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WO2024229682A1 - 促进生成具有线粒体的微囊泡的方法及其医疗用途 - Google Patents

促进生成具有线粒体的微囊泡的方法及其医疗用途 Download PDF

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Publication number
WO2024229682A1
WO2024229682A1 PCT/CN2023/092873 CN2023092873W WO2024229682A1 WO 2024229682 A1 WO2024229682 A1 WO 2024229682A1 CN 2023092873 W CN2023092873 W CN 2023092873W WO 2024229682 A1 WO2024229682 A1 WO 2024229682A1
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extracellular vesicle
mitochondria
production
microvesicles
factor
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PCT/CN2023/092873
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English (en)
French (fr)
Inventor
王以庄
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台湾君百有限公司
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Priority to PCT/CN2023/092873 priority Critical patent/WO2024229682A1/zh
Publication of WO2024229682A1 publication Critical patent/WO2024229682A1/zh

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a combined use of a factor promoting mitochondrial production and a factor promoting extracellular vesicle production, and also relates to a method for causing cells to generate an extracellular vesicle composition rich in microvesicles having mitochondria, an isolated extracellular vesicle composition, a pharmaceutical product containing the same, and its use.
  • Stem cells are undifferentiated mother cells that can differentiate into cells with different functions, and their ability to replicate can produce more identical stem cells. Because stem cells have the ability to replicate and differentiate into other types of cells, humans believe that they may be used to treat diseases, so they began to try to use stem cells as drugs.
  • the definition of stem cell therapy is to use stem cells to treat a variety of diseases. It can be traced back to bone marrow transplantation in 1968. However, the marrow replacement medical technology is to transplant "hematopoietic stem cells". Although it is different from today's mesenchymal stem cell transplantation therapy, it can still be regarded as the beginning of allogeneic stem cell therapy.
  • stem cells were implanted into the left ventricle to repair myocardial tissue. When applied to human spinal cord injuries, it can indeed significantly restore mobility. In the treatment of stroke, it can reduce acute inflammation and long-term brain degeneration and promote long-term functional recovery. In the study of brain injury, it has been proven that it can alleviate symptoms and improve mobility and long-term memory. Later, it was also proven that adult stem cells from fat are also effective in treating neurological diseases. Time and facts have proved that although stem cell therapy is effective, the original differentiation replacement theory has always been inconsistent with the results of experimental research, so the therapeutic principle of mesenchymal stem cells has never been determined.
  • stem cells with better mitochondrial quality have better therapeutic abilities. Later, it was directly confirmed that in treatment, it is not necessary to use the entire stem cell. Simply injecting the separated mitochondria directly into the lungs can treat lung inflammation and damage and restore fibrotic alveoli. Alternatively, injecting free mitochondria directly into the brain can treat stroke to improve the area of cerebral infarction, and multiple system atrophy (MSA) to reduce brain Impairment and improved behavioral abilities.
  • MSA multiple system atrophy
  • delivering healthy mitochondria to damaged cells can promote cell resurrection, cell growth and activation, and improve aging or various degenerative diseases caused by mitochondrial damage.
  • the biggest technical obstacle to such an effective treatment method is that there is still no method to mass-produce mitochondria, and the separated mitochondria will also be damaged and die quickly after leaving the intracellular environment, making it difficult to preserve.
  • purified mitochondrial preparations are always difficult to manufacture, mass-produce and preserve for a long time.
  • the innovation of the present invention lies in the simple, large-scale, and continuous mass production of mitochondrial preparations.
  • the mitochondria produced are easier to preserve for a long time. Due to the structure of the vesicles and the components therein, they can not only protect the mitochondria from damage, but also promote the mitochondria to enter damaged cells, thereby improving the therapeutic ability, and solving all technical obstacles to the practical application of purified mitochondrial preparations at one time.
  • the present invention provides a method of using a factor promoting mitochondria production in combination with a factor promoting extracellular vesicle production, which is used to promote cells having mitochondria to generate microvesicles having mitochondria.
  • the use according to the present invention may be for non-therapeutic purposes.
  • the present invention can produce a large number of microvesicles with mitochondria by using a factor that promotes the production of mitochondria and a factor that promotes the production of extracellular vesicles.
  • the microvesicle structure can promote the entry of mitochondria into damaged cells for repair.
  • the collected mitochondria are already covered by microvesicles and are protected by small molecule RNA, so they are easier to preserve.
  • the cells having mitochondria can produce extracellular vesicles and microvesicles having mitochondria.
  • the ratio of the amount of the factor promoting mitochondrial production to the amount of the factor promoting extracellular vesicle production is 1:1 to 1:1000, for example, 1:1 to 1:8, 1:1 to 1:6, 1:1 to 1:4, or 1:100, 1:300, 1:600, 1:900. More preferably, the ratio of the amount of the factor promoting mitochondrial production to the amount of the factor promoting extracellular vesicle production is 1:1 to 1:5.
  • the factors promoting mitochondrial production can be selected from the group consisting of: heme oxygenase-1 (HO-1), CoPPIX (Co-protoporphyrin IX), heme, heme derivatives (for example, heme chloride), hemoprotein, broken red blood cells, mitochondrial structure fragments, cell tissue fragments and 5%-20% oxygen concentration environment culture.
  • heme oxygenase-1 HO-1
  • CoPPIX Co-protoporphyrin IX
  • heme heme oxygenase-1
  • heme derivatives for example, heme chloride
  • hemoprotein broken red blood cells
  • mitochondrial structure fragments for example, cell tissue fragments and 5%-20% oxygen concentration environment culture.
  • the factor promoting the production of extracellular vesicles can be selected from: ethanol, EP4 receptor antagonist (EP4 antagonist), such as: GW627368X, endosomal protein sorting complex required for transport (ESCRT), ARRDC1 (arrestin domain-containing protein 1), associated protein's tumor suppressor gene 101 (TSG101) and cell relaxant
  • EP4 antagonist such as: GW627368X
  • ESCRT endosomal protein sorting complex required for transport
  • ARRDC1 arrestin domain-containing protein 1
  • TSG101 tumor suppressor gene 101
  • cell relaxant The group is composed of cytochalasin B.
  • the factor promoting the production of extracellular vesicles can promote cells to produce microvesicles and exosomes.
  • the cell having mitochondria is a mesenchymal stem cell, a hematopoietic stem cell, a bone marrow stem cell, a liver cell, or other somatic cells. More preferably, the cell having mitochondria is a mesenchymal stem cell.
  • the mesenchymal stem cell is a neonatal mesenchymal stem cell or an embryonic stem cell.
  • the present invention further provides a method for producing an extracellular vesicle composition rich in microvesicles having mitochondria, comprising:
  • Step (1) preparing cells with mitochondria
  • Step (2) adds a factor promoting mitochondrial production and a factor promoting extracellular vesicle production to the cells having mitochondria and co-cultivates them in a culture medium to obtain an extracellular vesicle composition rich in microvesicles having mitochondria, and the extracellular vesicle composition rich in microvesicles having mitochondria has more than 0.2% of microvesicles having mitochondria based on the total number of extracellular vesicles.
  • the step (2) comprises:
  • Step (2-1) adding a factor promoting mitochondrial production and a factor promoting extracellular vesicle production to the cells having mitochondria in a culture medium for co-culturing;
  • step (2-2) the extracellular vesicle composition is separated from the co-cultured culture medium to obtain an extracellular vesicle composition rich in microvesicles having mitochondria, and the extracellular vesicle composition rich in microvesicles having mitochondria has more than 0.2% of microvesicles having mitochondria based on the total number of extracellular vesicles. That is, the extracellular vesicle composition is obtained by removing cells and culture medium having mitochondria. That is, more than 0.2% of the extracellular vesicles are microvesicles having mitochondria.
  • the step (2) is to first add a factor promoting mitochondrial production to the cells having mitochondria, and then add a factor promoting extracellular vesicle production to the culture medium for co-culture, to obtain an extracellular vesicle composition rich in microvesicles having mitochondria, and the extracellular vesicle composition rich in microvesicles having mitochondria has more than 0.2% of microvesicles having mitochondria based on the total number of extracellular vesicles.
  • the step (2) is to first add a factor promoting extracellular vesicle production to the cells having mitochondria, and then add a factor promoting mitochondria production to the culture medium for co-culture, to obtain an extracellular vesicle composition rich in microvesicles having mitochondria, and the extracellular vesicle composition rich in microvesicles having mitochondria has more than 0.2% of microvesicles having mitochondria based on the total number of extracellular vesicles. That is, more than 0.2% of the extracellular vesicles are microvesicles having mitochondria.
  • the amount of the factor promoting mitochondrial production added is such that the concentration of the factor promoting mitochondrial production in the culture medium is 0.1 ng/mL to 100,000 ng/mL.
  • the amount of the factor promoting mitochondrial production added is such that the concentration of the factor promoting mitochondrial production in the culture medium is 1 ng/mL to 10,000 ng/mL, for example, 10 ng/mL, 50 ng/mL, 100 ng/mL, 500 ng/mL or 1,000 ng/mL.
  • the factor promoting mitochondrial production can be selected from the group consisting of: hemoglobin oxygenase 1, CoPPIX, heme, heme derivatives, such as heme chloride, heme protein, broken red blood cells, mitochondrial structure fragments, cell tissue fragments, and 5%-20% oxygen concentration environment culture.
  • the oxygen concentration can be 7% to 18% or 10% to 15%, or can be 12%, 13%, 14%.
  • the amount of hemoglobin oxygenase 1 added is such that the concentration of hemoglobin oxygenase 1 in the culture medium is 0.1 ng/mL to 300 ng/mL.
  • the amount of hemoglobin oxygenase 1 added is such that the concentration of hemoglobin oxygenase 1 in the culture medium is 1 ng/mL to 100 ng/mL, for example, 10 ng/mL, 30 ng/mL, 50 ng/mL or 70 ng/mL.
  • the amount of CoPPIX added is such that the concentration of CoPPIX in the culture medium is 0.2 micromoles/kilogram ( ⁇ mol/kg) to 100 ⁇ mol/kg.
  • the amount of CoPPIX added is such that the concentration of CoPPIX in the culture medium is 1 ⁇ mol/kg to 70 ⁇ mol/kg, for example, 5 ⁇ mol/kg, 10 ⁇ mol/kg, 30 ⁇ mol/kg or 50 ⁇ mol/kg.
  • the amount of heme or the heme derivative added is such that the concentration of heme or the heme derivative in the culture medium is 0.2 ⁇ mol/kg to 100 ⁇ mol/kg.
  • the amount of heme or the heme derivative added is such that the concentration of heme or the heme derivative in the culture medium is 1 ⁇ mol/kg to 80 ⁇ mol/kg, for example, 10 ⁇ mol/kg, 20 ⁇ mol/kg, 50 ⁇ mol/kg or 70 ⁇ mol/kg.
  • the amount of the factor promoting extracellular vesicle production is such that the concentration of the factor promoting extracellular vesicle production in the culture medium is 10-7 millimoles per liter (mM) to 800mM.
  • the amount of the factor promoting extracellular vesicle production is such that the concentration of the factor promoting extracellular vesicle production in the culture medium is 10-5 mM to 500mM, for example, 10-4 mM, 10-2 mM, 1mM, 10mM, 50mM, 100mM or 300mM.
  • the factor promoting the production of extracellular vesicles can be selected from the group consisting of ethanol, EP4 receptor antagonists, such as GW627368X, endosomal protein sorting and transport complex, ARRDC1, tumor suppressor genes of related proteins, and cytochalasin B.
  • the factor promoting the production of extracellular vesicles can promote cells to produce microvesicles and exosomes.
  • the amount of ethanol added is such that the ethanol concentration in the culture medium is 10 mM to 100 mM. More preferably, the amount of ethanol added is such that the ethanol concentration in the culture medium is 20 mM to 70 mM, for example, 30 mM, 40 mM, 50 mM or 60 mM.
  • the amount of the EP4 receptor antagonist added is such that the concentration of the EP4 receptor antagonist in the culture medium is 0.2 ⁇ g/mL to 500 ⁇ g/mL.
  • the amount of the EP4 receptor antagonist added is The concentration of the EP4 receptor antagonist in the culture medium is 1 ⁇ g/mL to 200 ⁇ g/mL, for example, 10 ⁇ g/mL, 50 ⁇ g/mL, 70 ⁇ g/mL, 100 ⁇ g/mL or 150 ⁇ g/mL.
  • the amount of the endosomal protein sorting and transport complex added is such that the concentration of the endosomal protein sorting and transport complex in the culture medium is 0.2 nanomoles per liter (nM) to 500 nM.
  • the amount of the endosomal protein sorting and transport complex added is such that the concentration of the endosomal protein sorting and transport complex in the culture medium is 1 nM to 300 nM, for example, 5 nM, 10 nM, 50 nM or 100 nM.
  • the amount of ARRDC1 added is such that the concentration of ARRDC1 in the culture medium is 0.3 ⁇ M to 600 ⁇ M.
  • the amount of ARRDC1 added is such that the concentration of ARRDC1 in the culture medium is 1 ⁇ M to 300 ⁇ M, for example, 10 ⁇ M, 50 ⁇ M or 150 ⁇ M.
  • the amount of the tumor suppressor gene of the related protein added is such that the concentration of the tumor suppressor gene of the related protein in the culture medium is 10-10 molar per liter (M) to 10-6 M.
  • the amount of the tumor suppressor gene of the related protein added is such that the concentration of the tumor suppressor gene of the related protein in the culture medium is 10-9 M to 10-7 M, for example, 10-8 M.
  • the amount of cytochalasin B added is such that the concentration of cytochalasin B in the culture medium is 10 -7 M to 10 -4 M.
  • the amount of cytochalasin B added is such that the concentration of cytochalasin B in the culture medium is 10 -6 M to 10 -5 M.
  • the culture conditions of cells having mitochondria can be adjusted as needed by those skilled in the art.
  • the co-cultivation is carried out at 5% carbon dioxide and 35° C. to 39° C. for 60 to 80 hours. Preferably, the co-cultivation is carried out for 70 to 75 hours.
  • the present invention further provides an isolated extracellular vesicle composition, which comprises more than 0.2% of microvesicles having mitochondria based on the total number of extracellular vesicles.
  • the aforementioned separated extracellular vesicle composition further comprises microvesicles and exosomes.
  • Extracellular vesicles that can encapsulate mitochondria are microvesicles.
  • Microvesicles with mitochondria can be combined with the above-mentioned other microvesicles or exosomes to increase their therapeutic effect.
  • the isolated extracellular vesicle composition has 0.5%, 1%, 2%, 3% or more microvesicles with mitochondria based on the total number of extracellular vesicles. More preferably, the isolated extracellular vesicle composition has 4% or more microvesicles with mitochondria based on the total number of extracellular vesicles, for example, 4.2%, 4.4%, 4.6% or 4.8% or more microvesicles with mitochondria.
  • the present invention further provides a pharmaceutical composition comprising the aforementioned isolated microvesicles containing mitochondria. Extracellular vesicle composition, and a pharmaceutically acceptable carrier.
  • the present invention further provides a use of an isolated extracellular vesicle composition, which is used as an application for preparing a drug for treating or alleviating diabetes.
  • the drug comprises an effective dose of the isolated extracellular vesicle composition and a pharmaceutically acceptable carrier, and the isolated extracellular vesicle composition, based on the total number of extracellular vesicles, has more than 0.2% of microvesicles with mitochondria.
  • the diabetes is type 2 diabetes.
  • the effective dose described in the present invention refers to an amount that is effective in terms of dosage and for the required time period to achieve the desired treatment or alleviation of diabetes; according to the present invention, it means that by administering a specific range of amounts of an isolated extracellular vesicle composition, the fasting blood glucose level and/or insulin concentration of diabetic rats can be reduced, thereby improving insulin resistance.
  • the drug is administered to a warm-blooded animal or a human being.
  • the warm-blooded animal is a mammal or a bird, and the mammal may be a rat or a mouse.
  • the present invention further provides a use of an isolated extracellular vesicle composition containing mitochondria, which is used as an application for preparing a drug for treating or alleviating kidney damage.
  • the drug comprises an effective dose of the isolated extracellular vesicle composition and a pharmaceutically acceptable carrier, and the isolated extracellular vesicle composition, based on the total number of extracellular vesicles, has more than 0.2% of microvesicles containing mitochondria.
  • the effective dose described in the present invention refers to an amount that is effective in terms of dosage and for the required time period to achieve the desired treatment or alleviation of chronic kidney disease; according to the present invention, it means that by administering a specific range of amounts of an isolated mitochondrial-containing extracellular vesicle composition, the blood creatinine and urea nitrogen levels of chronic kidney disease mice can be reduced.
  • the kidney injury may be chronic kidney disease or acute kidney injury.
  • the drug is administered to a warm-blooded animal or a human being.
  • the warm-blooded animal is a mammal or a bird, and the mammal may be a rat or a mouse.
  • composition or drug can exist in various forms, including, but not limited to, liquid, semi-solid and solid dosage forms, such as solution, emulsion, suspension and other dosage forms similar to or suitable for the present invention.
  • the pharmaceutical composition of the present invention is in the form of an injection, and the injection is administered intravenously.
  • the advantages of the present invention are that it can produce mitochondrial preparations in a simple and large-scale continuous manner; and microvesicles with mitochondria can be directly separated from the culture medium, without the need to break the cells to obtain mitochondria, and without the need for the high-cost and time-consuming mitochondrial high-speed centrifugation process, which can achieve the effect of saving costs and time.
  • the collected mitochondria are mitochondrial vesicles that have been coated by microvesicles, and are also protected by the small molecule RNA of exocrine factors, so they are easier to freeze and store.
  • the isolated extracellular vesicle composition of the present invention can indeed reduce the fasting blood glucose value and/or insulin concentration of diabetic rats, thereby improving insulin resistance, and can effectively treat or alleviate diabetes. It can also reduce creatinine and urea nitrogen in the blood of chronic kidney disease mice, and can effectively treat or alleviate kidney damage.
  • FIG1 shows the number of mitochondria in the extracellular vesicles produced by human mesenchymal stem cells after treatment with factors promoting mitochondrial production, factors promoting extracellular vesicle production or a combination thereof, * indicates a confidence interval of >95%;
  • FIG2 shows the ratio of microvesicles with mitochondria produced by human mesenchymal stem cells to extracellular vesicles produced in each treatment group after treatment with factors promoting mitochondria production, factors promoting extracellular vesicle production or a combination thereof, * indicates a confidence interval of >95%;
  • FIG3 shows the effects of the mitochondrial production-promoting factor extracellular vesicles, the mitochondrial production-promoting factor extracellular vesicles group and the co-treatment of extracellular vesicles on reducing the fasting blood glucose level in type II diabetic rats, * indicates a confidence interval of >95%;
  • FIG4 shows the effects of the mitochondrial production-promoting factor extracellular vesicles, the mitochondrial production-promoting factor extracellular vesicles group and the co-treatment extracellular vesicles on reducing the fasting insulin concentration in type II diabetic rats, * indicates a confidence interval of >95%, ** indicates a confidence interval of >90%;
  • FIG5 shows the effect of promoting mitochondrial production factor extracellular vesicles, promoting extracellular vesicle production factor extracellular vesicle group and co-treatment of extracellular vesicles on reducing blood creatinine in chronic kidney disease mice, * indicates a confidence interval of >95%;
  • FIG6 shows the effects of the extracellular vesicles that promote mitochondrial production factors, the extracellular vesicles group that promotes extracellular vesicle production factors, and the co-treated extracellular vesicles on reducing blood urea nitrogen in chronic kidney disease mice, and * indicates a confidence interval of >95%.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • glucose glucose concentration of 4500 mg/L
  • the cells were treated with the aforementioned DMEM supplemented with 10% FBS in four groups: the control group was cultured with the culture medium only without additional treatment; the group treated with mitochondrial production promoting factors was to add mitochondrial production promoting factors to the culture medium, and then the cells were cultured with 10% FBS.
  • the concentration of the mitochondrial production promoting factor is 40 ng/mL.
  • the mitochondrial production promoting factor is hemoglobin oxygenase 1 (Enzo LifeSciences, Ann Arbor, MI, USA); a group treated with factors for promoting extracellular vesicle production, wherein the concentration of the factors for promoting extracellular vesicle production in the culture medium was 50 mM after the factors for promoting extracellular vesicle production were added.
  • ethanol was selected as the factors for promoting extracellular vesicle production; or a group treated with factors for promoting mitochondria production and factors for promoting extracellular vesicle production together (hereinafter referred to as the co-treatment group).
  • the factors for promoting mitochondria production were added first, and then the factors for promoting extracellular vesicle production were added.
  • hemoglobin oxygenase 1 a factor for promoting mitochondria production
  • concentration of hemoglobin oxygenase 1 in the culture medium was 40 ng/mL.
  • ethanol a factor for promoting extracellular vesicle production
  • concentration of ethanol in the culture medium was 50 mM.
  • the supernatant was taken to collect the extracellular vesicles in the conditioned medium (CM) produced by each treatment group. Specifically, the conditioned medium produced by each treatment group was centrifuged at 3000g for 10 minutes to remove dead cells or larger cell fragments.
  • the supernatant of the conditioned medium of each group was pre-washed using a pre-washing column for isolating extracellular vesicles (Extracellular vesicle Isolation Pre-Clearing Column).
  • the pre-washed supernatant was transferred to an extracellular vesicle isolation column (CapturemTM) and centrifuged at 1,000g for 2 to 4 minutes at room temperature.
  • CapturemTM extracellular vesicle isolation column
  • the aforementioned column was washed once with extracellular vesicle isolation wash buffer, and then the extracellular vesicles of each group were washed with the extracellular vesicle elution buffer in the kit.
  • the extracellular vesicles obtained from each group of Example 1 were diluted with phosphate-buffered saline (PBS) to a dilution with 20-100 particles in the field of view.
  • PBS phosphate-buffered saline
  • the number of microvesicles with mitochondria was measured by Nanoparticle Tracking Analysis (NTA), and the final count of microvesicles with mitochondria was performed using fluorescent nanoparticle tracking analysis (fNTA).
  • NTA Nanoparticle Tracking Analysis
  • fNTA fluorescent nanoparticle tracking analysis
  • the extracellular vesicles separated from Example 1 were labeled with TMRE fluorescent dye (tetramethylrhodamine ethyl ester) to detect mitochondria in the extracellular vesicles, wherein the excitation light and emission light wavelengths of the TMRE fluorescent dye were 550 nanometers (nm) and 575 nm, respectively.
  • the sizes of the extracellular vesicles and microvesicles with mitochondria were measured using a nanoparticle size analyzer (NanoSight LM10-HS system, Malvern, UK), and the data were analyzed using Nanoparticle Tracking Software (version 2.3).
  • FIG1 The results of measuring the number of mitochondria in each treatment group are shown in FIG1 . It can be seen that the microvesicles with mitochondria obtained in the group treated with factors promoting mitochondria production, the group treated with factors promoting extracellular vesicle production, and the co-treatment group did promote the production of more microvesicles with mitochondria than in the control group. Therefore, human mesenchymal stem cells were treated with factors promoting mitochondria production, factors promoting extracellular vesicle production, or factors promoting mitochondria production and factors promoting extracellular vesicle production together.
  • both the treatment and co-treatment groups can promote the production of more mitochondrial microvesicles by human mesenchymal stem cells, and the treatment group with mitochondrial production promoting factors and the co-treatment group can promote the production of more mitochondrial microvesicles by human mesenchymal stem cells, which are 16 times and 132 times of the control group respectively.
  • the result of FIG2 was divided by the number of extracellular vesicles obtained from each treatment group to obtain the ratio of microvesicles with mitochondria to the extracellular vesicles produced in each treatment group.
  • the results are shown in FIG2.
  • the extracellular vesicles produced by the group treated with the factor promoting mitochondria production, the group treated with the factor promoting extracellular vesicle production, and the co-treatment group did have a higher proportion of mitochondria in the extracellular vesicles produced.
  • human mesenchymal stem cells treated with the factor promoting mitochondria production, the factor promoting extracellular vesicle production, or the factor promoting mitochondria production and the factor promoting extracellular vesicle production co-treatment can indeed promote a higher proportion of mitochondria in the extracellular vesicles produced by human mesenchymal stem cells.
  • the group treated with the factor promoting mitochondria production and the co-treatment group can make the extracellular vesicles produced by human mesenchymal stem cells have a higher proportion of mitochondria, which are 1.89% and 4.9%, respectively, which are 14.44 times and 37.40 times compared with the control group, respectively.
  • the combined use of factors promoting mitochondria production and factors promoting extracellular vesicle production can indeed promote cells with mitochondria to produce microvesicles with mitochondria, and the method of the present invention can indeed produce a large number of microvesicles with mitochondria.
  • HFD high-fat diet
  • Each group of rats was injected with PBS buffer solution (diabetic control group) by tail vein twice a week for a total of 2 weeks; or injected with the extracellular vesicles obtained in each treatment group in Example 1 by tail vein twice a week, wherein 3 ⁇ 10 8 extracellular vesicles per kilogram of body weight were administered each time for a total of two weeks.
  • PBS buffer solution diabetic control group
  • Example 1 injected with the extracellular vesicles obtained in each treatment group in Example 1 by tail vein twice a week, wherein 3 ⁇ 10 8 extracellular vesicles per kilogram of body weight were administered each time for a total of two weeks.
  • all rats were fed with general feed, and blood was collected from the rats three weeks after administration.
  • FIGs 3 and 4 show the improvement of fasting blood glucose and insulin resistance of diabetic rats after various treatments.
  • the fasting blood glucose values of diabetic rats in all treatment groups: the mitochondrial production factor extracellular vesicle group, the mitochondrial production factor extracellular vesicle group and the co-treatment extracellular vesicle group are lower than those in the diabetic control group, among which the mitochondrial production factor extracellular vesicle group and the co-treatment extracellular vesicle group have better effects.
  • the mitochondrial production factor extracellular vesicle group reduces the fasting blood glucose value by 61.78% compared with the diabetic control group, and the co-treatment extracellular vesicle group reduces the fasting blood glucose value by 65.35% compared with the diabetic control group, and is similar to the normal control group rats.
  • the fasting insulin concentrations of diabetic rats in all treatment groups were lower than those in the diabetic control group.
  • the mitochondrial production factor extracellular vesicle group and the co-treated extracellular vesicle group had better effects.
  • the mitochondrial production factor extracellular vesicle group reduced the fasting insulin concentration by 53.70% compared with the diabetic control group, and the co-treated extracellular vesicle group reduced the fasting insulin concentration by 56% compared with the diabetic control group, which was similar to that of the normal control group rats.
  • microvesicle and extracellular vesicle composition with mitochondria prepared by the method of the present invention can indeed treat type II diabetes, reduce fasting blood sugar levels, and improve high insulin concentrations caused by insulin resistance in diabetic rats.
  • mice All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.
  • Six-week-old BALB/c mice were housed in a controlled temperature (25°C) environment with a 12-hour light-dark cycle.
  • chronic kidney disease in mice was induced by feeding them with a feed containing 0.25% adenine.
  • mice One week later, each group of mice was injected with PBS buffer solution (chronic kidney disease control group) twice a week; or injected with extracellular vesicles obtained in each treatment group in Example 1 twice a week, wherein 3 ⁇ 10 8 extracellular vesicles per kg of body weight were administered each time for a total of two weeks. During the experiment, all mice were fed with normal feed, and blood was collected from the mice three weeks after administration.
  • PBS buffer solution chronic kidney disease control group
  • the collected mouse blood was centrifuged at 1500 rpm for 30 minutes, and the upper serum was taken for measuring the blood creatinine and blood urea nitrogen (BUN) values using enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the absorbance value at 450 nm was measured using a microplate analyzer (Thermo Fisher Scientific).
  • FIG5 and FIG6 respectively show the improvement of blood creatinine and urea nitrogen values of chronic kidney disease mice after various treatments.
  • Figure 5 shows that the blood creatinine values of the chronic kidney disease mice in all treatment groups: the mitochondrial production factor extracellular vesicle group, the extracellular vesicle production factor extracellular vesicle group and the co-treatment extracellular vesicle group were lower than those of the chronic kidney disease control group mice, among which the mitochondrial production factor extracellular vesicle group and the co-treatment extracellular vesicle group had the best effects.
  • the creatinine value of the mitochondrial production factor extracellular vesicle group was reduced by nearly 54.06% compared with the chronic kidney disease control group, and the co-treatment extracellular vesicle group was reduced by 67.96% compared with the chronic kidney disease control group, and was similar to that of the normal control group mice.
  • Figure 6 shows that the blood urea nitrogen values of the chronic kidney disease mice in all treatment groups: the mitochondrial production factor extracellular vesicle group, the extracellular vesicle production factor extracellular vesicle group and the co-treatment extracellular vesicle group were lower than those of the chronic kidney disease control group mice, among which the mitochondrial production factor extracellular vesicle group and the co-treatment extracellular vesicle group had the best effects.
  • the urea nitrogen value of the mitochondrial production factor extracellular vesicle group was reduced by nearly 66.80% compared with the chronic kidney disease control group, and the co-treatment extracellular vesicle group was reduced by 79.42% compared with the chronic kidney disease control group, and was similar to that of the normal control group mice.
  • the microvesicles with mitochondria prepared by the method of the present invention can indeed treat and improve diabetes; and can reduce the creatinine value and urea nitrogen value in the blood of mice with renal damage and chronic kidney disease.
  • the method of the present invention can indeed prepare a large number of microvesicles with mitochondria.
  • the isolated extracellular vesicle composition prepared by the method of the present invention can indeed treat or alleviate diabetes, such as type 2 diabetes, and kidney damage, such as chronic kidney disease, because it contains a large number of microvesicles with mitochondria.

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Abstract

一种促进生成具有线粒体的微囊泡的方法及其医疗用途。具体地，本发明提供一种联合使用促进线粒体产生因子及促进胞外囊泡产生因子的用途，是用于促进生成具有线粒体的微囊泡。并提供生成胞外囊泡组合物的方法：对细胞添加促进线粒体产生因子及促进胞外囊泡产生因子进行共培养，所得的胞外囊泡组合物富含具有线粒体的微囊泡。藉由添加促进线粒体产生因子及促进胞外囊泡产生因子，可促使细胞产生具有线粒体的微囊泡；且收集到的线粒体是由微囊泡所包覆的线粒体，因此更容易保存及传送线粒体至胞内。另提供一经分离的胞外囊泡组合物、包含其的医药组合物及其用于制备治疗糖尿病及/或肾损伤的药物的应用。

Description

促进生成具有线粒体的微囊泡的方法及其医疗用途 技术领域
本发明是关于一种联合使用促进线粒体产生因子及促进胞外囊泡产生因子的用途。此外，是有关于一种使细胞生成富含具有线粒体的微囊泡的胞外囊泡组合物的方法、一种经分离的胞外囊泡组合物、包含其的医药品及其用途。
背景技术
干细胞(stem cell)是未分化的母细胞，具有能分化成不同功能的细胞，且复制能力可产生更多一致干细胞。因干细胞具有复制与分化为其他种类细胞的能力，被人类认为可能用于治疗疾病，因而开始尝试以干细胞作为药物，而干细胞治疗的定义是使用干细胞来治疗多种疾病，最早可以追溯到1968年的骨髓移植，但是换髓医疗技术是移植“造血干细胞”，虽然与今日的间充质干细胞移植治疗有所不同，仍然可以算是一种异体干细胞治疗的开始。间充质干细胞经过了不少动物实验得到良好结果后，2000年后逐渐尝试用干细胞治疗人类疾病。例如2003年将“骨髓干细胞”植入左心室，修复心肌组织。而在应用于人体脊膸损伤，确实可使得行动能力明显恢复，在中风治疗方面，可以减少急性发炎与长期脑部退化，促进长期功能复原。在脑损伤的研究，证实可以减轻症状，并改善活动能力与长期记忆。后来也证实来自脂肪的成体干细胞对治疗神经相关疾病也有效果。时间与事实证明干细胞治疗虽然有效，但是原来的分化替代论，始终与实验研究结果有所矛盾，因此间充质干细胞的治疗原理始终没有被确定。
最新的研究已经证实干细胞的疗效原理，来自干细胞分泌的一些物质，其中线粒体通过通道(tunnel)、间隙连接(gap junction)、微囊泡(microvesicles)、细胞融合(cell fusion)等方式转移到受损细胞，而受损细胞获得了这些外源的线粒体，是干细胞产生疗效的主要原因。诸多论文，例如Paliwal et al.(2018)也验证了间充质干细胞的再生能力来自线粒体的传送。同年Wang et al.(2018)也提出干细胞的线粒体传输是组织受损的新颖治疗技术。
目前研究也证实线粒体质量较为优良的干细胞，会具有较佳的治疗能力，后来更直接证实了在治疗时，不需要使用整个干细胞，只需将分离出来的线粒体直接注射在肺部可以治疗肺部发炎损伤，将纤维化的肺泡复原。或是将游离线粒体直接注射在脑部，可以治疗中风以改善脑梗塞面积、多重脑退化(Multiple system atrophy，MSA)以减少脑部 损伤与改善行为能力。
因此，将健康的线粒体传送至受损细胞内，可以促进细胞复活、促进细胞生长与活化，改善因线粒体受损导致的老化或是多种退化性疾病。然而如此有效的治疗方法，目前最大的技术障碍是仍然没有大量生产线粒体的方法，且分离出的线粒体也会因离开胞内环境，使游离的线粒体很快受损死亡且难以保存，进而造成纯化的线粒体制剂始终难以制造、量产及长时间保存。
发明内容
有鉴于现有技术无法大量生产线粒体，即使生产出来也难以保存，本发明的创新在于简单、大量、连续式的量产线粒体制剂，且生产所得的线粒体更容易长时间保存，且因囊泡的结构与其中的成分，不但能保护线粒体不易受损，更能促进线粒体进入受损细胞，进而提升治疗能力，一次性的解决纯化线粒体制剂实际应用的所有技术障碍。
为达前述目的，本发明提供一种联合使用促进线粒体产生因子及促进胞外囊泡产生因子的用途，其是用于促进具有线粒体的细胞生成具有线粒体的微囊泡。
本发明所述的用途可以是非治疗目的的。
本发明藉由联合使用促进线粒体产生因子及促进胞外囊泡产生因子，可大量产生具有线粒体的微囊泡，微囊泡结构能促进线粒体进入受损细胞，进行修复。且收集到的线粒体是已经由微囊泡所包覆的线粒体，内有小分子核糖核酸的保护，因此更容易保存。
依据本发明，所述具有线粒体的细胞能产生胞外囊泡及具有线粒体的微囊泡。
较佳的，所述促进线粒体产生因子及所述促进胞外囊泡产生因子的添加量的比例为1：1至1：1000，例如可为1：1至1：8、1：1至1：6、1：1至1：4或1：100、1：300、1：600、1：900。更佳的，所述促进线粒体产生因子及所述促进胞外囊泡产生因子的添加量的比例为1：1至1：5。
较佳的，所述促进线粒体产生因子可选自由：血红加氧酶1(Heme oxygenase-1，HO-1)、CoPPIX(Co-protoporphyrin IX)、血红素(Hemin)、血红素衍生物(例如：氯化血红素)、血红素蛋白(Hemoprotein)、破碎红血球、线粒体结构破片、细胞组织破片及5％-20％氧浓度环境培养所组成的群组。
较佳的，所述促进胞外囊泡产生因子可选自由：乙醇、EP4受体拮抗剂(EP4antagonist)，例如：GW627368X、内体蛋白分选转运复合体(endosomal sorting complex required for transport，ESCRT)、ARRDC1(arrestin domain-containing protein 1)、相关蛋白的肿瘤抑制基因(associated protein’s tumor suppressor gene 101，TSG101)及细胞松弛 素B(cytochalasin B)所组成的群组。所述促进胞外囊泡产生因子可以促进细胞产生微囊泡及外泌体。
较佳的，所述具有线粒体的细胞为间充质干细胞、造血干细胞、骨髓干细胞、肝脏细胞、或其他体细胞。更佳的，所述具有线粒体的细胞为间充质干细胞。较佳的，所述间充质干细胞为新生儿的间充质干细胞或胚胎干细胞。
为达前述目的，本发明另外提供一种生成富含具有线粒体的微囊泡的胞外囊泡组合物的方法，其包含：
步骤(1)齐备具有线粒体的细胞；
步骤(2)对所述具有线粒体的细胞添加促进线粒体产生因子及促进胞外囊泡产生因子于培养基中进行共培养，得到富含具有线粒体的微囊泡的胞外囊泡组合物，且所述富含具有线粒体的微囊泡的胞外囊泡组合物以胞外囊泡总数为基准，具有0.2％以上的具有线粒体的微囊泡。
较佳的，所述步骤(2)包含：
步骤(2-1)对所述具有线粒体的细胞添加促进线粒体产生因子及促进胞外囊泡产生因子于培养基中进行共培养；及
步骤(2-2)自共培养的所述培养基中分离出胞外囊泡组合物，得到富含具有线粒体的微囊泡的胞外囊泡组合物，且所述富含具有线粒体的微囊泡的胞外囊泡组合物以胞外囊泡总数为基准，具有0.2％以上的具有线粒体的微囊泡。亦即移除具有线粒体的细胞及培养基而得到胞外囊泡组合物。亦即有0.2％以上的胞外囊泡为具有线粒体的微囊泡。
较佳的，所述步骤(2)为对所述具有线粒体的细胞先添加促进线粒体产生因子、再添加促进胞外囊泡产生因子于培养基进行共培养，得到富含具有线粒体的微囊泡的胞外囊泡组合物，且所述富含具有线粒体的微囊泡的胞外囊泡组合物以胞外囊泡总数为基准，具有0.2％以上的具有线粒体的微囊泡。在另一实施例中，所述步骤(2)为对所述具有线粒体的细胞先添加促进胞外囊泡产生因子、再添加促进线粒体产生因子于培养基进行共培养，得到富含具有线粒体的微囊泡的胞外囊泡组合物，且所述富含具有线粒体的微囊泡的胞外囊泡组合物以胞外囊泡总数为基准，具有0.2％以上的具有线粒体的微囊泡。亦即有0.2％以上的胞外囊泡为具有线粒体的微囊泡。
较佳的，所述促进线粒体产生因子的添加量是使所述培养基中的所述促进线粒体产生因子浓度为0.1纳克/毫升(ng/mL)至100,000ng/mL。较佳的，所述促进线粒体产生因子的添加量是使所述培养基中的所述促进线粒体产生因子浓度为1ng/mL至10,000 ng/mL，例如可为10ng/mL、50ng/mL、100ng/mL、500ng/mL或1,000ng/mL。
较佳的，所述促进线粒体产生因子可选自由：血红加氧酶1、CoPPIX、血红素、血红素衍生物，例如：氯化血红素、血红素蛋白、破碎红血球、线粒体结构破片、细胞组织破片及5％-20％氧浓度环境培养所组成的群组。其中，所述氧浓度可为7％至18％或10％至15％，或可为12％、13％、14％。
较佳的，所述血红加氧酶1的添加量是使所述培养基中的所述血红加氧酶1浓度为0.1ng/mL至300ng/mL。较佳的，所述血红加氧酶1的添加量是使所述培养基中的所述血红加氧酶1浓度为1ng/mL至100ng/mL，例如可为10ng/mL、30ng/mL、50ng/mL或70ng/mL。
较佳的，所述CoPPIX的添加量是使所述培养基中的所述CoPPIX浓度为0.2微摩尔/公斤(μmol/kg)至100μmol/kg。较佳的，所述CoPPIX的添加量是使所述培养基中的所述CoPPIX浓度为1μmol/kg至70μmol/kg，例如可为5μmol/kg、10μmol/kg、30μmol/kg或50μmol/kg。
较佳的，所述血红素或所述血红素衍生物的添加量是使所述培养基中的所述血红素或血红素衍生物浓度为0.2μmol/kg至100μmol/kg。较佳的，所述血红素或所述血红素衍生物的添加量是使所述培养基中的所述血红素或血红素衍生物浓度为1μmol/kg至80μmol/kg，例如可为10μmol/kg、20μmol/kg、50μmol/kg或70μmol/kg。
较佳的，所述促进胞外囊泡产生因子的添加量是使所述培养基中的所述促进胞外囊泡产生因子浓度为10-7毫摩尔每升(mM)至800mM。较佳的，所述促进胞外囊泡产生因子的添加量是使培养基中的所述促进胞外囊泡产生因子浓度为10-5mM至500mM，例如可为10-4mM、10-2mM、1mM、10mM、50mM、100mM或300mM。
较佳的，所述促进胞外囊泡产生因子可选自由：乙醇、EP4受体拮抗剂，例如：GW627368X、内体蛋白分选转运复合体、ARRDC1、相关蛋白的肿瘤抑制基因及细胞松弛素B所组成的群组。所述促进胞外囊泡产生因子可以促进细胞产生微囊泡及外泌体。
较佳的，所述乙醇的添加量是使所述培养基中的所述乙醇浓度为10mM至100mM。更佳的，所述乙醇的添加量是使所述培养基中的所述乙醇浓度为20mM至70mM，例如可为30mM、40mM、50mM或60mM。
较佳的，所述EP4受体拮抗剂的添加量是使所述培养基中的所述EP4受体拮抗剂浓度为0.2微克/毫升(μg/mL)至500μg/mL。较佳的，所述EP4受体拮抗剂的添加量 是使所述培养基中的所述EP4受体拮抗剂浓度为1μg/mL至200μg/mL，例如可为10μg/mL、50μg/mL、70μg/mL、100μg/mL或150μg/mL。
较佳的，所述内体蛋白分选转运复合体的添加量是使所述培养基中的所述内体蛋白分选转运复合体浓度为0.2纳摩尔每升(nM)至500nM。较佳的，所述内体蛋白分选转运复合体的添加量是使所述培养基中的所述内体蛋白分选转运复合体浓度为1nM至300nM，例如可为5nM、10nM、50nM或100nM。
较佳的，所述ARRDC1的添加量是使所述培养基中的所述ARRDC1浓度为0.3微摩尔每升(μM)至600μM。较佳的，所述ARRDC1的添加量是使所述培养基中的所述ARRDC1浓度为1μM至300μM，例如可为10μM、50μM或150μM。
较佳的，所述相关蛋白的肿瘤抑制基因的添加量是使所述培养基中的所述相关蛋白的肿瘤抑制基因浓度为10-10摩尔每升(M)至10-6M。较佳的，所述相关蛋白的肿瘤抑制基因的添加量是使所述培养基中的所述相关蛋白的肿瘤抑制基因浓度为10-9M至10-7M，例如可为10-8M。
较佳的，所述细胞松弛素B的添加量是使所述培养基中的所述细胞松弛素B浓度为10-7M至10-4M。较佳的，所述细胞松弛素B的添加量是使所述培养基中的所述细胞松弛素B浓度为10-6M至10-5M。
依据本发明，上述生成富含具有线粒体的微囊泡的胞外囊泡组合物的方法中，具有线粒体的细胞的培养条件是所属技术领域的技术人员可依所需而调整者。
较佳的，所述共培养是于5％二氧化碳、35℃至39℃下进行培养60至80小时。较佳的，培养70至75小时。
为达前述目的，本发明另外提供一种经分离的胞外囊泡组合物，以胞外囊泡总数为基准，其是具有0.2％以上的具有线粒体的微囊泡。
前述经分离的胞外囊泡组合物进一步包含有微囊泡及外泌体。而能包覆线粒体的胞外囊泡是微囊泡。具有线粒体的微囊泡搭配上述其他微囊泡或外泌体，能增加其治疗效果。
较佳的，所述经分离的胞外囊泡组合物，以胞外囊泡总数为基准，是具有0.5％、1％、2％、3％以上的具有线粒体的微囊泡。更佳的，所述经分离的胞外囊泡组合物，以胞外囊泡总数为基准，是具有4％以上的具有线粒体的微囊泡，例如可为4.2％、4.4％、4.6％或4.8％以上的具有线粒体的微囊泡。
本发明另外提供一种医药组合物，其包含前述的经分离的含具有线粒体的微囊泡的 胞外囊泡组合物，及药学上可接受的载剂。
本发明另外提供一种经分离胞外囊泡组合物的用途，其是作为制备治疗或减缓糖尿病药物的应用，所述药物中包含有效剂量的经分离的胞外囊泡组合物及药学上可接受的载剂，且所述经分离的胞外囊泡组合物，以胞外囊泡总数为基准，是具有0.2％以上的具有线粒体的微囊泡。
较佳的，所述糖尿病为第二型糖尿病。
发明所述的有效剂量是指在剂量上及对于所需要的时间段而言对达成所要治疗或减缓糖尿病的有效的量；依据本发明是指通过施予特定范围量的经分离的胞外囊泡组合物能够降低糖尿病大鼠的空腹血糖值及/或胰岛素浓度，而改善胰岛素抗性。
较佳的，所述药物的给药对象为恒温动物或人类。较佳的，所述恒温动物为哺乳动物或是鸟类，而所述哺乳动物可为：大鼠或小鼠。
本发明另外提供一种经分离含线粒体的胞外囊泡组合物的用途，其是作为制备治疗或减缓肾脏损伤药物的应用，所述药物中包含有效剂量的经分离的胞外囊泡组合物及药学上可接受的载剂，且所述经分离的胞外囊泡组合物，以胞外囊泡总数为基准，是具有0.2％以上的具有线粒体的微囊泡。
发明所述的有效剂量是指在剂量上及对于所需要的时间段而言对达成所要治疗或减缓慢性肾病的有效的量；依据本发明是指通过施予特定范围量的经分离含线粒体的胞外囊泡组合物能够降低慢性肾病小鼠的血液肌酸酐及尿素氮数值。
较佳的，所述肾脏损伤可为慢性肾病或急性肾损伤。
较佳的，所述药物的给药对象为恒温动物或人类。较佳的，所述恒温动物为哺乳动物或是鸟类，而所述哺乳动物可为：大鼠或小鼠。
本发明所述的“医药组合物或药物”可以多种形式存在，该等形式包含，但不限于液体、半固体及固体药剂形式，诸如溶液(solution)、乳剂(emulsion)、悬浮液(suspension)以及其他类似或适用本发明的剂型。
在一实施例中，本发明的医药组合物为注射剂型。且所述注射剂型是由静脉注射。
本发明的优点在于可简单、大量的连续生产具有线粒体制剂；且可直接从培养液中分离出具有线粒体的微囊泡，不需要额外打破细胞取线粒体，不需要高成本且耗时的线粒体高速离心过程，能达到节省成本、节省时间的效果。且收集到的线粒体，是已经由微囊泡包覆的线粒体囊泡，还有外泌因子的小分子核糖核酸的保护，因此更容易冷冻保存。
本发明的经分离的胞外囊泡组合物确实可以降低糖尿病大鼠的空腹血糖值及/或胰岛素浓度，而改善胰岛素抗性，而可有效治疗或减缓糖尿病。并能降低慢性肾病小鼠血液中肌酸酐、尿素氮，而可有效治疗或减缓肾损伤。
附图说明
图1为处理促进线粒体产生因子、促进胞外囊泡产生因子或其组合后人类间充质干细胞所生产的胞外囊泡中具有线粒体的数量，*表示信赖区间>95％；
图2为处理促进线粒体产生因子、促进胞外囊泡产生因子或其组合后人类间充质干细胞所生产具有线粒体的微囊泡占每一处理组中所产生的胞外囊泡的比例，*表示信赖区间>95％；
图3为促进线粒体产生因子胞外囊泡、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡降低第二型糖尿病大鼠的空腹血糖值的效果，*表示信赖区间>95％；
图4为促进线粒体产生因子胞外囊泡、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡降低第二型糖尿病大鼠的空腹胰岛素浓度效果，*表示信赖区间>95％，**表示信赖区间>90％；
图5为促进线粒体产生因子胞外囊泡、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡降低慢性肾病小鼠的血液肌酸酐效果，*表示信赖区间>95％；
图6为促进线粒体产生因子胞外囊泡、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡降低慢性肾病小鼠的血液尿素氮效果，*表示信赖区间>95％。
具体实施方式
本发明将由下列的实施例作为进一步说明，这些实施例并不限制本发明前面所揭示的内容。熟习本发明的技艺者，可以做些许的改良与修饰，但不脱离本发明的范畴。
实施例1培养间充质干细胞生成胞外囊泡(Extracellular Vesicles，EVs)
将健康捐赠者的分离自脐带的人类间充质干细胞以含有10％胎牛血清(Fetal bovine serum，FBS)的高糖(葡萄糖浓度为4500毫克(mg)/升(L))的达尔伯克氏改良伊格尔氏培养基(DMEM)培养于150毫米(mm)的培养皿中，且于5％二氧化碳、37℃下培养24至48小时直至细胞达40％至60％的满度。以前述的DMEM培养基冲洗细胞两次后，添加10％FBS的前述DMEM后对细胞分别进行四组处理：对照组仅以培养基培养细胞，未有额外处理；处理促进线粒体产生因子组，是使加入促进线粒体产生因子后，培养基中 的促进线粒体产生因子的浓度为40纳克/毫升(ng/mL)，在本实施例中促进线粒体产生因子是选用血红加氧酶1(Enzo LifeSciences,Ann Arbor,MI,USA)；处理促进胞外囊泡产生因子组，是使加入促进胞外囊泡产生因子后，培养基中的促进胞外囊泡产生因子的浓度为50mM，在本实施例中促进胞外囊泡产生因子是选用乙醇；或共同处理促进线粒体产生因子及促进胞外囊泡产生因子共同组(后简称共同处理组)，在本实施例中是先添加促进线粒体产生因子、再添加促进胞外囊泡产生因子，具体而言是在人类间充质干细胞培养24小时后，加入促进线粒体产生因子－血红加氧酶1，并使得培养基中之血红加氧酶1的浓度为40ng/mL，并在培养至第36小时时，加入促进胞外囊泡产生因子－乙醇，使得培养基中的乙醇的浓度为50mM，在培养72小时后取上清液，以搜集各处理组所产生的条件培养基(conditioned medium，CM)中的胞外囊泡。具体而言，将各处理组所产生的条件培养基以3000g离心10分钟以移除死细胞或较大的细胞片段，接着，将各组条件培养基的上清液利用分离胞外囊泡的预洗管柱进行预洗(Extracellular vesicle Isolation Pre-Clearing Column)。将预洗后的上清液移至胞外囊泡分离管柱(CapturemTM)，并于室温下以1,000g离心2至4分钟。将前述管柱以胞外囊泡分离冲洗缓冲液(Extracellular vesicle Isolation Wash Buffer)冲洗一次，再以套组中的胞外囊泡洗脱缓冲液(Extracellular vesicle Isolation Elution Buffer)洗下各组的胞外囊泡。
测试例1纳米颗粒追踪分析
将实施例1各组处理所得的胞外囊泡以磷酸盐缓冲生理食盐水(phosphate-buffered saline，PBS)稀释，使得视野中具有20-100颗粒的稀释液，以纳米颗粒追踪分析(Nanoparticle Tracking Analysis，NTA)测量具有线粒体的微囊泡数量，而对具有线粒体的微囊泡(microvesicle)的最终计数则是利用荧光纳米颗粒追踪分析(fluorescent NTA，fNTA)进行。将实施例1分离的胞外囊泡利用TMRE荧光染剂(tetramethylrhodamine ethyl ester)标记以侦测胞外囊泡中的线粒体，其中，TMRE荧光染剂的激发光及放射光波长分别为550纳米(nm)及575nm。而胞外囊泡及具有线粒体的微囊泡的尺寸则是利用纳米粒径分析仪(NanoSight LM10-HS system，Malvern，英国)进行测量，并以纳米颗粒追总软件(版本2.3)分析数据。
各处理组的线粒体数量测量结果如图1所示，可看到处理促进线粒体产生因子组、处理促进胞外囊泡产生因子组及共同处理组所得的具有线粒体的微囊泡，跟对照组相比，确实促进较多具有线粒体的微囊泡产生，因此以促进线粒体产生因子、促进胞外囊泡产生因子或促进线粒体产生因子及促进胞外囊泡产生因子共同处理人类间充质干细 胞确实均可促进人类间充质干细胞产生较多的具有线粒体的微囊泡。其中又以处理促进线粒体产生因子组及共同处理组较能促进人类间充质干细胞产生较多的具有线粒体的微囊泡，分别为对照组的16倍及132倍。
此外，将图2的结果与各处理组所得的胞外囊泡数量相除，以获得具有线粒体的微囊泡占每一处理组中所产生的胞外囊泡的比例，结果如图2所示，处理促进线粒体产生因子组、处理促进胞外囊泡产生因子组及共同处理组所得的胞外囊泡，跟对照组相比，所产生的胞外囊泡中确实有较高比例具有线粒体，因此以促进线粒体产生因子、促进胞外囊泡产生因子或促进线粒体产生因子及促进胞外囊泡产生因子共同处理人类间充质干细胞确实均可促进人类间充质干细胞产生的胞外囊泡中有较高比例具有线粒体。其中又以处理促进线粒体产生因子组及共同处理组，能使人类间充质干细胞产生的胞外囊泡中有较高比例具有线粒体，分别为1.89％及4.9％，而与对照组相比分别为14.44倍及37.40倍。
因此，联合使用促进线粒体产生因子及促进胞外囊泡产生因子，确实可促进具有线粒体的细胞生成具有线粒体的微囊泡、本发明方法确实可以大量生成具有线粒体的微囊泡。
测试例2第二型糖尿病大鼠模型的动物实验
将重量为150-200公克(g)的雄性白化大鼠每七只安置于1鼠笼中，饲养在控温(22℃至25℃)及12小时明暗循环(08:00-20:00进行光照)的环境下。实验动物能自由获取食物和水。于实验第0天，将实验动物随机分为两组：以标准实验食物饲养两周的正常对照组(n＝7)及以高脂饲料(High-fat diet，HFD；20％蛋白质、60％脂肪及20％碳水化合物)饲养两周的糖尿病组。在第14天，对糖尿病组实验动物以腹腔注射每公斤45毫克(mg/kg)的低剂量链佐霉素(streptozotocin，STZ)以诱发第二型糖尿病。低剂量链佐霉素及高脂饮食均为诱发第二型糖尿病胰岛素抗性的必要元素。因此，所有大鼠能自由获取组别对应的食物和水持续至试验结束。在实验第21天，测量空腹隔夜的对照组及糖尿病组的空腹血糖(fasting blood glucose，FBG)及胰岛素数值。第二型糖尿病模型大鼠再分为：糖尿病控制组(control组)、促进线粒体产生因子胞外囊泡组、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡组(每组n＝7)。对每组大鼠进行每周2次PBS缓冲溶液(糖尿病控制组)尾静脉注射，共投予2周；或每周2次、实施例1所得各处理组的胞外囊泡的尾静脉注射，其中，每次投予每公斤体重3×108胞外囊泡，共两周。实验期间，以一般饲料饲养所有大鼠，在给药后三周，对大鼠采血。
图3及图4分别为糖尿病大鼠经各处理的空腹血糖及胰岛素抗性改善情形。其中，图3可以看出所有处理组：促进线粒体产生因子胞外囊泡组、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡组的糖尿病大鼠的空腹血糖值均较糖尿病控制组大鼠要低，其中以促进线粒体产生因子胞外囊泡组及共同处理胞外囊泡组的效果较佳，促进线粒体产生因子胞外囊泡组相较糖尿病对控制降低61.78％的空腹血糖值，而共同处理胞外囊泡组相较糖尿病控制组更是降低了65.35％的空腹血糖值，且与正常对照组大鼠类似。
图4中可以看出所有处理组：促进线粒体产生因子胞外囊泡组、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡组的糖尿病大鼠的空腹胰岛素浓度均较糖尿病控制组大鼠要低，其中以促进线粒体产生因子胞外囊泡组及共同处理胞外囊泡组的效果较佳，促进线粒体产生因子胞外囊泡组相较糖尿病控制组降低53.70％的空腹胰岛素浓度，而共同处理胞外囊泡组相较糖尿病控制组更是降低了56％的空腹胰岛素浓度，且与正常对照组大鼠类似。
因此，本发明方法所制备的具有线粒体的微囊泡、胞外囊泡组合物，确实可以治疗第二型糖尿病。能降低空腹血糖值并改善糖尿病大鼠因胰岛素抗性造成的高胰岛素浓度。
测试例3慢性肾病小鼠模型的动物实验
所有动物实验均依照实验动物照护及使用指南进行。将六周龄的BALB/c小鼠饲养在控温(25℃)及12小时明暗循环的环境下。将小鼠分为下述五组(每组n＝5)：(1)正常对照组、(2)慢性肾病控制组(以PBS处理)、(3)促进线粒体产生因子胞外囊泡组、(4)促进胞外囊泡产生因子胞外囊泡组、(5)共同处理胞外囊泡组。其中，小鼠的慢性肾病是利用喂饲0.25％含有腺嘌呤的饲料以进行诱发。一周后，对每组小鼠进行每周2次的PBS缓冲溶液(慢性肾病控制组)尾静脉注射；或每周2次，实施例1所得各处理组的胞外囊泡的尾静脉注射，其中，每次投予每公斤体重3×108胞外囊泡，共两周。实验期间，以一般饲料饲养所有小鼠，在给药后三周，对小鼠采血。
将搜集后的小鼠血液以1500rpm离心30分钟后，取上层血清，用以搭配酵素免疫测定法(enzyme-linked immunosorbent assay，ELISA)测量其中的血液肌酸酐及尿素氮(blood urea nitrogen，BUN)数值，以利用微孔盘分析仪(Thermo Fisher Scientific)测量450nm下的吸光值。
图5及图6分别为慢性肾病小鼠经各处理的的血液肌酸酐及尿素氮数值改善情形。 图5可以看出所有处理组：促进线粒体产生因子胞外囊泡组、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡组的慢性肾病小鼠的血液肌酸酐数值均较慢性肾病控制组小鼠要低，其中以促进线粒体产生因子胞外囊泡组及共同处理胞外囊泡组的效果较佳。促进线粒体产生因子胞外囊泡组相较慢性肾病对照组降低近约54.06％的肌酸酐数值，而共同处理胞外囊泡组相较慢性肾病对照组更是降低了67.96％的肌酸酐数值，且与正常对照组小鼠类似。
图6可以看出所有处理组：促进线粒体产生因子胞外囊泡组、促进胞外囊泡产生因子胞外囊泡组及共同处理胞外囊泡组的慢性肾病小鼠的血液尿素氮数值均较慢性肾病控制组小鼠要低，其中以促进线粒体产生因子胞外囊泡组及共同处理胞外囊泡组的效果较佳。促进线粒体产生因子胞外囊泡组相较慢性肾病对照组降低近约66.80％的尿素氮数值，而共同处理胞外囊泡组相较慢性肾病对照组更是降低了79.42％的尿素氮数值，且与正常对照组小鼠类似。
因此，本发明方法所制备的具有线粒体的微囊泡，确实可以治疗、改善糖尿病；并能降低肾损伤、慢性肾病小鼠血液中的肌酸酐数值及尿素氮数值。
综上，本发明方法确实可以大量制备具有线粒体的微囊泡。且本发明方法制备所得的经分离的胞外囊泡组合物，因包含大量具有线粒体的微囊泡而确实可以治疗或减缓糖尿病，如第二型糖尿病以及肾脏损伤，如慢性肾病。
以上所述仅是为方便说明本发明的一较佳实施例，并非用于限定本发明的范围，本发明所主张的权利范围自应以申请专利范围所述为主。

Claims (16)

  1. 一种联合使用促进线粒体产生因子及促进胞外囊泡产生因子的用途，其是用于促进具有线粒体的细胞生成具有线粒体的微囊泡。
  2. 根据权利要求1所述的联合使用促进线粒体产生因子及促进胞外囊泡产生因子的用途，其中，所述促进线粒体产生因子可选自由：血红加氧酶1、CoPPIX、血红素、血红素衍生物、线粒体结构破片、细胞组织破片及5％-20％氧浓度环境培养所组成的群组。
  3. 根据权利要求1所述的联合使用促进线粒体产生因子及促进胞外囊泡产生因子的用途，其中，所述促进胞外囊泡产生因子可选自由：乙醇、EP4受体拮抗剂、内体蛋白分选转运复合体、ARRDC1、相关蛋白的肿瘤抑制基因及细胞松弛素B所组成的群组。
  4. 根据权利要求1所述的联合使用促进线粒体产生因子及促进胞外囊泡产生因子的用途，其中，所述具有线粒体的细胞为间充质干细胞、造血干细胞、骨髓干细胞或肝脏细胞。
  5. 一种生成富含具有线粒体的微囊泡的胞外囊泡组合物的方法，其包含：
    步骤(1)提供具有线粒体的细胞；及
    步骤(2)对所述具有线粒体的细胞添加促进线粒体产生因子及促进胞外囊泡产生因子于培养基中进行共培养，得到富含具有线粒体的微囊泡的胞外囊泡组合物，其中该富含具有线粒体的微囊泡的胞外囊泡组合物，以胞外囊泡总数为基准，具有0.2％以上的具有线粒体的微囊泡。
  6. 根据权利要求5所述的生成富含具有线粒体的微囊泡的胞外囊泡组合物的方法，其中，所述促进线粒体产生因子的添加量是使所述培养基中的所述促进线粒体产生因子的浓度为0.1纳克/毫升(ng/mL)至100000ng/mL。
  7. 根据权利要求5或6所述的生成富含具有线粒体的微囊泡的胞外囊泡组合物的方法，其中，所述促进胞外囊泡产生因子的添加量是使所述培养基中的所述促进胞外囊泡产生因子的浓度为10-7毫摩尔每升(mM)至800mM。
  8. 一种经分离的胞外囊泡组合物，其以胞外囊泡总数为基准，具有0.2％以上的具有线粒体的微囊泡。
  9. 根据权利要求8所述的经分离的胞外囊泡组合物，其中所述经分离的胞外囊泡组合物是经权利要求5至7任一项所述方法制备，再经分离后而得。
  10. 一种医药组合物，其包含根据权利要求8或9所述的经分离的胞外囊泡组合物， 及药学上可接受的载剂。
  11. 一种经分离的胞外囊泡组合物的用途，其是作为制备治疗或减缓糖尿病药物的应用，其中，所述药物中包含有效剂量的经分离的胞外囊泡组合物及药学上可接受的载剂，且所述经分离的胞外囊泡组合物以胞外囊泡总数为基准，具有0.2％以上的具有线粒体的微囊泡。
  12. 根据权利要求11所述的经分离的胞外囊泡组合物的用途，其中，所述糖尿病为第二型糖尿病。
  13. 根据权利要求11或12所述的经分离的胞外囊泡组合物的用途，其中，所述药物的给药对象为恒温动物或人类。
  14. 一种经分离的胞外囊泡组合物的用途，其是作为制备治疗或减缓肾脏损伤药物的应用，其中，所述药物中包含有效剂量的经分离的胞外囊泡组合物及药学上可接受的载剂，且所述经分离的胞外囊泡组合物以胞外囊泡总数为基准，具有0.2％以上的具有线粒体的微囊泡。
  15. 根据权利要求14所述的经分离的胞外囊泡组合物的用途，其中，所述肾脏损伤为慢性肾病。
  16. 根据权利要求14或15所述的经分离的胞外囊泡组合物的用途，其中，所述药物的给药对象为恒温动物或人类。
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