出羊水中的中性馏分，然后再通过凝胶层析将中性馏分中分子量在500-2000道尔顿范围内的活性成分分离出来，获得分子量在500-2000道尔顿之间的中性馏分。
步骤(2)中的主要目的是进一步对步骤(1)获得的中性馏分进行分离，获得分子量大小在500-1200道尔顿范围内的活性成分。本文中，可使用市售的凝胶层析柱来分离得到分子量在500-1200道尔顿范围内的馏分。合适的凝胶层析柱包括但不限于GE公司的HiLoad Superdex 16/600 Superdex75pg、Superdex Peptide、Superdex 200和Superdex 30等。应理解，也可以使用分离范围在500-10000道尔顿的其它品牌的凝胶层析填料。
通常，可先用ddH 2O平衡凝胶柱，流速可根据实际情况确定。例如，在某些实施方案中，流速可以是0.5-50ml/min，如1ml/min。通常，待280nm紫外吸收曲线平稳，回归基线后，结束平衡。平衡结束后，可上样。上样流速根据实际制备情况确定。上样结束后，可用脱气ddH 2O洗脱粗品，收集馏分，获得所含成分的分子量在500-1200道尔顿范围的馏分，即为本文所述的提取物。
采用上述方法获得的提取物，将其配制成pH为5.8-8.0的溶液后过多种离子交换柱(包括DEAE Sepharose、Q Sepharose、Mono Q、CM Sepharose、SP Sepharose和Mono S)，其所含活性成分均不与这些离子交换柱结合。
本文发现，本文的羊水和其提取物能促进细胞的生长(包括但不限于促进心肌梗死后心脏细胞再生，如心肌细胞)，提高或改善心脏功能。
因此，本文涉及使用羊水和其提取物来治疗由于缺血和缺氧引起的组织坏死。组织可以是人体或动物体的各种组织，包括但不限于软骨组织、半月板组织、韧带组织、肌腱组织、椎间盘组织、牙周组织、皮肤组织、血管组织、肌肉组织、筋膜组织、骨膜组织、眼组织、心包组织、肺组织、滑膜组织、神经组织、肾组织、骨髓、泌尿生殖组织、肠道组织、肝组织、胰腺组织、脾组织和脂肪组织中的任意一种或多种组织。在某些实施方案中，由于缺血和缺氧引起的组织坏死是心肌缺血坏死。在某些实施方案中，该组织坏死是急性、持续性缺血和缺氧所引起的心肌坏死，即心肌梗塞。本文的羊水和其提取物还可用于改善心脏功能，尤其是改善心力衰竭患者的心脏功能。在某些实施方案中，本文的羊水和其提取物可用于改善老年性心功能减退或心功能不全患者的心脏功能，提高其心脏功能。 本文中，心功能减退或心功能不全是由于各种原因造成心肌的收缩功能下降，使心脏前向性排血减少，造成血液淤滞在体循环或肺循环产生的症状。
可直接使用本文所述的羊水和/或其提取物用于本文所述的用途，给予需要的对象。给药方式可以是肠胃外给药，静脉注射给药或心腔注射。在某些实施方案中，可将治疗有效量的羊水和/或其提取物与适量的注射用生理盐水、注射用水或葡萄糖注射液混匀，然后通过例如静脉内输注或心腔注射给药。
给药的剂量和频次可根据具体的病情，患者的年龄和性别等情况由医护人员确定。通常，对于特定疾病的治疗，治疗有效量是指足以改善或以某些方式减轻与疾病有关的症状的药量。这样的药量可作为单一剂量施用，或者可依据有效的治疗方案给药。给药量也许可治愈疾病，但是给药通常是为了改善疾病的症状。一般需要反复给药来实现所需的症状改善。例如，对于给予人的剂量，通常可在1-200ml/次，可每天或每周注射给予。在某些实施方案中，给药频次可以是每二天、每三天、每四天、每五天或每六天给药一次，或每半个月给药一次，或者每月给药一次。
因此，在某些实施方案中，本文提供一种治疗缺血和缺氧引起的组织坏死的方法，该方法包括给予需要的对象治疗有效量的本文所述的羊水和/或其提取物或含所述的羊水和/或其提取物的药物组合物的步骤。
在某些实施方案中，本文还提供一种改善对象心脏功能的方法，所述方法包括给予需要的对象有效量的本文所述的羊水和/或其提取物或含所述的羊水和/或其提取物的药物组合物的步骤。
本文中，“对象”通常指动物，如哺乳动物，尤其是人。
尤其是，本文提供一种治疗心肌缺血坏死(尤其是心肌梗塞)的方法，以及改善对象心力衰竭(尤其是老年性心功能减退或心功能不全)的方法，该方法包括给予需要的对象治疗有效量的本文所述的羊水和/或其提取物或含所述的羊水和/或其提取物的药物组合物，尤其是禽蛋羊水和/或其提取物或含有所述禽蛋羊水和/或其提取物的药物组合物的步骤。优选地，所述禽蛋如本文所定义或如本文任一实施方案所述。
在某些实施方案中，禽蛋是家禽类的蛋，尤其是胚龄为5-12天、更优选为6-11天、更优选6-9、更优选7-8天的禽蛋，尤其是鸡蛋。
在其它实施方案中，羊水或其提取物是本文所述的来自怀孕8-20天、优选8-14天或11-16天、更优选13-14天的啮齿动物的羊水或其提取物。
在某些实施方案中，本文提供本文所述的羊水和/或其提取物在制备治疗缺血和缺氧引起的组织坏死的药物中的应用。还提供的是用于治疗缺血和缺氧引起的组织坏死的本文所述的羊水和/或其提取物。
在某些实施方案中，本文提供所述的羊水和/或其提取物在制备改善心脏功能用的药物中的应用。还提供的是用于改善心脏功能的本文所述的羊水和/或其提取物。
本文还提供一种药物组合物，该药物组合物含有本文所述的羊水和/或其提取物，尤其是家禽的蛋中的羊水和/或其提取物，更优选为胚龄为5-12天、更优选为6-11天、更优选6-9天、更优选7-8天的鸡蛋的羊水和/或其提取物。在一些实施方案中，羊水或其提取物是本文所述的来自怀孕8-20天、优选8-14天或11-16天、更优选13-14天的啮齿动物的羊水或其提取物。药物组合物可以是-60℃以下冷冻保存羊水和/或其提取物或其冻干试剂，例如冻干羊水和/或其提取物。药物组合物中还可含有其它药学上可接受的载体或赋形剂，例如注射用生理盐水、注射用水或葡萄糖注射液等。
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并不意图限制本发明的范围。实施例中所用到的方法、试剂和仪器，除非另有说明，否则为本领域常规的方法、试剂和仪器。
实施例一
1、材料
a)仪器和工具
微电脑全自动孵化器(正大 TMZF880)，洁净培养皿，1.0ml注射器(江西洪达 TM)，经70％酒精消毒的镊子，不锈钢筛子，无菌离心管(
Figure PCTCN2019099947-appb-000001
#SCT-50ML-25-S)和低速冷冻离心机(中佳KDC-2046)。
b)试剂和生物材料
胚龄7天的鸡蛋。
2、实验流程
取鸡蛋，敲击朝上放置的、较为扁平的钝端，使蛋壳碎裂，将蛋壳剥开形成一个直径约为2厘米的口子，边缘应尽量平整。用镊子小心撕开壳膜和卵黄膜，注意不要破坏羊膜。观察胚胎发育状况，只有发育良好且符合对应阶段标准的胚胎可用于抽提羊水。
将包裹着胚胎的羊膜和相连组织从壳中倾倒至培养皿中，用注射器刺入羊膜抽取羊水，针口斜面应背对胚胎，直至羊膜紧贴胚胎，然后将澄清、无色、无异物的羊水注入冰盒内的离心管中。
用镊子取出羊膜中的胚胎，收集于放置在冰上的不锈钢筛子中，每隔一小时将收集的胚胎用搅拌机匀质化，封装在无菌的塑料储物罐中，倾斜放置于-80℃冰箱中。冷冻后 可竖直放置。
通过美谱达 TM1800紫外分光光度仪测试收集的羊水抽提液，光度仪标准操作流程参见使用手册，合格的可用于混合、配平。
将收集羊水抽提液的离心管配平后用中佳 TMKDC-2046低速冷冻离心机于5℃，3500rpm离心21分钟(离心机标准操作流程参见使用手册)。将上清液倾析转移至洁净的塑料储物罐中，储存于-80℃冰箱中。每批次预留5ml小样用于后续测试。
所有步骤都在无菌条件下进行。
实施例二
1、材料
常用普通试剂如氢氧化钠、氯化钠、氯化钾、水合磷酸氢钠、磷酸二氢钾、碳酸氢钠、碳酸钠、氯化镁、丙酮、浓硫酸、浓盐酸、二甲苯、无水乙醇、石蜡和蔗糖等购自国药集团化学试剂有限公司；十二烷基硫酸钠和乙二胺四酸等购自美国Sigma公司；Triton X-100和肝素购自北京鼎国公司；Tween-20购自美国Thermo Fisher公司；水合氯醛购自北京索莱宝科技有限公司；多聚甲醛和Masson Masson三色染试剂盒购自谷歌生物科技有限公司；OCT包埋剂购自日本樱花公司；防荧光萃灭封片剂购自美国Vector公司。
兔抗人/鼠Aurora B抗体购自美国Sigma Aldrich公司；兔抗人/鼠磷酸化组蛋白H3多抗购自德国Merck Millipore公司；兔抗人/鼠cTnT多克隆抗体购自英国Abcam公司；Alexa Fluor 594标记山羊抗兔IgG、Alexa Fluor 488标记山羊抗兔IgG、Alexa Fluor 594标记山羊抗鼠IgG和Alexa Fluor 488标记山羊抗鼠IgG购自美国Life Technologies公司；DAPI购自美国Sigma Aldrich公司；山羊血清工作液购自武汉博士德生物工程有限公司。
Trizol购自美国Invitrogen公司；盐酸阿霉素购自上海生工生物工程股份有限公司。
实验动物为雄性C57BL/6J小鼠，购自上海斯莱克实验动物有限公司。
Leica Dmi8荧光显微镜和Leica IM50图像采集系统购自德国Leica公司；小动物超声诊断仪购自加拿大VisualSonics公司。
0.1mol/L磷酸盐缓冲液(1×PBS)的配制：NaCl 8.0g，KCl 0.2g，Na 2PO 4·H 2O 3.58g，KH 2PO 4 0.24，调pH值至7.4，去离子水定容至1000ml，高压灭菌，储存于4℃。
0.5％Triton X-100的配制：Triton X-100原液5ml，1×PBS 995ml。
2、试验方法
(1)免疫荧光
(a)按实验要求处理好细胞爬片或冷冻切片，PBS洗，5min×3次。
(b)0.5％Triton X-100室温通透15min，PBS洗，5min×3次。
(c)山羊血清37℃封闭30min。
(d)弃血清，将一抗按适当比例稀释，滴加覆盖组织，4℃湿盒过夜。
(e)取出湿盒，37℃复温30min，PBS洗玻片或组织切片，5min×3次。
(f)将二抗按适当比例稀释，滴加覆盖组织，37℃孵育30min～60min。
(g)PBS洗3次，每次5min，DAPI染核10min。
(h)PBS洗3次，每次5min，防萃灭封片剂封片后荧光显微镜下观察分析。
(2)H&E染色
(a)4μm厚度切片，42℃捞片，60℃拷片过夜，常温保存。
(b)石蜡切片脱蜡至水：二甲苯3次，每次20min；梯度酒精(100％，95％，95％，90％，80％)水化，分别为：2min，2min，2min，1min，1min，自来水洗5min。
(c)PBS洗3遍，每次5min。
(d)苏木素染色5min。
(e)自来水冲洗10min。
(f)1％盐酸酒精分化两下，自来水冲洗5min。
(g)1％氨水返蓝2min，自来水冲洗5分钟。
(h)伊红染色1-5min。
(i)分别用80％，90％，95％，95％，100％的酒精脱水，时间分别为1min，2min，2min，2min，2min。
(j)二甲苯透明3次，每次2min。
(k)中性树胶封片，镜下观查。
(3)马松三色染色
(a)石蜡切片脱蜡至水。
(b)铬化处理(重铬酸钾过夜处理)。
(c)依次自来水和蒸馏水洗。
(d)用Harris氏苏木素染液或Weigert苏木素液染核1-2min。
(e)充分水洗，如过染可盐酸酒精分化2-3s。
(f)氨水返蓝2min。
(g)用Masson丽春红酸性复红液5-10min。
(h)1％磷钼酸水溶液分化3-5min。
(i)1％苯胺蓝或光绿液染5min。
(j)1％冰醋酸水溶液分化几秒。
(k)95％酒精、无水酒精、二甲苯透明、中性树胶封固。
结果：胶原纤维、粘液、软骨呈蓝色(如光绿液染色为绿色)，胞浆、肌肉、纤维素、神经胶质呈红色，胞核黑蓝色。
(4)小鼠心肌梗塞模型的建立
8周C57BL/6J雄性小鼠经诱导箱中异氟烷气麻，呼吸机频率为115次/min，呼吸比1:1，潮气量为1.5ml。用20g留置针塑料管套经嘴气管插管，连接小动物呼吸机，用含有2.5％异氟烷的纯氧持续麻醉。备皮，3-4肋间开胸，暴露心脏，用7-0prolene线左前降支结扎，会看到心尖变白，缝合肋间，缝皮，消毒。关掉麻药，持续通气至小鼠苏醒。
(5)小鼠心力衰竭模型的建立
8周C57BL/6J雄性小鼠7天注射一次阿霉素(5mg/kg)，一共注射四次后即会导致小鼠心力衰竭，通过心脏超声验证。
(6)取材、固定和切片
(a)手术后治疗1周和8周，小鼠腹腔注射10％的水合氯醛(200mg/kg)处死，取出心脏，1周取材还包括肝肾，OCT包埋或石蜡包埋。
(b)冰冻切片用于做免疫荧光＝石蜡切片用于H&E和马松三色染。
(c)标本在做完马松三色染后，用Image J图像分析软件测量心梗大小。心梗面积计算公式为：
Figure PCTCN2019099947-appb-000002
每个标本取5个截面，计算平均值。
3、统计分析
所有实验结果均以Mean±SEM表示。两组之间比较用Two-tailed tailed t检验，多组之间比较用单因素方差分析(one way ANOVA)。P<0.05为有显著性统计学差异的标准。所有实验结果利用GraphPad Prism 5(Software,Inc.)和Image J软件作图、分析。
4、实验结果
(I)参照前述(4)所述的方法建立小鼠心肌梗塞模型。将建立的小鼠心肌梗塞模型分为对照组(NS)与鸡胚羊水(EE)治疗组(每组6只)。EE治疗组每两天通过尾静脉注射100微升实施例一制备得到的EE，到第三周21天时，共注射10次。对照组以相同方式注射生理盐水100微升10次。
左室射血分数(LVEF)是左心室功能的关键经典指标，左室射血分数提高表明小鼠心肌梗塞后的心脏功能得以提高。通过心脏超声测算出小鼠的射血分数，结果如图1所示。从图1可以看出，到第3周时，EE的治疗显著地提升了心梗小鼠的左室射血分数，表明EE的治疗显著提高了小鼠心肌梗塞后的心脏功能。
通过心脏超声测算出各组小鼠的左心室短轴缩短率(LVFS)，结果如图2所示。从图2可以看出，到第3周时，EE的治疗显著地提升了心梗小鼠的LVFS，也即提高了小鼠心肌梗塞后的心脏功能。
马松染色是判断心脏梗死组织与纤维组织的经典方法。处死经21天处理后的各组小鼠，制备心肌组织的石蜡切片，按前述第(3)点进行染色，结果如图3所示。图3中，蓝色的为梗死纤维化组织，红色为肌肉组织，从图中可以看到心梗小鼠有严重的纤维化，而EE治疗后纤维化显著减少；提示EE的治疗防止了小鼠心肌梗塞后的纤维化。另外，左心室腔的大小是判断心肌梗塞后有无心室扩张的依据，心室扩张是心脏功能下降的重要标志。从图3中还可以看出，对照组心肌梗塞小鼠心室腔严重扩大，而经过EE 3周治疗后，治疗组小鼠左心室腔没有明显扩大。
PH3染色是判断心脏内细胞再生情况的指标。处死经21天处理后的各组小鼠，制备心肌组织的冷冻切片，按照上述第(1)点所述方法进行PH3染色，结果如图4所示。从图4可明显看出，EE治疗组小鼠心脏组织中PH3染色阳性(绿色荧光点，箭头所指)细胞明显增加，表明EE的治疗促进了心脏组织内细胞的再生。AuroraB染色是判断心脏内细胞再生情况的指标。按照上述第(1)点所述方法进行AuroraB染色，结果如图5所示，从图5中可以明显看出EE治疗组小鼠心脏组织中AuroraB染色阳性(绿色荧光点，箭头所指)细胞明显增加，表明EE的治疗促进了心脏组织内细胞的再生。
(II)参照上述方法(5)构建小鼠心力衰竭模型。将建立的小鼠心力衰竭模型分为对照组与鸡胚成分萃取物(EE)治疗组(每组6只)。EE治疗组每两天通过尾静脉注射100微升实施例一制备得到的EE，到第三周21天时，共注射10次。对照组以相同方式注射生理盐水100微升10次。
左室射血分数(LVEF)是左心室功能的关键经典指标，左室射血分数提高表明小鼠心力衰竭后的心脏功能得以提高。通过心脏超声测算小鼠的射血分数，结果如图6所示。从图6可以看出，到第3周时，EE的治疗显著地提升了心衰小鼠的左室射血分数，表明EE的治疗显著地提升了心力衰竭小鼠的心脏功能。左室纤维化面积明显降低。
实施例三
本实施例使用日立Primaide型高效液相色谱仪检测不同胚龄的鸡蛋的羊水成分。按该色谱仪的使用说明书进行检测。其中，检测开始前先用100％乙腈洗涤30分钟，流速时间为0.8mL/min，之后用水平衡30分钟，流速0.8mL/min时间。抽取25μL样品并排除气泡，点击色谱仪自带的软件的“数据采集”按钮，选择“方法2”，点击屏幕下方“单分析开始”，待系统出现“等待进样”时，开始注射样品，注射要迅速，注射完后切换阀门。该方法2如下：
时间(min) 水(％) 乙腈(％) 流量(mL/min)
0.0 100.0 0.0 0.8
11.0 100.0 0.0 0.8
17.0 95.0 5.0 0.8
30.0 90.0 10.0 0.8
45.0 55.0 45.0 0.8
60.0 0.0 100.0 0.8
70.0 0.0 100.0 0.8
本实施例检测了胎龄为7天、11天和13天的羊水，结果如图7－9所示。
实施例四
DPPH即1,1-二苯基-2-苦基肼基自由基，其结构如下所示：
Figure PCTCN2019099947-appb-000003
DPPH的分子中，由于存在多个吸电子的-NO 2和苯环的大π键，所以，氮自由基能 稳定存在。
当DPPH自由基被清除，其最大吸收波长519nm处的吸光度A值随之减小。DPPH这种稳定的自由基为清除自由基活性的检测提供了一个理想而又简单的药理模型。本实施例利用DPPH来检测来自鸡胚羊水的抗自由基能力。
取0.8mg的DPPH，溶于20mL溶剂甲醇中，超声5min，充分振摇，使上下各部分均匀。取1mL该DPPH溶液，在519nm处测A 0值，A＝0.5-0.7。该DPPH溶液避光保存，3.5小时内用完。
采用实施例一所述的方法分别获得胚龄为6天、7天、8天、9天、10天和11天的鸡胚胎的羊水，离心后保存在4℃冰箱中待用。
以维生素C作为正对照，测定标准曲线。取不同体积的0.04mg/ml Vc样品加入0.6ml的DPPH，加入无水乙醇补足至1ml，混匀，以甲醇为对照调零，测定519nm波长下的吸光值。重复三次数据后作图。
取不同胚龄的羊水400μl至试管中，加入600μl中配制好的DPPH的甲醇溶液，混匀，反应10min，使气泡不再产生(测定前要混匀)。以甲醇为对照调零，测定519nm的吸光度。
各组的加样信息如下表所示：
实验组 样品液 95％乙醇(或无水乙醇) DPPH测试液 总体积
空白组 0mL 0.4mL 0.6mL 1mL
Vc nμL (400-n)μL 0.6mL 1mL
样品组 0.4mL 0mL 0.6mL 1mL
采用以下公司肌酸清除率(抑制率)：
清除率(％)＝(A 0-A)/A 0×100％。
结果如图10所示。
实施例五
本实施例测试实施例一的鸡蛋羊水(EE)对鸡胚胎成纤维细胞在不同培养条件下的生长的影响。本实施例使用的DMEM培养基的组成如下：
Figure PCTCN2019099947-appb-000004
#Cat.11960077，加入1％L-谷氨酰胺(
Figure PCTCN2019099947-appb-000005
#G0200)和5％FBS(
Figure PCTCN2019099947-appb-000006
#Cat.10099141))，0.25％胰酶(杭州科易生物 TM#CY003)，PBS(BI TM#02-024-1ACS)，0.4％台盼蓝染剂(BBI TM#72-57-1)。
1、鸡胚胎成纤维细胞的获得和培养
取胚龄7天的鸡蛋的胚胎，用PBS冲洗胚胎表面，将液体用移液枪吸干净。取出胚胎内脏，将其余组织剪碎至无肉眼可见的大颗粒、团块。加1ml的0.25％胰酶，用枪头使之与组织混匀，将悬液吸取至15ml离心管。用1ml的0.25％胰酶冲洗培养皿，将悬液吸取至同一支15ml离心管。将离心管放入37℃水浴，消化5-7分钟后，加入8ml的DMEM培养基(含PBS)中和胰酶。将离心管放入离心机，离心5-10秒。取出离心管，收集上清液。2000rpm离心该离心上清液2min。弃上清，加入4ml的DMEM培养基，用枪头使细胞重悬。分别取1ml细胞悬液注入10cm细胞培养皿，再加入10ml的DMEM培养基。十字方向晃动培养皿，每个方向至少20次，使细胞分布均匀。于37℃、5％CO 2条件下培养。当细胞覆盖70％-90％的培养皿底部时，将细胞传代。
将培养皿从培养箱取出，收集原有的培养基于离心管中。小心加入5ml的PBS清洗细胞。之后，加入500μl、0.25％胰酶，将培养皿放入培养箱，消化1分钟。轻轻拍打培养皿侧边加快消化过程，待细胞团块快速分解，大部分细胞呈漂浮状态，迅速加入9.5ml回收的原有培养基中和胰酶。用移液管吹打培养皿底部，将尽量多的细胞悬液收集至15ml离心管中，于2000rpm离心3min。弃上清，加入4ml的DMEM培养基，用枪头使细胞重悬。分别将1ml细胞悬液注入含有10ml含不同体积比的羊水的新鲜培养基的10cm细胞培养皿中。十字方向晃动培养皿，每个方向至少20次，使细胞分布均匀，于37℃、5％CO 2条件下培养。
取生长良好的鸡胚胎成纤维细胞，收集原有的培养基于离心管中。小心加入5ml的PBS清洗细胞，注意不可造成细胞层破损，轻轻晃动后倒除PBS。加入100μl的0.25％胰酶消化2-5分钟(24孔板)，用100μl培养基中和。用枪头使之成为单细胞悬液。按照一定倍数稀释该单细胞悬液，加入等量0.4％台盼蓝染液染色，稀释倍数以稀释后细胞数在20-200之间为宜。吸取适量(15μl)细胞悬液，从盖玻片上下边缘加样到血球计数板上，显微镜下计活细胞数。计算活细胞总数，调整细胞浓度至1×10 5个细胞/ml。每24小时取样一次，每次取3个孔细胞，进行常规胰酶消化、制备单细胞悬液、显微镜计数。以时间(天)为横轴，细胞浓度为纵轴绘制生长曲线。细胞个数＝细胞总计数/4×10 4×稀释倍数，细胞浓度＝该细胞个数/ml。
结果如图11所示。图11显示，在共培育96小时以后，添加EE的实验组中鸡胚胎成纤维细胞的数量明显高于与未添加EE的对照的细胞数量。
实施例六
采用与实施例一相同的方法获得胚龄为8天的鸭蛋的羊水。采用划痕实验测试鸡蛋羊水对鸡胚胎成纤维细胞和鸭蛋羊水对人脐静脉内皮细胞(HUVEC)的生长活力和迁移能力的影响。鸭蛋羊水获自胚龄8天的鸭蛋，采用实施例一的方法获得。鸡胚胎成纤维细胞采用实施例五所述的方法获得，人脐静脉内皮细胞从市售途径获得。
本实施例使用的DMEM培养基的组成如下：
Figure PCTCN2019099947-appb-000007
#Cat.11960077，加入1％L-谷氨酰胺(
Figure PCTCN2019099947-appb-000008
#G0200)和5％FBS(
Figure PCTCN2019099947-appb-000009
#Cat.10099141))，0.25％胰酶(杭州科易生物 TM#CY003)，PBS(BI TM#02-024-1ACS)，0.4％台盼蓝染剂(BBI TM#72-57-1)。
实验前第一天，准备6孔板，用马克笔在6孔板背后用直尺画出5-6条分布均匀的横线，横穿过孔；再在中线位置画一条垂直的竖线以指示划痕的位置。在每个孔中加入约5×10 5个处于对数生长期的细胞，原则上接种过夜后融合率达到90％。
实验当天，用枪头比着直尺，沿马克笔竖线，垂直于6孔板底面划线。尽量不要倾斜、弯曲，不同孔之间最好用同一支枪头，宽度在1000-2000μm为宜。每个孔用2ml PBS清洗3次，洗去划痕处的细胞。各个孔中分别加入2ml含不同含量的EE的培养基，常规培养，每48小时换液。从划痕计时为0h，每24小时定点拍照，测量划痕两侧细胞间距。观察每个孔中细胞生长状况；以时间(天)为横轴，每个孔中划痕距离为纵轴绘制图表；计算每个孔中划痕愈合的速度。
结果如图12和13所示。图12显示来自鸡蛋的羊水对人脐静脉内皮细胞(HUVEC)的生长活力和迁移能力的影响，添加5％(体积比)的羊水明显对HUVEC的愈合具有非常明显的促进作用。图13显示来自鸭蛋的羊水对鸡胚胎成纤维细胞的生长活力和迁移能力的影响，羊水的添加对鸡胚胎成纤维细胞的愈合也显示出非常明显的促进作用。
实施例七
本实施例的目的在于通过分析柱凝胶柱SephacrylS-200、阴离子交换柱HiPrep Q、脱盐柱HiPrep 26/10 Desalting、HiLoad 16/600 Superdex75pg逐步纯化鸡胚羊水中具有生物活性的化合物。
1、材料
1.1纯化样品：新鲜的胚龄为7天的鸡蛋羊水，50ml。
1.2主要实验设备及耗材
1)GE AKTA purifier；
2)凝胶柱GE Sephacryl S-200；
3)阴离子交换柱GEHiPrep Q；
4)脱盐柱GEHiPrep 26/10 Desalting；
5)凝胶柱GEHiLoad 16/600 Superdex75pg；
6)Superloop 10ml。
2、方法
2.1溶液制备
磷酸钠缓冲液A(50mM Na 2HPO 4+NaH 2PO 4，pH 8.0)的制备：46.6ml 1mol/l Na 2HPO 4与3.4ml 1mol/l NaH 2PO 4混合，加ddH 2O定容至1L。
2.2实验方法
2.2.2样本处理：新鲜羊水50ml，加入适量己烷，2500rpm、4℃离心20min，获得水相，0.22μm滤膜过滤。
2.2.3样品纯化
第一步：凝胶柱GE Sephacryl S-200
ddH 2O平衡凝胶柱：流速2ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：流速1ml/min，上样量10ml；
洗脱：用脱气ddH 2O洗脱粗品，流速2ml/min，等体积收集馏分，3ml/管。2柱体积(240ml)洗脱；
重复分离纯化5次，每一次中相同出峰时间的部分充分混合；
第二步：阴离子交换柱GE HiPrep Q
磷酸钠缓冲液A(50mM Na 2HPO 4+NaH 2PO 4，pH 8.0)平衡阴离子交换柱：流速2ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：取第一步纯化后具有生物活性的部分，用泵上样流速1.5ml/min，上样量250ml，同时等体积收集阴离子柱未结合部分，2ml/管；
脱盐：将离子柱中结合和不结合的馏分分别用GE HiPrep 26/10 Desalting置换到脱气ddH 2O中，收集脱盐后的部分；
第三步：凝胶柱GE HiLoad 16/600 Superdex75pg
ddH 2O平衡凝胶柱：流速1ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：流速1ml/min，上样量10ml；
洗脱：用脱气ddH 2O洗脱样品，流速1ml/min，等体积收集馏分，2ml/管。洗脱1.5柱体积(240ml)；
测细胞活性：将长势较好的AC16消化后，铺于96孔板中，8000个/孔，每一组五个复孔。在5％CO 2饱和湿度37℃培养箱培养2小时，细胞贴壁。用培养基DMEM饥饿培养24小时后，替换成10％FBS的DMEM、DMEM和添加20％(体积比)馏分的培养 基。培养24小时后，每孔加入10μl CCK-8试剂。孵育2小时后，于酶标仪在450nm检测吸收值。
3、实验结果
经凝胶柱GE HiLoad 16/600 Superdex75pg分离的未结合部分的色谱图如图14所示。细胞活力检测跟踪到具有生物活性的生长因子群，结果如图15所示。
实施例八
采用与实施例七相同的方法，实施以下分离纯化：
1、活性成分分离纯化
第一步：凝胶柱GE Sephacryl S-200
ddH 2O平衡凝胶柱：流速2ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：流速1ml/min，上样量10ml；
洗脱：用脱气ddH 2O洗脱粗品，流速2ml/min，收集分子量在500-2000道尔顿范围内的馏分；
重复分离纯化5次，每一次中相同出峰时间的部分充分混合；
第二步：阳离子交换柱GE HiPrep SP
磷酸钠缓冲液A(50mM Na 2HPO 4+NaH 2PO 4，pH 5.8)平衡阳离子交换柱：流速2ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：取第一步获得的分子量在500-2000道尔顿范围内的馏分，用泵上样流速1.5ml/min，上样量250ml，收集阳离子交换柱未结合部分；
第三步：凝胶柱GE HiLoad 16/600 Superdex75pg
ddH 2O平衡凝胶柱：流速1ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：取第二步获得的未结合部分上样，流速1ml/min，上样量10ml；
洗脱：用脱气ddH 2O洗脱样品，流速1ml/min，收集分子量在500-1200道尔顿范围内的馏分。
2、活性成分检测
将长势较好的AC16消化后，铺于96孔板中，8000个/孔，每一组五个复孔。在5％CO 2饱和湿度37℃培养箱培养2小时，细胞贴壁。用培养基DMEM饥饿培养24小时后，替换成10％FBS的DMEM、DMEM和添加20％(体积比)馏分的培养基。培养24小时后，每孔加入10μl CCK-8试剂。孵育2小时后，于酶标仪在450nm检测吸收值。阳离子 交换柱GE HiPrep SP处理后不结合区的细胞活力如图16所示。
实施例九
采用与实施例七相同的方法实施以下分离纯化：
1、活性成分分离纯化
第一步：离子交换柱，可使用阴离子交换柱HiPrep Q，使各溶液的pH分别为5.8和8.0，然后分别上样与离子交换柱，流速2ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：取羊水，用泵上样流速1.5ml/min，上样量50ml，收集离子柱未结合馏分；
第二步：凝胶柱GE Sephacryl S-200
ddH 2O平衡凝胶柱：流速2ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：样品为第一步未结合馏分，流速1ml/min，上样量10ml；
洗脱：用脱气ddH 2O洗脱粗品，流速2ml/min，收集分子量在500-2000道尔顿范围内的馏分；
第三步：凝胶柱GEHiLoad 16/600 Superdex75pg
ddH 2O平衡凝胶柱：流速1ml/min，直到280nm紫外吸收曲线平稳，回归基线；
上样：取第二步获得的500-2000道尔顿范围内的馏分上样，流速1ml/min，上样量10ml；
洗脱：用脱气ddH 2O洗脱样品，流速1ml/min，收集分子量在500-1200道尔顿范围内的馏分。
2、活性成分检测
将长势较好的AC16消化后，铺于96孔板中，8000个/孔，每一组五个复孔。在5％CO 2饱和湿度37℃培养箱培养2小时，细胞贴壁。用培养基DMEM饥饿培养24小时后，替换成10％FBS的DMEM、DMEM和添加20％(体积比)馏分的培养基。培养24小时后，每孔加入10μl CCK-8试剂。孵育2小时后，于酶标仪在450nm检测吸收值。阴离子交换柱GE HiPrep Q处理后不结合区的细胞活力如图16所示。
实施例十
1、分离原代心肌细胞(VM)
取乳鼠心室于预冷PBS清洗，之后在DMEM/F12中剪碎心脏组织。37℃水浴震荡，用0.04％胶原酶Ⅱ+0.08％胰酶消化。用筛网将已经消化下来的细胞过滤离心，1000r/min， 10min。加15％FBS细胞培养液铺板，在5％CO 2饱和湿度37℃培养箱培养。
2、细胞活力检测
将原代心肌细胞消化后，铺于96孔板中，6000个/孔，每一组五个复孔。在5％CO 2饱和湿度37℃培养箱培养24小时之后，分别用培养基DMEM/F12、含10％FBS的DMEM/F12、含10％FBS和5％EE(实施例一制备得到的羊水，体积比)的DMEM/F12代替原来培养基15％FBS的DMEM/F12。培养48小时后，每孔加入10μl CCK-8试剂。孵育2小时后，于酶标仪在450nm检测吸收值。
结果如图17所示。
实施例十一
用实验大白猪通过经皮动脉导管(PCI)至心脏冠状动脉前降支进行充气球囊堵塞50分钟后撤除，构建了大白猪的心脏缺血再灌注模型，术后立即通过静脉注射采用实施例一所述方法获得的鸡EE(1ml/kg)进行治疗。术前测定基础的心功能。结果如图18和19所示。
图18显示，鸡EE治疗心梗大白猪能提高心梗大白猪的左室射血分数和短轴缩短率，术后对照组大白猪的心功能呈现逐渐下降的趋势，而EE治疗组的左室功能有一定回升，术后2周、4周和8周的EF和FS显著高于对照组(图18，A和C)。用与术前基础值的差值进行ΔEF和ΔFS统计，发现EE治疗后1周能显著降低EF和FS相较于术前的下降数值，治疗组2周、4周和8周的下降数值显著低于对照组(图18，B和D)。治疗组的每搏输出量在术后的1-8周都显著高于对照组(图18，E)。对照的左心室收缩末期的容积和直径有上升趋势，治疗组较对照组低(图18，F和I)，说明EE提高了左心室收缩力。对照的左心室舒张末期的容积和直径有上升趋势，给药组呈现先上升后下降的趋势(图18，G和H)，说明EE逆转了部分心肌梗塞(myocardialinfarction，MI)导致的心室重构。
大白猪缺血再灌注(IR)模型后，给药组立即给以鸡EE治疗，对照组给以5％葡萄糖。通过饲养时观察和监控视频发现，对照组大白猪活动时间少，神情倦怠。术后一周，通过统计发现，治疗组大白猪的日常活动时间显著高于对照组(图19，D)。EE治疗后8周取材，通过心脏层切后氯化三苯基四氮唑(TTC)染色发现，对照组心尖到前壁的心肌变薄，染色后呈现白色，左心室轻度扩张；治疗组心尖到左心室前壁有轻度梗死，心室壁未见明显变薄，同时会发现对照组心脏组织中脂肪组织增多(图19，A)，通过统计发现治疗组TTC染色后梗死呈现白色的面积显著低于对照组(图19，B)。
取梗死区左心室前壁组织进行马松三色染色发现，对照组呈现透壁性梗死，心室壁 变薄；EE治疗组心脏纤维化穿插在心肌间隙，室壁未有明显变薄(图19，C)。
上述结果表明，EE能显著提升缺血再灌注大白猪的左心射血分数和每搏输出量，减轻由于心梗导致的左心室重构，减轻缺血再灌注大猪的肺淤血，提升每日活动量。此外，TTC染色结果表明，EE治疗组心脏梗死面积较对照组显著降低；组织马松染色染色结果表明，对照组大白猪左心前壁出现透壁性梗死，纤维化面积显著高于EE治疗组；荧光染色结果表明，EE能增加大白猪梗死区血管的新生。
实施例十二
参照实施例一所述的方法获得小鼠13-14天胎龄的羊水，将收集羊水抽提液的离心管配平后用中佳 TMKDC-2046低速冷冻离心机于5℃，3500rpm离心21分钟(离心机标准操作流程参见使用手册)。将上清液倾析转移至洁净的塑料储物罐中，储存于-80℃冰箱中。每批次预留5ml小样用于后续测试。所有步骤都在无菌条件下进行。
测细胞活性：将长势较好的AC16消化后，铺于96孔板中，8000个/孔，每一组五个复孔。在5％CO 2饱和湿度37℃培养箱培养2小时，细胞贴壁。用培养基DMEM饥饿培养24小时后，替换成10％FBS的DMEM、DMEM和分别添加2.5％、5％、10％和20％(体积比)小鼠EE的培养基。培养24小时后，每孔加入10μl CCK-8试剂。孵育2小时后，于酶标仪在450nm检测吸收值。
结果如图20所示。

Claims (10)

  1. 一种药物组合物，其特征在于，所述药物组合物含有：
    非人动物的羊水和/或其提取物，和
    任选的药学上可接受的载体；
    其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-20天、优选8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天、优选8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
  2. 如权利要求1所述的药物组合物，其特征在于，所述药物组合物为冷冻保存的羊水和/或其提取物，或为该羊水和/或其提取物的冻干试剂。
  3. 如权利要求1所述的药物组合物，其特征在于，所述药物组合物为输注液。
  4. 如权利要求3所述的药物组合物，其特征在于，所述药物组合物不含有或含有注射用生理盐水、注射用水或葡萄糖注射液。
  5. 如权利要求1-4中任一项所述的药物组合物，其特征在于，
    所述羊水来自鸡蛋、鸭蛋、鹅蛋或其组合；或
    所述羊水来自怀孕10-14天的啮齿类动物。
  6. 如权利要求1-5中任一项所述的药物组合物，其特征在于，所述提取物在pH5.8-8.0之间不和离子交换柱结合，且其所含成分的分子量在500-1200道尔顿范围。
  7. 羊水和/或其提取物在制备治疗缺血和缺氧引起的组织坏死的药物中的应用，或在制备改善心脏功能的药物中的应用；
    其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎 龄为8-20天、优选8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天、优选8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
  8. 如权利要求7所述的应用，其特征在于，所述药物用于治疗心肌缺血坏死，例如心肌梗塞；或所述药物用于改善心力衰竭，如老年性心功能减退或心功能不全。
  9. 如权利要求7-8中任一项所述的应用，其特征在于，
    所述羊水来自鸡蛋、鸭蛋、鹅蛋或其组合；或
    所述羊水来自怀孕10-14天的啮齿类动物。
  10. 如权利要求7-9中任一项所述的应用，其特征在于，所述提取物在pH5.8-8.0之间不和离子交换柱子结合，且其所含成分的分子量在500-1200道尔顿范围。
PCT/CN2019/099947 2018-08-10 2019-08-09 用于治疗组织坏死或改善心脏功能的药物 WO2020030091A1 (zh)

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