CN113143970A - Methods and compositions for promoting tissue repair in diabetic subjects - Google Patents

Abstract

The present invention relates to methods and compositions for treating diabetic foot, and to the use of amniotic fluid and/or extracts thereof from non-human animals, particularly avian and non-human mammals. The composition of the present invention may be a cell culture medium containing the amniotic fluid and/or an extract thereof, or a pharmaceutical composition.

Description

Methods and compositions for promoting tissue repair in diabetic subjects

Technical Field

The present invention relates to methods and compositions for promoting cell growth and tissue repair in a diabetic subject.

Background

Due to the limitations of traditional therapies and the aging population, there is an increasing need for new treatment regimens for tissue repair. Currently, cell-based therapies are commonly employed for the treatment of tissue and organ defects. These therapies involve the introduction of precursor cells, preferably stem cells, into the defect site, thereby expanding the endogenous cell population and increasing the rate of tissue regeneration and repair. These cells are autologous in nature, isolated from the patient in need of treatment, and expanded in vitro before being returned to the patient's defect site.

However, existing therapies also have some problems. First, cells cultured in vitro for long periods of time are mutated, unlike in vivo, which are currently unrecognizable and indistinguishable from the culture system, and thus the more tumorigenic cells that grow longer; cells cultured in vitro for long periods of time also differentiate, which reduces the ability of the cells to proliferate and repair tissue in vivo. Second, the cells themselves carry viruses, and in vitro culture of cells further exposes the cells to foreign substances that may contain contaminating particles (e.g., viruses and bacteria) or chemicals. These contaminants can potentially lead to significant disease and morbidity if not detected prior to implantation. Furthermore, the risk of cells becoming toxic increases with the length of cell culture.

Diabetes is a multi-system disease, and diabetic ulcers are a complication of diabetes, often found in the feet of patients, and are also called diabetic feet. The diabetic patients can cause the damage and the malformation of foot soft tissues and bone joint systems due to the combination of peripheral neuropathy and peripheral vascular diseases with overhigh mechanical pressure, thereby causing a series of foot problems from mild neurological symptoms to severe ulcer, infection, vascular diseases, Charcot joint diseases and neuropathic fracture. If active treatment fails to adequately address the symptoms and complications of the lower extremities, catastrophic results can result.

Unlike ulcers and lesions of other origin, diabetic foot complications arise from sensory neuropathy, autonomic and motor neuropathy, and vasculopathy. Sensory neuropathy, combined with excessive mechanical stress, is a major initiating factor in causing foot ulcers and infections. Inflammation and tissue damage are the result of a certain degree of repetitive stress on a particular area of loss of sensation. Pressure or shear forces from the ground, shoes, or other adjacent toes result in the formation of ulcers, which are often exacerbated by the presence of bony processes due to the lack of normal neuroprotective mechanisms. Lesions of the autonomic nervous system cause loss of the normal perspiration regulation function, skin temperature regulation function and blood transport regulation function of the skin, resulting in reduced flexibility of local tissues, formation of thick calluses and more susceptibility to breakage and cracking. The loss of normal perspiration blocking the rehydration of local tissues causes further destruction of tissues, making deep tissues more susceptible to bacterial colonization. Motor neuropathy also plays a role in the development of diabetic feet, where contractures of intrinsic muscles of the foot cause typical claw-toe deformities. Hyperextension of the metatarsophalangeal joint has also been shown to directly increase the pressure under the metatarsal heads, making the area more prone to ulceration. Flexion of the proximal interphalangeal joint results in an increased risk of ulcers forming on the dorsal side of the prominent interphalangeal joint and on the plantar side of the toe. While vascular lesions make it difficult for damaged tissue to heal.

In addition, autonomic dysfunction leads to skin soft tissue damage, causing foreign bacterial invasion. The change in chemotaxis leads to inefficient leukocyte responses. Moreover, hyperglycemia, reduced oxygen partial pressure, and malnutrition, among others, can collectively cause tissue edema, acid accumulation, hypertonicity, and inefficient anaerobic metabolism. Such environments hinder the function of leukocytes. In addition, vascular lesions can cause restricted antibiotic transport, further resulting in reduced bacterial clearance efficiency, leading to local soft tissue infections and even the development of osteomyelitis.

The overall goal of diabetes treatment is to prevent, control and delay the acute and chronic complications of diabetes, and to maximize or improve the quality of life of the patient. However, today there is still a lack of an exact and effective treatment regimen for the serious complications of diabetic foot ulcers, mainly relying on early prophylactic treatment. Once a diabetic foot ulcer develops, the vast majority of patients eventually undergo amputation due to impaired sensation, microcirculatory disturbance, difficulty in wound healing, wound infection, and gangrene. In the past, diabetic foot ulcers mainly depend on blood sugar control, reduction of load of affected limbs, wound dressing, local external application of antibiotics, negative pressure closed drainage technology and the like, but the amputation risk cannot be reduced.

Thus, there remains a need in the art for new methods and compositions for tissue repair that are capable of achieving tissue repair in a diabetic subject.

Disclosure of Invention

The invention provides application of amniotic fluid and/or an extract thereof in preparing a reagent for promoting cell growth, tissue repair and/or wound healing of a diabetic subject, wherein the amniotic fluid is derived from an egg with an embryo age of 5-12 days, preferably an egg with an embryo age of 6-11 days, more preferably an egg with an embryo age of 7-9 days, more preferably an egg with an embryo age of 7-8 days, or an egg from other birds except chickens with a development period corresponding to the development period of the egg with the embryo age; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

The invention also provides application of amniotic fluid and/or extract thereof in preparing a medicament for treating neuropathy and/or neuropathy induced neuropathic ulcer, vascular ulcer or neuro-vascular ulcer, wherein the amniotic fluid is from eggs with an embryo age of 5-12 days, preferably from eggs with an embryo age of 6-11 days, more preferably from eggs with an embryo age of 7-9 days, more preferably from eggs with an embryo age of 7-8 days, or from eggs from other birds except chickens with a development period corresponding to the development period of the eggs with the embryo age; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

The invention also provides application of amniotic fluid and/or extract thereof in preparing a medicament for treating vascular diseases of a diabetic subject, or recovering/improving skin perspiration regulation function, skin temperature regulation function and/or blood circulation regulation function of the diabetic subject, wherein the amniotic fluid is derived from eggs with an embryo age of 5-12 days, preferably 6-11 days, more preferably 7-9 days, more preferably 7-8 days, or from eggs of other birds except chickens with a development period corresponding to the development period of the eggs with the embryo age; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

The invention also provides application of amniotic fluid and/or extract thereof in preparing a medicament for treating diabetic foot or a condition related to diabetic foot, wherein the amniotic fluid is from an egg with an embryo age of 5-12 days, preferably an egg with an embryo age of 6-11 days, more preferably an egg with an embryo age of 7-9 days, more preferably an egg with an embryo age of 7-8 days, or an egg from other poultry except a chicken with a development period corresponding to the development period of the egg with the embryo age; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

In one or more embodiments, the agent or medicament is a cell culture comprising amniotic fluid and/or an extract thereof as described herein.

In one or more embodiments, the agent or medicament is a pharmaceutical composition comprising amniotic fluid and/or an extract thereof as described herein, and a pharmaceutically acceptable excipient.

In one or more embodiments, the major bioactive component of the extract is not bound to the ion exchange column at a pH between 7.0 and 8.0 and contains components having molecular weights in the range of 150-2000 daltons, but is not limited thereto.

In one or more embodiments, the tissue is from: any one or more tissues selected from cartilage tissue, meniscus tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascia tissue, periosteal tissue, nerve tissue, urogenital tissue, and adipose tissue; the cells are from any one or more of the tissues.

In one or more embodiments, the condition associated with a diabetic foot includes, but is not limited to, a neuropathic, vascular, or neuro-vascular ulcer caused by neuropathy and/or vasculopathy. In one or more embodiments, conditions associated with diabetic feet include, but are not limited to, chronic non-healing wound conditions in diabetic subjects caused by disease or trauma selected from the group consisting of: dry skin; the hair is separated by hair; skin temperature is decreased; pigmentation; the pulsation of the extremity arteries is reduced or eliminated; torn or broken tendons or ligaments; skin wounds, such as scars, traumatic wounds, ischemic wounds, diabetic wounds, surgical wounds, scalds, burns, skin ulcers, such as decubitus ulcers or pressure-induced ulcers, venous ulcers, diabetic ulcers, gangrene, necrosis; vascular conditions, such as peripheral arterial disease, venous disease, vascular defects, and vascular dysplasia; muscle diseases, such as muscle atrophy, inflammatory, neurological and myogenic muscle diseases; neurological disorders such as stinging, burning, numbness, dysesthesia or loss; bone diseases such as, for example, resting pain, osteomyelitis, Charcot's arthropathy, toe deformity; infection, tissue edema, intermittent claudication, and the like, and long-term non-healing wounds caused by skin tumors.

The present invention also provides a method of repairing tissue resulting from diabetes comprising: autologous or allogeneic cells are cultured in vitro by using amniotic fluid and/or extract thereof or a culture medium containing the amniotic fluid and/or extract thereof, or autologous cells or allogeneic cells or chicken embryonic stem cells are cultured by using the cell culture method described herein to form implantable tissues or matrixes, which are implanted into animals to repair the corresponding damaged tissues. Preferably at the site of the tissue defect.

Drawings

FIG. 1 shows HPLC analysis results of amniotic fluid of eggs 7 days old.

FIG. 2 shows HPLC analysis results of amniotic fluid of eggs 11 days old.

FIG. 3 shows HPLC analysis results of amniotic fluid of eggs 13 days old.

FIG. 4, free radical scavenging ability of egg saline of different embryo ages. Wherein the abscissa represents embryo age and the ordinate represents clearance.

FIG. 5, growth curves of chicken embryo fibroblasts under different culture conditions.

FIG. 6, effect of amniotic fluid from chicken eggs on growth viability and migration ability of Human Umbilical Vein Endothelial Cells (HUVEC). Wherein the abscissa represents the medium and the ordinate represents the OD450 value.

FIG. 7, effect of amniotic fluid from duck eggs on growth viability and migration ability of chicken embryonic fibroblasts. Wherein the abscissa represents the medium and the ordinate represents the OD450 value.

Figure 8 amniotic fluid from chicken eggs promoted the growth of mouse osteoblasts. Wherein the abscissa represents the medium and the ordinate represents the OD450 value.

Figure 9, amniotic fluid from chicken eggs promoted the growth of primary cardiomyocytes. Wherein the abscissa represents the medium and the ordinate represents the OD450 value.

FIG. 10, gel column GE HiLoad 16/600Superdex75pg separation chromatogram.

FIG. 11, cell viability assay gel column GE HiLoad 16/600Superdex75pg fractions. The abscissa represents the medium, wherein FBS represents fetal bovine serum; DMEM is Dulbecco's Modified Eagle Medium; EE represents amniotic fluid; "EE" refers to lyophilized amniotic fluid; S-200B represents the fraction of the B peak; q UNBOUND denotes the UNBOUND fraction of the anion column; 3-1 to 3-6 represent the equal volumes of fractions 1 to 6, respectively, in the third purification step.

FIG. 12, cell viability assay unbound fractions from cation exchange column GE HiPrep SP and anion exchange column HiPrep Q separations. The abscissa represents the medium, wherein FBS represents fetal bovine serum; DMEM is Dulbecco's Modified Eagle Medium; EE represents amniotic fluid; "EE" refers to lyophilized amniotic fluid; hiprep SP-UN represents fractions not bound to Hiprep SP column; hiprep Q-UN represents fractions not bound to a Hiprep Q column; hiprep Q-Bound represents fractions Bound to the Hiprep Q column.

FIG. 13 mean body weights (g) of groups D0-D20 after scalding in mice. Control was Control healthy mice; DM is a diabetes model mouse; DM _ HE was given HE treatment for diabetic model mice.

FIG. 14, mean wound healing rates of groups D0-D20 after scalding in mice. The figure is the same as that of FIG. 13.

Fig. 15, wound healing in diabetic subjects was significantly promoted using the amniotic fluid of the present invention. Average wound healing rates for groups D0-D20 after scald were observed in mice. Control (Control) was healthy mice, n ═ 5; DM is a diabetes model mouse, and n is 4; DM _ DWS was treated with DWS in diabetic model mice, with n being 6.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

The inventor finds that the growth factor group contained in the amniotic fluid and/or the extract of the amniotic fluid of the non-human animals can promote the cell growth or the wound healing of the diabetic subjects. Thus, the present disclosure relates to the use of amniotic fluid and/or extracts thereof for promoting cell growth, tissue repair or wound healing in a diabetic subject.

The amniotic fluid may be derived from poultry eggs and non-human mammals. Fowl eggs are referred to as poultry eggs. Preferred birds are poultry, such as chickens, ducks and geese. Preferably, the present invention uses eggs having an age of 5-20 days, preferably 6-15 days old. It will be appreciated that the appropriate age of the embryos need not be the same from egg to egg. For example, when eggs are used, eggs having an age of 5 to 12 days are preferably used, eggs having an age of 6 to 11 days are more preferably used, eggs having an age of 7 to 9 days are more preferably used, and eggs having an age of 7 to 8 days are more preferably used. When eggs of other birds are used, eggs whose development period corresponds to the development period of the above-mentioned embryonated egg may be used. For example, when using duck eggs, duck eggs having an embryo age of 8-10 days, especially 8-9 days, may be the best.

The poultry egg amniotic fluid can be obtained by adopting a conventional method. For example, the blunt end of an egg of the corresponding embryo age may be knocked to break the shell and peel it open to form an opening of approximately 2 cm in diameter. The shell membrane and yolk membrane were then carefully torn apart with forceps, taking care not to disrupt the amniotic membrane. The amniotic membrane and the associated tissue, which are wrapped with the embryo, are poured from the shell into a culture dish, and the amniotic membrane is punctured with an injector to extract amniotic fluid until the amniotic membrane is tightly attached to the embryo, thereby obtaining the amniotic fluid used in the present invention.

Herein, amniotic fluid may also be derived from a non-human mammal, particularly a rodent, such as from a mouse. Other non-human mammals may be common domestic animals such as cattle, sheep, dogs, cats, pigs, etc. In certain embodiments, the amniotic fluid is from an embryo from a rodent with a gestational age of 8-14 days, or from a non-human mammal with a developmental stage corresponding to the developmental stage in which a rodent with a gestational age of 8-14 days is located. The amniotic fluid can be obtained by conventional methods. For example, the amniotic fluid used in the present invention can be obtained by cutting the abdominal cavity of a mouse pregnant for 8-14 days with surgical scissors, carefully removing and cutting the uterus, and puncturing the amniotic membrane with a syringe to extract the amniotic fluid until the amniotic membrane is attached to the embryo.

It will be appreciated that, if necessary, the amniotic fluid may be centrifuged to separate impurities that may be contained, such as egg yolk and the like, to obtain as pure an amniotic fluid as possible. The supernatant obtained after centrifugation is the amniotic fluid used in the invention. It is understood that all the steps of obtaining the amniotic fluid need not be performed under sterile conditions; in addition, as used herein, "amniotic fluid" shall mean "pure" amniotic fluid, i.e., amniotic fluid isolated from an avian egg or a non-human mammalian embryo that does not contain other components within the avian egg or the non-human mammalian embryo and that is also not contaminated with foreign matter. Pure amniotic fluid can be stored in a refrigerator below-60 deg.C, thawed and used.

In certain embodiments, the present invention uses extracts of amniotic fluid. Preferably, the major bioactive component of the extract is not bound to the ion exchange column at a pH of 7.0-8.0 and contains components having molecular weights in the range of 150-2000 daltons, but is not limited thereto. The extract can be obtained by separating a neutral fraction having a molecular weight of 150-. Gel columns and ion exchange columns known in the art may be used to carry out the methods herein. For example, a fraction having a molecular weight of 150-2000 daltons can be separated from amniotic fluid using a well-known gel chromatography column (e.g., various gel chromatography columns described below), and then a neutral fraction can be separated from the fraction using an ion exchange method (e.g., using an ion exchange column described below). Alternatively, the neutral fraction may be separated from the amniotic fluid by ion exchange means (e.g., using an ion exchange column as described below) and then the fraction having a molecular weight in the range of 150-2000 daltons in the neutral fraction may be separated using a gel chromatography column (e.g., various gel chromatography columns as described below).

In certain embodiments, a neutral fraction having a molecular weight in the range of 150-. In particular, the method may comprise the steps of:

(1) separating neutral fraction with molecular weight of 150-; and

(2) the neutral fraction with the molecular weight of 200-1200 Dalton is separated from the neutral fraction with the molecular weight of 150-2000 Dalton.

Step (1) can be achieved by using gel chromatography and ion exchange methods. The fractions with molecular weight of 150-2000 Dalton were separated from the amniotic fluid by means of a gel chromatography column, whereas the fraction without electric charge (neutral) was obtained by means of ion exchange.

Herein, gel chromatography may be carried out using various commercially available gel chromatography columns including, but not limited to, Sephacryl S-100, Sephacryl S-200, Sephacryl S-300, Sephacryl S-400, Superose 12, Superose 6, Superdex 12, Superdex 6, and the like from GE. It is understood that any other gel chromatography packing with a separation range of 100-. In general, when a gel column is used, ddH may be used first₂The flow rate of the O-balanced gel chromatographic column can be determined according to actual conditions. For example, in certain embodiments, the flow rate may be 0.5 to 50ml/min, such as 1 ml/min. Typically, the UV absorption is between 200 and 300nm, such as 280 nm. And after the ultraviolet absorption curve is stable and the base line is returned, ending the balance. After the balance is over, the sample can be loaded. The sample flow rate is determined according to the actual preparation conditions. After the sample loading is finished, degassing ddH can be used₂The crude product was eluted and fractions with molecular weights between 150 and 2000 daltons were collected. When needed, the deviceThe separation by gel chromatography can be repeated several times, and fractions with the same peak time in each separation are mixed.

Herein, charged components can be separated from uncharged components using methods well known in the art. This can be achieved, for example, using ion exchange methods. Both anion exchange and cation exchange can be used in the process of the present invention. In certain embodiments, an anion exchange process is employed 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 corporation. It should be understood that other brands of anion exchange packing may also be used. Alternatively, commercially available cation exchange columns may be used, including but not limited to CM Sepharose, SP Sepharose, Capto S, Mono S, Mini S, and the like.

Typically, when ion exchange is performed, the ion exchange column is first equilibrated with a buffer. The buffer may be a buffer conventional in the art, for example, a phosphate buffer, especially a sodium phosphate buffer, may be used. 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 anion exchange column may be equilibrated with a buffer solution having a pH of 7.5 to 8.5, preferably 7.5 to 8.0; when a cation exchange column is used, the cation exchange column can be equilibrated with a buffer solution having a pH of 5.8 to 7.0, preferably 5.8 to 6.5. In certain embodiments, the sodium phosphate buffer contains Na₂HPO₄And NaH₂PO₄The pH was about 5.8 or 8.0. The present invention preferably uses an anion exchange column for the separation. The flow rate may be determined according to actual conditions. For example, in certain embodiments, the flow rate may be 0.5 to 50ml/min, such as 1 ml/min. Generally, after the 280nm UV absorption curve has stabilized and the baseline has returned, the equilibrium is terminated. After equilibration is complete, the sample can be loaded and the effluent fraction (i.e., the fraction not bound to the column) collected. The sample flow rate is determined according to the actual preparation conditions.

In the step (1), gel chromatography can be firstly carried out to separate out the fraction with the molecular weight of 150-; alternatively, ion exchange can be carried out to separate the neutral fraction from the amniotic fluid, and then the active ingredients with the molecular weight in the range of 150-.

The main purpose in step (2) is to further separate the neutral fraction obtained in step (1) to obtain active ingredients with molecular weight in the range of 150-1200 daltons. Here, a commercially available gel chromatography column may be used to separate the fractions having molecular weights in the range of 150-1200 daltons. Suitable gel chromatography columns include, but are not limited to, HiLoad Superdex 16/600Superdex75pg, Superdex Peptide, Superdex 200, Superdex 30, and the like from GE. It is understood that other brands of gel chromatography packing with separation ranges of 500-.

In general, ddH can be used first₂O balance gel column, the flow rate can be determined according to actual conditions. For example, in certain embodiments, the flow rate may be 0.5 to 50ml/min, such as 1 ml/min. Generally, after the 280nm UV absorption curve has stabilized and the baseline has returned, the equilibrium is terminated. After the balance is over, the sample can be loaded. The sample flow rate is determined according to the actual preparation conditions. After the sample loading is finished, degassing ddH can be used₂And O eluting the crude product, and collecting fractions to obtain fractions containing components with molecular weights in the range of 150-1200 daltons, namely the extract. In one or more embodiments, the amniotic fluid and/or extract thereof or a composite dressing formulated from the amniotic fluid as a primary material is referred to as a DWS.

The extract obtained by the above method is prepared into a solution with pH of 5.8-8.0, and then is passed through various ion exchange columns (including DEAE Sepharose, Q Sepharose, Mono Q, CM Sepharose, SP Sepharose and Mono S), and active ingredients contained in the extract are not combined with the ion exchange columns.

The amniotic fluid and/or the extract thereof described herein can be used as an active ingredient of a medicament for in vivo administration to a subject in need thereof for promoting cell growth and tissue repair in vivo. For example, an effective amount of amniotic fluid and/or extract thereof as described herein, or a pharmaceutical composition containing the same, may be administered to a subject in need thereof.

Herein, the animal may be a mammal, in particular a human.

"repair" as used herein refers to the formation of new tissue sufficient to at least partially fill a tissue defect site that is ineffective or structurally discontinuous. "tissue defect" or "tissue defect site" refers to the destruction of epithelial, connective, or muscle tissue. Tissue defects result in tissue functioning at an undesirable level or under undesirable conditions. For example, a tissue defect may be a partial or complete tear in a tendon, or local cell death resulting from a myocardial infarction. A tissue defect may form a "void," which may be understood as a three-dimensional defect, such as a tear, cavity, hole, or other substantial disruption in the integrity of epithelial, connective, or muscle tissue. In certain embodiments, a tissue defect refers to those tissues that are not capable of endogenous repair or spontaneous repair. The tissue defect may be caused by an accident, disease and/or surgical procedure. For example, cartilage defects may be the result of trauma to a joint, such as movement of torn meniscal tissue into the joint. The tissue defect may also be caused by degenerative diseases such as osteoarthritis. In certain embodiments, the invention is particularly directed to repair of tissue defects caused by neuropathy and/or vasculopathy.

Tissues described herein include, but are not limited to, muscle tissue, epithelial tissue, connective tissue, and neural 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, fascia tissue, periosteal tissue, synovial tissue, neural tissue, bone marrow, and adipose tissue. Thus, the cells described herein may be cells from any of the above tissues.

In certain embodiments, conditions associated with tissue damage, including but not limited to conditions caused by disease or trauma or the failure of tissues to develop normally, such as dry skin; the hair is separated by hair; skin temperature is decreased; pigmentation; the pulsation of the extremity arteries is reduced or eliminated; torn or broken tendons or ligaments; skin wound (such as scar, traumatic wound, ischemic wound, diabetic wound, and surgical wound), scald, burn, skin ulcer (such as decubital ulcer or pressure-induced ulcer, venous ulcer, diabetic ulcer, gangrene, and necrosis); vascular conditions (e.g., peripheral arterial disease, venous disease, vascular defects, vascular dysplasia); muscle diseases (e.g., muscle atrophy, inflammatory, neurological and myogenic muscle diseases); neurological conditions (e.g., tingling, burning, numbness, dysesthesia or loss); bone diseases (e.g., rest pain, osteomyelitis, Charcot's arthropathy, toe deformity); infection, tissue edema, intermittent claudication, and chronic non-healing wounds caused by skin tumors.

Accordingly, provided herein is the use of an amniotic fluid and/or an extract thereof as described herein for promoting cell growth, tissue repair and/or wound healing in a diabetic subject. Also provided herein is a method for repairing a tissue of a subject with diabetes, the method comprising culturing tissue cells of interest in vitro using amniotic fluid and/or an extract thereof as described herein or a composite dressing formulated from the same as a main material or a cell culture medium comprising the amniotic fluid or the extract as described herein to form a tissue matrix, and implanting the tissue matrix into the site of tissue injury or defect. Also provided herein is a method of treating a neuropathy and/or vasculopathy-induced neuropathic, vascular, or neuro-vascular ulcer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of an amniotic fluid and/or extract thereof as described herein or a pharmaceutical composition comprising the amniotic fluid and/or extract thereof. The invention also provides methods of treating vasculopathy in a diabetic subject, and restoring the skin perspiration regulation function, skin temperature regulation function, and blood circulation regulation function of the diabetic subject. The invention also provides methods of treating diabetic feet or conditions associated with diabetic feet.

A therapeutically effective amount refers to a dose that achieves treatment, prevention, alleviation, and/or amelioration of a disease or disorder in a subject. The therapeutically effective amount may be determined based on factors such as the age, sex, condition and severity of the condition, other physical conditions of the patient, etc. Herein, a subject or patient generally refers to a mammal, in particular a human.

Herein, autologous cells or allogeneic cells may be cultured in vitro using amniotic fluid and/or an extract thereof or a culture medium containing the amniotic fluid and/or the extract thereof, or cultured using a cell culture method described herein to form an implantable tissue or matrix, which is implanted into an animal, particularly a human, particularly at a tissue defect site to repair the corresponding damaged tissue. Autologous cells are cells isolated from the animal body itself in need of tissue repair or treatment, in particular from the tissue itself in need of repair or treatment.

The present disclosure also relates to the use of amniotic fluid and/or extracts thereof as described herein or a composite dressing formulated from the same as a primary material for administration to a subject in need thereof, for methods or uses as described herein. The administration mode can be parenteral administration, intravenous injection, cardiac cavity injection and the like. In certain embodiments, a therapeutically effective amount of amniotic fluid and/or extract thereof may be mixed with an appropriate amount of saline for injection, water for injection, or dextrose injection, and then administered by a suitable means, such as intravenous infusion, intracardiac injection, or focal area, for example, by application to the wound surface. In a preferred embodiment, the present invention relates to the administration of amniotic fluid and/or an extract thereof as described herein or a composite dressing formulated from the same as a primary material directly at a site of injury to promote the proliferation of normal cells at the site and thereby effect tissue repair of the defective portion.

Pharmaceutical compositions containing amniotic fluid and/or extracts thereof as described herein will typically further contain pharmaceutically acceptable excipients. Herein, "pharmaceutically acceptable excipients" refer to carriers, diluents and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient, including but not limited to: antibiotics, humectants, pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, flavoring agents, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers. In some embodiments, the pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients in combination with the pharmaceutically effective compound. More specifically, suitable pharmaceutically acceptable excipients may be those commonly used in the art for diabetic feet. In one or more embodiments, suitable pharmaceutically acceptable excipients suitable for use in sprays are selected from one or more of the following: water, gluconolactone, sodium benzoate, arbutin, sodium hyaluronate, nicotinamide and glycerol. In one or more embodiments, suitable pharmaceutically acceptable excipients suitable for use in the coating or dressing are selected from one or more of the following: water, glycerin, panthenol, magnesium ascorbyl phosphate, nicotinamide, sodium hyaluronate, phenoxyethanol, caprylyl glycol and sorbic acid. The content of pharmaceutically acceptable auxiliary materials can be determined according to actual conditions in the field.

Dressing is generally required for application of the medicament to the wound. A "dressing" is an article for dressing a wound to cover a sore, wound, or other damaged material. The types of wound dressings are passive, interactive and bioactive. Wound dressings suitable for use in the present invention are known in the art.

The pharmaceutical compositions described herein may contain, in addition to amniotic fluid and/or extracts thereof, other active ingredients that aid in wound healing in diabetic patients, including, but not limited to, centella asiatica extract, rose hydrosol, licorice root extract, olive leaf extract, calendula officinalis extract, willow bark extract, lavender extract, lemon fruit extract, hydrolyzed soy protein, mugwort leaf extract, tea leaf extract, thyme extract, echinacea purpurea extract, hypericum perforatum/leaf extract, aloe vera leaf powder, yeast extract.

The dosage and frequency of administration can be determined by the health care provider according to the particular condition, age and sex of the patient, etc. Generally, for the treatment of a particular disease, a therapeutically effective amount refers to an amount sufficient to ameliorate or in some way reduce the symptoms associated with the disease. Such amounts may be administered as a single dose or may be administered according to an effective treatment regimen. The amount administered may be sufficient to cure the disease, but is generally administered to ameliorate the symptoms of the disease. Repeated administration is generally required to achieve the desired improvement in symptoms. For example, for a dose administered to a human, it can be generally administered in the range of 1-200 ml/time, and can be administered by daily or weekly injections. In certain embodiments, the frequency of administration may be multiple times daily, twice daily, every two days, every three days, every four days, every five days, or every six days, or once every half month, or once monthly.

Also provided herein is a pharmaceutical composition comprising amniotic fluid and/or an extract thereof as described herein, particularly amniotic fluid and/or an extract thereof from an egg of poultry, more preferably an egg having an embryo age of 5-12 days, more preferably 6-11 days, more preferably 6-9 days, more preferably 7-8 days. The pharmaceutical composition may be a lyophilized liquid for cryopreservation of amniotic fluid and/or extract thereof or lyophilized reagent thereof, e.g. lyophilized amniotic fluid and/or extract thereof, at a temperature below-60 ℃. The pharmaceutical composition may further comprise other pharmaceutically acceptable carriers or excipients, such as physiological saline for injection, water for injection, or glucose injection. Preferably, the pharmaceutical composition comprises 5-40% (v/v) or 10% -35% amniotic fluid and/or extract thereof, preferably 15-30%.

In certain embodiments, also provided herein is a cell culture medium comprising an amount of amniotic fluid and/or an extract thereof as described herein. The content of the amniotic fluid and/or the extract thereof in the cell culture medium can be determined according to the type of cells to be cultured, for example, the addition amount of the amniotic fluid or the extract can be 0.1-30%, such as 1-25% or 3-20% by weight of the cell culture medium. Suitable cell culture media 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.

The present invention will be illustrated below by way of specific examples. It is to be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods, reagents and apparatus used in the examples are, unless otherwise indicated, conventional in the art.

Examples

Example 1: materials and methods

1. Material

a) Instrument and tool

Microcomputer full-automatic incubator (Zhengda)^TMZF880), clean culture dish, 1.0ml syringe (Jiangxi Honda)^TM) Tweezers sterilized by 70% alcohol, stainless steel sieve, sterile centrifuge tube: (

# SCT-50ML-25-S) and a low-speed refrigerated centrifuge (medium-preferred KDC-2046).

b) Reagent and biomaterial

Eggs aged 7 days old.

2. Experimental procedure

The egg is taken and the relatively flat blunt end placed upwards is knocked to break the eggshell, the eggshell is peeled to form an opening with the diameter of about 2 cm, and the edge is required to be as flat as possible. The shell membrane and yolk membrane were carefully torn apart with forceps, taking care not to break the amnion. And observing the development condition of the embryo, wherein only the embryo which is well developed and meets the standard of the corresponding stage can be used for extracting the amniotic fluid.

Pouring the amnion and the connected tissue wrapped with the embryo into a culture dish from the shell, puncturing the amnion by using an injector to extract amniotic fluid, enabling the bevel of the needle opening to face back to the embryo until the amnion is tightly attached to the embryo, and then injecting the clear, colorless and foreign-matter-free amniotic fluid into a centrifuge tube in an ice box.

Taking out embryo from amnion with tweezers, collecting in stainless steel sieve placed on ice, homogenizing every hour, packaging in sterile plastic storage tank, and placing in refrigerator at-80 deg.C. The frozen food can be placed vertically.

Can be used for treating diseases by beautifying the spectrum^TMThe collected amniotic fluid extract is tested by 1800 ultraviolet spectrophotometer, and the standard operation process of the spectrophotometer is shown in the instruction manual.

The centrifugal tube for collecting the amniotic fluid extract is used after being balanced^TMKDC-2046 low-speed freezing centrifuge at 5 deg.C, 3500rpmHeart 20 minutes (see manual for standard centrifuge operating procedures). The supernatant was decanted and transferred to a clean plastic storage tank and stored in a-80 ℃ refrigerator. A small 5ml sample was reserved per batch for subsequent testing.

All steps are performed under sterile conditions.

Example 2: component detection

In this example, amniotic fluid components of eggs of different embryo ages were detected by Hitachi Primaide-type HPLC. Detection was performed according to the instruction of the chromatograph. Wherein, before the detection, 100% acetonitrile is used for washing for 30 minutes, the flow rate time is 0.8ml/min, and then water is used for balancing for 30 minutes, and the flow rate is 0.8 ml/min. Extracting 25 mul of sample and removing air bubbles, clicking a 'data acquisition' button of software carried by a chromatograph, selecting 'method 2', clicking 'single analysis start' below a screen, starting to inject the sample when a 'waiting for sample injection' appears in the system, wherein the injection is rapid, and switching valves after the injection is finished. The method 2 comprises the following steps:

time (min)	Water (%)	Acetonitrile (%)	Flow (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

Amniotic fluid with embryo ages of 7 days, 11 days and 13 days was examined in this example, and the results are shown in FIGS. 1 to 3.

Example 3: detection of anti-free radical capability

DPPH, a1, 1-diphenyl-2-picrylhydrazino radical, has the following structure:

DPPH is present in moleculesMultiple electron-withdrawing-NO₂And a large pi bond of a benzene ring, so that a nitrogen radical can exist stably.

When DPPH radicals are eliminated, the absorbance A at 519nm, the absorption maximum, decreases. DPPH, a stable free radical, provides an ideal and simple pharmacological model for the detection of free radical scavenging activity. This example uses DPPH to test anti-free radical ability from chick embryo amniotic fluid.

0.8mg of DPPH is taken and dissolved in 20ml of solvent methanol, ultrasonic treatment is carried out for 5min, and the mixture is fully shaken to ensure that the upper part and the lower part are uniform. Taking 1ml of the DPPH solution, measuring A at 519nm₀The value of a is 0.5 to 0.7. The DPPH solution was stored in the dark and used up within 3.5 hours.

Amniotic fluid of eggs having embryo ages of 6 days, 7 days, 8 days, 9 days, 10 days and 11 days was obtained by the method described in example one, and the eggs were stored in a refrigerator at 4 ℃ after centrifugation.

The standard curve was measured with vitamin C as a positive control. Taking Vc samples of 0.04mg/ml with different volumes, adding 0.6ml of DPPH, adding absolute ethyl alcohol to complement to 1ml, mixing uniformly, adjusting zero by taking methyl alcohol as a contrast, and measuring the light absorption value under the 519nm wavelength. Data were plotted after triplicate.

Adding 400 μ l of amniotic fluid with different embryo ages into a test tube, adding 600 μ l of prepared DPPH methanol solution, mixing, reacting for 10min to prevent bubbles from generating (mixing before measurement). The absorbance at 519nm was measured by zeroing with methanol as a control.

The loading information for each group is shown in the following table:

experimental group	Sample liquid	95% ethanol (or none)Water ethanol	DPPH test solution	Total volume
					Blank group	0ml	0.4ml	0.6ml	1ml
Vc	nμl	(400-n)μl	0.6ml	1ml
					Sample set	0.4ml	0ml	0.6ml	1ml

Clearance (inhibition) was calculated using the following formula:

clearance (%) ═ a₀-A)/A₀×100％。

The results are shown in FIG. 4.

Example 4: effect on chicken embryo fibroblasts

This example tests the effect of the chicken egg amniotic fluid (EE) of example 1 on the growth of chicken embryo fibroblasts in different culture conditions. The composition of the DMEM medium used in this example was as follows:

# Cat.11960077, 1% L-Glutamine (b)

# G0200) and 5% FBS (FBS: (FBS) ((R))

# Cat.10099141)), 0.25% pancreatin-EDTA (Hangzhou Biopsis of Hangzhou Ke^TM#CY003)，PBS(BI^TM#02-024-1ACS), 0.4% Trypan blue dye (BBI)^TM#72-57-1)。

Taking the embryos of eggs with embryo age of 7 days, washing the surface of the embryos by PBS, and completely sucking the liquid by a liquid transfer gun. The embryonic viscera were removed and the remaining tissue was minced to no macroscopic large particles, clumps. 1ml of 0.25% pancreatin-EDTA was added, the mixture was mixed with the tissue by means of a pipette tip, and the suspension was pipetted into a 15ml centrifuge tube. The petri dish was rinsed with 1ml of 0.25% pancreatin-EDTA and the suspension was pipetted into the same 15ml centrifuge tube. The centrifuge tube was placed in a 37 ℃ water bath, digested for 5-7 minutes, and then neutralized with pancreatin-EDTA by adding 8ml of DMEM medium (containing PBS). And (5) placing the centrifugal tube into a centrifugal machine, and centrifuging for 5-10 seconds. Taking out the centrifuge tube and collecting the supernatant. The centrifuged supernatant was centrifuged at 2000rpm for 2 min. The supernatant was discarded, 4ml of DMEM medium was added, and the cells were resuspended with a pipette tip. 1ml of the cell suspension was injected into a10 cm cell culture dish, and 10ml of DMEM medium was added thereto. The culture dish was shaken in the cross direction at least 20 times per direction to distribute the cells evenly. At 37 ℃ with 5% CO₂Culturing under the condition. Cells were passaged when they covered 70% -90% of the bottom of the dish.

The petri dish was taken out of the incubator and the original culture medium was collected in a centrifuge tube. Cells were washed carefully with 5ml of PBS. Thereafter, 500. mu.l of 0.25% pancreatin-EDTA was added, and the plate was placed in an incubator and digested for 1 minute. Gently patting the side of the culture dish to accelerate the digestion process, and quickly adding 9.5ml of recovered original culture medium to neutralize pancreatin-EDTA when most cells are in a floating state after the cell mass is quickly decomposed. The bottom of the dish was pipetted and as much cell suspension as possible was collected in a 15ml centrifuge tube and centrifuged at 2000rpm for 3 min. The supernatant was discarded, 4ml of DMEM medium was added, and the cells were resuspended with a pipette tip. 1ml of each cell suspension was poured into 10cm cell culture dishes containing 10ml of fresh medium containing amniotic fluid in different volume ratios. Cross direction shaking culture dishAt least 20 times per direction to distribute the cells evenly at 37 deg.C and 5% CO₂Culturing under the condition.

Collecting well-grown chicken embryo fibroblasts, and collecting the original culture medium in a centrifuge tube. The cells were washed carefully with 5ml PBS, taking care that no damage could be caused to the cell layer, and after gentle shaking the PBS was removed. Mu.l of 0.25% pancreatin-EDTA was added for digestion for 2-5 minutes (24 well plates) and neutralized with 100. mu.l of medium. The tip was used to make a single cell suspension. Diluting the single cell suspension according to a certain multiple, adding an equal amount of 0.4% trypan blue staining solution for staining, and preferably, the dilution multiple is that the number of the diluted cells is between 20 and 200. Appropriate amount (15. mu.l) of cell suspension was aspirated, loaded onto a hemocytometer from the upper and lower edges of the cover glass, and the viable cell count was counted under a microscope. Calculating the total number of living cells, and adjusting the cell concentration to 1 × 10⁵Individual cells/ml. Sampling is carried out once every 24 hours, 3 pore cells are taken each time, conventional pancreatin-EDTA digestion is carried out, single cell suspension is prepared, and counting is carried out by a microscope. The growth curve was plotted with time (days) as the horizontal axis and cell concentration as the vertical axis. Cell count (total cell count/4 × 10)⁴X dilution factor, cell concentration ═ number of cells/ml.

The results are shown in FIG. 5. FIG. 5 shows that the number of chicken embryo fibroblasts in the experimental group to which EE was added was significantly higher than that of the control to which EE was not added, after 96 hours of co-incubation.

Example 5: viability and migration Capacity of cells in amniotic fluid extract

Amniotic fluid of 8-day embryo-aged duck eggs was obtained in the same manner as in example 1. The effect of the egg amniotic fluid on the growth activity and migration capability of chicken embryo fibroblasts and duck egg amniotic fluid on Human Umbilical Vein Endothelial Cells (HUVEC) is tested by a scratch experiment. Duck egg amniotic fluid was obtained from 8 day old duck eggs using the method of example one. Chicken embryo fibroblasts were obtained by the method described in example five, and human umbilical vein endothelial cells were obtained from a commercially available source.

The composition of the DMEM medium used in this example was as follows:

# Cat.11960077, 1% L-Glutamine (b)

# G0200) and 5% FBS (FBS: (FBS) ((R))

# Cat.10099141)), 0.25% pancreatin-EDTA (Hangzhou Biopsis of Hangzhou Ke^TM#CY003)，PBS(BI^TM#02-024-1ACS), 0.4% Trypan blue dye (BBI)^TM#72-57-1)。

On the first day before the experiment, preparing a 6-hole plate, drawing 5-6 uniformly distributed transverse lines on the back of the 6-hole plate by using a mark pen and using a ruler to transversely penetrate through the holes; then, a vertical line is drawn at the center line position to indicate the position of the scratch. About 5X 10 additions to each well⁵In principle, the fusion rate reached 90% after overnight inoculation of several cells in logarithmic growth phase.

On the day of the experiment, the straight ruler is compared with the gun head, and the line is drawn along the vertical line of the Mark pen and vertical to the bottom surface of the 6-hole plate. The inclination and bending are avoided as much as possible, the same branch tip is preferably used between different holes, and the width is preferably 1000-. Each well was washed 3 times with 2ml PBS and the cells at the scratch were washed away. 2ml of culture medium containing different amounts of EE was added to each well, and the culture was performed conventionally, with the medium changed every 48 hours. The time from scratch was 0h, and the distance between cells on both sides of the scratch was measured by taking a photograph every 24 hours at a fixed point. Observing the growth of the cells in each well; plotting the time (days) as the horizontal axis and the scratch distance in each hole as the vertical axis; the rate of healing of the scratch in each well was calculated.

The results are shown in FIGS. 6 and 7. FIG. 6 shows the effect of amniotic fluid from chicken eggs on the growth viability and migration ability of Human Umbilical Vein Endothelial Cells (HUVEC), and the addition of 5% (by volume) of amniotic fluid clearly has a very clear promoting effect on the healing of HUVEC. FIG. 7 shows the effect of amniotic fluid from duck eggs on the growth viability and migration ability of chicken embryo fibroblasts, and the addition of amniotic fluid also shows a very significant promoting effect on the healing of chicken embryo fibroblasts.

Example 6: effect on mammalian osteoblasts

Egg amniotic fluid (EE) with embryo age of 7 days was prepared as described in example 1 and used in this experiment.

Osteoblasts were isolated from adult mice and cultured in DMEM supplemented with 10% FBS at 37 deg.C and 5% CO₂Cultured in an incubator to the third generation (P3). 400 cells were seeded per well in 96-well plates. After 24 hours, the cells were cultured in DMEM-only medium (starvation) for 24 hours, and then in the following medium (EE was added in a volume of DMEM) for 72 hours, after which the growth of the cells was measured using CCK-8 kit:

1) DMEM (serum free);

2)DMEM+2.5％EE；

3)DMEM+5％EE；

4)DMEM+7.5％EE；

5)DMEM+10％EE。

the results are shown in FIG. 8. FIG. 8 shows that chick embryo amniotic fluid can significantly promote the growth of mouse osteoblasts.

Example 7: effect of amniotic fluid on cardiomyocytes.

1. Isolation of Primary cardiomyocytes (VM)

The ventricles of the suckling mice were washed with pre-cooled PBS, after which the heart tissue was minced in DMEM/F12. Shaking in water bath at 37 deg.C, and digesting with 0.04% collagenase II + 0.08% pancreatin. The digested cells were centrifuged through a screen at 1000rpm/min for 10 min. Plating with 15% FBS cell culture medium in 5% CO₂The saturated humidity is cultured in an incubator at 37 ℃.

2. Cell viability assay

Primary cardiomyocytes were digested and plated in 96-well plates at 6000/well, five replicates per set. At 5% CO₂After 24 hours of incubation in a 37 ℃ incubator at saturated humidity, DMEM/F12 with 15% FBS in the original medium was replaced with DMEM/F12, DMEM/F12 with 10% FBS, DMEM/F12 with 10% FBS and 5% EE, respectively. After 48 hours of incubation, 10. mu.l of CCK-8 reagent was added to each well. After 2 hours of incubation, absorbance was measured at 450nm in a microplate reader.

The results are shown in FIG. 9.

Examples 8 to 10: purification of active Compounds in amniotic fluid

The purpose of this example is to purify stepwise bioactive compounds from chick embryo amniotic fluid by Sephacryl S-200 column, HiPrep Q anion exchange column, HiPrep 26/10 desaling column, HiLoad 16/600Superdex75pg column.

1. Material

1.1 purification of samples: fresh eggs aged 7 days were amniotic fluid, 50 ml.

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 desaling;

5) gel column GEHiLoad 16/600Superdex75 pg;

6)Superloop 10ml。

2. method of producing a composite material

2.1 preparation of the solution

Sodium phosphate buffer A (50mM Na)₂HPO₄+NaH₂PO₄pH 8.0): 46.6ml of 1M Na₂HPO₄With 3.4ml of 1M NaH₂PO₄Mixing, adding ddH₂And O is metered to 1L.

2.2 Experimental methods

2.2.2 sample treatment: adding appropriate amount of hexane into fresh amniotic fluid 50ml, centrifuging at 2500rpm and 4 deg.C for 20min to obtain water phase, and filtering with 0.22 μm filter membrane.

2.2.3 sample purification

The first step is as follows: gel column GE Sephacryl S-200

ddH₂O-equilibrium gel column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;

loading: the flow rate is 1ml/min, and the sample loading amount is 10 ml;

and (3) elution: by degassing ddH₂The crude product was eluted with a flow rate of 2ml/min and the fractions were collected in equal volumes, 3 ml/tube. 2 column volumes (240ml) elute;

repeating the separation and purification for 5 times, and fully mixing the parts with the same peak-off time in each time;

the second step is that: anion exchange column GE HiPrep Q

Sodium phosphate buffer A (50mM Na)₂HPO₄+NaH₂PO₄pH 8.0) equilibrium anion exchange column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;

loading: taking the part with biological activity after the first step of purification, using a pump to load the sample with the flow rate of 1.5ml/min and the sample loading amount of 250ml, and simultaneously collecting the non-binding part of the anion column with the same volume, 2 ml/tube;

desalting: the bound and unbound fractions from the ion column were separately replaced with GE HiPrep 26/10 desaling to degassed ddH₂Collecting desalted part in O;

the third step: gel column GE HiLoad 16/600Superdex75pg

ddH₂O-equilibrium gel column: the flow rate is 1ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;

loading: the flow rate is 1ml/min, and the sample loading amount is 10 ml;

and (3) elution: by degassing ddH₂The sample was eluted with a flow rate of 1ml/min and fractions were collected in equal volumes, 2 ml/tube. Elute 1.5 column volumes (240 ml);

and (3) measuring the cell activity: better growing AC16 was digested and plated in 96-well plates at 8000 wells, five replicates per group. At 5% CO₂The cells are cultured in an incubator with the saturation humidity of 37 ℃ for 2 hours and adhere to the wall. After 24 hours of starvation culture with the medium DMEM, DMEM with 10% FBS, DMEM and a medium containing 20% of the fraction were replaced. After 24 hours of incubation, 10. mu.l of CCK-8 reagent was added per well. After 2 hours of incubation, absorbance was measured at 450nm in a microplate reader.

3. Results of the experiment

The chromatogram of the unbound fraction separated by gel column GE HiLoad 16/600Superdex75pg is shown in FIG. 10. Cell viability assays followed groups of biologically active growth factors, the results are shown in figure 11.

Example 9

The following separation and purification were carried out in the same manner as in example 8:

1. separating and purifying active ingredients

The first step is as follows: gel column GE Sephacryl S-200

ddH₂O-equilibrium gel column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;

loading: the flow rate is 1ml/min, and the sample loading amount is 10 ml;

and (3) elution: by degassing ddH₂Eluting the crude product with O at a flow rate of 2ml/min, and collecting fractions with molecular weights in the range of 500-;

repeating the separation and purification for 5 times, and fully mixing the parts with the same peak-off time in each time;

the second step is that: cation exchange column GE HiPrep SP

Sodium phosphate buffer A (50mM Na)₂HPO₄+NaH₂PO₄pH 5.8) equilibrium cation exchange column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;

loading: taking the fraction with the molecular weight within the range of 150-;

the third step: gel column GE HiLoad 16/600Superdex75pg

ddH₂O-equilibrium gel column: the flow rate is 1ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;

loading: sampling the non-combined part obtained in the second step, wherein the flow rate is 1ml/min, and the sampling amount is 10 ml;

and (3) elution: by degassing ddH₂The sample was eluted with a flow rate of 1ml/min and fractions with molecular weights in the range of 500-.

2. Active ingredient detection

Better growing AC16 was digested and plated in 96-well plates at 8000 wells, five replicates per group. At 5% CO₂The cells are cultured in an incubator with the saturation humidity of 37 ℃ for 2 hours and adhere to the wall. After 24 hours of starvation culture with the medium DMEM, DMEM with 10% FBS, DMEM and a medium containing 20% of the fraction were replaced. After 24 hours of incubation, 10. mu.l of CCK-8 reagent was added per well. After 2 hours of incubation, inThe microplate reader detects the absorption value at 450 nm. Cell viability of the unbound regions after cation exchange column GE HiPrep SP treatment is shown in figure 12.

Example 10

The following separation and purification were carried out in the same manner as in example 8:

1. separating and purifying active ingredients

The first step is as follows: an ion exchange column, namely an anion exchange column HiPrep Q can be used, the pH of each solution is respectively 5.8 and 8.0, then the solutions are respectively loaded on the ion exchange column, the flow rate is 2ml/min, until the ultraviolet absorption curve of 280nm is stable, and the base line is returned;

loading: sampling amniotic fluid with a pump at a flow rate of 1.5ml/min and a sample volume of 50ml, and collecting unbound fraction from the ion column;

the second step is that: gel column GE Sephacryl S-200

ddH₂O-equilibrium gel column: the flow rate is 2ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;

loading: the sample is the unbound fraction of the first step, the flow rate is 1ml/min, and the sample loading amount is 10 ml;

and (3) elution: by degassing ddH₂Eluting the crude product with O at a flow rate of 2ml/min, and collecting fractions with molecular weights in the range of 150-;

the third step: gel column GEHiLoad 16/600Superdex75pg

ddH₂O-equilibrium gel column: the flow rate is 1ml/min until the ultraviolet absorption curve of 280nm is stable, and the baseline is returned;

loading: sampling the fraction obtained in the second step within the range of 150-2000 daltons at a flow rate of 1ml/min and a sample loading amount of 10 ml;

and (3) elution: by degassing ddH₂The sample was eluted with a flow rate of 1ml/min and fractions with molecular weights in the range of 150-.

2. Active ingredient detection

Better growing AC16 was digested and plated in 96-well plates at 8000 wells, five replicates per group. At 5% CO₂The cells are cultured in an incubator with the saturation humidity of 37 ℃ for 2 hours and adhere to the wall. DME replaced with 10% FBS after 24 hours of starvation culture with DMEM mediumM, DMEM and a medium containing 20% of the distillate. After 24 hours of incubation, 10. mu.l of CCK-8 reagent was added per well. After 2 hours of incubation, absorbance was measured at 450nm in a microplate reader. Cell viability of the unbound regions after anion exchange column GE HiPrep Q treatment is shown in fig. 12.

Example 11: therapeutic effect of amniotic fluid on diabetic foot

1. Material

Biological material: c57BL6 mice (Male, raised at 6-8 weeks old with high fat, experimental 11-13 weeks old)

Feed: mouse feed with 36% fat content

Reagents and drugs: 75% ethanol, isoflurane

Anhydrous citric acid (c)

A100529-0250), trisodium citrate dihydrate (

A100101-0500), streptozotocin (STZ,

) Sodium hyaluronate (HA,

1706221), egg amniotic fluid (EE) of example 1, 5% chloral hydrate (E: (E)

A600288-0250), cefquinome (f)

50ml，1.25g)

Instruments and tools: glucometer and test paper (

Onetouch), stereoscopic microscope (

SMZ168), constant temperature electric iron, ophthalmic scissors, tweezers, electric shaver (C)

HC1055), mouse holder, 1ml syringe

100 mul pipette

Anesthesia apparatus (

ABM type)

2. Method of producing a composite material

2.1 model building of diabetes mellitus

6 weeks

Healthy 6-8 week old male mice were fed in 3 cages to provide a sufficient amount of high calorie feed with 36% fat content and secondary water for voluntary feeding. Controlling the room temperature between 22-24 deg.C, following natural rhythm (light simulating natural illumination when natural illumination is poor), replacing wood chip padding every week, and continuously raising for 6 weeks.

16 hours at

16 hours prior to STZ injection, all drinking water and feed were removed and replaced with clean bedding, one cage per mouse and numbered individually.

Day 0

The weight of each mouse was weighed and recorded. Cutting the tail to collect blood, measuring fasting blood glucose with a glucometer, timely compressing to stop bleeding, and wiping off residual blood stain with a medical cotton ball.

Using a 0.1M citric acid (MW 192) mother liquor and a 0.1M trisodium citrate dihydrate (MW 294) mother liquor in a ratio of 1.3: a0.1 mM citric acid buffer solution having a pH of 4.4 to 4.5 was prepared at a volume ratio of 1 ℃ and stored at 4 ℃.

The STZ solution is weighed, prepared into 20mg/ml STZ solution by using citric acid buffer solution and used within 15 minutes, and the STZ solution is placed on ice in a dark place during use.

The STZ solution was injected intraperitoneally at a dose of 140mg/kg into # S01- # S26 mice. The control groups # C01- # C05 were injected with 0.20ml of citric acid buffer solution.

One hour after STZ injection, sufficient sterile water and 36% fat-rich diet were given to keep the bedding dry. After injection, D4, D8 and D11 (scald D0) are fasted for 16 hours before water deprivation, and then fasting blood sugar and body weight of the mice are measured. The fasting time and the measurement time were the same for each time. Those with fasting plasma glucose lasting greater than 11.1mM were considered to be a model of diabetes for further experiments.

2.2 Scald experiment

Groups (shown in the following table) are set according to the modeling success rate and the types of the dressings to be tested, the control group is a mouse without diabetes, and the experimental group is a mouse which meets the blood sugar standard of diabetes after being induced by STZ. All mice had the same growth stage and feeding conditions. Scald wounds with the same area and the same degree were made in each mouse and tested with different dressings.

Control group: diabetic-free mice without dressing

Experimental groups: diabetic mice without dressing, diabetic mice coated HE dressing (0.8% HA, 20% EE, balance water)

Grouping and numbering of laboratory mice

Group of	Numbering
		Non-diabetic mouse, no dressing (Control)	C 1-7
Diabetic mouse, no Dressing (DM)	DM 1-9
		Diabetic mouse, HE (DM _ HE)	DM_HE 1-9

Clean cages were prepared and mice were individually weighed and placed individually in the cages, this time D0.

Mice were injected with 5% chloral hydrate (0.007ml/g) intraperitoneally, and after the righting reflex disappeared, their bodies were fixed on a surgical cloth, and back hair was shaved with an electric shaver.

The temperature-controlled electric welding pen is heated to 150 ℃, and stably contacts the skin of the middle back of the mouse for 60 seconds after being kept at a constant temperature, so that a round scald wound is manufactured. After scald, infection is prevented by injecting cefquinome injection suspension (2.5mg/kg) into thigh muscles.

And (4) taking a wound picture by using a stereoscopic microscope at the same magnification, measuring the area of the burn, and recording data. The burnt part should be parallel to the lens as much as possible, so that the measurement error is reduced.

The dressing is dripped on the wound of the corresponding mouse by a medical cotton swab, and the wound is covered by the area of the dressing. The auxiliary materials are dripped once a day in the morning and at night.

The medical cotton is used for replacing wood chips in the feeding boxes, and one box is used for feeding alone, so that the mice are prevented from scratching wounds. Sufficient sterilized water and 36% fat-content high-fat feed are given, medical cotton is changed every 2-3 days, and padding is kept clean. D2, D4, D7, D9, D14, D17, D20 examined and measured the wounds and recorded photographs and data.

Differences in wound healing were observed in normal groups of mice, and the effects of the dressing on wound healing rate and effect were calculated and compared. Data from Graph Pad Prism

And (6) processing.

3. Results

3.1 model modeling of diabetes

After STZ induction, mice whose blood glucose continued to meet the molding standard within 10 days were used as scald experiments. In this experiment, the total number of the molds was 35, and fasting blood glucose was continuously observed on days 4 and 10 after the molding. After scald, D7, D14 and D20 continuously monitor blood sugar, and all meet the setting requirements of the experimental group.

3.2 Scald experiment

3.2.1 body weight

In the experimental process, especially after scalding, diabetic mice have the manifestations of poor states in different degrees, mainly manifested as slow movement and partial death.

Average body weights of D0-D20 groups

Body weight (g)	D0	D2	D4	D7	D9	D14	D17	D20
									Control	28.6	27.6	27.8	27.8	28.1	28.8	28.6	29.0
DM	23.2	21.7	21.3	21.3	21.0	21.3	21.1	20.9
									DM_HE	22.5	20.0	20.2	23.0	22.2	21.4	20.8	21.3

The weight of the diabetes model mouse caused by drug-induced modeling is obviously lower than that of a healthy mouse in a positive reference group, and the diabetes model mouse has no obvious difference among all groups and belongs to a normal condition. The body weight of each group of mice decreased slightly after scald (D0) and was relatively stable, as expected, as shown in fig. 13.

2.2 wound healing Rate

The healing rate (epsilon) of wounds of each group D0-D20 after scald was calculated by the following formula,

the average values of the wound healing rates for the groups D0-D20 are shown in the following table:

healing (%)

D0

D2

D4

D7

D9

D14

D17

D20

Control

0.00％

-49.35％

-98.14％

-60.08％

-29.60％

57.92％

82.41％

94.20％

DM

0.00％

-29.50％

-86.94％

-67.40％

-63.80％

-7.70％

22.58％

55.30％

DM_HE

0.00％

-31.72％

-46.66％

-41.09％

-23.93％

29.85％

49.54％

94.89％

As shown in FIG. 14, following scald, D0-D4, each group had a different degree of enlargement of the scald wound, manifested as redness, blisters, and ulcers; D4-D9, the wound gradually starts to heal, and the healing rate is slower; after D9, the healing rate was highest in the control group, better than in the DM _ HE group, and lowest in the DM group; the posterior control and DM _ HE groups healed closer, reaching 94.20% and 94.89% at D20, respectively.

Comparing two groups of Control and DM in the wound healing rates of the groups to prove that the wound healing capacity of the diabetic mice is lower than that of healthy mice; DM _ HE compared with DM, HE has the function of promoting wound healing of diabetic mice. The HE dressing can effectively reduce the expansion range of the wound at the early stage of scald.

EXAMPLE 12 therapeutic Effect of composite dressings on diabetic foot

1. Material

Biological materials and feeds: as described in example 11.

Reagents and drugs: 75% ethanol, isoflurane

Anhydrous citric acid (c)

A100529-0250), trisodium citrate dihydrate (

A100101-0500), streptozotocin (STZ,

) Sodium hyaluronate (HA,

1706221), 5% chloral hydrate (C: (A)

A600288-0250), composite formula dressings (DWS, containing 15% egg amniotic fluid (EE) of example 1, plant extracts including licorice root extract, olive leaf extract, centella asiatica extract, and calendula extract, and coating adjuvants).

Instruments and tools: as described in example 11.

2. Method of producing a composite material

2.1 modeling of diabetes mellitus model as described in example 11.

2.2 Scald experiment

Groups (shown in the following table) are set according to the modeling success rate and the types of the dressings to be tested, the control group is 5 healthy mice which are not injected with STZ, and the experimental group is mice which meet the blood sugar standard of diabetes after the STZ is induced. All mice had the same growth stage and feeding conditions. Scald wounds with the same area and the same degree were made in each mouse and tested with different dressings.

Positive control group: non-diabetic mice, no dressing (control, 1-5)

Experimental groups: diabetic mice no dressing (DM,1-8), diabetic mice coated with DWS dressing (DM _ DWS,1-8), diabetic mice coated with hyaluronic acid (0.8% HA-PBS) for treatment (DM _ HA-PBS,1-6)

Grouping and numbering of laboratory mice

Clean cages were prepared and diabetic mice were weighed separately and placed individually in the cages.

Mice were injected intraperitoneally with 5% chloral hydrate (0.007ml/g), and after the righting reflex disappeared, their bodies were fixed with a mouse dissecting plate and the back hair was shaved with an electric shaver.

The temperature-controlled electric welding pen is heated to 150 ℃, and stably contacts the skin of the middle back of the mouse for 60 seconds after being kept at a constant temperature, so that a round scald wound is manufactured.

And (4) taking a wound picture by using a stereoscopic microscope at the same magnification, measuring the area of the burn, and recording data. The burnt part should be parallel to the lens as much as possible, so that the measurement error is reduced.

And (3) coating the dressing qualified in quality inspection on the corresponding mouse wound by using a medical cotton swab, wherein the wound is covered by the area of the dressing. The dressing is applied once a day, morning and evening.

Medical cotton is used for replacing sawdust in the feeding box, and the mice are fed independently, so that the mice are prevented from being scratched and injured. Thereafter D2, D4, D7, D10, D14, D17, D20 examined and measured the wounds and recorded photographs and data.

Mice were periodically starved and monitored for fasting blood glucose levels.

Differences in wound healing between normal and diabetic mice were observed and the effects of DWS dressings on wound healing rate and effect were calculated and compared.

3. Results

3.1 model modeling of diabetes

Mice with blood glucose that consistently met the molding criteria for at least 10 days after STZ induction were used as scald trials. In the experiment, the total number of the molded products is 23, the fasting blood glucose is continuously observed after the molded products are D4, D8 and D11 (scald D0), the number of the standard-reaching samples is 22, and the molding success rate is 95.7%. After scald, D4, D10, D14 and D21 continuously monitor fasting blood glucose, and all meet the setting requirements of the experimental group.

3.2 Scald experiment

3.2.1 survival Rate

During the experiment, especially after scalding, each group of DM mice showed a more or less poor performance, with individual samples dying, as shown in the table below. The DM _ HA-PBS group had a sample size of only 2 at D10, and thus the subsequent results were not included.

Survival rates of groups by day 20 post-scald (D20)

As shown in the above table, the survival rate of the control group was up to 100%. DM _ DWS had significantly higher survival rates than the DM group and the DM _ HA-PBS group, and consistent with the sample status observed during the experiment, the DM _ DWS status was significantly better than the DM group.

3.2.2 wound healing Rate

The healing rate (epsilon) of wounds of each group D0-D20 after scald was calculated by the following formula,

the average values of the wound healing rates for the groups D0-D20 are shown in the following table:

D0

D2

D4

D7

D10

D14

D17

D20

control

0

-34.18％

-88.52％

-39.34％

10.05％

79.65％

96.50％

99.90％

DM

0

-61.38％

-78.32％

-46.86％

0.77％

21.96％

31.25％

54.37％

DM_DWS

0

-35.00％

-69.89％

-40.26％

-25.09％

33.40％

65.89％

74.48％

As shown in fig. 15, following scald, D0-D4, the scald wounds in each group had different degrees of enlargement, manifested as red swelling, blisters and ulcers; D4-D10, the wound healed gradually, and the healing rate of the control group has no significant difference from the DM group and the DM _ DWS group; after D10, the healing rate was better in the DM and DM _ DWS groups than in the D20 by 99.90%, 54.37% and 74.48%, respectively.

In conclusion, the DWS has a sedative effect on scald wounds of the mice by combining observation of the states of the mice and the survival rates of all groups of samples in the experimental process, and can relieve pain caused by scald to a certain extent; comparing the control group and the DM group in the wound healing rate of each group to prove that the wound healing capacity of the diabetic mice is lower than that of healthy mice; comparing the two groups, DM _ DWS and DM, DWS has the function of promoting the wound healing of diabetic mice.

Claims (10)

1. Use of amniotic fluid and/or an extract thereof for the preparation of a reagent for promoting cell growth, tissue repair and/or wound healing in a diabetic subject,

wherein the amniotic fluid is derived from an egg with an embryo age of 5-12 days, preferably an egg with an embryo age of 6-11 days, more preferably an egg with an embryo age of 7-9 days, more preferably an egg with an embryo age of 7-8 days, or an egg from a bird other than a chicken whose development period corresponds to the development period in which the embryo-aged egg is present; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

2. Application of amniotic fluid and/or its extract in preparing medicine for treating nervous ulcer, vascular ulcer or neuro-vascular ulcer caused by neuropathy and/or vasculopathy,

wherein the amniotic fluid is derived from an egg with an embryo age of 5-12 days, preferably an egg with an embryo age of 6-11 days, more preferably an egg with an embryo age of 7-9 days, more preferably an egg with an embryo age of 7-8 days, or an egg from a bird other than a chicken whose development period corresponds to the development period in which the embryo-aged egg is present; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

3. Use of amniotic fluid and/or an extract thereof for the manufacture of a medicament for the treatment of neuropathy or vasculopathy in a subject suffering from diabetes,

wherein the amniotic fluid is derived from an egg with an embryo age of 5-12 days, preferably an egg with an embryo age of 6-11 days, more preferably an egg with an embryo age of 7-9 days, more preferably an egg with an embryo age of 7-8 days, or an egg from a bird other than a chicken whose development period corresponds to the development period in which the embryo-aged egg is present; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

4. Use of amniotic fluid and/or an extract thereof for the manufacture of a medicament for restoring the skin perspiration regulating function, the skin temperature regulating function or the blood circulation regulating function of a diabetic subject,

wherein the amniotic fluid is derived from an egg with an embryo age of 5-12 days, preferably an egg with an embryo age of 6-11 days, more preferably an egg with an embryo age of 7-9 days, more preferably an egg with an embryo age of 7-8 days, or an egg from a bird other than a chicken whose development period corresponds to the development period in which the embryo-aged egg is present; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

5. The use of amniotic fluid and/or an extract thereof for the manufacture of a medicament for the treatment of diabetic foot or a condition associated with diabetic foot,

wherein the amniotic fluid is derived from an egg with an embryo age of 5-12 days, preferably an egg with an embryo age of 6-11 days, more preferably an egg with an embryo age of 7-9 days, more preferably an egg with an embryo age of 7-8 days, or an egg from a bird other than a chicken whose development period corresponds to the development period in which the embryo-aged egg is present; or embryos from rodents with gestational age of 8-14 days, or embryos from non-human mammals other than rodents with developmental stages corresponding to the developmental stages of rodents with gestational age of 8-14 days.

6. The use according to any one of claims 1 to 5, wherein the agent or medicament is a cell culture and/or embryonic stem cells comprising amniotic fluid and/or an extract thereof as described herein.

7. The use according to any one of claims 1 to 5, wherein the agent or medicament is a pharmaceutical composition comprising amniotic fluid and/or an extract thereof and/or chicken embryonic stem cells as described herein and a pharmaceutically acceptable excipient.

8. The use as claimed in any one of claims 1 to 5, wherein the major biologically active component of the extract is not bound to the ion exchange column at a pH of between 7.0 and 8.0 and comprises components having molecular weights in the range of 150 and 2000 daltons.

9. The use of claim 1, wherein the tissue is from: any one or more tissues selected from cartilage tissue, meniscus tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascia tissue, periosteal tissue, nerve tissue, urogenital tissue, and adipose tissue; the cells are from any one or more of the tissues.

10. The use of claim 5, wherein the condition associated with diabetic foot includes, but is not limited to, a long-term non-healing wound condition in a diabetic subject caused by disease or trauma selected from the group consisting of: dry skin; the hair is separated by hair; skin temperature is decreased; pigmentation; the pulsation of the extremity arteries is reduced or eliminated; torn or broken tendons or ligaments; skin wounds, such as scars, traumatic wounds, ischemic wounds, diabetic wounds, surgical wounds, scalds, burns, skin ulcers, such as decubitus ulcers or pressure-induced ulcers, venous ulcers, diabetic ulcers, gangrene, necrosis; vascular conditions, such as peripheral arterial disease, venous disease, vascular defects, and vascular dysplasia; muscle diseases, such as muscle atrophy, inflammatory, neurological and myogenic muscle diseases; neurological disorders such as stinging, burning, numbness, dysesthesia or loss; bone diseases such as, for example, resting pain, osteomyelitis, Charcot's arthropathy, toe deformity; infection, tissue edema, intermittent claudication, and chronic non-healing wounds caused by skin tumors.

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