WO2020030097A1 - 促进细胞生长和组织修复的方法及组合物 - Google Patents
促进细胞生长和组织修复的方法及组合物 Download PDFInfo
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Definitions
- the present invention relates to methods and compositions for promoting cell growth and tissue repair.
- tissue and organ defects are treated with cell-based therapies.
- These therapies include introducing precursor cells, preferably stem cells, to the defect site, thereby expanding the endogenous cell population and increasing the rate of tissue regeneration and repair.
- These cells are inherently autologous, isolated from the patient in need of treatment, and expanded in vitro before returning to the patient's defect site.
- the invention provides a cell culture method, which comprises the step of culturing the cells in vitro using a cell culture medium containing amniotic fluid and / or an extract thereof from a non-human animal; wherein the amniotic fluid is derived from an embryonic age of 5-12 days Eggs, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or eggs from the developmental period and the embryonic age Eggs from birds other than chickens corresponding to the developmental stage; or embryos from rodents with a gestational age of 8-20 days, preferably 8-14 days, or from developmental stages with a gestational age of 8-20 days, Embryos of non-human mammals other than rodents corresponding to rodent development periods of 8-14 days are preferred. This method promotes cell growth.
- the present invention also provides a cell culture medium containing amniotic fluid and / or extracts thereof of non-human animals; 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-day eggs, more preferably 7-9 days of embryonic age, more preferably 7-8 days of embryonic age, or from chickens with a developmental period that corresponds to the developmental period in which the embryonic age eggs are located Eggs from other birds; or embryos from rodents with a 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 developmental stage corresponds to embryos of non-human mammals other than rodents.
- the invention also provides the use of amniotic fluid and / or its extract in the preparation of a reagent for promoting the growth of animal cells and / or tissue repair, 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-day eggs, more preferably 7-9 days of embryonic age, more preferably 7-8 days of embryonic age, or from other chickens at a developmental stage corresponding to the developmental stage in which the embryonic age eggs are located Eggs from other birds; or embryos from rodents with a 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 developmental stage corresponds to embryos of non-human mammals other than rodents.
- the invention also provides the use of amniotic fluid and / or its extract in the preparation of a medicament for treating a condition related to tissue damage, 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 Eggs, more preferably eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or eggs from a development stage other than chickens corresponding to the developmental stage in which the eggs of the embryonic age are located Avian eggs; or embryos from rodents with a gestational age of 8-20 days, preferably 8-14 days, or development from rodents with developmental and gestational ages of 8-20 days, preferably 8-14 days The embryos of non-human mammals other than rodents corresponding to the period.
- the extract is not bound to an ion exchange column between pH 5.8 and 8.0, and the molecular weight of the ingredients contained in the extract is in the range of 500 to 1200 Daltons.
- the tissue is from: cartilage tissue, meniscus tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascial tissue, periosteal tissue, Any one or more of ocular 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.
- the animal cells are from the any one or more tissues.
- the cells are cardiac cells, such as cardiomyocytes.
- the conditions related to tissue damage include, but are not limited to, conditions caused by disease or trauma or failure of normal tissue development, selected from the group consisting of: hernia; pelvic floor defect; torn or broken Tendons or ligaments; skin wounds such as scars, traumatic wounds, ischemic wounds, diabetic wounds, severe burns, skin ulcers such as bedsores or pressure-induced ulcers, venous ulcers and diabetic ulcers, surgical wounds related to the removal of skin cancer
- Vascular conditions such as peripheral arterial disease, abdominal aortic aneurysm, carotid disease and venous disease, vascular defects, improper vascular development; and muscle diseases, such as congenital myopathy, myasthenia gravis, inflammatory, neurological, and myogenic muscles Muscular dystrophy, and muscular dystrophy, such as Duchenne muscular dystrophy, Baker muscular dystrophy, myotonic dystrophy, limb girdle muscular dystrophy, facial and shoulder brachial muscular dystrophy,
- amniotic fluid and / or extract thereof in the preparation of a medicament for promoting wound healing is also provided herein, 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, and more preferably 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 Embryos from rodents with gestational age of 8-20 days, preferably 8-14 days, or from developmental rods corresponding to the developmental period of rodents with gestational age 8-20 days, preferably 8-14 days Embryo of a non-human mammal other than an animal.
- the extract is not bound to an ion exchange column between pH 5.8 and 8.0, and the molecular weight of the ingredients contained in the extract is in the range of 500 to 1200 Daltons.
- Figure 1 HPLC results of amniotic fluid of eggs at 7 days of embryonic age.
- Figure 2 HPLC results of amniotic fluid of eggs at 11 days of age.
- Figure 3 HPLC results of amniotic fluid of eggs at 13 days of embryo age.
- Figure 4 Anti-free radical capacity of egg brine at different embryo ages. Among them, the abscissa represents the embryonic age, and the ordinate represents the clearance rate.
- Figure 5 Growth curves of chicken embryo fibroblasts under different culture conditions.
- Figure 6 Effect of amniotic fluid from eggs on the growth viability and migration ability of human umbilical vein endothelial cells (HUVEC). Among them, the abscissa indicates the culture medium, and the ordinate indicates the OD450 value.
- HAVEC umbilical vein endothelial cells
- Figure 7 Effect of amniotic fluid from duck eggs on the growth viability and migration capacity of chicken embryo fibroblasts. Among them, the abscissa indicates the culture medium, and the ordinate indicates the OD450 value.
- Figure 8 Amniotic fluid from eggs promotes growth of mouse osteoblasts. Among them, the abscissa indicates the culture medium, and the ordinate indicates the OD450 value.
- Figure 9 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 10 Gel column GE HiLoad 16/600 Superdex 75pg separation chromatogram.
- FIG. 11 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 12 Unbound fractions separated from 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 are not bound to 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 13 The use of the amniotic fluid of the present invention can significantly promote wound healing. Data are expressed as the ratio (%) of the wound area on day 0 minus the wound area on day 2 or 4 to the wound area on day 0.
- FIG. 14 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
- Figure 15 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. 16 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. 17 Immunofluorescent staining (PH3, cTnT, DAPI) of the heart of mice with myocardial infarction.
- FIG. 18 Immunofluorescent 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 19 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 20 EE improves cardiac function and reduces left ventricular remodeling in MI pigs.
- Figure 21 EE reduces myocardial infarct size and prolongs activity time in IR pigs.
- Figure 22 The effect of amniotic fluid from mice on the growth viability of AC16 cells.
- the inventors have discovered that the growth factor population contained in non-human animal amniotic fluid and / or its extract can promote cell growth or migration, including, but not limited to, promoting the regeneration of heart cells such as cardiomyocytes after myocardial infarction. Therefore, this article relates to the use of amniotic fluid and / or its extracts to promote cell growth and promote tissue repair.
- Amniotic fluid can come from 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.
- 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 whose development period corresponds to the development period of the above-mentioned embryonic age eggs may be used.
- duck eggs 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 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.
- the amniotic fluid and / or its extracts herein can be used to culture cells in vitro. Specifically, various cells of various tissues and various origins from various animals can be cultured using the amniotic fluid and / or extracts thereof described herein.
- the amniotic fluid can be added to a suitable cell culture medium for culturing cells of interest.
- An appropriate cell culture medium can be selected according to the cells to be cultured, and then an appropriate amount of amniotic fluid and / or its extract of the present invention is added to the cell culture medium.
- Exemplary cell culture media include, but are not limited to, various commercially available media such as DMEM, RPMI, 1640, MEM, DMEM / F12, and the like.
- the added amount of amniotic fluid or extract may be 0.1 to 30% of the weight of the cell culture medium, such as 1 to 25% or 3 to 20%.
- amniotic fluid and / or its extract described herein can be used as the active ingredient of a medicament for in vivo administration to a subject in need, and promote cell growth and tissue repair in vivo.
- an effective amount of amniotic fluid and / or extract thereof described herein, or a pharmaceutical composition containing the amniotic fluid and / or extract thereof may be administered to a subject in need thereof.
- the animal may be a mammal, especially a human.
- tissue defect or "tissue defect site” refers to the destruction of epithelial, connective or muscle tissue. Tissue defects cause the organization to operate at suboptimal levels or under suboptimal conditions.
- a tissue defect can be a torn layer or whole of a tendon, or local cell death caused by a myocardial infarction.
- Tissue defects can form a "void", which can be understood as a three-dimensional defect, such as the formation of nicks, cavities, holes or other substantial damage in the integrity structure of epithelium, connectives, or muscle tissue.
- tissue defects are those tissues that are not capable of endogenous or spontaneous repair.
- Tissue defects can be caused by accidents, diseases and / or surgical procedures.
- a cartilage defect may be caused by a joint trauma, such as a torn meniscus tissue moving into the joint.
- Tissue defects can also be caused by degenerative diseases such as osteoarthritis.
- the invention relates particularly to the repair of cartilage.
- Tissues described herein include, but are not limited to, muscle tissue, epithelial tissue, connective tissue, and neural tissue.
- the tissues described herein include, but are not limited to: cartilage tissue, meniscus tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascia tissue, Periosteal tissue, eye tissue, pericardial tissue, lung tissue, synovial tissue, nerve tissue, kidney tissue, bone marrow, urogenital tissue, intestinal tissue, liver tissue, pancreatic tissue, spleen tissue and adipose tissue.
- the cells described herein may be cells from any of the tissues described above.
- the cells may be autologous cells or allogeneic cells.
- the cells are autologous cells, that is, isolated from the body of an animal in need of tissue repair or treatment, especially from the tissue itself in need of repair or treatment. Therefore, autologous cells can be cultured in vitro using amniotic fluid and / or extracts thereof or a medium containing said amniotic fluid and / or extracts thereof, or autologous cells can be cultured using the cell culture methods described herein to form
- the implanted tissue or matrix is implanted in animals, especially in humans, and especially in tissue defect sites to repair corresponding damaged tissues. These cells expand the endogenous cell population and increase the rate of tissue regeneration and repair.
- the present invention relates to the administration of amniotic fluid and / or extracts thereof as described herein directly at a damaged site to promote the proliferation of normal cells at the site, thereby achieving tissue repair of the defective portion.
- conditions associated with tissue damage including, but not limited to, conditions caused by disease or trauma or the failure of normal tissue development, such as a hernia, can be treated with amniotic fluid and / or extracts thereof as described herein.
- vascular conditions such as peripheral arterial disease, abdominal aortic aneurysm, carotid artery disease, and venous disease; vascular defects, improperly developed blood vessels
- muscle diseases such as congenital Myopathy; myasthenia gravis; inflammatory, neurological, and myogenic muscle disorders; and muscular dystrophies, such as Duchenne muscular dystrophy, Baker muscular dystrophy, myotonic dystrophy, limb girdle muscular atrophy, facial shoulder humerus Muscular dystrophy, congenital muscular dystrophy, ocular pharyngeal muscular dystrophy, distal muscular dystrophy, and Emory Dreyfus muscular dystrophy); and Senile diseases, such as senile dementia and senile joint degeneration.
- tissue repair method comprising culturing cells of interest in vitro using amniotic fluid and / or extracts thereof as described herein, or a cell culture medium containing amniotic fluid or extracts described herein to form tissue After the stroma, the tissue matrix is implanted at the site of tissue injury or defect.
- Also provided herein is a method of treating a condition associated with tissue damage comprising administering to a subject in need thereof a therapeutically effective amount of the amniotic fluid and / or its extract or a pharmaceutical composition containing the amniotic fluid and / or its extract.
- a step of. The present invention also provides a method for promoting wound healing, the method comprising administering to a subject in need thereof a therapeutically effective amount of amniotic fluid and / or an extract thereof described herein or a pharmaceutical composition containing the amniotic fluid and / or an extract thereof A step of.
- a therapeutically effective amount can be determined based on the specific condition.
- amniotic fluid and / or its extracts described herein can be used directly for the methods or uses 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 a suitable method, such as intravenous infusion. 3. Intracardiac injection or in the lesion area.
- 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.
- 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.
- a cell culture medium containing an appropriate amount of amniotic fluid and / or extracts thereof as described herein.
- the content of amniotic fluid and / or its extract in the cell culture medium can be determined according to the type of cells being cultured.
- the amount of amniotic fluid or extract can be 0.1 to 30% of the weight of the cell culture medium, such as 1 to 25. % Or 3 to 20%; or, when amniotic fluid is used, the addition amount (volume ratio) in the cell culture medium may be 1-30%, such as 5-20%, of the basal cell culture medium.
- a suitable basal cell culture medium can be selected according to the cells to be cultured, and exemplary cell culture media include, but are not limited to, various commercially available media such as DMEM, RPMI, 1640, MEM, DMEM / F12, and the like.
- 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 Spectrometer TM 1800 UV spectrophotometer.
- Spectrometer TM 1800 UV spectrophotometer For standard operating procedures of the photometer, refer to the instruction manual.
- 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 embryonic ages of 7 days, 11 days, and 13 days is detected, and the results are shown in Figures 1-3.
- 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 eggs with embryo ages of 6 days, 7 days, 8 days, 9 days, 10 days and 11 days. After centrifugation, the amniotic fluid was stored in a refrigerator at 4 ° C for use.
- 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, zero with methanol as a control, and measure the absorbance at 519nm. 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 5 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) .
- Figure 6 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 promotes the healing of HUVEC.
- Figure 7 shows the effect of amniotic fluid from duck eggs on the growth viability and migration ability 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
- E Egg amniotic fluid with an embryonic age of 7 days was prepared according to the method described in Example 1 and used in this experiment.
- Osteoblasts were isolated from adult mice, and cultured to the third generation (P3) with a DMEM culture solution containing 10% FBS in a 37 ° C, 5% CO 2 incubator. 400 cells were seeded in each well in a 96-well plate. After 24 hours, change to only DMEM culture medium (starved), culture for 24 hours, and then change to the following culture medium (the amount of EE added is based on the volume of DMEM), continue to culture for 72 hours, and then use the CCK-8 kit to detect cell growth :
- Figure 8 shows that chicken embryo amniotic fluid can significantly promote the growth of mouse osteoblasts.
- 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.
- DMEM / F12 Primary myocardial cells were digested and plated in a 96-well plate at 6000 cells / well with five replicates in each group. After incubation in a 5% CO 2 saturated humidity 37 ° C incubator for 24 hours, the medium was replaced with DMEM / F12, DMEM / F12 containing 10% FBS, DMEM / F12 containing 10% FBS and 5% (volume ratio) EE. DMEM / F12 in original medium 15% FBS. After 48 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 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. 12.
- 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 Daltons obtained in the second step, load the sample at a flow rate of 1ml / min, and load 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 unbound region after anion exchange column GE HiPrep Q treatment is shown in FIG. 12.
- a mouse full-thickness skin injury model (Murine full-thickness wound model) is used to study the wound healing in mice by the amniotic fluid and / or its extract described herein.
- a chloral hydrate having a concentration of 5.00% is dispensed into a mouse intraperitoneally with a 1ml syringe at a ratio of 0.07ml / 10g (anesthesia induction time is about 5-10 minutes, and anesthesia maintenance time is about 35 minutes).
- the wound area of the mice was measured on the 2nd and 4th days, respectively.
- the wound area of both groups was reduced, and the EE group had a higher healing rate and no scar formation.
- 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. 14. It can be seen from FIG. 14 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. 15. It can be seen from FIG. 15 that by the third week, the treatment of EE significantly improved the LVFS of myocardial infarction mice, that is, the cardiac function of the 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 staining was performed according to the above point (3).
- the results are shown in FIG. 16.
- the infarcted fibrotic tissue is blue and the muscle tissue is red. From the figure, it can be seen that 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 Figure 16 that the ventricular cavity of the myocardial infarction mice in the control group was severely enlarged, but after three 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. 17.
- FIG. 17 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.
- FIG. 18 From FIG. 18, it can be clearly seen that AuroraB staining (green fluorescent dots, arrows) cells in the heart tissue of the EE-treated mice. 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.
- LVEF Left ventricular ejection fraction
- 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. 19. It can be seen from FIG. 19 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.
- Figure 20 shows that chicken EE can improve left ventricular ejection fraction and short-axis shortening rate of myocardial infarcted large white pigs, and the heart function of large white pigs in the control group showed a gradual decline after operation.
- 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 20, A and C).
- ⁇ EF and ⁇ FS statistics were performed 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. Lower than the control group ( Figure 20, B and D).
- the stroke output of the treatment group was significantly higher than that of the control group at 1-8 weeks after operation (Figure 20, E).
- the volume and diameter of the left ventricular systole at the end of the control tended to increase, and the treatment group was lower than the control group ( Figure 20, F and I), indicating that EE increased the left ventricular contractility.
- the volume and diameter of the left ventricle at the end of diastole in the control showed an upward trend, and the administration group showed an upward trend and then a downward trend (Figure 20, G and H), indicating that EE reversed the ventricular remodeling caused by partial myocardial infarction (MI) .
- MI myocardial infarction
- the control group showed transmural infarction and the ventricular wall became thinner.
- the EE treatment group had cardiac fibrosis interspersed in the myocardial space without significant thinning of the ventricular wall ( Figure 21 , C).
- 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
Description
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 |
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 |
Claims (10)
- 一种细胞培养方法,其特征在于,所述方法包括体外使用含有非人动物的羊水和/或其提取物的细胞培养基培养该细胞的步骤;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。
- 如权利要求1所述的方法,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。
- 如权利要求1或2所述的方法,其特征在于,所述细胞来自: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。
- 一种细胞培养基,其特征在于,所述细胞培养基含有非人动物的羊水和/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。
- 如权利要求4所述的细胞培养基,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。
- 羊水和/或其提取物在制备促进细胞生长和/或组织修复的试剂中的应用,或在制备治疗与组织损伤相关的病情的药物中的应用;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。
- 如权利要求6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结合,且其所含成分的分子量在500-1200道尔顿范围。
- 如权利要求6或7所述的应用,其特征在于,所述组织来自: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;所述动物细胞来自所述任意一种或多种组织。
- 如权利要求6-8中任一项所述的应用,其特征在于,所述与组织损伤相关的病情包括由疾病或外伤或组织未能正常发育所引起的病情,选自:疝气;盆底缺损;撕裂或断裂的肌腱或韧带;皮肤伤口,如疤痕、外伤性创伤、缺血性伤口、糖尿病伤口、严重烧伤、皮肤溃疡如褥疮或压力导致的溃疡、静脉溃疡和糖尿病溃疡、与皮肤癌的切除相关的手术伤口;血管病情,如外周动脉疾病、腹主动脉瘤、颈动脉疾病和静脉疾病、血管缺损、血管发育不当;以及肌肉疾病,如先天性肌病,重症肌无力,炎性、神经性和生肌性肌肉疾病,和肌肉萎缩症,例如杜氏肌肉萎缩症、贝克肌肉萎缩症、肌强直性萎缩症、肢带肌萎缩症、面肩肱型肌萎缩症、先天性肌肉萎缩症、眼咽肌肉萎缩症、远端肌肉萎缩症和埃默里-德赖富斯肌肉萎缩症;以及老年性疾病,如老年性痴呆和老年性关节退化。
- 羊水和/或其提取物在制备促进伤口愈合的药物中的应用;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;优选地,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。
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EP19848242.4A EP3835413A4 (en) | 2018-08-10 | 2019-08-09 | METHOD AND COMPOSITION FOR PROMOTING CELL GROWTH AND TISSUE REPAIR |
JP2021531169A JP2021533820A (ja) | 2018-08-10 | 2019-08-09 | 細胞増殖と組織修復の促進方法及び組成物 |
AU2019317696A AU2019317696A1 (en) | 2018-08-10 | 2019-08-09 | Method and composition for promoting cell growth and tissue repair |
CN201980054221.8A CN112639076A (zh) | 2018-08-10 | 2019-08-09 | 促进细胞生长和组织修复的方法及组合物 |
KR1020217006668A KR20210042343A (ko) | 2018-08-10 | 2019-08-09 | 세포 생장을 촉진하고 조직을 복구하는 방법 및 조합물이다 |
CA3109198A CA3109198A1 (en) | 2018-08-10 | 2019-08-09 | Method and composition for promoting cell growth and tissue repair |
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