Methods and compositions for promoting cell proliferation and tissue repair

Abstract

The present invention relates to a method and composition for promoting cell proliferation and tissue repair, and relates to the use of amniotic fluid and / or an extract thereof derived from non-human animals, particularly birds and non-human mammals. The composition of the present invention may be a cell culture medium containing amniotic fluid and / or an extract thereof, or may be a pharmaceutical composition.

Description

[Technical field]

[0001] The present invention relates to methods and compositions for promoting cell proliferation and tissue repair. [Background technology]

[0002] Due to the limitations of conventional treatments and the aging of the population, there is a gradually increasing demand for new therapies for tissue repair. Currently, tissue and organ defect treatments usually use cell therapy. These therapies involve the introduction of progenitor cells, preferably stem cells, into the defect site to expand the endogenous cell population and increase the rate of tissue regeneration and repair. These cells are essentially autologous cells, isolated from the patient in need of treatment and expanded ex vivo before being returned to the patient's defect site.

[0003] However, there are several problems with existing treatments. First, cells cultured outside the body for a long time can develop mutations, and unlike in vivo, these mutations cannot currently be identified and differentiated by the culture system, so the longer the culture time, the more tumorous the cells become. Cells cultured outside the body for a long time also differentiate, reducing the ability of cells to grow and repair tissue in the body. Second, in vitro cell culture exposes cells to foreign substances that may include contaminating particles (such as viruses and bacteria) or chemicals. If undetected before transplantation, these contaminants can lead to serious diseases and illnesses. Finally, the risk of cells becoming toxic increases the longer the cell culture time. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there remains a need in the art for new tissue repair methods and compositions that can directly promote cell proliferation within the human or animal body to achieve tissue repair. [Means for solving the problem]

[0005] The present invention provides a cell culture method. The method includes a step of in vitro culturing the cells in a cell culture medium containing amniotic fluid of a non-human animal and / or an extract thereof. The amniotic fluid is derived from eggs with an embryonic age of 5 to 12 days, preferably from eggs with an embryonic age of 6 to 11 days, more preferably from eggs with an embryonic age of 7 to 9 days, and even more preferably from eggs with an embryonic age of 7 to 8 days, or from eggs of birds other than chickens corresponding to the developmental stage of the eggs, or from rodent embryos with an embryonic age of 8 to 20 days, preferably from 8 to 14 days, or from embryos of non-human mammals other than rodents corresponding to the developmental stage of rodents with an embryonic age of 8 to 20 days, preferably from 8 to 14 days. The method can promote cell proliferation.

[0006] The present invention also provides a cell culture medium, which contains amniotic fluid of a non-human animal and / or an extract thereof, and the amniotic fluid is derived from eggs with an embryonic age of 5 to 12 days, preferably from eggs with an embryonic age of 6 to 11 days, more preferably from eggs with an embryonic age of 7 to 9 days, and even more preferably from eggs with an embryonic age of 7 to 8 days, or from eggs of birds other than chickens that correspond to the developmental stage of the eggs, or from rodent embryos with an embryonic age of 8 to 20 days, preferably from 8 to 14 days, or from embryos of non-human mammals other than rodents that correspond to the developmental stage of rodents with an embryonic age of 8 to 20 days, preferably from 8 to 14 days.

[0007] The present invention also provides the use of amniotic fluid and / or an extract thereof in the preparation of a medicament for promoting cell proliferation and / or tissue repair in an animal, wherein the amniotic fluid is derived from an egg with an embryonic age of 5 to 12 days, preferably an egg with an embryonic age of 6 to 11 days, more preferably an egg with an embryonic age of 7 to 9 days, and even more preferably an egg with an embryonic age of 7 to 8 days, or an egg of a bird other than a chicken corresponding to the developmental stage of the egg, or a rodent embryo with an embryonic age of 8 to 20 days, preferably an embryonic age of 8 to 14 days, or an embryo of a non-human mammal other than a rodent corresponding to the developmental stage of a rodent with an embryonic age of 8 to 20 days, preferably an embryonic age of 8 to 14 days.

[0008] The present invention also provides the use of amniotic fluid and / or an extract thereof in the treatment of pathologies related to tissue damage, wherein the amniotic fluid is derived from eggs with an embryonic age of 5-12 days, preferably from eggs with an embryonic age of 6-11 days, more preferably from eggs with an embryonic age of 7-9 days, even more preferably from eggs with an embryonic age of 7-8 days, or from eggs of birds other than chickens corresponding to the developmental stage of the eggs, or from rodent embryos with an embryonic age of 8-20 days, preferably from 8-14 days, or from non-human mammalian embryos other than rodents corresponding to the developmental stage of rodents with an embryonic age of 8-20 days, preferably from 8-14 days.

[0009] In one or more embodiments, the extract does not bind to an ion exchange column between pH 5.8-8.0 and contains components with molecular weights in the range of 500-1200 daltons.

[0010] In one or more embodiments, the tissue originates from one or more of the following tissues: cartilage tissue, meniscus tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascial tissue, periosteum tissue, eye tissue, pericardial tissue, lung tissue, synovial tissue, nervous tissue, kidney tissue, bone marrow, urogenital tissue, intestinal tissue, liver tissue, pancreatic tissue, spleen tissue, and adipose tissue. The animal cell originates from one or more of the above tissues. In certain embodiments, the cell is a cardiac cell, such as a cardiomyocyte.

[0011] In one or more embodiments, the conditions associated with tissue damage include: hernias, pelvic floor defects, tears and ruptures of tendons and ligaments, skin wounds (including scars, traumatic wounds, ischemic wounds, diabetic wounds, severe burns, skin ulcers (including pressure sores, venous ulcers and diabetic ulcers)), surgical wounds associated with the removal of skin cancer, vascular conditions (including peripheral arterial disease, abdominal aortic aneurysms, carotid artery and venous disease, vascular deficiencies, vascular dysplasias, etc.), muscle diseases (including congenital myopathies, myasthenia gravis, inflammatory, neurogenic and biomuscular myopathies, etc.). and conditions resulting from diseases, trauma, or abnormalities in tissue development, including, but not limited to, muscular dystrophies (Duchenne muscular dystrophy, Baker muscular dystrophy, myotonic muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophies, oculopharyngeal muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, etc.), geriatric diseases (senile dementia and geriatric osteoarthritis, etc.).

[0012] The present specification also provides the application of amniotic fluid and / or an extract thereof in wound healing, wherein the amniotic fluid is derived from eggs with an embryonic age of 5 to 12 days, preferably from eggs with an embryonic age of 6 to 11 days, more preferably from eggs with an embryonic age of 7 to 9 days, and even more preferably from eggs with an embryonic age of 7 to 8 days, or from eggs of birds other than chickens that correspond to the developmental stage of the eggs, or from rodent embryos with an embryonic age of 8 to 20 days, preferably from 8 to 14 days, or from non-human mammalian embryos other than rodents that correspond to the developmental stage of rodents with an embryonic age of 8 to 20 days, preferably from 8 to 14 days.

[0013] In one or more embodiments, the extract does not bind to an ion exchange column between pH 5.8-8.0 and contains components with molecular weights in the range of 500-1200 daltons. [Brief description of the drawings]

[0014] [Figure 1] HPLC analysis of amniotic fluid from 7-day-old embryos. [Diagram 2] HPLC analysis of amniotic fluid from 11-day-old eggs. [Diagram 3] HPLC analysis of amniotic fluid from 13-day-old eggs. [Figure 4] Anti-free radical capacity of amniotic fluid from eggs of different embryonic ages. The horizontal axis represents the embryonic age, and the vertical axis represents the removal rate. [Diagram 5] Growth curves of chicken embryo fibroblasts under different culture conditions. [Figure 6] The effect of amniotic fluid from eggs on the growth vitality and migration ability of human umbilical vein endothelial cells (HUVEC). The horizontal axis represents the medium, and the vertical axis represents the OD450 value. [Figure 7] The effect of amniotic fluid from duck eggs on the growth activity and migration ability of chicken embryo fibroblasts. The horizontal axis represents the medium, and the vertical axis represents the OD450 value. [Figure 8] Amniotic fluid from eggs promotes the proliferation of mouse osteoblasts. The horizontal axis shows the medium, and the vertical axis shows the OD450 value. [Figure 9] Amniotic fluid from eggs promotes the proliferation of primary cardiomyocytes. In this figure, the horizontal axis shows the medium and the vertical axis shows the OD450 value. [Figure 10] Gel column GE HiLoad 16 / 600 Superdex75 pg separation chromatogram. [Figure 11] Cell viability assay of the fraction separated from the gel column GE HiLoad 16 / 600 Superdex 75 pg. The horizontal axis represents the medium, in which FBS is fetal bovine serum, DMEM is Dulbecco's Modified Eagle Medium, EE is amniotic fluid, "EE" is lyophilized amniotic fluid, S-200B is the fraction of peak B, Q UNBOUND is the anion column unbound fraction, and 3-1 to 3-6 are equivalent fractions 1 to 6 from the purification step (III), respectively. [Figure 12] Cell viability assay of the unbound fraction from separation on cation exchange column GE HiPrep SP and anion exchange column HiPrep Q. The horizontal axis represents the medium, in which FBS is fetal bovine serum, DMEM is Dulbecco's Modified Eagle Medium, EE is amniotic fluid, "EE" is lyophilized amniotic fluid, Hiprep SP-UN is the fraction from the unbound Hiprep SP column, Hiprep Q-UN is the fraction from the unbound Hiprep Q column, and HiprepQ-Bound is the fraction from the bound Hiprep Q column. [Figure 13]The use of the amniotic fluid of the present invention significantly promotes wound healing. The data shows the percentage of the wound area on day 0 minus the wound area on day 2 or day 4. [Figure 14] Ejection fraction of mice with myocardial infarction. Ejection fraction and left ventricular short axis shortening fraction of mice can be measured by cardiac ultrasound. As shown in the figure, amniotic fluid (EE) treatment significantly increased the ejection fraction and significantly improved cardiac function in mice with myocardial infarction. [Figure 15] Left ventricular short-axis shortening fraction in mice with myocardial infarction. Ejection fraction and left ventricular short-axis shortening fraction can be measured in mice using cardiac ultrasound. As shown in the figure, amniotic fluid (EE) treatment significantly increased left ventricular short-axis shortening fraction and significantly improved cardiac function. [Figure 16] Masson's trichrome staining of the heart of a mouse with myocardial infarction. As shown in the figure, the mouse with myocardial infarction had severe fibrosis and the left ventricular wall was significantly thinned. After amniotic fluid (EE) treatment, the thinning of the left ventricular wall was not observed and fibrosis was significantly reduced. [Figure 17] Immunofluorescence staining (PH3, cTnT, DAPI) of myocardial infarcted mouse hearts. [Figure 18] Immunofluorescence staining (Aurora B, cTnT, DAPI) of myocardial infarction mouse hearts. As shown in the figure, PH3-positive and Aurora B-positive cells in the mouse hearts of the treatment group were significantly increased. This demonstrated that EE treatment significantly induced cardiac cell regeneration in myocardial infarction mice. [Figure 19] The area of ​​cardiac fibrosis in mice with myocardial infarction was significantly reduced after amniotic fluid (EE) treatment compared with the untreated group (NS). [Figure 20] EE improves cardiac function and reduces left ventricular remodeling in pigs with myocardial infarction. [Figure 21] EE reduced myocardial infarct size and extended activity time in IR pigs. [Figure 22] Effect of amniotic fluid from mice on the proliferation activity of AC16 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] It shall be understood that within the scope of the present invention, each of the above technical features of the present invention can be combined with each of the technical features specifically described below (such as in the examples) to form a preferred technical solution.

[0016] The present inventors have found that growth factors contained in amniotic fluid of non-human animals and / or extracts thereof can promote cell proliferation or migration (including, but not limited to, promoting the regeneration of cardiac cells, such as cardiomyocytes, following myocardial infarction). Thus, the present specification relates to the use of amniotic fluid and extracts thereof for promoting cell proliferation and tissue repair.

[0017] Amniotic fluid can be obtained from avian eggs and non-human mammals. Avian eggs refer to avian eggs. Preferred birds are poultry such as chickens, ducks, and geese. In the present invention, it is preferable to use avian eggs with an embryonic age of 5 to 20 days, preferably 6 to 15 days. It is understood that different avian eggs and suitable embryonic ages are not necessarily the same. For example, when eggs are used, eggs with an embryonic age of 5 to 12 days are preferred, more preferably eggs with an embryonic age of 6 to 11 days, even more preferably eggs with an embryonic age of 7 to 9 days, and most preferably eggs with an embryonic age of 7 to 8 days. When eggs of other birds are used, eggs of other birds corresponding to the developmental period of eggs of the above embryonic ages can be used. For example, when duck eggs are used, duck eggs with an embryonic age of 8 to 10 days, particularly 8 to 9 days, may be most preferred.

[0018] Amniotic fluid from avian eggs can be obtained by conventional methods. For example, the blunt end of an egg of suitable embryonic age is struck to break the shell, and the eggshell is peeled off to form an incision of about 2 cm in diameter. Next, the shell membrane and the vitelline membrane are carefully torn apart with tweezers so as not to break the amniotic membrane. The amniotic membrane and connective tissue encasing the embryo are injected from the shell into a culture dish, and the amniotic membrane is pierced with a syringe to extract the amniotic fluid, which is then extracted until the amniotic membrane is in close contact with the embryo, thereby obtaining the amniotic fluid used in the present invention.

[0019] Herein, the source of amniotic fluid can also be obtained from non-human mammals, particularly rodents such as mice. Other non-human mammals may be common livestock such as cows, sheep, dogs, cats, pigs, etc. In a particular embodiment, the source of amniotic fluid is a rodent embryo at embryonic days 8-20, preferably embryonic days 8-14 or 11-16, more preferably embryonic days 13-14, or a non-human mammal embryo corresponding to the developmental period of a rodent at embryonic days 8-20, preferably embryonic days 8-14 or 11-16, more preferably embryonic days 13-14. Amniotic fluid can be obtained by conventional methods. For example, the abdominal cavity of a mouse that is 8 to 20 days, preferably 8 to 14 or 11 to 16 days, and more preferably 13 to 14 days, of pregnancy is opened with surgical scissors, the uterus is carefully incised, and the amniotic membrane is punctured with a syringe to extract amniotic fluid, which is extracted until the amniotic membrane adheres closely to the embryo, thereby obtaining the amniotic fluid to be used in the present invention.

[0020] It is understood that the amniotic fluid may be centrifuged as necessary to separate impurities such as egg yolk, to obtain amniotic fluid as pure as possible. The supernatant obtained after centrifugation is the amniotic fluid used in the present invention. All steps for obtaining amniotic fluid must be performed under sterile conditions, and it is understood that the term "amniotic fluid" used in this specification refers to "pure" amniotic fluid, i.e., amniotic fluid that is free from other components of avian eggs or non-human mammalian embryos separated from avian eggs or non-human mammalian embryos and is not contaminated by foreign substances. Pure amniotic fluid may be stored in a refrigerator at -60°C or below, and may be thawed and reused.

[0021] In a specific embodiment, the present invention uses an extract of amniotic fluid. The extract does not bind to an ion exchange column at a pH between 5.8 and 8.0, and the molecular weight of the components contained therein is preferably controlled to be within the range of 500 to 1200 daltons. The extract can be obtained by separating a neutral fraction having a molecular weight of 500 to 1200 daltons from the amniotic fluid. The method of the present specification can be carried out using a gel column and an ion exchange column known in the art. For example, a known gel chromatography column (various gel chromatography columns described below) may be used to separate a fraction having a molecular weight of 500 to 1200 daltons from the amniotic fluid, and then the neutral fraction may be separated using an ion exchange method (ion exchange column described below). Alternatively, the neutral fraction may be separated from the amniotic fluid by an ion exchange method (using an ion exchange column described below), and then a fraction having a molecular weight of 500 to 1200 daltons from the neutral fraction may be separated using a gel chromatography column (various gel chromatography columns described below).

[0022] In a specific embodiment, a neutral fraction having a molecular weight of 500 to 2000 daltons may be separated from the amniotic fluid, and then a fraction having a molecular weight in the range of 500 to 1200 daltons may be separated therefrom. Specifically, the method may include the following steps:

[0023] (I) A neutral fraction with a molecular weight of 500 to 2,000 daltons is isolated from amniotic fluid. (II) Separating a neutral fraction having a molecular weight of 500 to 1200 daltons from a neutral fraction having a molecular weight of 500 to 2000 daltons.

[0024] Step (I) can be carried out by gel chromatography and ion exchange. Components with a molecular weight of 500 to 2000 daltons in the amniotic fluid can be separated using a gel chromatography column, and an uncharged (neutral) fraction can be obtained by ion exchange.

[0025] In the present specification, gel chromatography can be performed using various commercially available gel chromatography columns. Such gel chromatography columns include, but are not limited to, Sephacryl S-100, Sephacryl S-200, Sephacryl S-300, Sephacryl S-400, Superose 12, Superose 6, Superdex 12 and Superdex 6 from GE. It is understood that any other gel chromatography packing with a separation range of 500 to 10,000 daltons may be used. Typically, when using a gel chromatography column, first, ddH 2 The gel chromatography column may be equilibrated with 200. The flow rate may be determined according to the actual situation. For example, in a specific embodiment, the flow rate may be 0.5-50 ml / min, e.g., 1 ml / min. Usually, the ultraviolet absorption is 200-300 nm, e.g., 280 nm. After the ultraviolet absorption curve stabilizes and returns to the baseline, the equilibration is completed. After the equilibration is completed, the sample can be loaded. The sample loading flow rate is determined according to the actual preparation situation. After the sample loading is completed, the degassed ddH 2 The crude product can be eluted with O and fractions in the molecular weight range of 500-2000 daltons can be collected. If necessary, the gel chromatography separation can be repeated several times and fractions with equivalent peak detection times in each separation can be mixed.

[0026] Herein, the charged and uncharged components can be separated by methods known in the art. For example, it can be performed using ion exchange methods. Anion exchange and cation exchange can be used in the method of the present invention. In a particular embodiment, the present invention uses anion exchange methods. Commercially available anion exchange columns can be used. Examples of suitable anion exchange columns include, but are not limited to, DEAE Sepharose, ANX Sepharose, Q Sepharose, Capto DEAE, Capto Q, Mono Q and Mini Q, etc. from GE. It is understood that other brands of anion exchange packings can be used. Alternatively, commercially available cation exchange columns can be used. Examples of suitable cation exchange columns include, but are not limited to, CM Sepharose, SP Sepharose, Capto S, Mono S and Mini S, etc.

[0027] Typically, when ion exchange is performed, the ion exchange column is equilibrated with a buffer. The buffer may be a conventional buffer in the art, for example, a phosphate buffer, particularly a sodium phosphate buffer. The pH of the buffer can be determined according to the ion exchange column used. For example, when an anion exchange column is used, the column may be equilibrated with a buffer of pH 7.5 to 8.5, preferably 7.5 to 8.0. When a cation exchange column is used, the column may be equilibrated with a buffer of pH 5.8 to 7.0, preferably 5.8 to 6.5. In a particular embodiment, the sodium phosphate buffer is Na 2 HPO 4 and NaH 2 PO 4and the pH is about 5.8 or 8.0. In the present invention, it is preferable to separate using an anion exchange column. The flow rate may be determined according to the actual situation. For example, in a specific embodiment, the flow rate may be 0.5 to 50 ml / min, e.g., 1 ml / min. Usually, the equilibration is completed after the 280 nm ultraviolet absorption curve is stabilized and returns to the baseline. After the equilibration is completed, the sample is loaded and the effluent portion (i.e., the portion not bound to the column) can be collected. The sample loading flow rate is determined according to the actual preparation situation.

[0028] In step (I), gel chromatography may be performed to separate a fraction having a molecular weight of 500 to 2000 daltons, followed by ion exchange to separate a neutral fraction. Alternatively, the neutral fraction in the amniotic fluid may be separated by ion exchange, and then an active ingredient having a molecular weight of 500 to 2000 daltons in the neutral fraction may be separated by gel chromatography to obtain a neutral fraction having a molecular weight of 500 to 2000 daltons.

[0029] The main purpose of step (II) is to further separate the neutral fraction obtained in step (I) to obtain active ingredients with molecular weights in the range of 500-1200 Daltons. In this specification, the fraction with molecular weights in the range of 500-1200 Daltons can be separated by a commercially available gel chromatography column. Suitable gel chromatography columns include, but are not limited to, HiLoad Superdex 16 / 600 Superdex75 pg, Superdex Peptide, Superdex 200 and Superdex 30 from GE. It is understood that other brands of gel chromatography packings within the separation range of 500-10000 Daltons may also be used.

[0030] Usually, ddH 2A 2000 equilibration gel column can be used, and the flow rate can be determined according to the actual situation. For example, in a specific embodiment, the flow rate can be 0.5-50 ml / min, e.g., 1 ml / min. Usually, the equilibration is completed after the 280 nm ultraviolet absorption curve stabilizes and returns to the baseline. After the equilibration is completed, the sample can be loaded. The sample loading flow rate is determined according to the actual preparation situation. After the sample loading is completed, the degassed ddH 2 The crude product is eluted with O, and fractions are collected to obtain the fraction containing components with molecular weights in the range of 500 to 1200 daltons, i.e., the extract described in this specification.

[0031] The extract obtained by the above method was adjusted to a solution of pH 5.8-8.0 and then passed through several ion exchange columns (DEAE Sepharose, Q Sepharose, Mono Q, CM Sepharose, SP Sepharose and Mono S), but none of the active ingredients contained therein could be bound to these ion exchange columns.

[0032] The amniotic fluid and / or extract thereof of the present invention may be used for in vitro cell culture. Specifically, the amniotic fluid and / or extract thereof described herein may be used to culture various cells derived from various tissues of various animals. For example, the amniotic fluid may be added to a suitable cell culture medium for culturing the cells of interest. A suitable cell culture medium may be selected according to the cells to be cultured, and an appropriate amount of the amniotic fluid and / or extract thereof of the present invention may be added to this cell culture medium. Exemplary cell culture media include, but are not limited to, various commercially available media such as DMEM, RPMI 1640, MEM, and DMEM / F12. For example, the amount of amniotic fluid or extract added may be 0.1 to 30% (e.g., 1 to 25% or 3 to 20%) of the weight of the cell culture medium.

[0033] Alternatively, the amniotic fluid and / or extract thereof described herein may be administered to a subject in need thereof and used as an active ingredient of a pharmaceutical for promoting cell proliferation and tissue repair in the body. For example, an effective amount of the 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.

[0034] In this specification, the animal may be a mammal, particularly a human.

[0035] As used herein, "repair" refers to the formation of new tissue sufficient to at least partially compensate for an ineffective or structurally discontinuous tissue defect site. "Tissue defect" or "tissue defect site" refers to the destruction of epithelial, connective or muscle tissue. A tissue defect causes the tissue to operate at an undesirable level or under undesirable conditions. For example, a tissue defect may be a partial or entire layer of a tendon rupture, or localized cell death due to myocardial infarction. A tissue defect may form a "void", which may be understood as a three-dimensional defect (e.g., the formation of a cleft, cavity, hole, or other substantial disruption in the intact structure of epithelial, connective or muscle tissue). In certain embodiments, a tissue defect refers to a tissue that does not have the capacity to undergo endogenous or natural repair. A tissue defect may be caused by accident, disease, and / or surgical manipulation. For example, a cartilage defect may be caused by joint trauma (e.g., a torn meniscus tissue migrates into the joint). A tissue defect may also be caused by a degenerative disease, such as osteoarthritis. In certain embodiments, the present invention is specifically directed to the repair of cartilage.

[0036] The tissues described herein include, but are not limited to, muscle tissue, epithelial tissue, connective tissue, and nerve tissue.In certain embodiments, 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, fascial tissue, periosteum 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.Therefore, the origin of the cells described herein can be any of the above tissues.

[0037] The cells herein may be autologous or allogeneic. In a preferred embodiment, the cells are autologous, i.e., isolated from the animal body itself in need of tissue repair or treatment, and in particular from the tissue itself in need of repair or treatment. Thus, the amniotic fluid and / or extracts thereof as described herein, and the media containing amniotic fluid and / or extracts thereof may be used to culture autologous cells outside the body, or the autologous cells may be cultured by the cell culture method described herein to form a transplantable tissue or matrix, and may be transplanted into an animal body, particularly a human body, and in particular into a tissue defect, to repair the corresponding damaged tissue. These cells, the endogenous cell population, are expanded, and tissue regeneration and repair rates are increased. In a particular embodiment, the present specification relates to administering the amniotic fluid and / or extracts thereof as described herein directly to the site of injury to promote the proliferation of normal cells at the site of injury, thereby achieving tissue repair at the defect site.

[0038] In certain embodiments, the amniotic fluid and / or extracts thereof described herein may be used to treat conditions associated with tissue damage, including but not limited to: hernias, pelvic floor defects, tears and ruptures of tendons and ligaments, skin wounds (including scars, traumatic wounds, ischemic wounds, diabetic wounds, severe burns, skin ulcers (including pressure ulcers, venous ulcers and diabetic ulcers), surgical wounds following removal of skin cancer, vascular conditions (including peripheral arterial disease, abdominal aortic aneurysms, carotid artery and venous disease, vascular defects, vascular dysplasia, etc.), muscle diseases (including congenital myopathies, myasthenia gravis, inflammatory, neuropathic and biopathic muscle diseases, and muscular dysplasia). muscular dystrophy (Duchenne muscular dystrophy, Baker muscular dystrophy, myotonic muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, etc.), geriatric diseases (senile dementia, geriatric osteoarthritis, etc.), trauma, or conditions due to abnormal tissue development

[0039] Thus, the present specification provides the use of amniotic fluid and / or extracts thereof as described herein in the preparation of reagents or agents for use in promoting cell proliferation and / or tissue repair, and in the preparation of medicaments for treating conditions related to tissue damage. The present specification also provides a method for tissue repair, which comprises in vitro culturing of cells of interest with said amniotic fluid and / or extracts thereof, or with a cell culture medium containing said amniotic fluid or extracts, forming a tissue matrix, and then transplanting said tissue matrix into the site of tissue damage or defect. The present specification also provides a method for treating conditions related to tissue damage, which comprises administering to a subject in need thereof a therapeutically effective amount of amniotic fluid and / or extracts thereof as described herein, or a pharmaceutical composition containing amniotic fluid and / or extracts thereof as described herein. The present specification also provides a method for promoting wound healing, which comprises administering to a subject in need thereof a therapeutically effective amount of amniotic fluid and / or extracts thereof as described herein, or a pharmaceutical composition containing amniotic fluid and / or extracts thereof as described herein. The therapeutically effective amount may be determined depending on the specific condition.

[0040] The amniotic fluid and / or its extract described herein may be directly used in the methods and applications described herein and administered to a subject in need. The administration method may be parenteral administration, intravenous injection, or intracardiac injection. In a specific embodiment, a therapeutically effective amount of amniotic fluid and / or its extract is mixed with an appropriate amount of saline for injection, water for injection, or glucose injection. Then, it is administered by a suitable method (intravenous injection or intracardiac injection, intralesional administration, etc.).

[0041] The dosage and frequency of administration may be determined by medical staff depending on the specific condition, age, and sex of the patient, etc. Typically, for the treatment of a particular disease, a therapeutically effective amount is an amount of drug sufficient to improve or in some way alleviate the symptoms associated with that disease. This amount may be administered as a single dose or according to an effective treatment protocol. The dosage may be administered to improve symptoms of the disease, although it may cure the disease. Repeated administration is usually necessary to achieve the desired improvement of symptoms. For example, the dosage administered to humans may be administered by daily or weekly injection, usually 1-200 ml / time. In certain embodiments, the frequency of administration may be every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every half month, or once a month.

[0042] The present invention also provides a pharmaceutical composition comprising amniotic fluid and / or extracts thereof as described herein, in particular amniotic fluid and / or extracts thereof in poultry eggs, preferably amniotic fluid and / or extracts thereof from chicken eggs at embryonic days 5-12, more preferably 6-11, even more preferably 6-9, and most preferably 7-8. In a particular embodiment, the source of the amniotic fluid or extracts thereof is amniotic fluid or extracts thereof from rodents at gestation days 8-20, preferably 8-14 or 11-16, more preferably 13-14, as described herein. The pharmaceutical composition may be amniotic fluid and / or extracts thereof frozen and stored at -60°C or below, or may be a lyophilized agent such as lyophilized amniotic fluid and / or extracts thereof. The pharmaceutical composition may contain other pharma- ceutical acceptable carriers or excipients, such as saline for injection, water for injection, or glucose injection.

[0043] In a specific embodiment, the present specification provides a cell culture medium. The medium contains an appropriate amount of the amniotic fluid and / or an extract thereof. The content of the amniotic fluid and / or an extract thereof in the cell culture medium may be determined according to the type of cells to be cultured. For example, the amount of amniotic fluid or extract added may be 0.1 to 30% (e.g., 1 to 25% or 3 to 20%) of the weight of the cell culture medium. When amniotic fluid is used, the amount (volume ratio) added to the cell culture medium may be 1 to 30% (e.g., 5 to 20%) of the basal cell culture medium. An appropriate basal cell culture medium may be selected according to the cells to be cultured. Exemplary cell culture media include, but are not limited to, various media commercially available, such as DMEM, RPMI 1640, MEM, and DMEM / F12.

[0044] The present invention will be described below with reference to specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods, reagents and equipment used in the examples are conventional methods, reagents and equipment in the art unless otherwise specified. EXAMPLES

[0045] 1, Material a) Equipment and tools A microcomputer-controlled fully automatic incubator (Zhengda® ZF880), clean culture dishes, a 1.0 ml syringe (Jiangxi Hongda®), tweezers disinfected with 70% ethanol, a stainless steel sieve, sterile centrifuge tubes (Axygen® #SCT-50ML-25-S), and a low-speed refrigerated centrifuge (Zhongjia KDC-2046).

[0046] b) Reagents and biological materials Eggs are 7 days old.

[0047] 2. Experimental Procedure Take an egg and break the shell by hitting it with a flat blunt end placed upwards, then peel the shell to form a cut about 2 cm in diameter, with the edges as flat as possible. Carefully tear the shell membrane and vitelline membrane with tweezers, so as not to break the amniotic membrane. Observe the development of the embryo, and only embryos that are well developed and meet the criteria of the corresponding stage can be used for amniotic fluid extraction.

[0048] The amniotic membrane and connective tissue encasing the embryo are then injected from the shell into a culture dish, the amniotic membrane is pierced with a syringe to extract amniotic fluid, with the needle bevel facing away from the embryo, until the amniotic membrane is tightly attached to the embryo, and the clear, colorless, contaminant-free fluid is then injected into a centrifuge tube in an icebox.

[0049] Embryos were removed from the amniotic membrane with forceps and collected on a stainless steel sieve placed on ice. The embryos collected every hour were homogenized in a blender, sealed in a sterile plastic storage tank, and placed at an angle in a refrigerator at -80°C. After freezing, they may be placed vertically.

[0050] The collected amniotic fluid extracts can be tested in a Mapada™ 1800 UV spectrophotometer (see instruction manual for spectrophotometer standard operating procedures) and extracts that pass can be used for mixing and equilibration.

[0051] The centrifuge tubes containing the amniotic fluid extracts were equilibrated and then centrifuged at 3500 rpm for 21 minutes at 5°C using a Zhongjia™ KDC-2046 slow refrigerated centrifuge (see centrifuge user manual for standard operating procedures). The supernatants were decanted and transferred to clean plastic storage tanks and stored in a -80°C refrigerator. A 5 ml subsample of each lot was pre-stored for subsequent testing.

[0052] The entire procedure is performed under sterile conditions. EXAMPLES

[0053] In this embodiment, a Hitachi Primaide high performance liquid chromatograph is used to detect amniotic fluid components of eggs of different embryonic ages. Detection is performed according to the instruction manual of this chromatograph. In this, before the start of detection, washing is performed with 100% acetonitrile for 30 minutes, the flow rate time is 0.8 mL / min, and then equilibration with water for 30 minutes, the flow rate time is 0.8 mL / min. Extract a 25 μL sample to eliminate air bubbles, click the "Data Collection" button of the software attached to the chromatograph to select "Method 2", click "Start Single Analysis" at the bottom of the screen, start sample injection when the system displays "Waiting for Sample Injection", perform the injection quickly, and switch the valve after the injection is completed. This method 2 is as follows.

[0054] [Table 1]

[0055] In this example, the results of detecting amniotic fluid on fetal days 7, 11, and 13 are shown in Figures 1-3. EXAMPLES

[0056] DPPH, or 1,1-diphenyl-2-picrylhydrazyl radical, the structure of which is shown below. [ka]

[0057] In the DPPH molecule, there are multiple delocalized π bonds between the electron-withdrawing -NO2 and the benzene ring, so the nitrogen radical exists stably.

[0058] As the DPPH radical is scavenged, the absorbance A at its maximum absorption wavelength of 519 nm decreases. DPPH, a stable free radical, provides an ideal and simple pharmacological model for measuring free radical scavenging ability. In this example, DPPH is used to examine the free radical scavenging ability derived from chicken embryo amniotic fluid.

[0059] Take 0.8mg of DPPH, dissolve it in 20mL of solvent methanol, and vibrate it sufficiently with ultrasonic waves for 5min to make each part uniform. Take 1mL of this DPPH solution and measure the A0 value at 519nm. A=0.5-0.7. Store this DPPH solution away from light and use it up within 3.5 hours.

[0060] Using the method described in Example 1, amniotic fluid was obtained from chicken embryos on days 6, 7, 8, 9, 10, and 11 of embryonic age, respectively, and after centrifugation, was stored in a refrigerator at 4°C in preparation for use.

[0061] The calibration curve was measured using vitamin C as a positive control. Different volumes of 0.04mg / ml Vc samples were added with 0.6ml DPPH, and then made up to 1ml with absolute ethanol, mixed uniformly, zero-adjusted with methanol as a control, and the absorbance value at 519nm was measured. The data were measured three times and plotted.

[0062] Inject 400μl of amniotic fluid of different embryo ages into a test tube, add 600μl of the prepared DPPH methanol solution, mix, and react for 10 minutes until no bubbles are generated (mix uniformly before measurement). Zero adjustment is performed using methanol as a control, and the absorbance at 519nm is measured.

[0063] The sample loading information for each group is shown in the following table:

[0064] [Table 2]

[0065] The creatine clearance rate (inhibition rate) is calculated using the following formula.

[0066] Creatine clearance rate (%) = (A 0 -A) / A 0 ×100%.

[0067] The results are shown in Figure 4. EXAMPLES

[0068] This example tests the effect of the egg amniotic fluid (EE) of Example 1 on the growth of chicken embryo fibroblasts under different culture conditions. In this example, the composition of the DMEM medium used is as follows: Gibco® #Cat.11960077, supplemented with 1% L-glutamine (Solarbio® #G0200) and 5% FBS (Gibco® #Cat.10099141), 0.25% pancreatin (Hangzhou Keyi Bio® #CY003), PBS (BI® #02-024-1ACS), 0.4% trypan blue dye (BBI® #72-57-1).

[0069] 1. Obtaining and Culturing Chicken Embryo Fibroblasts Take an embryo from an egg at 7 days of embryonic age, wash the surface of the embryo with PBS, and aspirate the liquid with a pipette gun until it is clean. Remove the internal organs of the embryo and cut the remaining tissue into granules and nodules of a size invisible to the naked eye. Add 1 ml of 0.25% pancreatin, mix it with the tissue uniformly with the gun head, and inject the suspension into a 15 ml centrifuge tube. Wash the culture dish with 1 ml of 0.25% pancreatin, and inject the suspension into the same 15 ml centrifuge tube. Place the centrifuge tube in a 37°C water bath and digest for 5-7 minutes, then add 8 ml of DMEM medium (containing PBS) to neutralize the pancreatin. Place the centrifuge tube in a centrifuge and centrifuge for 5-10 seconds. Remove the centrifuge tube and collect the supernatant. Centrifuge this supernatant at 2000 rpm for 2 minutes. Discard the supernatant, add 4 ml of DMEM medium, and resuspend the cells with the gun head. Inject 1 ml of each cell suspension into a 10 cm cell culture dish, and add 10 ml of DMEM medium. Rock the culture dish in a crosswise direction at least 20 times to distribute the cells evenly. Incubate at 37°C, 5% CO 2 The cells are cultured under the conditions described above. The cells are passaged when they cover 70-90% of the bottom plate of the culture dish.

[0070] Remove the culture dish from the incubator and collect the primary medium in a centrifuge tube. Carefully add 5 ml of PBS to wash the cells. Then add 500 μl of 0.25% pancreatin, place the culture dish in the incubator, and digest for 1 minute. Tap the side edge of the culture dish lightly to speed up the digestion process, so that the cell clumps are rapidly decomposed and most of the cells are in a floating state. Add 9.5 ml of the collected primary medium quickly to neutralize the pancreatin. Blow the bottom plate of the culture dish with a pipette to collect as much cell suspension as possible into a 15 ml centrifuge tube and centrifuge at 2000 rpm for 3 min. Discard the supernatant, add 4 ml of DMEM medium, and resuspend the cells with a gun head. Inject 1 ml of cell suspension into a 10 cm cell culture dish containing 10 ml of fresh medium containing amniotic fluid at different volume ratios. Rock the culture dish in a cross direction at least 20 times each to ensure uniform cell distribution, and incubate at 37 °C, 5% CO 2 The mixture is cultured under the following conditions.

[0071] Take well-grown chicken embryo fibroblasts and inject primary medium into a centrifuge tube. Carefully add 5 ml of PBS to wash the cells, and gently shake to avoid damaging the cell layer before removing the PBS. Add 100 μl of 0.25% pancreatin, digest for 2-5 minutes (24 orifice plate), and neutralize with 100 μl medium. Use a gun head to form a single-cell suspension. Dilute this single-cell suspension at a certain multiple, add an equal amount of 0.4% trypan blue staining solution to stain, and the number of cells after dilution is preferably within the range of 20 to 200. Aspirate an appropriate amount (15 μl) of cell suspension, add the sample from the upper and lower edges of the cover glass to a hemocytometer, and calculate the number of live cells under a microscope. Calculate the total number of live cells and adjust the cell concentration to 1 × 105 cells / ml. Sample once every 24 hours, take 3 orifice cells each time, perform regular pancreatin digestion, prepare a single-cell suspension, and perform microscopic counting. Growth curves are plotted with time (days) on the horizontal axis and cell concentration on the vertical axis. Cell count = total number of cells / 4 x 104 x dilution factor, cell concentration = number of cells / ml.

[0072] The results are shown in Figure 5. Figure 5 shows that after a total of 96 hours of culture, the number of chicken embryonic fibroblasts in the experimental group with added EE was significantly higher than that in the control group without added EE. EXAMPLES

[0073] Amniotic fluid from duck eggs at embryonic day 8 is obtained by the same method as in Example 1. The effects of chicken embryo fibroblasts and duck egg amniotic fluid on the proliferation and migration ability of human umbilical vein endothelial cells (HUVEC) are examined by scratch assay. Duck amniotic fluid is obtained from duck eggs at embryonic day 8 by the method in Example 1. Chicken embryo fibroblasts are obtained and human umbilical vein endothelial cells are commercially obtained by the method in Example 5.

[0074] In this example, the composition of the DMEM medium used is as follows: Gibco® #Cat.11960077, supplemented with 1% L-glutamine (Solarbio® #G0200) and 5% FBS (Gibco® #Cat.10099141), 0.25% pancreatin (Hangzhou Keyi Bio® #CY003), PBS (BI® #02-024-1ACS), and 0.4% trypan blue dye (BBI® #72-57-1).

[0075] On the day before the experiment, prepare a 6-well microplate and draw 5-6 uniformly spaced horizontal lines across the wells on the back of the 6-well microplate using a marker and ruler. Also draw a vertical line perpendicular to the center line to indicate the scratch position. Add approximately 5 x 105 cells in logarithmic growth phase to each well, and the confluence rate usually reaches 90% overnight after inoculation.

[0076] On the day of the experiment, align the gun head with the ruler and draw a line vertically along the vertical line of the marker on the bottom of a 6-well microplate. Use the same gun head between different wells to avoid tilting and bending as much as possible, and the width is preferably 1000-2000 μm. Wash each well three times with 2 ml of PBS to wash away the cells in the scratch area. Add 2 ml of medium containing different amounts of EE to each well, culture normally, and change the liquid every 48 hours. Adjust to 0 h from the scratch, take regular photos every 24 hours, and measure the cell spacing on both sides of the scratch. Observe the cell growth status in each well. Plot a graph with time (days) on the horizontal axis and the distance of the scratch in each well on the vertical axis. Calculate the healing rate of the scratch in each well.

[0077] The results are shown in Figures 6 and 7. Figure 6 shows the effect of amniotic fluid from eggs on the proliferation and migration of human umbilical vein endothelial cells (HUVECs), and the addition of 5% (volume ratio) amniotic fluid has a significant promoting effect on the fusion of HUVECs. Figure 7 shows the effect of amniotic fluid from duck eggs on the proliferation and migration of chicken embryo fibroblasts, and the addition of amniotic fluid also has a significant promoting effect on the fusion of chicken embryo fibroblasts. EXAMPLES

[0078] According to the method described in Example 1, amniotic fluid (EE) from embryos at embryonic day 7 is prepared and used in this experiment.

[0079] Osteoblasts were isolated from adult mice and cultured in DMEM supplemented with 10% FBS at 37°C and 5% CO. 2 The cells were cultured in an incubator until the third generation (P3). 400 cells were seeded in each well of a 96-well plate. After 24 hours, the culture medium was replaced with DMEM only (starvation), and after 24 hours of culture, the culture medium was replaced with the following culture medium (added EE by the volume of DMEM), and after 72 hours of continuous culture, cell proliferation was detected using a CCK-8 reagent kit: 1)DMEM (serum-free) 2) DMEM + 2.5% EE 3) DMEM + 5% EE 4) DMEM + 7.5% EE 5) DMEM + 10% EE

[0080] The results are shown in Figure 8. Figure 8 shows that chicken embryo amniotic fluid can significantly promote the proliferation of mouse osteoblasts. EXAMPLES

[0081] 1. Isolation of Primary Cardiomyocytes (VM) Neonatal rat ventricles were washed with pre-cooled PBS, and then the cardiac tissue was dissected in DMEM / F12. The tissue was digested with 0.04% collagenase II + 0.08% pancreatin in a shaking water bath at 37°C. The digested cells were filtered through a sieve and centrifuged at 1000r / min x 10min. 15% FBS cell culture medium was added and the tissue was poured into a plate. The plate was then incubated at 5% CO 2 Cultured in an incubator at saturated humidity and 37°C.

[0082] 2. Cell viability assay After digestion of primary cardiomyocytes, they were injected into a 96-well plate, 6000 cells / well, 5 duplicate wells / group. Incubated in 5% CO 2 After 24 hours of incubation in a saturated humidity 37°C incubator, the primary medium DMEM / F12 with 15% FBS is replaced with medium DMEM / F12, DMEM / F12 with 10% FBS, and DMEM / F12 with 10% FBS and 5% EE (amniotic fluid from Example 1, by volume), respectively. After 48 hours of incubation, 10 μl of CCK-8 reagent is added to each well. After 2 hours of incubation, the absorbance value at 450 nm is measured by an enzyme labeling device.

[0083] The results are shown in Figure 9. EXAMPLES

[0084] The objective of this example is to purify biologically active compounds in chicken embryo amniotic fluid stepwise using analytical gel column Sephacryl S-200, anion exchange column HiPrep Q, desalting column HiPrep 26 / 10 Desalting, and HiLoad 16 / 600 Superdex 75 pg.

[0085] 1, Material 1.1 Purification sample: fresh amniotic fluid from 7-day-old embryos, 50 ml.

[0086] 1.2 Main experimental equipment and consumables 1) GE AKTA purifier 2) Gel column GE Sephacryl S-200 3) Anion Exchange Column GEHiPrep Q 4) Desalting column GEHiPrep 26 / 10 Desalting 5) Gel column GEHiLoad 16 / 600 Superdex75pg 6) Superloop 10ml

[0087] 2, method 2.1 Preparation of solutions Sodium phosphate buffer A (50 mM Na 2 HPO 4 +NaH 2 PO 4 Preparation of 46.6 ml 1 mol / l NaCl (pH 8.0) 2 HPO 4 and 3.4 ml 1 mol / l NaH 2 PO 4 Mix and add ddHO 2 Add O to bring the volume to 1 L.

[0088] 2.2 Experimental Method 2.2.2 Sample processing: 50 ml of fresh amniotic fluid was added with an appropriate amount of hexane, centrifuged at 2500 rpm and 4°C for 20 minutes to obtain the aqueous phase, which was then filtered through a 0.22 μm filter membrane.

[0089] 2.2.3 Sample purification Step 1: Gel column GE Sephacryl S-200 ddH 2 O equilibration gel column: Flow rate 2 ml / min. The UV absorption curve is gentle up to 280 nm and returns to the baseline. Sample filling: flow rate 1ml / min, sample filling volume 10ml. Elution: degassed ddH 2The crude product was eluted with O at a flow rate of 2 ml / min, and fractions were collected in equal amounts, 3 ml per tube. Two columns of 240 ml were eluted. The separation and purification was repeated five times, and the fractions having the same peak detection time in each run were thoroughly mixed.

[0090] Step 2: Anion exchange column GE HiPrep Q Sodium phosphate buffer A (50 mM Na 2 HPO 4 +NaH 2 PO 4 , pH 8.0) Equilibrated anion exchange column: Flow rate 2 ml / min, UV absorption curve is gentle up to 280 nm and returns to baseline. Sample loading: Take the fraction with biological activity after step 1 purification, and load the sample with a pump. Flow rate is 1.5 ml / min, sample loading volume is 250 ml, and at the same time, collect an equal volume of anion column unbound fraction, 2 ml / tube. Desalting: The bound and unbound fractions in the ion column were degassed with GE HiPrep 26 / 10 Desalting ddH 2 Replace with O and collect the desalted fractions.

[0091] Step 3: Gel column GE HiLoad 16 / 600 Superdex 75pg ddH 2 O equilibration gel column: Flow rate 1 ml / min. The UV absorption curve is gentle up to 280 nm and returns to the baseline. Sample filling: flow rate 1ml / min, sample filling volume 10ml. Elution: degassed ddH 2 The sample was eluted with O at a flow rate of 1 ml / min, and fractions were collected in equal amounts, 2 ml / tube. Column volume (240 ml) was eluted into 1.5 tubes. Measurement of cell activity: After AC16 digestion, cells were grown well, in a 96-well plate, 8000 cells / well, 5 duplicate wells / group. 5% CO 2Incubate in a saturated humidity 37℃ incubator for 2 hours, and allow cells to adhere. After starvation culture in DMEM medium for 24 hours, replace with DMEM medium containing 10% FBS and DMEM and 20% (volume ratio) fraction. After 24 hours of culture, add 10μl of CCK-8 reagent to each well. After 2 hours of incubation, measure the absorbance at 450nm using an enzyme labeling device.

[0092] 3. Experimental results The chromatogram of the unbound fraction separated on the gel column GE HiLoad 16 / 600 Superdex75 pg is shown in Figure 10. The cell viability assay detected a group of growth factors with biological activity, and the results are shown in Figure 11. EXAMPLES

[0093] The following separation and purification are carried out in the same manner as in Example 8.

[0094] 1. Separation and purification of active ingredients Step 1: Gel column GE Sephacryl S-200 ddH 2 O equilibration gel column: Flow rate 2 ml / min. The UV absorption curve is gentle up to 280 nm and returns to the baseline. Sample filling: flow rate 1ml / min, sample filling volume 10ml. Elution: degassed ddH 2 The crude product was eluted with O at a flow rate of 2 ml / min, and fractions in the molecular weight range of 500-2000 daltons were collected. The separation and purification was repeated five times, and the fractions having the same peak detection time in each run were thoroughly mixed.

[0095] Step 2: Cation exchange column GE HiPrep SP Sodium phosphate buffer A (50 mM Na 2 HPO 4 +NaH 2 PO 4 , pH 5.8) Equilibrated cation exchange column: Flow rate 2 ml / min, UV absorption curve is gentle up to 280 nm and returns to baseline. Sample loading: Take the fraction from the first step in the molecular weight range of 500-2000 Daltons and collect the unbound fraction from the cation exchange column using a pump for sample loading, a flow rate of 1.5 ml / min, and a sample loading volume of 250 ml.

[0096] Step 3: Gel column GE HiLoad 16 / 600 Superdex 75pg ddH 2 O equilibration gel column: Flow rate 1 ml / min. The UV absorption curve is gentle up to 280 nm and returns to the baseline. Sample loading: take the unbound fraction from the second step to load the sample, flow rate 1 ml / min, sample loading volume 10 ml. Elution: degassed ddH 2 The sample was eluted with O at a flow rate of 1 ml / min and fractions in the molecular weight range of 500-1200 daltons were collected.

[0097] 2. Active ingredient detection After AC16 digestion, well-grown cells were cultured in a 96-well plate, 8000 cells / well, 5 duplicate wells / group. 5% CO 2 Incubate in a saturated humidity 37℃ incubator for 2 hours, and then adhere to the cells. After starvation culture in DMEM medium for 24 hours, replace with DMEM medium containing 10% FBS and DMEM and 20% (volume ratio) fraction. After 24 hours of culture, add 10μl of CCK-8 reagent to each well. After 2 hours of incubation, measure the absorbance at 450nm using an enzyme labeling device. Figure 12 shows the cell vitality in the non-binding area after treatment with a cation exchange column GE HiPrep SP. EXAMPLES

[0098] The following separation and purification are carried out in the same manner as in Example 8.

[0099] 1. Separation and purification of active ingredients Step 1: Using an ion exchange column and an anion exchange column HiPrep Q, adjust the pH of each solution to 5.8 and 8.0, respectively, and load the sample and ion exchange column. The flow rate is 2 ml / min, and the 280 nm UV absorption curve is gentle and returns to the baseline. Sample loading: Take amniotic fluid and load the sample using a pump. Flow rate: 1.5 ml / min. Sample loading volume: 50 ml. Collect the unbound fraction from the ion column. Step 2: Gel column GE Sephacryl S-200 ddH 2 O equilibration gel column: Flow rate 2 ml / min. The UV absorption curve is gentle up to 280 nm and returns to the baseline. Sample loading: The sample was the unbound fraction from step 1, flow rate 1 ml / min, sample loading volume 10 ml. Elution: degassed ddH 2 The crude product was eluted with O at a flow rate of 2 ml / min, and fractions in the molecular weight range of 500-2000 daltons were collected.

[0100] Step 3: Gel column GEHiLoad 16 / 600 Superdex 75pg ddH 2 O equilibration gel column: Flow rate 1 ml / min. The UV absorption curve is gentle up to 280 nm and returns to the baseline. Sample loading: Sample loading with fractions in the range of 500-2000 Daltons from step 2, flow rate 1 ml / min, sample loading volume 10 ml. Elution: degassed ddH 2 The sample was eluted with O at a flow rate of 1 ml / min and fractions in the molecular weight range of 500-1200 daltons were collected.

[0101] 2. Active ingredient detection After AC16 digestion, well-grown cells were cultured in a 96-well plate, 8000 cells / well, 5 duplicate wells / group. 5% CO 2 Incubate in a saturated humidity 37℃ incubator for 2 hours, and then adhere to the cells. After starvation culture in DMEM medium for 24 hours, replace with DMEM medium containing 10% FBS and DMEM and 20% (volume ratio) fraction. After 24 hours of culture, add 10μl of CCK-8 reagent to each well. After 2 hours of incubation, measure the absorbance at 450nm using an enzyme labeling device. Figure 12 shows the cell vitality in the non-binding area after treatment with an anion exchange column GE HiPrep Q. EXAMPLES

[0102] This example uses a Murine full-thickness wound model to examine the effect of amniotic fluid and / or an extract thereof as described herein on wound healing.

[0103] 1, Material Anesthetic (5.00% chloral hydrate), 75% ethanol, sterile EE from Example 1, PBS, mouse (C57BL / 6, 8 weeks old).

[0104] 2. Experimental Procedure (1) Prepare nine clean cages and weigh nine healthy adult female mice of the same age (6 to 8 weeks or older) and place them in the cages. (2) Prepare 5.00% chloral hydrate and inject it intraperitoneally into the mouse at a rate of 0.07 ml / 10 g using a 1 ml syringe (anesthesia induction time: approximately 5-10 minutes, anesthesia maintenance time: approximately 35 minutes). (3) Once the mouse is anesthetized (after loss of righting reflex), fix the body on a mouse dissection board, shave the hair on the neck and back with an electric shaver, and disinfect the skin by wiping it with 75% ethanol. (4) Using the index finger and thumb of one hand, lift the skin on the back of the mouse's neck to form a fold of sufficient area, and place the skin fold on a dissection board. With the other hand, hold a biopsy needle (hole diameter 6 mm) and apply pressure to the center of the fold, rotating it until the skin on both sides of the biopsy area is penetrated and falls off. (5) If any skin remains, clean the edges of the incised cylindrical tissue mass with tweezers and ophthalmic scissors, and shear the full-thickness skin along the mark left by the skin biopsy needle to create two identical wounds. (6) Photograph the wound with a stereomicroscope, measure the wound area, and record the data. The wound should be parallel to the horizontal line as much as possible, otherwise the measurement error will be large. (7) 100 μl of EE and PBS (blank control group) that have passed the quality inspection are dropped into the corresponding wounds of the mice using a pipette gun, respectively, and the wounds are covered with auxiliary materials. (8) In the rearing box, medical cotton was used instead of wood chips, and the mice were reared singly to prevent them from scratching the wound. After that, the wound was inspected and measured every 24 hours, and photographs and data were recorded. (9) Calculate and compare the effects of different auxiliary materials on wound healing speed.

[0105] 3. Experimental results As shown in FIG. 13, the wound areas of the mice were measured on the 2nd and 4th days, respectively, and the wound areas of both groups were reduced, and the wound healing rate of the EE group was faster and no scars were formed. EXAMPLES

[0106] 1, Material Examples of commonly used common reagents include sodium hydroxide, sodium chloride, potassium chloride, hydrated sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, sodium carbonate, magnesium chloride, acetone, concentrated sulfuric acid, concentrated hydrochloric acid, xylene, absolute ethanol, paraffin, and sucrose from China National Pharmaceutical Group Chemical Reagents Co., Ltd., sodium dodecyl sulfate and ethylenediaminetetraacetic acid from Sigma, USA, Triton X-100 and heparin from Beijing Dingguo Company, Tween-20 from Thermo Fisher, USA, chloral hydrate from Beijing Solabao Technology Co., Ltd., paraformaldehyde and Masson Trichrome Staining Kit from Google Biotechnology Co., Ltd., OCT embedding medium from Sakura Finetech Japan, and fluorescent antifade mounting medium from Vector, USA.

[0107] Rabbit anti-human / mouse Aurora B antibody from Sigma Aldrich, USA; rabbit anti-human / mouse phosphorylated histone H3 polyclonal antibody from Merck Millipore, Germany; rabbit anti-human / mouse cTnT polyclonal antibody from Abcam, UK; goat anti-rabbit secondary antibody (Alexa Fluor 594 conjugated), goat anti-rabbit secondary antibody (Alexa Fluor 488 conjugated), goat anti-mouse secondary antibody (Alexa Fluor 594 conjugated) and goat anti-mouse secondary antibody (Alexa Fluor 488 conjugated) from Life Technologies, USA; DAPI from Sigma Aldrich, USA; goat serum working fluid from Wuhan Doctor Bio-Engineering Co., Ltd.

[0108] Trizol from Invitrogen, USA, and doxorubicin hydrochloride from Shanghai Synchro Biotechnology Co., Ltd.

[0109] The experimental animals were male C57BL / 6J mice purchased from Shanghai Silica Laboratory Animal Co., Ltd.

[0110] a Leica Dmi8 fluorescence microscope and Leica IM50 image acquisition system from Leica, Germany, and a small animal ultrasound diagnostic device from VisualSonics, Canada.

[0111] Preparation of 0.1 mol / L phosphate buffer (1x PBS): NaCl 8.0 g, KCl 0.2 g, Na 2 PO 4 H 2 O 3.58g, KH 2 PO 4 Adjust pH to 7.4, make up to 1000 ml with deionized water, autoclave, and store at 4°C.

[0112] Preparation of 0.5% Triton X-100: 5 ml of Triton X-100 stock solution, 995 ml of 1x PBS.

[0113] II. Test method (1) Immunofluorescence (a) Treat coverglass, cell cultures or frozen sections according to experimental requirements, wash with PBS, 5 min × 3 times. (b) Permeabilized with 0.5% Triton X-100 at room temperature for 15 min, then washed with PBS for 5 min x 3 times. (c) Goat serum was sealed at 37°C for 30 min. (d) Discard the serum, dilute the primary antibody at an appropriate ratio, add it dropwise to the coated tissue, and store it in a humid box at 4°C overnight. (e) Remove the humidity chamber, reheat at 37°C for 30 min, and wash the glass or tissue slices with PBS for 5 min x 3 times. (f) Dilute the secondary antibody at an appropriate ratio, add dropwise to the coated tissue, and incubate at 37°C for 30-60 min. (g) Wash with PBS, 5 min x 3 times, and stain nuclei with DAPI for 10 min. (h) Wash with PBS, 5 min x 3 times, mount with anti-fading mountant, and then observe and analyze with a fluorescence microscope.

[0114] (2) H&E staining (a) 4-μm-thick sections, scooped at 42°C, dried at 60°C overnight, and stored at room temperature. (b) Paraffin sections, deparaffinized, and immersed in xylene three times for 20 min each time, then immersed in descending concentrations of ethanol (100%, 95%, 95%, 90%, 80%) for 2 min, 2 min, 2 min, 1 min, and 1 min, respectively, and washed in tap water for 5 min. (c) Wash with PBS, 5 min × 3 times. (d) Hematoxylin staining, 5 min. (e) Wash with tap water for 10 min. (f) 1% hydrochloric acid ethanol differentiation, washed twice with tap water, 5 min. (g) Return to blue color with 1% ammonia water for 2 minutes, wash with tap water for 5 minutes. (h) Eosin staining, 1-5 min. (i) Dehydrate in 80%, 90%, 95%, 95%, and 100% ethanol for 1 min, 2 min, 2 min, 2 min, and 2 min, respectively. (j) Clear with xylene, 2 min x 3 times. (k) Encapsulated with neutral rubber and observed under a microscope.

[0115] (3) Masson's trichrome staining (a) Paraffin section, deparaffinized, and immersed in water. (b) Chromate treatment (overnight treatment with potassium dichromate) (c) Wash with tap water and distilled water successively. (d) Stain nuclei with hematoxylin (Harris) stain or hematoxylin (Weigert) stain for 1-2 min. (e) Wash thoroughly with water. If overstained, differentiate with hydrochloric acid-ethanol for 2-3 s. (f) Return to blue color with ammonia water for 2 minutes. (g) Masson Lishun Red Complex Red Liquid was used for 5-10 minutes. (h) Differentiation with 1% phosphomolybdic acid solution for 3-5 min. (i) Stained with 1% aniline blue or light green solution for 5 min. (j) Differentiation in 1% glacial acetic acid solution for a few seconds. (k) 95% ethanol, absolute ethanol, xylene clearing, neutral rubber sealing.

[0116] Results: Collagen fibers, mucus, and cartilage appeared blue (e.g., light green solution stained green), cytoplasm, muscle, fibrin, and neuroglial tissue appeared red, and cell nuclei appeared black-blue.

[0117] (4) Creation of a mouse myocardial infarction model Eight-week-old C57BL / 6J male mice were anesthetized with isoflurane via induction chamber, with a respiratory frequency of 115 breaths / min, respiratory ratio of 1:1, and humidity of 1.5 ml. A 20 g plastic tube was orally intubated into the trachea, connected to a small animal respirator, and continuously anesthetized with pure oxygen containing 2.5% isoflurane. Skin was prepared, 3-4 intercostal space was opened, the heart was exposed, the left anterior descending artery was ligated with a 7-0 prolene line, the apex was blanch, the intercostal space was sutured, the skin was sutured, and disinfected. Narcotics were stopped, and ventilation was continued until the mouse woke up.

[0118] (5) Creation of a mouse model of heart failure C57BL / 6J male mice were injected with doxorubicin (5 mg / kg) once every 7 days for 8 weeks. After a total of 4 injections, mouse heart failure was induced and verified by cardiac ultrasound.

[0119] (6) Examination, fixation and sectioning (a) After 1 week and 8 weeks of treatment after surgery, mice were killed by intraperitoneal injection of 10% chloral hydrate (200 mg / kg), and the hearts were removed, and the 1-week collection also included liver and kidneys, embedded in OCT or paraffin. (b) Frozen sections are used for immunofluorescence = Paraffin sections are used for H&E and Masson's trichrome staining. (c) After Masson's trichrome staining, the myocardial infarction size was measured using Image J image analysis software. The formula for calculating the myocardial infarction area is shown below.

number

[0120] 3, statistical analysis All experimental results are shown as mean + SEM. Comparisons between groups were performed using two-tailed tailed t-tests, and comparisons between multiple groups were performed using one-way ANOVA. P<0.05 was the criterion for statistical significance. All experimental results were plotted and analyzed using GraphPad Prism 5 (Software, Inc.) and Image J software.

[0121] 4. Experimental results (I) A mouse myocardial infarction model is prepared by referring to the method of (4) above. The prepared mouse myocardial infarction model is divided into a control group (NS) and a chicken embryo amniotic fluid (EE) treatment group (6 mice per group). The EE treatment group is injected with 100 microliters of EE prepared in Example 1 via the tail vein every two days, for a total of 10 injections by the 21st day of the third week. The control group is injected with 100 microliters of saline 10 times in the same manner.

[0122] Left ventricular ejection fraction (LVEF) is a conventional important indicator of left ventricular function, and improvement in LVEF indicates improved cardiac function after myocardial infarction in mice. The results of measuring the ejection fraction of mice using cardiac ultrasound are shown in Figure 14. As shown in Figure 14, by the third week, EE treatment significantly improved the left ventricular ejection fraction of mice with myocardial infarction, and EE treatment significantly improved cardiac function after myocardial infarction in mice.

[0123] The left ventricular fractional shortening (LVFS) of mice in each group was measured by cardiac ultrasound and the results are shown in Figure 15. As shown in Figure 15, EE treatment significantly improved the LVFS of mice with myocardial infarction, i.e., cardiac function after myocardial infarction, by the third week.

[0124] Masson staining is a conventional method to judge myocardial infarction tissue and fibrous tissue. After 21 days of treatment, mice from each group were killed, and paraffin sections of myocardial tissue were prepared and stained as described above at point (3). The results are shown in Figure 16. In Figure 16, blue indicates infarcted fibrotic tissue, and red indicates muscle tissue. It can be seen from the figure that the mice with myocardial infarction had severe fibrosis, and fibrosis was significantly reduced after EE treatment. This shows that EE treatment prevented fibrosis after myocardial infarction in mice. In addition, the size of the left ventricular cavity is the basis for judging the presence or absence of ventricular dilation after myocardial infarction, and ventricular dilation is an important marker for cardiac dysfunction. In addition, as shown in Figure 16, the ventricular cavity of the control group mice with myocardial infarction was severely dilated, but after 3 weeks of EE treatment, the left ventricular cavity of the treated group mice did not have significant dilation.

[0125] PH3 staining is an index for judging the regeneration status of intracardiac cells. Mice from each group treated for 21 days were killed, frozen sections of myocardial tissue were prepared, and the results of PH3 staining were performed by the method described in point (1) above. The results are shown in FIG. 17. As shown in FIG. 17, the number of PH3-stained positive (green fluorescent dots, indicated by arrows) cells in the cardiac tissue of the EE-treated mice was significantly increased, and it is clear that the EE treatment promoted the regeneration of cells in the cardiac tissue. Aurora B staining is an index for judging the regeneration status of intracardiac cells. As shown in FIG. 18, the results of Aurora B staining performed by the method described in point (1) above were significantly increased in the cardiac tissue of the EE-treated mice, and it is clear that the EE treatment promoted the regeneration of cells in the cardiac tissue.

[0126] (II) A mouse heart failure model is prepared by referring to the method of (5) above. The prepared mouse heart failure model is divided into a control group and a chicken embryo extract (EE) treatment group (6 mice per group). The EE treatment group is injected with 100 microliters of EE prepared in Example 1 via the tail vein every two days, for a total of 10 injections by the 21st of the third week. The control group is injected with 100 microliters of saline 10 times in the same manner.

[0127] Left ventricular ejection fraction (LVEF) is a traditional important indicator of left ventricular function, and an improvement in LVEF indicates that the cardiac function of mice with heart failure has improved. The results of measuring the ejection fraction of mice using cardiac ultrasound are shown in Figure 19. As shown in Figure 19, by the third week, EE treatment significantly increased the left ventricular ejection fraction of mice with heart failure, and EE treatment significantly improved the cardiac function of mice with heart failure. The left ventricular fibrosis area was obviously reduced. EXAMPLES

[0128] A percutaneous arterial catheter (PCI) was inserted into the anterior descending coronary artery of the heart using an experimental large white pig, and the catheter was removed 50 minutes after the balloon was occluded. A cardiac ischemia-reperfusion model was then prepared in the large white pig. Chicken EE (1 ml / kg) collected by the method described in Example 1 was then administered intravenously immediately after the operation. Basal cardiac function was measured before the operation. The results are shown in Figures 20 and 21.

[0129] Figure 20 shows that treatment of myocardial infarction in large white pigs with chicken EE can improve the left ventricular ejection fraction and short axis shortening fraction of large white pigs with myocardial infarction. After surgery, the cardiac function of the large white pigs in the control group gradually declined, while the left ventricular function of the EE treatment group recovered to a certain extent, and the EF and FS at 2, 4 and 8 weeks after surgery were significantly higher than those of the control group (Figure 20, A and C). The statistical analysis of ΔEF and ΔFS based on the difference from the baseline values ​​before surgery showed that the decrease in EF and FS at 1 week after EE treatment was significantly lower than that before surgery, and the decrease at 2, 4 and 8 weeks in the treatment group was significantly lower than that of the control group (Figure 20, B and D). The stroke volume of the treatment group was significantly higher than that of the control group at 1-8 weeks after surgery (Figure 18, E). The left ventricular end-systolic volume and diameter of the control tended to increase, while those of the treatment group were lower than those of the control group (Figure 20, F and I), indicating that EE improves the left ventricular contractile force. Left ventricular end-diastolic volume and diameter in the controls tended to increase and then decline in the treated groups (Fig. 20, G and H), indicating that EE reversed ventricular remodeling due to partial myocardial infarction (MI).

[0130] After preparing the large white pig ischemia-reperfusion (IR) model, the treatment group was immediately treated with chicken EE, and the control group was administered 5% glucose. Through observation and surveillance footage during rearing, it was found that the large white pigs in the control group had less activity time and looked tired. One week after surgery, statistics showed that the daily activity time of the large white pigs in the treatment group was significantly longer than that of the control group (Figure 21, D). Interviews were conducted 8 weeks after EE treatment, and the myocardium from the apex to the anterior wall of the heart in the control group was thinned and white after staining with triphenyltetrazolium chloride (TTC). The treatment group had mild infarction from the apex to the anterior wall of the left ventricle, and no significant thinning of the ventricular wall was observed. At the same time, an increase in fat tissue in the cardiac tissue of the control group was observed (Figure 21, A), and statistics showed that the area of ​​infarction white after TTC staining in the treatment group was significantly lower than that of the control group (Figure 21, B).

[0131] The left ventricular anterior wall tissue from the infarcted area was collected and stained with Masson's trichrome staining, and it was found that the control group exhibited transmural infarction and thinning of the ventricular wall, whereas the cardiac fibrosis in the EE-treated group extended into the myocardial space, and no significant thinning of the ventricular wall was observed (Figure 21, C).

[0132] The above results show that EE can significantly improve the left ventricular ejection fraction and stroke volume of ischemia-reperfused large white pigs, reduce the left ventricular remodeling caused by myocardial infarction, reduce pulmonary congestion in ischemia-reperfused large white pigs, and improve daily activity. In addition, the TTC staining results show that the myocardial infarction area of ​​the EE treatment group was significantly smaller than that of the control group. The tissue Masson's Trichrome staining results show that transmural infarction appeared in the left anterior wall of the large white pigs in the control group, and the fibrosis area was significantly higher than that of the EE treatment group. The fluorescent staining results show that EE can increase the angiogenesis of the infarcted area of ​​the large white pigs. EXAMPLES

[0133] With reference to the method described in Example 1, amniotic fluid from mice of 13-14 days of gestation is obtained, and the centrifuge tubes in which the amniotic fluid extracts are collected are equilibrated and then centrifuged at 5°C, 3500 rpm for 21 minutes using a Zhongjia KDC-2046 low-speed refrigerated centrifuge (see the instruction manual for the standard operating procedure of the centrifuge). The supernatant is decanted and transferred to a clean plastic storage tank and stored in a refrigerator at -80°C. A 5 ml subsample of each lot is pre-stored for subsequent testing. The entire procedure is performed under aseptic conditions.

[0134] Measurement of cell activity: After AC16 digestion, cells were grown well, in a 96-well plate, 8000 cells / well, 5 duplicate wells / group. 5% CO 2 Incubate in a saturated humidity 37℃ incubator for 2 hours to allow cell adhesion. After 24 hours of starvation in DMEM medium, replace with DMEM containing 10% FBS, or DMEM with 2.5%, 5%, 10% and 20% (volume ratio) of mouse EE. After 24 hours of incubation, add 10μl of CCK-8 reagent to each well. After 2 hours of incubation, measure the absorbance at 450nm using an enzyme labeling device.

[0135] The results are shown in Figure 22.

Claims

1. 1. Use of an extract of amniotic fluid in the preparation of a reagent for promoting cell growth and / or tissue repair, comprising: the amniotic fluid is derived from a chicken egg having an embryonic age of 5-12 days, or from a duck or goose egg of a developmental stage corresponding to that of a chicken egg of said embryonic age, or from a rodent embryo having an embryonic age of 8-20 days, the extract does not bind to an ion exchange column between pH 5.8 and 8.0, and the molecular weight of the components is within the range of 500 to 1200 daltons; and The tissue is derived from any one or more of: cartilage tissue, meniscus tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, vascular tissue, periosteum tissue, pericardial tissue, lung tissue, synovial tissue, nervous tissue, renal tissue, bone marrow, urogenital tissue, intestinal tissue, liver tissue, pancreatic tissue, spleen tissue and adipose tissue, and the cells are derived from any one or more of said tissues.

2. 2. The use according to claim 1, wherein the amniotic fluid is derived from a chicken egg having an embryonic age of 6-11 days, or from a chicken egg having an embryonic age of 7-9 days, or from a chicken egg having an embryonic age of 7-8 days, or the amniotic fluid is derived from a rodent embryo having an embryonic age of 8-14 days, or the amniotic fluid is derived from a duck egg having an embryonic age of 8-10 days, or from an embryonic age of 8-9 days.

3. 3. The use according to claim 1 or claim 2, wherein the agent is used to promote the regeneration of cardiac cells after myocardial infarction.

4. The use according to claim 3, wherein the cardiac cells are cardiomyocytes.

5. 1. Use of an extract of amniotic fluid in the preparation of a medicament for the treatment of a pathology relating to tissue damage, comprising: the amniotic fluid is derived from a chicken egg having an embryonic age of 5-12 days, or from a duck or goose egg of a developmental stage corresponding to that of a chicken egg of said embryonic age, or from a rodent embryo having an embryonic age of 8-20 days, The extract does not bind to an ion exchange column at a pH between 5.8 and 8.0, and the molecular weight of the components contained therein is within the range of 500 to 1200 daltons; Conditions relating to tissue damage include conditions caused by disease, trauma, or failure of normal development of tissue, the condition being selected from the group consisting of hernias, pelvic floor defects, tears or ruptures of tendons and ligaments, venous ulcers, vascular diseases, and geriatric diseases.

6. 6. The use according to claim 5, wherein the amniotic fluid is derived from a chicken egg having an embryonic age of 6-11 days, or from a chicken egg having an embryonic age of 7-9 days, or from a chicken egg having an embryonic age of 7-8 days, or the amniotic fluid is derived from a rodent embryo having an embryonic age of 8-14 days, or the amniotic fluid is derived from a duck egg having an embryonic age of 8-10 days, or from an embryonic age of 8-9 days.

7. 7. The use according to claim 5 or claim 6, wherein the vascular disease is selected from the group consisting of peripheral arterial disease, abdominal aortic aneurysm, carotid artery and venous disease, vascular defects and vascular dysplasia.

8. 7. The use according to claim 5 or claim 6, wherein the geriatric disease is selected from the group consisting of geriatric dementia and geriatric osteoarthritis.

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