WO2022170939A1 - 无细胞脂肪提取物对视神经损伤的治疗用途 - Google Patents
无细胞脂肪提取物对视神经损伤的治疗用途 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the invention relates to the field of medicine, in particular to the therapeutic use of acellular fat extract for optic nerve damage.
- Optic nerve injury is a serious disease, which can be caused by various factors, such as traumatic stress, pathological mechanism and inflammatory response, which can seriously lead to blindness and endanger human health.
- traumatic stress a serious disease
- pathological mechanism a mechanism for inflammatory response
- inflammatory response a mechanism for inflammatory response
- the development of an effective drug for the treatment of the optic nerve has become a hot research topic today.
- the purpose of the present invention is to provide the use of a cell-free fat extract in preventing and/or treating optic nerve damage.
- the first aspect of the present invention provides the use of a cell-free fat extract for preparing a composition or preparation for preventing and/or treating optic nerve damage.
- the optic nerve injury includes optic nerve injury caused by traumatic stress.
- the prevention and/or treatment of optic nerve damage includes prevention and/or treatment in one or more ways selected from the following group:
- RRCs retinal neural detail cells
- the prevention and/or treatment of optic nerve injury has one or more features selected from the group consisting of:
- Optic nerve axons can grow to about 1.5-1.8 mm after 2 weeks of injury;
- the number of optic nerve axons at 0.25mm is about 2300-2500 after 2 weeks of injury;
- the cell-free fat extract is a cell-free fat extract prepared from human or non-human mammalian fat.
- the non-human mammal is monkey, orangutan, cow, pig, dog, sheep, mouse or rabbit.
- compositions or preparations include pharmaceutical compositions or preparations, food compositions or preparations, health product compositions or preparations, or dietary supplements.
- composition or preparation further includes a pharmaceutically, food, health product or dietary acceptable carrier.
- composition or preparation further includes other drugs for preventing and/or treating optic nerve damage.
- the other drugs for preventing and/or treating optic nerve injury are selected from the group consisting of: mecobalamin, citicoline, inosine, neurotrophic agent, neurotrophic factor, glucocorticoid, blood vessel Dilators, calcium channel blockers, vitamins, stem cells, or a combination thereof.
- the neurotrophic agent is selected from the group consisting of vitamin B1, vitamin B12, or a combination thereof.
- the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation.
- the injection preparation is an intravenous injection or an intramuscular injection.
- the injection preparation is an ophthalmic injection preparation.
- the injection preparation is intravitreal injection preparation.
- the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as solution, gel, cream, lotion, ointment, cream, paste, cake, powder, Patches etc.
- the dosage form of the composition or preparation is powder, granule, capsule, injection, tincture, oral liquid, tablet or lozenge.
- composition or preparation is administered externally, topically, or by injection.
- the cell-free fat extract does not contain cells and does not contain lipid droplets.
- the lipid droplets are oil droplets released after fat cells are disrupted.
- the "does not contain lipid droplets" means that in the cell-free fat extract, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, more preferably less than 0.1%.
- the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.
- the "cell-free” refers to the average number of cells in 1 ml of cell-free fat extract ⁇ 1, preferably ⁇ 0.5, more preferably ⁇ 0.1, or 0.
- the cell-free fat extract is a naturally-obtained nano-fat extract without added components.
- the "no added components” means that, except for the rinsing step, no solution, solvent, small molecule, chemical agent, and biological additive are added during the preparation of the fat extract.
- the cell-free adipose extract is prepared by centrifuging adipose tissue after emulsification.
- the cell-free fat extract contains one or more components selected from the group consisting of IGF-1, BDNF, GDNF, TGF- ⁇ 1, HGF, bFGF, VEGF, TGF- ⁇ 1 , PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.
- the cell-free fat extract contains, but is not limited to, one or more components selected from the group consisting of IGF-1, BDNF, GDNF, bFGF, VEGF, TGF- ⁇ 1, HGF , PDGF, or a combination thereof.
- the cell-free fat extract is a cell-free fat extract.
- the concentration of the IGF-1 is 5000-30000pg/ml, preferably 6000-20000pg/ml, more preferably 7000-15000pg/ml , more preferably 8000-12000pg/ml, more preferably 9000-11000pg/ml, more preferably 9500-10500pg/ml.
- the concentration of BDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more Preferably 1400-2000 pg/ml, more preferably 1600-2000 pg/ml, more preferably 1700-1850 pg/ml.
- the concentration of GDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more Preferably 1400-2000 pg/ml, more preferably 1600-2000 pg/ml, more preferably 1700-1900 pg/ml.
- the concentration of the bFGF is 50-600pg/ml, preferably 100-500pg/ml, more preferably 120-400pg/ml, more Preferably 150-300 pg/ml, more preferably 200-280 pg/ml, more preferably 220-260 pg/ml.
- the concentration of the VEGF is 50-500pg/ml, preferably 100-400pg/ml, more preferably 120-300pg/ml, more Preferably 150-250 pg/ml, more preferably 170-230 pg/ml, more preferably 190-210 pg/ml.
- the concentration of TGF- ⁇ 1 is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml , more preferably 800-1200pg/ml, more preferably 800-1100pg/ml, more preferably 900-1000pg/ml.
- the concentration of the HGF is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more Preferably 600-1200 pg/ml, more preferably 800-1000 pg/ml, more preferably 850-950 pg/ml.
- the concentration of PDGF is 50-600pg/ml, preferably 80-400pg/ml, more preferably 100-300pg/ml, more Preferably 140-220 pg/ml, more preferably 160-200 pg/ml, more preferably 170-190 pg/ml.
- the weight ratio of IGF-1 to VEGF is 20-100:1, preferably 30-70:1, more preferably 40-60:1, most preferably 45-55: 1.
- the weight ratio of BDNF to VEGF is 2-20:1, preferably 4-15:1, more preferably 6-12:1, and most preferably 8-9.5:1.
- the weight ratio of GDNF to VEGF is 2-20:1, preferably 4-15:1, more preferably 6-12:1, and most preferably 8.5-9.5:1.
- the weight ratio of bFGF to VEGF is 0.2-8:1, preferably 0.5-5:1, more preferably 0.6-2:1, more preferably 0.8-1.6:1, Optimally 1-1.5:1.
- the weight ratio of TGF- ⁇ 1 to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8: 1, preferably 4-6:1.
- the weight ratio of HGF to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8:1, More preferably 4-5.5:1.
- the weight ratio of PDGF to VEGF is 0.1-3:1, preferably 0.2-2:1, more preferably 0.4-1.5:1, and most preferably 0.7-1.2:1.
- the cell-free fat extract is prepared by the following method:
- Emulsifying the intermediate layer to obtain an emulsified fat mixture also referred to as nano-fat
- a second aspect of the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps of:
- Emulsifying the intermediate layer to obtain an emulsified fat mixture also referred to as nano-fat
- the cell-free fat extract is as described in the first aspect of the present invention.
- the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.
- the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 1-8 minutes, and optimally 1-5 minutes.
- the temperature of the centrifugation is 2-6°C.
- the emulsification is mechanical emulsification.
- the mechanical emulsification is performed mechanically by repeated blowing through a syringe (eg 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times). emulsification.
- the blowing method is to repeatedly push and beat at a constant speed with two 10ml injection syringes connected to a tee tube.
- the emulsification is a method of crushing by a tissue homogenizer.
- the emulsified fat mixture is further frozen and then thawed.
- the thawed mixture is used for centrifugation after thawing after freezing.
- the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, more preferably -70°C to -90°C.
- the thawing temperature is 20-40°C, preferably 25-40°C, more preferably 37°C.
- the number of cycles of thawing after freezing is 1-5 times (preferably 1, 2, 3 or 4 times).
- the emulsified fat mixture is layered into four layers, the first layer is an oil layer, the second layer is a residual adipose tissue layer, and the third layer is a liquid layer layer (ie, the middle liquid layer), and the fourth layer is the cell/tissue debris sedimentation layer.
- the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.
- the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 2-8 minutes, and optimally 3-7 minutes.
- the temperature of the centrifugation is 2-6°C.
- the first layer, the second layer, the third layer and the fourth layer are arranged in order from top to bottom.
- the intermediate liquid layer is a transparent or substantially transparent layer.
- the filter bag in the step (6), can remove the adipocytes in the primary fat extract.
- the filtration and sterilization are performed through a filter (eg, a 0.22 ⁇ m microporous membrane).
- a filter eg, a 0.22 ⁇ m microporous membrane
- the filter is a microporous membrane filter.
- the pore size of the microporous filter membrane is 0.05-0.8 ⁇ m, preferably 0.1-0.5 ⁇ m, more preferably 0.1-0.4 ⁇ m, more preferably 0.15-0.3 ⁇ m, more preferably 0.2-0.25 ⁇ m, optimally 0.22 ⁇ m.
- the filtration and sterilization are firstly passed through a first filter that can filter out cells, and then passed through a second filter that can filter out pathogens (such as bacteria).
- filter eg, 0.22 ⁇ m filter.
- the step (6) further includes sub-packaging the fat extract to form a sub-packaged product.
- the subpackaged extract can be stored at -20°C for later use; it can be used directly after thawing at low temperature (such as -4°C) or normal temperature, or it can be stored at low temperature (such as 4°C) for a period of time after thawing, and then used ).
- the third aspect of the present invention provides a cell-free fat extract, which is prepared by the method described in the second aspect of the present invention.
- the fourth aspect of the present invention provides a composition or preparation for preventing and/or treating optic nerve damage, the composition or preparation comprising (a) the cell-free adipose extract according to the third aspect of the present invention; and (b) A pharmaceutically, food, health product or dietary acceptable carrier or excipient.
- the composition is a pharmaceutical composition, a food composition, a health product composition or a dietary supplement.
- the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation.
- the dosage form of the composition or preparation is powder, granule, capsule, injection, tincture, oral liquid, tablet or lozenge.
- the injection is an intravenous injection or an intramuscular injection.
- the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as solution, gel, cream, lotion, ointment, cream, paste, cake, powder, Patches etc.
- the mass percentage of the cell-free fat extract is 5 wt %, preferably 1-20 wt %, based on the total weight of the composition or preparation.
- the fifth aspect of the present invention provides a method for preparing the composition or preparation according to the fourth aspect of the present invention, the method comprising the steps of: mixing the cell-free fat extract according to the third aspect of the present invention with a pharmaceutical It is mixed with acceptable carriers or excipients on food, health product or diet to form a composition or preparation.
- the sixth aspect of the present invention provides a method for preventing and/or treating optic nerve damage, by administering the cell-free fat extract according to the third aspect of the present invention to a subject in need.
- the subject is a human or a non-human mammal.
- the non-human mammals include rodents, such as rats and mice.
- the administration mode is oral administration, topical administration or injection administration.
- the dose administered by injection is 3-5 ⁇ g/ ⁇ L.
- Figure 1 is the experimental flow chart.
- Figure 2 shows that CEFFE can promote axonal regeneration after optic nerve injury.
- Figure 2A shows the regenerated axons traced by CTB-55 in different administration groups after 14 days of injury (* is the clamp injury), and
- Figure 2B shows the statistics of the number of regenerated axons at different distances from the injury in Figure 2A (* **p ⁇ 0.001).
- PBS is phosphate buffered saline blank control
- CEFFE Thrice is the intravitreal injection of 3 ⁇ l CEFFE immediately after optic nerve injury, 3 days after injury and 7 days after injury
- CEFFE Once is 3 ⁇ l CEFFE injected into the vitreous cavity immediately after optic nerve injury.
- FIG. 3 shows that CEFFE and BDNF cytokines promote axonal regeneration after optic nerve injury (* is the clamp injury).
- Fig. 3A shows the axonal regeneration traced by CTB-55 after intravitreal injection of PBS, BDNF and CEFFE, respectively.
- Figure 3B is the statistics of the number of regenerated axons in Figure 3A (**p ⁇ 0.01).
- Figure 4 is an immunofluorescence image of retinal slides.
- Fig. 4A is a graph showing the statistical survival of RGCs in the uninjured group
- Fig. 4B shows the survival of RGCs after intravitreal injection of PBS in an animal model of optic nerve injury.
- Figure 4C shows the survival of RGCs after intravitreal injection of CEFFE in an animal model of optic nerve injury. (Retinal mounts were immunofluorescently stained for TUJ1 to label viable RGCs).
- Figure 4D is the quantification statistics of surviving RGCs in Figures 4A-C (**p ⁇ 0.05).
- the terms “comprising,” “including,” and “containing” are used interchangeably to include not only open definitions, but also semi-closed, and closed definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
- prevention refers to a method of preventing the onset of a disease and/or its attendant symptoms or protecting a subject from acquiring a disease.
- prevention also includes delaying the onset of the disease and/or its attendant symptoms and reducing the risk of the disease in a subject.
- Treatment includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal.
- the cell-free fat extract of the present invention reduces, inhibits and/or reverses optic nerve damage, eg, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%.
- IGF-1 insulin-like growth factors-1
- BDNF brain-derived neurotrophic factor
- GDNF glial cellline-derived neurotrophic factor
- bFGF basic fibroblast growth factor
- VEGF vascular endothelial growth factor
- TGF- ⁇ 1 is referred to as transforming growth factor- ⁇ 1.
- HGF Hepatocyte Growth Factor
- PDGF Platelet derived growth factor
- EGF Epidermal Growth Factor
- NT-3 As used in the text, the term "NT-3" is referred to as neurotrophins-3.
- GH Growth Hormone
- G-CSF granulocyte colony stimulating factor
- CEFFE Cell free fat extract
- the terms "cell-free adipose extract of the present invention”, “extract of the present invention”, “fat extract of the present invention” and the like are used interchangeably to refer to the process during the preparation of the fat extract (other than the rinsing step) )
- An adipose tissue-derived extract (or extract) prepared without the addition of any solutions, solvents, small molecules, chemicals, and biological additives.
- a typical method for preparing the extract of the present invention is as described above in the second aspect of the present invention.
- the extract of the present invention does not have to add any additives (or added components) during the preparation process, some or small amounts of safe substances (such as small amounts) that do not negatively or adversely affect the activity of the extract of the present invention may also be added. water).
- the cell-free fat extract of the present invention can be derived from human adipose tissue, which is purified from nano-fat by removing oil and cell/extracellular matrix fractions after centrifugation, and is a cell-free, easy-to-prepare, rich in various growth factor liquid.
- the cell-free fat extract is a cell-free fat extract.
- the cell-free adipose extract of the present invention various cytokines may be included.
- the cell-free adipose extract comprises IGF-1, BDNF, GDNF, TGF- ⁇ , HGF, bFGF, VEGF, TGF- ⁇ 1, PDGF, EGF, NT-3, GH and G-CSF one or more.
- the concentration of the IGF-1 is 5000-30000pg/ml, preferably 6000-20000pg/ml, more preferably 7000-15000pg/ml , more preferably 8000-12000pg/ml, more preferably 9000-11000pg/ml, more preferably 9500-10500pg/ml.
- the concentration of BDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more Preferably 1400-2000 pg/ml, more preferably 1600-2000 pg/ml, more preferably 1700-1850 pg/ml.
- the concentration of GDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more Preferably 1400-2000 pg/ml, more preferably 1600-2000 pg/ml, more preferably 1700-1900 pg/ml.
- the concentration of the bFGF is 50-600pg/ml, preferably 100-500pg/ml, more preferably 120-400pg/ml, more Preferably 150-300 pg/ml, more preferably 200-280 pg/ml, more preferably 220-260 pg/ml.
- the concentration of the VEGF is 50-500pg/ml, preferably 100-400pg/ml, more preferably 120-300pg/ml, more Preferably 150-250 pg/ml, more preferably 170-230 pg/ml, more preferably 190-210 pg/ml.
- the concentration of TGF- ⁇ 1 is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml , more preferably 800-1200pg/ml, more preferably 800-1100pg/ml, more preferably 900-1000pg/ml.
- the concentration of the HGF is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more Preferably 600-1200 pg/ml, more preferably 800-1000 pg/ml, more preferably 850-950 pg/ml.
- the concentration of PDGF is 50-600pg/ml, preferably 80-400pg/ml, more preferably 100-300pg/ml, more Preferably 140-220 pg/ml, more preferably 160-200 pg/ml, more preferably 170-190 pg/ml.
- the weight ratio of IGF-1 to VEGF is 20-100:1, preferably 30-70:1, more preferably 40-60:1, most preferably 45-55: 1.
- the weight ratio of BDNF to VEGF is 2-20:1, preferably 4-15:1, more preferably 6-12:1, and most preferably 8-9.5:1.
- the weight ratio of GDNF to VEGF is 2-20:1, preferably 4-15:1, more preferably 6-12:1, and most preferably 8.5-9.5:1.
- the weight ratio of bFGF to VEGF is 0.2-8:1, preferably 0.5-5:1, more preferably 0.6-2:1, more preferably 0.8-1.6:1, Optimally 1-1.5:1.
- the weight ratio of TGF- ⁇ 1 to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8: 1, preferably 4-6:1.
- the weight ratio of HGF to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8:1, More preferably 4-5.5:1.
- the weight ratio of PDGF to VEGF is 0.1-3:1, preferably 0.2-2:1, more preferably 0.4-1.5:1, and most preferably 0.7-1.2:1.
- the cell-free fat extract of the present invention is prepared by the method as described in the second aspect of the present invention.
- the cell-free fat extracts of the present invention are prepared by the following methods:
- Emulsifying the intermediate layer to obtain an emulsified fat mixture also referred to as nano-fat
- the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.
- the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 1-8 minutes, and optimally 1-5 minutes.
- the emulsification is mechanical emulsification.
- the mechanical emulsification is performed mechanically by repeated blowing through a syringe (eg 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times). emulsification.
- the blowing method is to repeatedly push and beat at a constant speed with two 10ml injection syringes connected to a tee tube.
- the emulsification is a method of crushing by a tissue homogenizer.
- the emulsified fat mixture is further frozen and then thawed.
- the thawed mixture is used for centrifugation after thawing after freezing.
- the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, more preferably -70°C to -90°C.
- the thawing temperature is 20-40°C, preferably 25-40°C, more preferably 37°C.
- the number of cycles of thawing after freezing is 1-5 times (preferably 1, 2, 3 or 4 times).
- the emulsified fat mixture is layered into four layers, the first layer is an oil layer, the second layer is a residual adipose tissue layer, and the third layer is a liquid layer layer (ie, the middle liquid layer), and the fourth layer is the cell/tissue debris sedimentation layer.
- the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.
- the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 2-8 minutes, and optimally 3-7 minutes.
- the first layer, the second layer, the third layer and the fourth layer are arranged in order from top to bottom.
- the intermediate liquid layer is a transparent or substantially transparent layer.
- the filter bag in the step (6), can remove the adipocytes in the primary fat extract.
- the filtration and sterilization are performed through a filter (eg, a 0.22 ⁇ m microporous membrane).
- a filter eg, a 0.22 ⁇ m microporous membrane
- the filter is a microporous membrane filter.
- the pore size of the microporous filter membrane is 0.05-0.8 ⁇ m, preferably 0.1-0.5 ⁇ m, more preferably 0.1-0.4 ⁇ m, more preferably 0.15-0.3 ⁇ m, more preferably 0.2-0.25 ⁇ m, optimally 0.22 ⁇ m.
- the filtration and sterilization are firstly passed through a first filter that can filter out cells, and then passed through a second filter that can filter out pathogens (such as bacteria).
- filter eg, 0.22 ⁇ m filter.
- the step (6) further includes sub-packaging the fat extract to form a sub-packaged product.
- the subpackaged extract can be stored at -20°C for later use; it can be used directly after thawing at low temperature (such as -4°C) or normal temperature, or it can be stored at low temperature (such as 4°C) for a period of time after thawing, and then used ).
- the present invention provides the use of a cell-free adipose extract for preparing a composition or formulation for preventing and/or treating optic nerve damage.
- the optic nerve injury includes optic nerve injury caused by traumatic stress.
- the prevention and/or treatment of optic nerve damage includes prevention and/or treatment in one or more ways selected from the following group:
- RRCs retinal neural detail cells
- the present invention also provides a method for preventing and/or treating optic nerve damage, by administering the cell-free fat extract of the present invention to a subject in need.
- the subject is a human or a non-human mammal.
- the non-human mammals include rodents, such as rats and mice.
- the administration mode is oral administration, topical administration or injection administration.
- the injection administration is ocular injection administration.
- the injection administration is intravitreal injection administration.
- compositions described in the present invention include (but are not limited to): pharmaceutical compositions, food compositions, health care compositions, dietary supplements, and the like.
- the cell-free fat extracts of the present invention can be prepared into pharmaceutical compositions such as tablets, capsules, powders, microparticles, solutions, lozenges, jellies, creams, elixirs, suspensions, Dosage forms such as tinctures, poultices, liniments, lotions, and aerosols.
- Pharmaceutical compositions can be prepared by generally known preparation techniques, and suitable pharmaceutical additives can be added to the medicament.
- composition of the present invention may also include a pharmaceutically, food, health product or dietary acceptable carrier.
- “Pharmaceutically, food, nutraceutical or dietary acceptable carrier” means: one or more compatible solid or liquid filler or gel substances, which are suitable for human use and must be of sufficient purity and sufficiently low toxicity.
- “Compatibility” as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds.
- acceptable carrier moieties are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.) , gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol) etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
- solid lubricants such as stearic acid, magnesium stearate
- calcium sulfate such as soybean oil, sesame oil, peanut oil, olive oil, etc.
- polyols such as propylene glycol, glycerin,
- the mode of administration of the composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular), topical, and preferred modes of administration are oral administration and injection.
- the injection administration is an ocular injection administration, preferably a vitreous intravitreal injection formulation.
- the dosage form of the composition or preparation of the present invention is an oral preparation, an external preparation or an injection preparation.
- solid dosage forms for oral administration or administration include capsules, tablets, pills, powders and granules.
- the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as sodium citrate
- Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifiers.
- Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
- liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.
- inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, di
- compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
- adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
- suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.
- suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.
- compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
- Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
- Dosage forms for topical administration or administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants.
- the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
- the cell-free adipose extract of the present invention may be administered or administered alone, or in combination with other drugs for preventing and/or treating fatty liver and/or its complications.
- a safe and effective amount of the cell-free fat extract of the present invention is suitable for human or non-human animals (such as rats, mice, dogs, cats, cows, chickens, ducks, etc.) in need of treatment, wherein the administration
- the current dose is the effective dose that can be considered as acceptable in pharmacy, food or health care products.
- the term "safe and effective amount” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals. Those of ordinary skill in the art should understand that the "safe and effective amount” may vary with the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs, etc. different.
- the daily dosage is usually 0.1 to 1000 mg, preferably 1 to 600 mg, and more preferably 2 to 300 mg.
- the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
- the present invention finds for the first time that the acellular fat extract has an excellent therapeutic effect on optic nerve injury, so as to be used for the treatment of optic nerve injury.
- the cell-free fat extract of the present invention is a cell-free component that can avoid cell-related problems in clinical applications, including, for example, genetic stability after cell processing, cell viability and viability after injection, The multiple administration and storage of cells, and the immunogenicity of cells when using allogeneic fat, the cell-free fat extract of the present invention has the advantages of higher safety and lower side effects in the preparation and prevention of optic nerve injury .
- Adipose tissue was obtained from 6 healthy women who underwent conventional liposuction, with an average age of 31 years (24-36 years). After local injection of tumescent fluid anesthesia, a 3mm liposuction cannula with a large lateral hole (2mm x 7mm) was used to connect a 20mL syringe, and radial suction was performed under artificial negative pressure. Rinse 3 times with normal saline.
- the middle layer ie, the fat layer containing adipocytes
- the mechanically emulsified fat mixture was placed in a -80°C refrigerator for freezing, and then thawed in a 37°C water bath. After a single freeze-thaw cycle, the thawed fat mixture was centrifuged at 1200g at 4°C for 5 minutes to obtain fractions.
- the layered mixture is divided into 4 layers, the first layer is the oil layer, the second layer is the residual adipose tissue layer, the third layer is the liquid layer, and the fourth layer is the cell/tissue debris precipitation layer, remove the oil layer and For the residual adipose tissue layer, the liquid layer is sucked, and the contamination of the cell/tissue debris sediment layer is avoided during the sucking process, so as to obtain the initial fat extraction solution.
- the content of cytokines including IGF-1, BDNF, GDNF, bFGF, VEGF, TGF- ⁇ 1, HGF and PDGF, was detected by ELISA immunosorbent assay kit.
- the average concentrations of 6 samples were as follows: IGF-1 (9840.6pg/ml), BDNF (1764.5pg/ml), GDNF (1831.9pg/ml), bFGF (242.3pg/ml), VEGF (202.9pg/ml), TGF- ⁇ 1 (954.5 pg/ml), HGF (898.4 pg/ml) and PDGF (179.9 pg/ml).
- the retina was placed in a 24-well plate containing blocking solution and blocked at room temperature for 2 hours. After blocking, remove the blocking solution, add primary antibody (TUJ1 1:300) and incubate at 4°C for 48 hours.
- Graphpad statistical software was used for statistical processing. The data in each group were expressed as mean ⁇ S.D., and the homogeneity of variance was tested. The comparison between two groups was performed by t test, and the comparison between multiple groups was performed by one-way analysis of variance. P ⁇ 0.05 represents statistical significance. learning differences.
- 2.1 CEFFE can promote the regeneration of injured axons
- Figure 1A is a 5-week-old C57BL/6 mouse, and the optic nerve was clamped at 1 mm posterior to the optic disc for 5 s to establish an optic nerve injury model.
- Figure 1B shows the intravitreal injection of 3 ⁇ l CEFFE immediately after injury, 3 days after injury, and 7 days after injury.
- Figure 1C shows regenerated axons traced by intravitreal injection of 1.5 ⁇ l of CTB-55 two weeks later.
- CEFFE can promote the regeneration of axons after optic nerve injury as shown in Figure 2.
- Figure 2A shows the regenerated axons traced by CTB-55 in different administration groups after 14 days of injury (* is the clamp injury), in Figure 2B , 250, 500, 750, 1000, and 1500 mm from the injury site were taken to count the number of axons. It can be seen that the experimental group with intravitreal injection of CEFFE could grow up to about 1.5 mm after 2 weeks of injury, and The number of axon regeneration at different distances was significantly higher than that of the control group. The above results indicate that CEEFE can promote the regeneration of optic nerve axons after injury.
- BDNF cytokines and CEFFE were used to measure axonal regeneration after optic nerve injury under the same experimental method, and PBS was the blank control of phosphate buffer.
- BDNF did not promote axon regeneration, but at the same concentration (5ug/ul), CEFFE could significantly promote axon regeneration compared with BDNF, indicating that in promoting axon regeneration after nerve injury, various CEFFE The factors play a coordinating role with each other.
- FIG. 4 The immunofluorescence images of retinal mounts are shown in Figure 4, and it can be seen from Figures 4A-4D that in an animal model of optic nerve injury, CEFFE can significantly promote the survival of injured RGCs (retinal ganglion cells) compared with PBS administration to treat optic nerve damage.
- RGCs retinal ganglion cells
- CEFFE can promote the regeneration of damaged optic nerve and promote the survival of damaged RGCs, so as to prevent and treat optic nerve damage.
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Abstract
提供了一种无细胞脂肪提取物在制备用于预防和/或治疗视神经损伤的组合物或制剂中的用途。
Description
本发明涉及药物领域,具体涉及无细胞脂肪提取物对视神经损伤的治疗用途。
由创伤、缺血、疾病等原因造成的神经细胞受损,缺失或死亡,常使神经功能严重受损而导致偏瘫,失语,智力障碍或昏迷,甚至死亡.轴突作为神经元结构和功能的基础,起到传递信号、物质交流的作用。与外周神经系统相比,中枢神经系统(central nervous system,CNS)受到损伤后,往往很难再生,这就造成了中枢神经系统功能的恢复障碍。传统的药物治疗及功能性电刺激虽显示了一定的效果,但重建与修复神经功能仍是亟待解决的问题。
视神经损伤是一种严重疾病,可由多种因素导致如创伤应激、病理机制和炎症反应,严重导致失明,从而危害人体健康。然而,现有技术中缺乏一种有效治疗视神经损伤的药物,因此,开发一种有效治疗视神经的药物成为当今研究的热点。
因此,本领域需要开发一种能够有效治疗视神经损伤
发明内容
本发明的目在于提供一种无细胞脂肪提取物在预防和/或治疗视神经损伤方面中的用途。
本发明第一方面,提供一种无细胞脂肪提取物的用途,用于制备组合物或制剂,所述组合物或制剂用于预防和/或治疗视神经损伤。
在另一优选例中,所述的视神经损伤包括创伤应激导致的视神经损伤。
在另一优选例中,所述的预防和/或治疗视神经损伤包括选自下组的一种或多种方式进行预防和/或治疗:
(i)促进损伤后的视神经轴突再生;和/或
(ii)促进视网膜神经细节细胞(RGC)的存活和/或再生。
在另一优选例中,所述的预防和/或治疗视神经损伤具有选自下组的一个或多个特征:
(i)在损伤2周后视神经轴突可长至约1.5-1.8mm;
(ii)在损伤2周后视神经轴突在0.25mm处的数量有约2300-2500个;
(iii)视网膜神经细节细胞存活率超过40%。
在另一优选例中,所述的无细胞脂肪提取物为从人或非人哺乳动物中的脂肪中提取制备获得的无细胞脂肪提取物。
在另一优选例中,所述的非人哺乳动物为猴、猩猩、牛、猪、狗、羊、鼠或兔。
在另一优选例中,所述的组合物或制剂包括药物组合物或制剂、食品组合物或制剂、保健品组合物或制剂或膳食补充剂。
在另一优选例中,所述的组合物或制剂还包括药学上、食品上、保健品或膳食上可接受的载体。
在另一优选例中,所述的组合物或制剂还包括其它预防和/或治疗视神经损伤的药物。
在另一优选例中,所述的其它预防和/或治疗视神经损伤的药物选自下组:甲钴胺、胞磷胆碱、肌苷、神经营养剂、神经营养因子、糖皮质激素、血管扩张剂、钙通道阻滞剂、维生素类药物,干细胞,或其组合。
在另一优选例中,所述的神经营养剂选自下组:维生素B1、维生素B12,或其组合。
在另一优选例中,所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。
在另一优选例中,所述的注射制剂为静脉注射剂或肌肉注射剂。
在另一优选例中,所述的注射制剂为眼部注射制剂。
在另一优选例中,所述的注射制剂为玻璃体腔注射制剂。
在另一优选例中,所述组合物或制剂的剂型为固体剂型、半固体剂型、或液体剂型,如溶液、凝胶、膏霜、乳液、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
在另一优选例中,所述组合物或制剂的剂型为粉剂、颗粒剂、胶囊剂、注射 剂、酊剂、口服液、片剂或含片。
在另一优选例中,所述的组合物或制剂通过外用、局部、或注射方式施用。
在另一优选例中,所述无细胞脂肪提取物不含有细胞且不含有脂滴。
在另一优选例中,所述脂滴为脂肪细胞破碎后释放的油滴。
在另一优选例中,所述“不含有脂滴”指所述无细胞脂肪提取物中,油滴体积占总液体百分比小于1%,优选地小于0.5%,更优选地小于0.1%。
在另一优选例中,所述细胞选自下组:内皮细胞、脂肪干细胞、巨噬血细胞、基质细胞。
在另一优选例中,所述“无细胞”指1ml无细胞脂肪提取物中的细胞平均数量≤1个,优选地≤0.5个,更佳地≤0.1个,或为0个。
在另一优选例中,所述无细胞脂肪提取物为天然获得的无添加成分的纳米脂肪提取物。
在另一优选例中,所述“无添加成分的”指除漂洗步骤外,在所述脂肪提取物的制备过程中未添加任何溶液、溶剂、小分子、化学制剂、和生物添加剂。
在另一优选例中,所述种无细胞脂肪提取物是通过将脂肪组织经过乳化后离心制备获得。
在另一优选例中,所述的无细胞脂肪提取物含有一种或多种选自下组的组分:IGF-1、BDNF、GDNF、TGF-β1、HGF、bFGF、VEGF、TGF-β1、PDGF、EGF、NT-3、GH、G-CSF,或其组合。
在另一优选例中,所述的种无细胞脂肪提取物含有但不限于一种或多种选自下组的组分:IGF-1、BDNF、GDNF、bFGF、VEGF、TGF-β1、HGF、PDGF,或其组合。
在另一优选例中,所述的无细胞脂肪提取物为无细胞脂肪提取液。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的IGF-1的浓度为5000-30000pg/ml,较佳地6000-20000pg/ml,更佳地7000-15000pg/ml,更佳地8000-12000pg/ml,更佳地9000-11000pg/ml,更佳地9500-10500pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的BDNF的浓度为800-5000pg/ml,较佳地1000-4000pg/ml,更佳地1200-2500pg/ml,更佳地1400-2000 pg/ml,更佳地1600-2000pg/ml,更佳地1700-1850pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的GDNF的浓度为800-5000pg/ml,较佳地1000-4000pg/ml,更佳地1200-2500pg/ml,更佳地1400-2000pg/ml,更佳地1600-2000pg/ml,更佳地1700-1900pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的bFGF的浓度为50-600pg/ml,较佳地100-500pg/ml,更佳地120-400pg/ml,更佳地150-300pg/ml,更佳地200-280pg/ml,更佳地220-260pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的VEGF的浓度为50-500pg/ml,较佳地100-400pg/ml,更佳地120-300pg/ml,更佳地150-250pg/ml,更佳地170-230pg/ml,更佳地190-210pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的TGF-β1的浓度为200-3000pg/ml,较佳地400-2000pg/ml,更佳地600-1500pg/ml,更佳地800-1200pg/ml,更佳地800-1100pg/ml,更佳地900-1000pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的HGF的浓度为200-3000pg/ml,较佳地400-2000pg/ml,更佳地600-1500pg/ml,更佳地600-1200pg/ml,更佳地800-1000pg/ml,更佳地850-950pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的PDGF的浓度为50-600pg/ml,较佳地80-400pg/ml,更佳地100-300pg/ml,更佳地140-220pg/ml,更佳地160-200pg/ml,更佳地170-190pg/ml。
在另一优选例中,所述的IGF-1与VEGF的重量比为20-100:1,较佳地30-70:1,更佳地40-60:1,最佳地45-55:1。
在另一优选例中,所述的BDNF与VEGF的重量比为2-20:1,较佳地4-15:1,更佳地6-12:1,最佳地8-9.5:1。
在另一优选例中,所述的GDNF与VEGF的重量比为2-20:1,较佳地4-15:1,更佳地6-12:1,最佳地8.5-9.5:1。
在另一优选例中,所述的bFGF与VEGF的重量比为0.2-8:1,较佳地0.5-5:1,更佳地0.6-2:1,更佳地0.8-1.6:1,最佳地1-1.5:1。
在另一优选例中,所述的TGF-β1与VEGF的重量比为1-20:1,较佳地1-15:1, 更佳地1-10:1,更佳地2-8:1,更佳地4-6:1。
在另一优选例中,所述的HGF与VEGF的重量比为1-20:1,较佳地1-15:1,更佳地1-10:1,更佳地2-8:1,更佳地4-5.5:1。
在另一优选例中,所述的PDGF与VEGF的重量比为0.1-3:1,较佳地0.2-2:1,更佳地0.4-1.5:1,最佳地0.7-1.2:1。
在另一优选例中,所述的无细胞脂肪提取物通过以下方法制备:
(1)提供一脂肪组织原料,将所述脂肪组织原料破碎,并进行漂洗(如用生理盐水),从而获得经漂洗的脂肪组织;
(2)对所述经漂洗后的脂肪组织进行离心,获得分层的混合物;
(3)对所述分层的混合物,去除上层油层和下层水层,收集中间层(即含脂肪细胞的脂肪层);
(4)对所述中间层进行乳化,获得乳化的脂肪混合物(也称为纳米脂肪);
(5)将所述乳化的脂肪混合物通过离心处理,从而获得中间液体层,即为脂肪初提物;和
(6)对所述脂肪初提物进行过滤和除菌,从而获得无细胞的脂肪提取物。
本发明第二方面,提供一种制备无细胞脂肪提取物的方法,所述的方法包括步骤:
(1)提供一脂肪组织原料,将所述脂肪组织原料破碎,并进行漂洗(如用生理盐水),从而获得经漂洗的脂肪组织;
(2)对所述经漂洗后的脂肪组织进行离心,获得分层的混合物;
(3)对所述分层的混合物,去除上层油层和下层水层,收集中间层(即含脂肪细胞的脂肪层);
(4)对所述中间层进行乳化,获得乳化的脂肪混合物(也称为纳米脂肪);
(5)将所述乳化的脂肪混合物通过离心处理,从而获得中间液体层,即为脂肪初提物;和
(6)对所述脂肪初提物进行过滤和除菌,从而获得无细胞的脂肪提取物。
在另一优选例中,所述的无细胞脂肪提取物如本发明第一方面所述。
在另一优选例中,所述的步骤(2)中,所述离心在800-2500g下离心,较佳地800-2000g,更佳地1000-1500g,最佳地1100-1300g。
在另一优选例中,所述的步骤(2)中,所述离心的时间为1-15min,较佳地1-10min,更佳地1-8min,最佳地1-5min。
在另一优选例中,所述的离心的温度为2-6℃。
在另一优选例中,所述的步骤(4)中,所述的乳化为机械乳化。
在另一优选例中,所述机械乳化为经注射器反复吹打(如吹打20-200次,较佳地20-150次,更佳地20-100次,更佳地30-50次)进行机械乳化。
在另一优选例中,所述的吹打的方式为2个10ml注射针筒连接三通管反复匀速推打。
在另一优选例中,,所述的步骤(4)中,所述乳化为通过组织匀浆机打碎的方法。
在另一优选例中,所述的步骤(5)中,在将所述乳化的脂肪混合物通过离心处理前,还包括对所述乳化的脂肪混合物冷冻后解冻处理。
在另一优选例中,冷冻后解冻处理后,将解冻后的混合物用于离心。
在另一优选例中,所述的冷冻的温度为-50℃至-120℃,较佳地-60℃至-100℃,更佳地-70℃至-90℃。
在另一优选例中,所述的解冻的温度为20-40℃,较佳地25-40℃,更佳地37℃。
在另一优选例中,所述的冷冻后解冻的循环次数为1-5次(优选为1、2、3或4次)。
在另一优选例中,所述的步骤(5)中,离心后,所述乳化的脂肪混合物分层4层,第一层为油层,第二层为残余脂肪组织层,第三层为液体层(即为中间液体层),第四层为细胞/组织碎片沉淀层。
在另一优选例中,所述的步骤(5)中,所述离心在800-2500g下离心,较佳地800-2000g,更佳地1000-1500g,最佳地1100-1300g。
在另一优选例中,所述的步骤(5)中,所述离心的时间为1-15min,较佳地1-10min,更佳地2-8min,最佳地3-7min。
在另一优选例中,所述的离心的温度为2-6℃。
在另一优选例中,所述的步骤(5)中,第一层、第二层、第三层和第四层从上到下依次排列。
在另一优选例中,所述的步骤(5)中,所述的中间液体层为透明或基本透明层。
在另一优选例中,所述的步骤(6)中,所述的过滤包能够将脂肪初提物中的脂肪细胞除去。
在另一优选例中,所述的步骤(6)中,所述的过滤和除菌是通过滤器(如0.22μm微孔滤膜)进行。
在另一优选例中,所述的过滤器为微孔滤膜过滤器。
在另一优选例中,所述的微孔滤膜的孔径大小为0.05-0.8μm,较佳地0.1-0.5μm,更佳地0.1-0.4μm,更佳地0.15-0.3μm,更佳地0.2-0.25μm,最佳地0.22μm。
在另一优选例中,所述的步骤(6)中,所述的过滤和除菌是先通过可滤去细胞的第一过滤器,然后再通过可滤去病原体(如细菌)的第二滤器(如0.22μm的滤器)进行的。
在另一优选例中,所述的步骤(6)中,还包括对所述脂肪提取物进行分装,形成分装的产品。(所述分装后的提取物可于-20℃保存待用;可低温(如-4℃)或常温解冻后直接使用,或解冻后置于低温(如4℃)保存一段时间,然后使用)。
本发明第三方面,提供一种无细胞脂肪提取物,所述的无细胞脂肪提取物通过如本发明第二方面所述的方法制备获得。
本发明第四方面,提供一种用于预防和/或治疗视神经损伤组合物或制剂,所述的组合物或制剂包含(a)如本发明第三方面所述的无细胞脂肪提取物;和(b)药学上、食品上、保健品或膳食上可接受的载体或赋形剂。
在另一优选例中,所述的组合物为药物组合物、食品组合物、保健品组合物或膳食补充剂。
在另一优选例中,所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。
在另一优选例中,所述组合物或制剂的剂型为粉剂、颗粒剂、胶囊剂、注射剂、酊剂、口服液、片剂或含片。
在另一优选例中,所述的注射剂为静脉注射剂或肌肉注射剂。
在另一优选例中,所述组合物或制剂的剂型为固体剂型、半固体剂型、或液体剂型,如溶液、凝胶、膏霜、乳液、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
在另一优选例中,在所述组合物或制剂中,无细胞脂肪提取物的质量百分比为5wt%,较佳地1-20wt%,以合物或制剂的总重量计。
本发明第五方面,提供一种制备如本发明第四方面所述的组合物或制剂的方法,所述的方法包括步骤:将如本发明第三方面所述的无细胞脂肪提取物与药学上、食品上、保健品或膳食上可接受的载体或赋形剂混合,从而形成组合物或制剂。
本发明第六方面,提供一种预防和/或治疗视神经损伤的方法,对需要的对象施用如本发明第三方面所述的的无细胞脂肪提取物。
在另一优选例中,所述的对象为人或非人哺乳动物。
在另一优选例中,所述非人哺乳动物包括啮齿动物,如大鼠、小鼠。
在另一优选例中,所述的施用方式为口服、外用或注射施用。
在另一优选例中,所述注射施用的剂量为3-5μg/μL。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
图1为实验流程图。
图2为CEFFE可促进视神经损伤后轴突的再生。图2A为损伤14天后,不同给药组中CTB-55示踪的再生轴突(*为钳夹损伤处),图2B为图2A中距损伤不同距离处,再生轴突数量的统计(***p<0.001)。其中,PBS为磷酸 盐缓冲液空白对照,CEFFE
Thrice为分别在视神经损伤即刻、损伤3天后和损伤7天后玻璃体腔内注射3μl CEFFE,CEFFE
Once为在视神经损伤即刻玻璃体腔内注射3μl CEFFE。
图3为CEFFE和BDNF细胞因子促进视神经损伤后的轴突再生(*为钳夹损伤处)。其中,图3A为玻璃体腔分别注射PBS、BDNF及CEFFE后CTB-55示踪的轴突再生情况。图3B为图3A图中再生轴突数目的统计(**p<0.01)。
图4为视网膜铺片免疫荧光图。其中,图4A为未损伤组视网膜铺片统计存活RGC图;图4B为在视神经损伤动物模型中玻璃体腔注射PBS后,RGC的存活情况。图4C为在视神经损伤动物模型中玻璃体腔注射CEFFE后,RGC的存活情况。(视网膜铺片免疫荧光染色TUJ1来标记存活RGC)。图4D为图4A-C图中存活的RGC量化统计(**p<0.05)。
本发明人经过广泛而深入的研究,首次开发了无细胞脂肪提取物对视神经损伤具有优异的治疗作用。本发明实施例研究表明,本发明所述的无细胞脂肪提取液对视神经损伤具有优异的治疗效果。在此基础上完成了本发明。
术语
除非另有定义,否则本文中所用的所有技术和科学术语的含义与本发明所属领域普通技术人员普遍理解的含义相同。
如本文所用,术语“包括”、“包含”与“含有”可互换使用,不仅包括开放式定义,还包括半封闭式、和封闭式定义。换言之,所述术语包括了“由……构成”、“基本上由……构成”。
在本发明中,术语“预防”表示预防疾病和/或它的附随症状的发作或者保护对象免于获得疾病的方法。本文中使用的"预防"还包括延迟疾病和/或它的附随症状的发作和降低对象的得病的风险。
本发明所述的“治疗”包括延缓和终止疾病的进展,或消除疾病,并不需要100%抑制、消灭和逆转。在一些实施方案中,与不存在本发明所述的无细胞脂 肪提取物观相比,本发明所述无细胞脂肪提取物减轻、抑制和/或逆转了视神经损伤例如至少约10%、至少约30%、至少约50%、或至少约80%。
如文本所用,术语“IGF-1”称为胰岛素样生长因子1(insulin-like growth factors-1)。
如文本所用,术语“BDNF”称为脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)。
如文本所用,术语“GDNF”称为胶质细胞源性神经营养因子(glialcellline-derivedneurotrophicfactor)。
如文本所用,术语“bFGF”称为碱性成纤维细胞生长因子(basic fibroblast growth factor)。
如文本所用,术语“VEGF”称为血管内皮生长因子(vascular endothelial growth factor)。
如文本所用,术语“TGF-β1”称为转化生长因子-β1(transforming growth factor-β1)。
如文本所用,术语“HGF”称为肝细胞生长因子
如文本所用,术语“PDGF”称为血小板衍生生长因子(Platelet derived growth factor)
如文本所用,术语“EGF”称为表皮细胞生长因子(Epidermal Growth Factor)
如文本所用,术语“NT-3”称为神经营养因子3(neurotrophins-3)。
如文本所用,术语“GH”称为生长激素(Growth Hormone)。
如文本所用,术语“G-CSF”称为粒细胞集落刺激因子(granulocyte colony stimulating factor)。
无细胞脂肪提取物(Cell free fat extract,CEFFE)及其制备方法
如本文所用,术语“本发明的无细胞脂肪提取物”、“本发明提取物”、“本发明的脂肪提取物”等可互换使用,指在脂肪提取物制备过程中(除漂洗步骤外)未添加任何溶液、溶剂、小分子、化学制剂、和生物添加剂所制备的源自脂肪组织的提取物(或提取液)。一种典型的制备本发明提取物的方法如上本发明第二 方面中所述。此外,应理解,虽然本发明提取物在制备过程中不必添加任何添加剂(或添加成分),但是也可以添加一些或少量的对本发明提取物的活性无负面或不利影响的安全的物质(如少量水)。
本发明的无细胞脂肪提取物可来源于人类脂肪组织,它是通过离心后除去油和细胞/细胞外基质部分而从纳米脂肪中提纯出来的,是一种无细胞、易于制备、富含各种生长因子的液体。
在本发明的一个优选例中,所述的无细胞脂肪提取物为无细胞脂肪提取液。
在本发明所述的无细胞脂肪提取物,可以包括多种细胞因子。代表性地,所述的无细胞脂肪提取物包括IGF-1、BDNF、GDNF、TGF-β、HGF、bFGF、VEGF、TGF-β1、PDGF、EGF、NT-3、GH和G-CSF中的一种或多种。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的IGF-1的浓度为5000-30000pg/ml,较佳地6000-20000pg/ml,更佳地7000-15000pg/ml,更佳地8000-12000pg/ml,更佳地9000-11000pg/ml,更佳地9500-10500pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的BDNF的浓度为800-5000pg/ml,较佳地1000-4000pg/ml,更佳地1200-2500pg/ml,更佳地1400-2000pg/ml,更佳地1600-2000pg/ml,更佳地1700-1850pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的GDNF的浓度为800-5000pg/ml,较佳地1000-4000pg/ml,更佳地1200-2500pg/ml,更佳地1400-2000pg/ml,更佳地1600-2000pg/ml,更佳地1700-1900pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的bFGF的浓度为50-600pg/ml,较佳地100-500pg/ml,更佳地120-400pg/ml,更佳地150-300pg/ml,更佳地200-280pg/ml,更佳地220-260pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的VEGF的浓度为50-500pg/ml,较佳地100-400pg/ml,更佳地120-300pg/ml,更佳地150-250pg/ml,更佳地170-230pg/ml,更佳地190-210pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的TGF-β1的浓度为200-3000pg/ml,较佳地400-2000pg/ml,更佳地600-1500pg/ml,更佳地800-1200pg/ml,更佳地800-1100pg/ml,更佳地900-1000pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的HGF的浓度为200-3000pg/ml,较佳地400-2000pg/ml,更佳地600-1500pg/ml,更佳地600-1200pg/ml,更佳地800-1000pg/ml,更佳地850-950pg/ml。
在另一优选例中,在所述的无细胞脂肪提取物中,所述的PDGF的浓度为50-600pg/ml,较佳地80-400pg/ml,更佳地100-300pg/ml,更佳地140-220pg/ml,更佳地160-200pg/ml,更佳地170-190pg/ml。
在另一优选例中,所述的IGF-1与VEGF的重量比为20-100:1,较佳地30-70:1,更佳地40-60:1,最佳地45-55:1。
在另一优选例中,所述的BDNF与VEGF的重量比为2-20:1,较佳地4-15:1,更佳地6-12:1,最佳地8-9.5:1。
在另一优选例中,所述的GDNF与VEGF的重量比为2-20:1,较佳地4-15:1,更佳地6-12:1,最佳地8.5-9.5:1。
在另一优选例中,所述的bFGF与VEGF的重量比为0.2-8:1,较佳地0.5-5:1,更佳地0.6-2:1,更佳地0.8-1.6:1,最佳地1-1.5:1。
在另一优选例中,所述的TGF-β1与VEGF的重量比为1-20:1,较佳地1-15:1,更佳地1-10:1,更佳地2-8:1,更佳地4-6:1。
在另一优选例中,所述的HGF与VEGF的重量比为1-20:1,较佳地1-15:1,更佳地1-10:1,更佳地2-8:1,更佳地4-5.5:1。
在另一优选例中,所述的PDGF与VEGF的重量比为0.1-3:1,较佳地0.2-2:1,更佳地0.4-1.5:1,最佳地0.7-1.2:1。
优选地,本发明所述的无细胞脂肪提取物通过如上述本发明第二方面所述的方法制备获得。
代表性地,本发明所述的无细胞脂肪提取物通过以下方法制备:
(1)提供一脂肪组织原料,将所述脂肪组织原料破碎,并进行漂洗(如用生理盐水),从而获得经漂洗的脂肪组织;
(2)对所述经漂洗后的脂肪组织进行离心,获得分层的混合物;
(3)对所述分层的混合物,去除上层油层和下层水层,收集中间层(即含脂肪细胞的脂肪层);
(4)对所述中间层进行乳化,获得乳化的脂肪混合物(也称为纳米脂肪);
(5)将所述乳化的脂肪混合物通过离心处理,从而获得中间液体层,即为脂肪初提物;和
(6)对所述脂肪初提物进行过滤和除菌,从而获得无细胞的脂肪提取物。
在另一优选例中,所述的步骤(2)中,所述离心在800-2500g下离心,较佳地800-2000g,更佳地1000-1500g,最佳地1100-1300g。
在另一优选例中,所述的步骤(2)中,所述离心的时间为1-15min,较佳地1-10min,更佳地1-8min,最佳地1-5min。
在另一优选例中,所述的步骤(4)中,所述的乳化为机械乳化。
在另一优选例中,所述机械乳化为经注射器反复吹打(如吹打20-200次,较佳地20-150次,更佳地20-100次,更佳地30-50次)进行机械乳化。
在另一优选例中,所述的吹打的方式为2个10ml注射针筒连接三通管反复匀速推打。
在另一优选例中,所述的步骤(4)中,所述乳化为通过组织匀浆机打碎的方法。
在另一优选例中,所述的步骤(5)中,在将所述乳化的脂肪混合物通过离心处理前,还包括对所述乳化的脂肪混合物冷冻后解冻处理。
在另一优选例中,冷冻后解冻处理后,将解冻后的混合物用于离心。
在另一优选例中,所述的冷冻的温度为-50℃至-120℃,较佳地-60℃至-100℃,更佳地-70℃至-90℃。
在另一优选例中,所述的解冻的温度为20-40℃,较佳地25-40℃,更佳地37℃。
在另一优选例中,所述的冷冻后解冻的循环次数为1-5次(优选为1、2、3或4次)。
在另一优选例中,所述的步骤(5)中,离心后,所述乳化的脂肪混合物分层4层,第一层为油层,第二层为残余脂肪组织层,第三层为液体层(即为中间液体层),第四层为细胞/组织碎片沉淀层。
在另一优选例中,所述的步骤(5)中,所述离心在800-2500g下离心,较佳地800-2000g,更佳地1000-1500g,最佳地1100-1300g。
在另一优选例中,所述的步骤(5)中,所述离心的时间为1-15min,较佳地1-10min,更佳地2-8min,最佳地3-7min。
在另一优选例中,所述的步骤(5)中,第一层、第二层、第三层和第四层从上到下依次排列。
在另一优选例中,所述的步骤(5)中,所述的中间液体层为透明或基本透明层。
在另一优选例中,所述的步骤(6)中,所述的过滤包能够将脂肪初提物中的脂肪细胞除去。
在另一优选例中,所述的步骤(6)中,所述的过滤和除菌是通过滤器(如0.22μm微孔滤膜)进行。
在另一优选例中,所述的过滤器为微孔滤膜过滤器。
在另一优选例中,所述的微孔滤膜的孔径大小为0.05-0.8μm,较佳地0.1-0.5μm,更佳地0.1-0.4μm,更佳地0.15-0.3μm,更佳地0.2-0.25μm,最佳地0.22μm。
在另一优选例中,所述的步骤(6)中,所述的过滤和除菌是先通过可滤去细胞的第一过滤器,然后再通过可滤去病原体(如细菌)的第二滤器(如0.22μm的滤器)进行的。
在另一优选例中,所述的步骤(6)中,还包括对所述脂肪提取物进行分装,形成分装的产品。(所述分装后的提取物可于-20℃保存待用;可低温(如-4℃)或常温解冻后直接使用,或解冻后置于低温(如4℃)保存一段时间,然后使用)。
用途
本发明提供一种无细胞脂肪提取物的用途,用于制备组合物或制剂,所述组合物或制剂用于预防和/或治疗视神经损伤。
在本发明一个优选例中,所述的视神经损伤包括创伤应激导致的视神经损伤。
在本发明另一优选例中,所述的预防和/或治疗视神经损伤包括选自下组的一种或多种方式进行预防和/或治疗:
(i)促进损伤后的视神经轴突再生;和/或
(ii)促进视网膜神经细节细胞(RGC)的存活和/或再生。
本发明还提供一种一种预防和/或治疗视神经损伤的方法,对需要的对象施用如本发明所述的的无细胞脂肪提取物。
在另一优选例中,所述的对象为人或非人哺乳动物。
在另一优选例中,所述非人哺乳动物包括啮齿动物,如大鼠、小鼠。
在另一优选例中,所述的施用方式为口服、外用或注射施用。
在另一优选例中,所述的注射施用为眼部注射施用。
在另一优选例中,所述的注射施用为玻璃体腔注射施用。
组合物和施用
本发明所述的组合物包括(但并不限于):药物组合物、食品组合物、保健组合物、膳食补充剂等。
代表性地,可将本发明的无细胞脂肪提取物制备成药物组合物,诸如片剂、胶囊、粉剂、微粒剂、溶液剂、锭剂、胶冻、乳膏制剂、醑剂、悬液、酊、泥敷剂、搽剂、洗剂、和气雾剂之类的剂型。药物组合物能够由通常已知的制备技术来制备,并且合适的药物添加剂能够被添加到该药物中。
本发明的组合物还可以包括药学上、食品上、保健品或膳食上可接受的载体。“药学上、食品上、保健品或膳食上可接受的载体”指的是:一种或多种相容性固体或液体填料或凝胶物质,它们适合于人使用,而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和,而不明显降低化合物的药效。药学上、食品上、保健品或膳食上可接受的载体可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明组合物施用方式没有特别限制,代表性的施用方式包括(但并不限 于):口服、肠胃外(静脉内、肌肉内)、局部施用,优选的施用方式为口服施用和注射施用。例如,所述的注射施用为眼部注射施用,优选为璃体腔注射制剂。
本发明所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。代表性地,用于口服施用或给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中,活性化合物与至少一种常规惰性赋形剂(或载体)混合,如柠檬酸钠或磷酸二钙,或与下述成分混合:(a)填料或增容剂,例如,淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸;(b)粘合剂,例如,羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶;(c)保湿剂,例如,甘油;(d)崩解剂,例如,琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠;(e)缓溶剂,例如石蜡;(f)吸收加速剂,例如,季胺化合物;(g)润湿剂,例如鲸蜡醇和单硬脂酸甘油酯;(h)吸附剂,例如,高岭土;和(i)润滑剂,例如,滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠,或其混合物。胶囊剂、片剂和丸剂中,剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备,如肠衣和其它本领域公知的材料。它们可包含不透明剂,。
用于口服施用或给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外,液体剂型可包含本领域中常规采用的惰性稀释剂,如水或其它溶剂,增溶剂和乳化剂,例知,乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油,特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外,组合物也可包含助剂,如润湿剂、乳化剂和悬浮剂、甜味剂、娇味剂和香料。
除了活性成分外,悬浮液可包含悬浮剂,例如,乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液,和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及 其适宜的混合物。
用于局部施用或给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂,或必要时可能需要的推进剂一起混合。
本发明无细胞脂肪提取物可以单独施用或给药,或者与其它预防和/或治疗脂肪肝和/或其并发症的药物联合施用或给药。
施用组合物时,是将安全有效量的本发明无细胞脂肪提取物适用于需要治疗的人或非人动物(如大鼠、小鼠、狗、猫、牛、鸡、鸭等),其中施用时剂量为药学上、食品上或保健品上可接受认为的有效给药剂量。如本文所用,术语“安全有效量”,是指对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。本领域的普通技术人员应该理解,所述的“安全有效量”可随着药物组合物的形式、给药途径、所用药物的辅料、疾病的严重程度以及与其他药物联合用药等情况的不同而有所不同。例如,对于60kg体重的人而言,日给药剂量通常为0.1~1000mg,优选1~600mg,更优选为2-300mg。当然,具体剂量还应考虑给药途径、病人健康状况等因素,这些都是熟练医师技能范围之内的。
本发明的主要优点包括:
1.本发明首次发现无细胞脂肪提取物对视神经损伤具有优异的治疗效果,从而用于治疗视神经损伤。
2.本发明所述的无细胞脂肪提取物是一种无细胞组分,可以避免临床应用中与细胞相关的问题,例如包括细胞加工后的遗传稳定性,注射后的细胞活性和存活率,细胞的多次给药储存,以及使用同种异体脂肪时细胞的免疫原性,本发明所述的无细胞脂肪提取物在作为制备防治视神经损伤中有着较高的安全性和较低副作用的优势。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非另外说明,否则 百分比和份数按重量计算。
实施例1
1.实验方法
1.1.无细胞脂肪提取液(CEFFE)的制备
脂肪由自愿者在获得知情同意的条件下获得。无细胞脂肪组织提取液的制备方法如下:
(1)脂肪组织获取自6名常规脂肪抽吸术的健康女性,平均年龄31岁(24-36岁)。局部注射肿胀液麻醉后,使用具有大侧孔(2mm x 7mm)的3mm吸脂抽脂套管连接20mL注射器,人工负压下放射状抽吸,将获得的脂肪直立静止,去除肿胀液后,用生理盐水漂洗3遍。
(2)取经漂洗后的脂肪组织,置于离心管中,放入离心机中以1200g 4℃离心3分钟后,获得分层的混合物。
(3)对所述分层的混合物,去除上层油层和下层水层,收集中间层(即含脂肪细胞的脂肪层)。
(4)对所述中间层,用2个10ml注射针筒连接三通管反复匀速推打30次,从而进行机械乳化,并获得经机械乳化的脂肪混合物(也称为纳米脂肪)。
(5)将所述经机械乳化的脂肪混合物置入-80℃冰箱冷冻,再进行37℃水浴解冻,单次冻融循环后,将解冻后的脂肪混合物以1200g 4℃离心5分钟,获得分层的混合物,分层的混合物共分为4层,第一层为油层,第二层为残余脂肪组织层,第三层为液体层,第四层为细胞/组织碎片沉淀层,去除油层和残余脂肪组织层,吸取液体层,吸取过程中避免细胞/组织碎片沉淀层污染,从而得到脂肪初提取液。
(6)将得到的脂肪初提取液经0.22μm滤器过滤除菌,从而灭菌并去除可能混有的活细胞,从而获得无细胞脂肪提取液(CEFFE),分装冻存于-20℃保存,使用时4℃解冻。
对制备得到的无细胞脂肪提取液,使用ELISA免疫吸附测定试剂盒检测细胞因子含量,包括IGF-1、BDNF、GDNF、bFGF、VEGF、TGF-β1、HGF和 PDGF等细胞因子。6例样本检测平均浓度如下:IGF-1(9840.6pg/ml)、BDNF(1764.5pg/ml)、GDNF(1831.9pg/ml)、bFGF(242.3pg/ml)、VEGF(202.9pg/ml)、TGF-β1(954.5pg/ml)、HGF(898.4pg/ml)和PDGF(179.9pg/ml)。
1.2小鼠视神经损伤模型的建立
1)选取5周龄雄性C57BL/6小鼠,使用1%戊巴比妥钠注射液(10μl/g)行腹腔注射麻醉,待小鼠麻醉后在左侧眼球处滴加盐酸奥布卡因角膜表面麻醉剂后置于解剖显微镜下。取一大小合适物体置于小鼠头下,使小鼠头部处于水平位置。
2)选取外眦处作为手术入路,先用精细镊提起结膜,向外牵拉眼球,用venus剪在外眦结膜处剪开一小口直至球结膜层,向下向后钝性分离球后组织,暴露视神经。操作应轻柔仔细,避免损伤球后动脉。
3)仔细剥开视神经鞘膜,于球后1mm处用Dumont#5自闭合镊夹持视神经5秒钟。切勿损伤鞘内血管以免影响眼底血供和回流。对照组暴露视神经但不进行钳夹。
4)消毒伤口,在切口缘涂抹红霉素眼膏。观察眼底血供,将出现眼底动脉苍白、晶状体受损的小鼠剔除出组,另选补充。
1.3再生轴突示踪
1)配制神经示踪剂,用预冷的PBS溶解CTB-555(是指霍乱毒素B亚基)粉末,使终浓度为2μg/ul,分装冻存在-20℃冰箱备用,使用及储存过程中注意避光,避免反复冻融。
2)视神经损伤2周后,再次用1%戊巴比妥钠注射液(10μl/g)行腹腔注射麻醉,待小鼠麻醉后在左侧眼球处滴加复方托吡卡胺滴眼液放大瞳孔。
3)同样的方法在角巩膜缘后约1mm处用微量注射器斜行45°向下进针,注意不要损伤晶状体及视网膜,抽取1.5ul玻璃体液后拔出注射器,这有助于接下来的玻璃体腔注射并能预防因注射引起的眼内压升高。
4)再次使用微量注射器从原针口进针,将准备好的CTB-555溶液1.5ul缓慢 地注射到小鼠玻璃体腔内。注射完毕后停留5min待试剂充分浸润玻璃体腔再拔针,以防回流。
5)术后涂抹妥布霉素地塞米松眼膏预防感染,转入温育箱待麻醉苏醒后回笼饲养。术后密切观察,出现晶状体浑浊、玻璃体腔大量出血及视网膜大范围剥离者剔除。
6)3天后麻醉处死小鼠取材。
1.4视网膜铺片免疫荧光
1)实验小鼠麻醉后,使用提前预冷的PBS从左心室冲洗灌流全身血管。3-5min待血全部冲洗完毕,换用提前预冷的4%PFA(1xPBS)灌流5-10min。
2)使用显微剪刀、显微镊轻轻分离取出眼球和视神经,仔细去除周围软组织,3)在角膜中央用针头戳一小口。将修剪好的组织放入4%PFA中4℃后固定2小时。接着再用PBS冲洗两遍。然后将组织放入30%的蔗糖溶液中于4℃冰箱中脱水48小时。
4)取出脱水后组织,在PBS中小心解剖分离出完整的视网膜,操作要仔细轻柔,避免损伤视网膜。分离出视网膜后,用1xPBS润洗3遍,每次10min。
5)将视网膜置于含有封闭液的24孔板中,室温封闭2小时。封闭结束后,吸去封闭液,加入一抗(TUJ1 1:300)4℃孵育48小时。
6)两天后,从4℃冰箱中取出铺片,常温复性30min,吸去一抗。1xPBST洗三遍,每次10min。
7)加入相应的荧光二抗及DAPI(二抗1:500,DAPI 1:1000),室温避光孵育2h。1xPBST洗3遍,每次10min。
8)将视网膜沿颞侧、鼻侧、背侧、腹侧四个方向用剪刀小心剪成花瓣状,GCL层向上,平铺在载玻片上。
9)吸弃玻片及铺片上残留的PBST,待稍干燥后,滴加适量抗荧光淬灭剂,封片。
激光共聚焦荧光显微镜拍照观察、拍片。
1.5存活RGC细胞(视网膜神经节细胞)及再生轴突统计
对于损伤后的视神经再生轴突的统计采用已知报道过的方法,介绍如下:损伤位置后CTB-555标记上的轴突视为再生轴突,取距损伤点不同位置处的再生轴突进行统计,取每根视神经相同的切片层数进行统计并取平均数。距损伤点某处的再生轴突数目统计公式为:Σad=πr
2x[average axons/mm]/t。其中d为距损伤处的距离,r为该处视神经的直径,t为切片的厚度(本实验中均为14um)。对于损伤后存活的RGC采用如下方法进行统计简单介绍如下:在视网膜铺片的每个象限内随机选区一个视野采用Image J软件对TUJ1+的RGCs进行计数。
1.6统计学方法
采用Graphpad统计软件进行统计学处理,各组数据均以mean±S.D.表示,进行方差齐性检测,两组之间比较采用t检验,多组间比较采用单因素方差分析,p<0.05代表有统计学差异。
2.结果
2.1CEFFE可促进损伤轴突再生
实验流程如图1所示,其中图1A为取5周大小C57BL/6小鼠,在视乳头后1mm处钳夹视神经5s,建立视神经损伤模型。图1B为分别在损伤即刻、损伤3天后及损伤7天后玻璃体腔内注射3μl CEFFE。图1C为两周后,玻璃体腔内注射1.5μl的CTB-55示踪再生轴突。
CEFFE可促进视神经损伤后轴突的再生如图2所示,图2A为损伤14天后,不同给药组中CTB-55示踪的再生轴突(*为钳夹损伤处),在图2B中,取距离损伤处250、500、750、1000、1500mm处对轴突数目进行统计,可以看出,玻璃体腔内注射CEFFE的实验组在损伤2周后最远可长至约1.5mm处,而且在不同距离其轴突再生的数量都显著高于对照组。以上结果说明CEEFE对损伤后的视神经轴突再生具有促进作用。
2.2CEFFE比单独应用生长因子更能促进轴突再生
分别采用BDNF细胞因子和CEFFE在同样实验方法下测定视神经损伤后 轴突再生,同时PBS为磷酸盐缓冲液空白对照,结果如图3所示,从图3A-3B中可以看出,低浓度的BDNF并没有促进轴突再生,而在同等浓度的情况下(5ug/ul),与BDNF相比,CEFFE能够显著促进轴突再生,表明在促进神经损伤后的轴突再生方面,CEFFE中各种因子之间起到相互协调的作用。
2.3CEFFE可促进视网膜神经细节细胞(RGC)存活
视网膜铺片免疫荧光图如图4所示,从图4A-4D中可以看出,在视神经损伤动物模型中,与PBS给药相比,CEFFE能够显著促进损伤RGC(视网膜神经节细胞)的存活,从而治疗视神经损伤。
结论:
在本实施例中可以看出,CEFFE可促进受损视神经的再生,促进损伤RGC存活,从而用于预防和治疗视神经损伤。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (15)
- 一种无细胞脂肪提取物的用途,其特征在于,用于制备组合物或制剂,所述组合物或制剂用于预防和/或治疗视神经损伤。
- 如权利要求1所述的用途,其特征在于,所述的视神经损伤包括创伤应激导致的视神经损伤。
- 如权利要求1所述的用途,其特征在于,所述的预防和/或治疗视神经损伤包括选自下组的一种或多种方式进行预防和/或治疗:(i)促进损伤后的视神经轴突再生;和/或(ii)促进视网膜神经细节细胞(RGC)的存活和/或再生。
- 如权利要求1所述的用途,其特征在于,所述的预防和/或治疗视神经损伤具有选自下组的一个或多个特征:(i)在损伤2周后视神经轴突可长至约1.5-1.8mm;(ii)在损伤2周后视神经轴突在0.25mm处的数量有约2300-2500个;(iii)视网膜神经细节细胞存活率超过40%。
- 如权利要求1所述的用途,其特征在于,所述的组合物或制剂包括药物组合物或制剂、食品组合物或制剂、保健品组合物或制剂或膳食补充剂。
- 如权利要求1所述的用途,其特征在于,所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。
- 如权利要求6所述的用途,其特征在于,所述的注射制剂为眼部注射制剂。
- 如权利要求1所述的用途,其特征在于,所述的组合物或制剂的剂型为玻璃体腔注射制剂。
- 如权利要求1所述的用途,其特征在于,所述的无细胞脂肪提取物含有一种或多种选自下组的组分:IGF-1、BDNF、GDNF、TGF-β1、HGF、bFGF、VEGF、TGF-β1、HGF、PDGF、EGF、NT-3、GH、G-CSF,或其组合。
- 如权利要求1所述的用途,其特征在于,所述的无细胞脂肪提取物含有一种或多种选自下组的组分:IGF-1、BDNF、GDNF、bFGF、VEGF、TGF-β1、HGF、PDGF,或其组合。
- 如权利要求9所述的用途,其特征在于,所示的无细胞脂肪提取物包括选自下组的一种或多种特征:在所述的无细胞脂肪提取物中,所述的IGF-1的浓度为5000-30000pg/ml,较佳地6000-20000pg/ml,更佳地7000-15000pg/ml,更佳地8000-12000pg/ml,更佳地9000-11000pg/ml,更佳地9500-10500pg/ml;在所述的无细胞脂肪提取物中,所述的BDNF的浓度为800-5000pg/ml,较佳地1000-4000pg/ml,更佳地1200-2500pg/ml,更佳地1400-2000pg/ml,更佳地1600-2000pg/ml,更佳地1700-1850pg/ml;在所述的无细胞脂肪提取物中,所述的GDNF的浓度为800-5000pg/ml,较佳地1000-4000pg/ml,更佳地1200-2500pg/ml,更佳地1400-2000pg/ml,更佳地1600-2000pg/ml,更佳地1700-1900pg/ml;在所述的无细胞脂肪提取物中,所述的bFGF的浓度为50-600pg/ml,较佳地100-500pg/ml,更佳地120-400pg/ml,更佳地150-300pg/ml,更佳地200-280pg/ml,更佳地220-260pg/ml;在所述的无细胞脂肪提取物中,所述的VEGF的浓度为50-500pg/ml,较佳地100-400pg/ml,更佳地120-300pg/ml,更佳地150-250pg/ml,更佳地170-230pg/ml,更佳地190-210pg/ml;在所述的无细胞脂肪提取物中,所述的TGF-β1的浓度为200-3000pg/ml,较佳地400-2000pg/ml,更佳地600-1500pg/ml,更佳地800-1200pg/ml,更佳地800-1100pg/ml,更佳地900-1000pg/ml;在所述的无细胞脂肪提取物中,所述的HGF的浓度为200-3000pg/ml,较佳地400-2000pg/ml,更佳地600-1500pg/ml,更佳地600-1200pg/ml,更佳地800-1000pg/ml,更佳地850-950pg/ml;和/或在所述的无细胞脂肪提取物中,所述的PDGF的浓度为50-600pg/ml,较佳地80-400pg/ml,更佳地100-300pg/ml,更佳地140-220pg/ml,更佳地160-200pg/ml,更佳地170-190pg/ml。
- 如权利要求9所述的用途,其特征在于,所示的无细胞脂肪提取物包括选自下组的一种或多种特征:所述的IGF-1与VEGF的重量比为20-100:1,较佳地30-70:1,更佳地40-60:1,最佳地45-55:1;所述的BDNF与VEGF的重量比为2-20:1,较佳地4-15:1,更佳地6-12:1,最佳地8-9.5:1;所述的GDNF与VEGF的重量比为2-20:1,较佳地4-15:1,更佳地6-12:1,最佳地8.5-9.5:1;所述的bFGF与VEGF的重量比为0.2-8:1,较佳地0.5-5:1,更佳地0.6-2:1,更佳地0.8-1.6:1,最佳地1-1.5:1;所述的TGF-β1与VEGF的重量比为1-20:1,较佳地1-15:1,更佳地1-10:1,更佳地2-8:1,更佳地4-6:1;所述的HGF与VEGF的重量比为1-20:1,较佳地1-15:1,更佳地1-10:1,更佳地2-8:1,更佳地4-5.5:1;和/或所述的PDGF与VEGF的重量比为0.1-3:1,较佳地0.2-2:1,更佳地0.4-1.5:1,最佳地0.7-1.2:1。
- 如权利要求1所述的用途,其特征在于,所述的无细胞脂肪提取物通过以下方法制备:(1)提供一脂肪组织原料,将所述脂肪组织原料破碎,并进行漂洗(如用生理盐水),从而获得经漂洗的脂肪组织;(2)对所述经漂洗后的脂肪组织进行离心,获得分层的混合物;(3)对所述分层的混合物,去除上层油层和下层水层,收集中间层(即含脂肪细胞的脂肪层);(4)对所述中间层进行乳化,获得乳化的脂肪混合物(也称为纳米脂肪);(5)将所述乳化的脂肪混合物通过离心处理,从而获得中间液体层,即为脂肪初提物;和(6)对所述脂肪初提物进行过滤和除菌,从而获得无细胞的脂肪提取物。
- 一种用于预防和/或治疗视神经损伤的组合物或制剂,其特征在于,所述的组合物或制剂包含(a)无细胞脂肪提取物;和(b)药学上、食品上、保健品或膳食上可接受的载体或赋形剂。
- 一种预防和/或治疗视神经损伤的方法,其特征在于,对需要的对象施用无细胞脂肪提取物。
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