Medicine for treating spinal cord injury and application thereof

Abstract

The present invention relates to methods and compositions for the treatment of spinal cord injury, to the use of amniotic fluid from non-human animals, in particular poultry and non-human mammals. The composition provided by the invention can be a cell culture medium containing the amniotic fluid and can also be a pharmaceutical composition.

Description

Technical Field

[0001] The invention relates to a medicine for treating spinal cord injury and application thereof. Background Art

[0002] Spinal cord injury (SCI) is a neurological disease that seriously affects the quality of life of patients. It often occurs after accidents such as car accidents and falls from heights, which damage the integrity of the spine and cause compression or contusion of the spinal cord. It often leads to sensory, motor and autonomic dysfunction, and in severe cases, it can lead to permanent disability or even death. According to statistics from the World Health Organization, about 200,000 to 250,000 people worldwide become disabled due to spinal cord injury each year. In China, the incidence of spinal cord injury has shown an upward trend in different genders and age groups. The high-incidence age of spinal cord injury varies in different regions. Generally speaking, the age of traumatic spinal cord injury is more common in the elderly (65-74 years old). Spinal cord injury brings heavy economic burden and psychological pressure to patients and their families. In-depth research on the pathophysiological mechanism of spinal cord injury is an important process in the treatment of spinal cord injury.

[0003] According to the different stages of injury, spinal cord injury can be divided into primary injury and secondary injury. Primary injury refers to the direct or indirect action of external force on the spinal cord, and the injury is mostly local and incomplete. The entire primary stage includes axonal injury, vascular rupture and cell membrane rupture. After early reactions such as bleeding and edema, the scope of injury gradually expands on the basis of primary injury, and secondary injury occurs, including free radical production, electrolyte changes, cell apoptosis, inflammatory response, etc. Secondary injury can be divided into acute, subacute and chronic stages. The injury gradually progresses at different stages, and early intervention treatment should be performed to improve the prognosis of spinal cord injury. In the past few decades, scientists have made a series of research progress on the repair of spinal cord injury, but there are still many unresolved issues. Therefore, it is of great significance to seek more effective treatment methods to promote the recovery of neurological function after spinal cord injury.

[0004] Chick early amniotic fluid (ceAF) is the amniotic fluid collected in the amniotic sac of chick embryos 5-12 days after the egg is fertilized. Amniotic fluid is the environment for embryonic growth. It provides fluid and nutrients for the embryo and provides necessary growth factors for normal development and growth. As the source of many key components, signaling molecules, cytokines and peptides, chick early amniotic fluid has been widely used in a variety of clinical medical studies, including skin wound healing, repair after myocardial infarction, and other organ repair and regeneration. Some studies have shown that chick early amniotic fluid can promote cell proliferation and angiogenesis by downregulating the hippo-yap signaling pathway. It can also inhibit cellular inflammatory responses by inactivating the TLR4 / NF-κB axis and activating the Nrf2 axis, and improve inflammation and oxidative stress responses by promoting M2 macrophage polarization. However, there are no studies on the role of chick early amniotic fluid in the functional recovery of spinal cord injury. Summary of the invention

[0005] The present invention first provides the use of amniotic fluid in preparing a medicine for treating and / or repairing complete or incomplete spinal cord injury and related diseases; wherein the amniotic fluid comes from: eggs with an embryonic age of 5-12 days; eggs of other birds other than chickens whose developmental period corresponds to the developmental period of the eggs with the embryonic age; embryos of rodents with a gestational age of 8-14 days; or embryos of other non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-14 days.

[0006] In one or more embodiments, the amniotic fluid is from eggs with an embryonic age of 6-11 days, preferably from eggs with an embryonic age of 7-9 days, more preferably from eggs with an embryonic age of 7-8 days, or from duck eggs with an embryonic age of 8-10 days, preferably 8-9 days.

[0007] In one or more embodiments, the medicament is a pharmaceutical composition comprising the amniotic fluid and a pharmaceutically acceptable excipient.

[0008] In one or more embodiments, the complete or incomplete spinal cord injury and its related disorders include damage to the structural integrity of the spine, damage to the neural structure and function within the spinal canal, and spinal cord dysfunction at and below the injury level.

[0009] In one or more embodiments, the complete or incomplete spinal cord injury and its related conditions also include paraplegia, flaccid paralysis, spastic paralysis, loss of motor sensation, loss of saddle sensation of the perineal skin, loss of sphincter function, urinary and bowel dysfunction, sexual dysfunction and one or more of complications.

[0010] In one or more embodiments, the complications include respiratory failure and respiratory tract infections caused by autonomic dysfunction due to spinal cord injury, infections and stones of the genitourinary tract, bedsores or pressure sores, and temperature disorders.

[0011] In one or more embodiments, the repair of complete or incomplete spinal cord injury and related disorders includes promoting nerve regeneration and nerve function recovery, inhibiting excessive immune response, repairing damaged cells, and promoting cell regeneration.

[0012] In one or more embodiments, the promoting nerve regeneration and nerve function recovery includes promoting recovery of motor function after injury, inhibiting the expansion of the injured area, and reducing motor neuron loss.

[0013] The first aspect of the present invention also provides the use of amniotic fluid in the preparation of a drug for promoting the recovery of motor function after spinal cord injury in patients, inhibiting the expansion of the injured area of ​​the spinal cord in patients, and / or reducing the loss of motor neurons around the injured area of ​​the spinal cord in patients; wherein the amniotic fluid comes from: eggs with an embryonic age of 5-12 days; eggs of other birds other than chickens whose developmental period corresponds to the developmental period of the eggs with the embryonic age; embryos of rodents with a gestational age of 8-14 days; or embryos of other non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-14 days.

[0014] In one or more embodiments, the amniotic fluid is from eggs with an embryonic age of 6-11 days, preferably from eggs with an embryonic age of 7-9 days, more preferably from eggs with an embryonic age of 7-8 days, or from duck eggs with an embryonic age of 8-10 days, preferably 8-9 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of ceAF promoting the recovery of motor function after spinal cord injury in mice. Figure 1 (A) is a typical image of the footprint experiment on day 28 after spinal cord injury. The red line is the length between the front and back footprints of the forepaws, and the black line is the width between the two forepaws. Figure 1 (B) is a statistical diagram of the length of mouse forepaw footprints (n=4). Figure 1 (C) is a statistical diagram of mouse front paw footprint width (n=4). Figure 1 (D) is the statistical diagram of the total BMS score of mice (n=4). Figure 1 (E) is a statistical diagram of the BMS score of the left foot of mice (n=4). Figure 1 (F) is a statistical diagram of the BMS score of the right foot of mice (n=4). Figure 1 (A) and Figure 1 The scale bar length of (B) is 0.5 cm. Values ​​are mean ± standard error. * indicates p < 0.05, ** indicates p < 0.01.

[0016] Figure 2 Schematic diagram of the expansion of spinal cord injury area in ceAF-inhibited mice. Figure 2 (A) is a typical picture of the HE staining results of the spinal cord 28 days after spinal cord injury. The dotted line indicates the injured area. Figure 2 (B) is a typical picture of the spinal cord Nissl staining results 28 days after spinal cord injury. The dotted line indicates the injured area. Figure 2 (C) is the statistical diagram of the damaged area by HE staining (n=4). Figure 2 (D) is a statistical diagram of the damaged area by Nissl staining (n=4). Figure 2 (A) and Figure 1 The scale bar length of (B) is 500 μm. Values ​​are mean ± standard error. * indicates p < 0.05, ** indicates p < 0.01.

[0017] Figure 3 Schematic diagram of how ceAF reduces the loss of peripheral motor neurons in the spinal cord after injury in mice. Figure 3 (A) is a typical image of NeuN fluorescence staining results of the spinal cord 28 days after spinal cord injury. The dotted line indicates the injured area. Figure 3 (B) is a statistical diagram of the density of NeuN+ cells around spinal cord injury stained with fluorescence (n=4). Figure 3 The scale bar in (A) is 200 μm. * indicates p < 0.01, ** indicates p < 0.001. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0019] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0020] Herein, “comprising”, “including”, “containing” and similar terms encompass the meanings of “consisting essentially of” and “consisting of”. For example, when “A comprises B and C” is disclosed herein, “A consists essentially of B and C” and “A consists of B and C” should be deemed to be disclosed herein.

[0021] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0022] In this document, unless otherwise specified, percentage refers to mass percentage and ratio refers to mass ratio.

[0023] Herein, the sum of the percentages of the various components of the composition is 100%.

[0024] Herein, when describing embodiments or examples, it should be understood that they are not used to limit the present invention to these embodiments or examples. On the contrary, all substitutes, improvements and equivalents of the methods and materials described in the present invention can be included in the scope limited by the claims.

[0025] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0026] The inventors combined previous studies and found that the amniotic fluid described herein can be used to treat spinal cord injury and related diseases, thereby completing the present invention.

[0027] Amniotic fluid

[0028] The amniotic fluid used in this article can come from eggs and non-human mammals. Eggs refer to eggs of birds. Preferred birds are poultry, such as chickens, ducks, ostriches and geese. Preferably, the present invention uses eggs with an embryonic age of 5-20 days, preferably 6-15 days. It should be understood that different eggs, suitable embryonic ages are not necessarily the same. For example, when using eggs, it is preferred to use eggs with an embryonic age of 5-12 days, more preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, and more preferably eggs with an embryonic age of 7-8 days. When using eggs of other birds, eggs corresponding to the developmental period of the eggs of the above-mentioned embryonic age can be used. For example, when using duck eggs, duck eggs with an embryonic age of 8-10 days, especially 8-9 days, may be the best. In some specific embodiments, ceAF refers to amniotic fluid extracted from chicken embryos incubated for 6-8 days.

[0029] The amniotic fluid of poultry eggs can be obtained by conventional methods. For example, the blunt end of an egg of the corresponding embryonic age can be tapped to break the eggshell, and the eggshell can be peeled open to form a hole with a diameter of about 2 cm. Then, the shell membrane and the yolk membrane are carefully torn apart with tweezers, and care is taken not to damage the amnion. The amnion and the connected tissues enveloping the embryo are poured from the shell into a culture dish, and the amnion is pierced with a syringe to extract the amnion until the amnion is close to the embryo, thereby obtaining the amniotic fluid used in the present invention.

[0030] Herein, amniotic fluid can also be from non-human mammals, especially rodents, such as from mice. Other non-human mammals can be common livestock, such as cattle, sheep, dogs, cats, pigs, etc. In certain embodiments, amniotic fluid is from the embryo of a rodent with a gestational age of 8-14 days, or from the embryo of a non-human mammal corresponding to the developmental period of a rodent with a gestational age of 8-14 days. Conventional methods can be used to obtain amniotic fluid. For example, the abdominal cavity of a mouse with a gestational age of 8-14 days is cut open with surgical scissors, the uterus is carefully removed and cut open, and the amniotic membrane is pierced with a syringe to extract amniotic fluid until the amniotic membrane is close to the embryo, thus obtaining the amniotic fluid used for the present invention.

[0031] It should be understood that, if necessary, the amniotic fluid can be centrifuged to separate possible impurities, such as egg yolk, etc., to obtain as pure amniotic fluid as possible. The supernatant obtained after centrifugation is the amniotic fluid used in the present invention. It should be understood that all steps of obtaining amniotic fluid must be performed under sterile conditions; in addition, the "amniotic fluid" shown herein should refer to "pure" amniotic fluid, that is, amniotic fluid separated from poultry eggs or non-human mammal embryos that does not contain other components in poultry eggs or non-human mammal embryos and is not contaminated by exogenous substances. Pure amniotic fluid can be stored in a refrigerator below -60°C and used after thawing.

[0032] The amniotic fluid described herein can be used as an active ingredient of a drug for in vivo administration to a subject in need thereof. For example, an effective amount of the amniotic fluid described herein, or a pharmaceutical composition containing the amniotic fluid, can be administered to a subject in need thereof.

[0033] It should be understood that, in combination with CN 201810911038.2, CN 201810909485.4, CN 201810909193.0 and CN 201910887556.X, the amniotic fluid from various sources described in the present invention has the same or similar biological activity, and when used in the present invention, can be used to treat and / or repair complete or incomplete spinal cord injury and related diseases.

[0034] Herein, the animal may be a mammal, such as a mouse or a human, in particular a human.

[0035] Pharmaceutical composition

[0036] The present invention also provides a pharmaceutical composition, which contains the amniotic fluid described herein, especially the amniotic fluid in poultry eggs, more preferably the amniotic fluid of eggs with an embryonic age of 5-12 days, more preferably 6-11 days, more preferably 6-9 days, and more preferably 7-8 days. The pharmaceutical composition can be amniotic fluid or a lyophilized agent thereof frozen at below -60°C, such as lyophilized amniotic fluid. The pharmaceutical composition can also contain other pharmaceutically acceptable carriers or excipients, such as physiological saline for injection, water for injection, or glucose injection.

[0037] Methods and applications for treating spinal cord injury

[0038] Based on the foregoing findings, the present invention also provides a method for treating spinal cord injury, the method comprising administering to a subject a therapeutically effective amount of the amniotic fluid as described in any embodiment herein, the subject suffering from spinal cord injury and its related conditions. In some embodiments, the present invention also provides the use of the amniotic fluid as described in any embodiment herein in treating spinal cord injury, or the use of the amniotic fluid as described in any embodiment herein in preparing a medicament for treating spinal cord injury.

[0039] The "spinal cord injury" mentioned in this article refers to the destruction of the integrity of the spinal structure caused by various traumatic factors, such as the destruction of vertebrae, intervertebral discs and / or spinal connective tissue (damage to the ligaments and paraspinal muscles that stabilize the spine), resulting in damage to the neural structure in the spinal canal (including the spinal cord, i.e., nerve roots) and its function. Usually, patients with spinal cord injury will have disorders of spinal cord function (such as movement, sensation, reflexes, etc.) at the level of injury and below, paraplegia, flaccid paralysis, spastic paralysis, loss of motor sensation, loss of saddle sensation in the perineal skin, loss of sphincter function, urinary and bowel dysfunction, sexual dysfunction, and complications such as respiratory failure and respiratory tract infections, infections and stones of the urogenital tract, bedsores or pressure sores, and temperature disorders.

[0040] "Treatment" or "repair" as described herein refers to partial or complete restoration of the physiological state and vital signs of the subject before the onset of primary spinal cord injury and secondary spinal cord injury. Generally, treatment or repair of complete or incomplete spinal cord injury and its related conditions include promoting nerve regeneration and nerve function recovery (such as promoting motor function recovery after injury, inhibiting the expansion of the injured area and reducing motor neuron loss), inhibiting excessive immune response, repairing damaged cells, and promoting cell regeneration.

[0041] Herein, a therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation and / or relief of a disease or condition in a subject. The therapeutically effective amount can be determined based on factors such as the patient's age, gender, the disease and its severity, and other physical conditions of the patient. Herein, a subject or patient generally refers to a mammal, particularly a human.

[0042] Herein, the dosage and frequency of administration can be determined by medical staff according to the specific condition, the age and gender of the patient, etc. Generally, for the treatment of a specific disease, a therapeutically effective amount refers to a dose sufficient to improve or alleviate the symptoms associated with the disease in some way. Such a dose can be administered as a single dose, or can be administered according to an effective treatment regimen. The dosage may cure the disease, but the administration is usually to improve the symptoms of the disease. Generally, repeated administration is required to achieve the desired symptom improvement, and multiple treatment courses can be implemented according to the actual cure. For example, for the dose given to a person, it can usually be 1-200ml / time, and can be injected daily or weekly. In certain embodiments, the frequency of administration can be multiple times a day, twice a day, every two days, every three days, every four days, every five days or every six days, or once every half a month, or once a month. In some embodiments, the frequency of administration is once a day, and the administration cycle is at least 1 to 4 weeks. In some embodiments, the administration is once a day, and the continuous administration is at least 1 to 4 weeks, which is a treatment course.

[0043] The amniotic fluid described herein can be used directly as an active ingredient for the purposes described herein and administered to a subject in need thereof. The administration method can be parenteral administration, such as intravenous injection or local injection administration at the desired site. In certain embodiments, a therapeutically effective amount of amniotic fluid (or with other drugs) can be mixed with an appropriate amount of physiological saline for injection, water for injection or glucose injection, and then administered by, for example, intravenous infusion.

[0044] In the present invention, mice are used as experimental animals. It is easy for those skilled in the art to convert the dosage of mice into the dosage suitable for humans. For example, the dosage can be calculated according to the Meeh-Rubner formula:

[0045] A=k×(W 2 / 3 ) / 10000.

[0046] Where A is the body surface area, expressed in m 2 Calculation: W is body weight, in g; k is a constant, which varies with the animal species, 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that the dosage conversion may vary depending on the drug and clinical situation, based on the evaluation of an experienced pharmacist.

[0047] In an exemplary embodiment, when the subject of administration of the present invention is a mammal, especially a mouse, the dosage of amniotic fluid in the pharmaceutical composition of the present invention is generally about 5 times that of humans, and the dosage is 1 to 20 ml / kgBw, such as 1 to 5 ml / kgBw or 5 to 15 ml / kgBw. In an exemplary embodiment, when the subject of administration of the present invention is a mammal, especially a human, the dosage of amniotic fluid in the pharmaceutical composition of the present invention is 1 to 10 ml / kgBw, such as 1 to 3 ml / kgBw or 3 to 5 ml / kgBw.

[0048] The present invention has the following beneficial effects:

[0049] The amniotic fluid of the present invention can promote the recovery of motor function of mammals (such as humans and mice) after spinal cord injury, inhibit the expansion of the spinal cord injury area and reduce the loss of motor neurons around the spinal cord injury area.

[0050] The present invention will be further described below by way of 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 and reagents used in the examples are, unless otherwise stated, conventional methods and reagents in the art.

[0051] Experimental methods:

[0052] 1. Animal Experiment Grouping

[0053] 1) Sham operation: only laminectomy was performed without spinal cord injury, and the same volume of saline was used for treatment.

[0054] 2) Spinal cord injury + saline group (SCI): spinal cord injury model was established and treated with the same volume of saline.

[0055] 3) Spinal cord injury + medium dose group (SCI + ceAF 5ml / kg): Spinal cord injury model was established and treated with ceAF.

[0056] 4) Spinal cord injury + high-dose group (SCI + ceAF 15ml / kg): Spinal cord injury model was established and treated with ceAF.

[0057] 2. Spinal Cord Injury Impact Model

[0058] After weighing, wild-type C57BL / 6 male mice were generally anesthetized by intraperitoneal injection of 1.2% tribromoethanol (20ml / kg). When there was no obvious reaction to pinching the hind limbs of the mice, the anesthesia of the mice was successful. The skin of the back of the mice was prepared with a shaver and disinfected with iodine. A 1cm long midline incision was made at the level of T8 to T10 on the back with a scalpel. After separating and removing the paravertebral muscles of the mice, the mice were fixed on the spinal cord adapter and the lamina was knocked out with microscissors to expose the spinal cord. The mouse spinal cord impactor (RWD, 68097, 10g) hit the mouse spinal cord from a height of 5cm, causing spinal cord injury. Paralysis of both lower limbs indicated that the spinal cord injury model was successfully established. The surgical wound was disinfected layer by layer and sutured. After modeling, the mice were placed on a 37℃ heating pad for recovery. After spinal cord injury, manual urination was performed twice a day until the urination function of the mice was restored. The sham operation group only underwent laminectomy. All animals were randomly divided into experimental groups. These mice were used to evaluate histological, biochemical, and behavioral functions.

[0059] 3. Drug intervention methods in animals

[0060] After the spinal cord injury model was established in mice, the target drug was injected into the tail vein according to the grouping and dosage, and the time points were 0.5h, 2d, 4d, and 6d after the model was established. The sham group and the SCI group were injected with the same volume of normal saline as the control.

[0061] 4. Frozen Sections

[0062] 1) First, the mouse was anesthetized with tribromoethanol, fixed on the dissecting table, and the apex of the heart was exposed. The scalp needle was used to perfuse the liver through the apex of the heart with 1×PBS. When the liver turned white, the perfusion was considered complete. Then 4% PFA was used for perfusion. After the body of the mouse became stiff, the spinal tissue was taken and immersed in 4% PFA for 48 hours;

[0063] 2) Replace 4% PFA with 30% sucrose to soak the tissue, and after the tissue sinks, it can be used for OCT embedding;

[0064] 3) Embed the spinal cord tissue with OCT at -20°C;

[0065] 4) The tissue was sliced ​​on a freezing microtome with a thickness of 20 μm and the slices were stored in a -20°C refrigerator.

[0066] 5. Nissl staining of frozen sections

[0067] 1) Place the slices at room temperature for 5-10 minutes, then dry them in a 55°C oven for 30 minutes;

[0068] 2) Wash the sections three times with 1× PBS, 5 min each time, to wash away OCT;

[0069] 3) Place the slices in anhydrous ethanol for 1 min, then place them in 95% ethanol for 1 min;

[0070] 4) Soak the sections in Nissl solution for staining, and adjust the time according to the staining effect;

[0071] 5) Place the slices in 95% ethanol for decolorization. The decolorization time depends on the decolorization situation.

[0072] 6) Place the slices in anhydrous ethanol twice, 5 minutes each time;

[0073] 7) Place the sections in xylene twice, 5 min each time;

[0074] 8) Take out the slices and seal them with neutral resin. Let the slides dry at room temperature and then store them.

[0075] 6. HE staining of frozen sections

[0076] 1) Take out the frozen sections and place them at room temperature for 5-10 minutes, then place them in a 55°C oven for 30 minutes to dry the tissue and further adhere it to the slide;

[0077] 2) Wash the tissue with 1×PBS for 5 min × 3 times to remove OCT on the tissue surface;

[0078] 3) Stain in hematoxylin staining solution for 5-10 minutes, and the time can be adjusted according to the staining effect and requirements;

[0079] 4) Wash the slides under running water to remove any residual hematoxylin dye, and then soak them in ddH2O for 15 minutes;

[0080] 5) Differentiation in differentiation medium for 30 seconds;

[0081] 6) Stain in eosin staining solution for 2-30 seconds, and the time can be adjusted according to the staining effect and requirements;

[0082] 7) Wash the slides under running water to remove any residual eosin dye, and then soak them in ddH2O for 5 minutes;

[0083] 8) Dehydrate the slides in 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 15 seconds each;

[0084] 9) Place the slide in a xylene dye tank for 5 min x 2 times;

[0085] 10) Take out the slices and quickly seal them with neutral resin, taking care to avoid rapid volatilization of xylene and the generation of bubbles. After drying, store the slides at room temperature.

[0086] 7. Immunofluorescence Staining of Frozen Sections

[0087] 1) Take out the frozen sections and place them at room temperature for 5-10 minutes, then place them in a 60°C oven for 30 minutes;

[0088] 2) Wash with 1×PBS solution for 5 min × 3 times to remove OCT from the tissue surface;

[0089] 3) Cover the tissue with 4% PFA solution and fix for 30 minutes;

[0090] 4) Wash with 1×PBS solution for 5 min×3 times

[0091] 5) Dilute 50× sodium citrate antigen repair solution with ddH2O to 1× sodium citrate antigen repair solution and place it in a glass slide staining jar. After heating it to 90°C in a constant temperature water bath, place the sections in it and perform antigen repair for 30 minutes.

[0092] 6) Wash with 1×PBS solution for 5 min × 3 times;

[0093] 7) 0.3% Triton punch solution at room temperature for 90 minutes.

[0094] 8) After removing the punching solution, add primary antibodies prepared with 5% BSA and incubate overnight at 4°C. The primary antibodies include rabbit anti-MBP (1:1000,), mouse anti-NF (1:1000), rabbit anti-NeuN (1:1000, MAB377, Millipore), and rabbit anti-CD43 (1:1000).

[0095] 9) Take out the slices and warm them up at room temperature for 10 minutes, recover the primary antibody, wash with 1×PBS for 5 minutes × 3 times, add 5% BSA to prepare the secondary antibody of the corresponding species and incubate at room temperature in the dark for 60 minutes (nucleus DAPI 1:1000, secondary antibody dilution 1:1000)

[0096] 10) Remove the secondary antibody, wash with 1×PBS for 5 min × 3 times, add appropriate amount of anti-fluorescence quencher to seal the slides, dry them, place them in a slice box and store them at 4℃ for observation under a microscope.

[0097] 8. Mouse footprint analysis test:

[0098] 1) Three days before the experiment, the experimental animals were placed on a track covered with white paper in the footprint meter to familiarize themselves with the environment and practice crawling;

[0099] 2) Before the experiment, the front and rear limbs of all mice were painted with red (forelimbs) and blue (hind limbs) non-toxic ink respectively;

[0100] 3) The experiment was conducted in a quiet environment. The mice were placed on a track with white paper on the footprint meter and crawled. The paper was removed and the track was cleaned.

[0101] 4) Between two experiments, the runway needs to be cleaned (hair, feces, urine, etc.), and then the same size paper is placed and the next mouse is replaced for the experiment;

[0102] 5) Take the gait map, select the stable walking gait in the middle section of the strip, and qualitatively and quantitatively analyze the foot length and width of the mouse.

[0103] 9. Basso Mouse Scale (BMS) scoring

[0104] BMS scores were performed on mice before surgery and on days 3, 7, 14, 21, and 28 after surgery. Scoring was performed independently by a non-experimental staff member familiar with the scoring criteria, and each mouse was observed for 4 minutes. All mice had their bladders manually emptied before scoring to avoid affecting the experimental results due to their full bladders.

[0105] The BMS scoring criteria for mice are as follows:

[0106]

[0107]

[0108] 10. Statistical methods

[0109] The footprint test and BMS score were analyzed by two-way ANOVA, the HE staining injury area and Nissl staining injury area were analyzed by Student's t-test, and the NeuN+ cell density was analyzed by one-way ANOVA.

[0110] 11. ceAF Preparation

[0111] Fertilized chicken eggs were incubated at 38°C and 50% humidity. ceAFs were isolated from them between days 6 and 8 of incubation. After centrifugation (2500×g, 20 min), the supernatant was filtered using a 0.22 μm sterile filter (Millipore, USA), and the filtered samples were aliquoted and kept at −80°C.

[0112] Example 1: ceAF promotes recovery of motor function after spinal cord injury in mice

[0113] Figure 1 Schematic diagram of ceAF promoting the recovery of motor function after spinal cord injury in mice. Figure 1 (A) is a typical image of the footprint experiment on day 28 after spinal cord injury. The red line is the length between the front and back footprints of the forepaws, and the black line is the width between the two forepaws. Figure 1 (B) is a statistical graph of mouse front paw footprint length (n=4). Figure 1 (C) is a statistical graph of mouse forepaw footprint width (n=4). Figure 1(D) is a statistical graph of the total BMS score of mice (n=4). Figure 1 (E) is a statistical graph of BMS scores of the left foot of mice (n=4). Figure 1 (F) is a statistical diagram of the BMS score of the right foot of mice (n=4). Figure 1 (A) and Figure 1 The scale bar length of (B) is 0.5 cm. Values ​​are mean ± standard error. * indicates p < 0.05, ** indicates p < 0.01.

[0114] The results showed that after 28 days of ceAF treatment, the motor function of the treatment group was significantly restored compared with the control group, and there was a dose effect.

[0115] Example 2: ceAF inhibits the expansion of spinal cord injury area in mice

[0116] Figure 2 Schematic diagram of the expansion of spinal cord injury area in ceAF-inhibited mice. Figure 2 (A) is a typical image of HE staining results of the spinal cord 28 days after spinal cord injury, and the dotted line indicates the injured area. Figure 2 (B) is a typical image of Nissl staining of the spinal cord 28 days after spinal cord injury, with the dotted line indicating the injured area. Figure 2 (C) is a statistical diagram of the damaged area by HE staining (n=4). Figure 2 (D) is a statistical diagram of the damaged area by Nissl staining (n=4). Figure 2 (A) and Figure 1 The scale bar length of (B) is 500 μm. Values ​​are mean ± standard error. * indicates p < 0.05, ** indicates p < 0.01.

[0117] The results showed that after 28 days of ceAF treatment, the spinal cord injury area in the treatment group was significantly smaller than that in the control group, and there was a dose effect.

[0118] Example 3: ceAF reduces the loss of peripheral motor neurons in the spinal cord after injury in mice

[0119] Figure 3 Schematic diagram of how ceAF reduces the loss of peripheral motor neurons in the spinal cord after injury in mice. Figure 3 (A) is a typical image of NeuN fluorescence staining results in the spinal cord 28 days after spinal cord injury. The dotted line indicates the injured area. Figure 3 (B) is a statistical diagram of the density of NeuN+ cells around spinal cord injury stained with fluorescence (n=4). Figure 3 The scale bar in (A) is 200 μm. * indicates p < 0.01, ** indicates p < 0.001.

[0120] The results showed that after 28 days of ceAF treatment, the loss of motor neurons around the posterior spinal cord injury area in the treatment group was significantly less than that in the control group, and there was a dose effect.

Claims

1. Use of amniotic fluid in the preparation of a drug for treating and / or repairing complete or incomplete spinal cord injury and related diseases; wherein: The amniotic fluid comes from: eggs with an embryonic age of 5-12 days; eggs of poultry other than chickens whose developmental period corresponds to the developmental period of the eggs with the embryonic age; embryos of rodents with a gestational age of 8-14 days; or embryos of non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-14 days.

2. The use according to claim 1, characterized in that The amniotic fluid comes from eggs with an embryonic age of 6-11 days, preferably from eggs with an embryonic age of 7-9 days, more preferably from eggs with an embryonic age of 7-8 days, or from duck eggs with an embryonic age of 8-10 days, preferably 8-9 days.

3. The use according to any one of claims 1 to 2, characterized in that: The medicine is a pharmaceutical composition comprising the amniotic fluid and pharmaceutically acceptable excipients.

4. The use according to any one of claims 1 to 3, characterized in that The complete or incomplete spinal cord injury and its related diseases include damage to the integrity of the spinal structure, damage to the structure and function of the nerves in the spinal canal, and dysfunction of the spinal cord at the injury level and below.

5. The use according to claim 4, characterized in that The complete or incomplete spinal cord injury and its related diseases also include paraplegia, flaccid paralysis, spastic paralysis, loss of motor sensation, loss of saddle sensation of perineal skin, loss of sphincter function, urinary and bowel dysfunction, sexual dysfunction and one or more of complications.

6. The use according to claim 5, characterized in that The complications include respiratory failure and respiratory tract infections caused by autonomic dysfunction of spinal cord injury, infections and stones of the genitourinary tract, bedsores or pressure sores, and temperature disorders.

7. The use according to claim 1, characterized in that The repair of complete or incomplete spinal cord injury and its related diseases includes promoting nerve regeneration and nerve function recovery, inhibiting excessive immune response, repairing damaged cells and promoting cell regeneration.

8. The use according to claim 7, characterized in that The promotion of nerve regeneration and nerve function recovery includes promoting recovery of motor function after injury, inhibiting the expansion of the injury area and reducing the loss of motor neurons.

9. Use of amniotic fluid in the preparation of a drug for promoting the recovery of motor function after spinal cord injury, inhibiting the expansion of the spinal cord injury area, and / or reducing the loss of motor neurons around the spinal cord injury area after the patient's spinal cord injury; wherein, The amniotic fluid comes from: eggs with an embryonic age of 5-12 days; eggs of poultry other than chickens whose developmental period corresponds to the developmental period of the eggs with the embryonic age; embryos of rodents with a gestational age of 8-14 days; or embryos of non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-14 days.

10. The use according to claim 9, characterized in that The amniotic fluid comes from eggs with an embryonic age of 6-11 days, preferably from eggs with an embryonic age of 7-9 days, more preferably from eggs with an embryonic age of 7-8 days, or from duck eggs with an embryonic age of 8-10 days, preferably 8-9 days.

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