WO2021185376A1 - 含有线粒体的组合物及其修复软骨损伤或改善退化性关节炎的用途 - Google Patents

含有线粒体的组合物及其修复软骨损伤或改善退化性关节炎的用途 Download PDF

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WO2021185376A1
WO2021185376A1 PCT/CN2021/082094 CN2021082094W WO2021185376A1 WO 2021185376 A1 WO2021185376 A1 WO 2021185376A1 CN 2021082094 W CN2021082094 W CN 2021082094W WO 2021185376 A1 WO2021185376 A1 WO 2021185376A1
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mitochondria
cells
composition
cartilage
degenerative arthritis
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PCT/CN2021/082094
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French (fr)
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郑汉中
许智凯
杨舜杰
郑安玲
凃启堂
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台湾粒线体应用技术股份有限公司
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Priority to CN202180022422.7A priority Critical patent/CN115297872B/zh
Priority to EP21770632.4A priority patent/EP4122444A4/en
Publication of WO2021185376A1 publication Critical patent/WO2021185376A1/zh
Priority to US17/948,007 priority patent/US20230023438A1/en

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Definitions

  • the present invention relates to the use of mitochondria and a composition containing mitochondria, in particular to the use of mitochondria for preparing a composition for repairing cartilage damage or improving degenerative arthritis and a composition containing mitochondria and high-concentration platelet plasma.
  • Degenerative arthritis is the most common joint disease in human aging.
  • the main lesion is the joint contact surface between the bones, which is the so-called articular cartilage.
  • damage to cartilage, uneven joint surfaces, narrowing of the joint cavity, and bone spurs can be observed.
  • the main symptoms of degenerative arthritis include pain, stiffness, swelling, inflammation, or deformation.
  • Traditional degenerative arthritis treatment methods include drug therapy, physical therapy, hyaluronic acid injection, artificial joint replacement and so on.
  • mitochondria are provided to cartilage to repair cartilage damage, thereby improving and treating degenerative arthritis.
  • An embodiment of the present invention provides a use of mitochondria for preparing a composition for repairing cartilage damage.
  • An embodiment of the present invention provides a use of mitochondria for preparing a composition for improving degenerative arthritis.
  • An embodiment of the present invention provides a composition comprising mitochondria and blood-derived growth factors.
  • An embodiment of the present invention provides a composition containing mitochondria.
  • mitochondria By providing mitochondria to cartilage, the function of mitochondria in cartilage cells can be improved to improve the repair ability of cartilage cells and repair cartilage damage caused by aging of cartilage cells, thereby improving cartilage damage or degradation. Pain, inflammation, swelling, or stiffness caused by arthritis can improve and treat degenerative arthritis.
  • Figure 1 is a graph of the cell survival rate of human chondrosarcoma cells treated with 75 ⁇ M tBHP and repaired with the composition of the example.
  • Figure 2 is a graph showing the degree of aging of human chondrosarcoma cells treated with 50 ⁇ M tBHP and repaired with the composition of the example.
  • Figure 3 is a stained photograph of human chondrosarcoma cells treated with 50 ⁇ M tBHP and repaired with the composition of the example.
  • Fig. 4 is an analysis diagram of mitochondrial membrane potential of human chondrosarcoma cells treated with 50 ⁇ M tBHP and repaired with the composition of the example.
  • Mitochondria are the sites for oxidative phosphorylation and the synthesis of adenosine triphosphate (ATP) in cells.
  • mitochondria are also responsible for regulating oxidative stress processing and signal transmission in cells.
  • the function of mitochondria in damaged or aging chondrocytes is significantly reduced, which is manifested in the decrease of ATP production efficiency and the decrease of membrane potential.
  • the efficiency of the electron transport chain becomes worse.
  • various biological responses will begin to decline, that is, cartilage cells begin to age, making cartilage cells gradually unable to maintain normal operations, and symptoms of inflammation or arthritis will appear. Therefore, by supplying healthy mitochondria to cartilage cells, cartilage aging can be improved and cartilage repair ability can be improved, and further treatment of diseases caused by aging such as degenerative arthritis.
  • the concentration of mitochondria may range from 1 microgram per milliliter to 4000 micrograms ( ⁇ g/mL), but is not limited thereto.
  • the concentration of mitochondria may range from 1 microgram to 200 micrograms per milliliter.
  • the concentration of mitochondria may range from 1 microgram to 100 micrograms per milliliter.
  • the concentration of mitochondria may be 1 microgram to 40 micrograms per milliliter.
  • the concentration of mitochondria may range from 15 micrograms to 40 micrograms per milliliter.
  • the effective dose of mitochondria can be 7.9 mg to 2.11 g per 1 square centimeter of the joint cavity, but it is not limited to this. In other embodiments, the effective dose of mitochondria may be 7.9 mg to 21.1 mg per 1 square centimeter of the joint cavity.
  • the mitochondria included in the composition may be exogenous mitochondria.
  • Exogenous mitochondria are cells that are taken from a different person than the person who administers the composition.
  • the person applying the composition and the person providing the exogenous mitochondria are more preferably of the same genus, and more preferably of the same species.
  • the mitochondria may be autologous mitochondria taken from the cells of the person administering the composition. After the cells that provide mitochondria are taken out, the mitochondria can be separated directly from the cells or the cells can be cultured and proliferated in vitro to separate the mitochondria.
  • the aforementioned cells that provide exogenous mitochondria or provide autologous mitochondria may be adipose stem cells, monocytes, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, CD34+ stem cells, bone marrow stem cells and other cells with mitochondria.
  • composition of this embodiment can be administered to the joint by oral, injection or other methods.
  • the composition is injected into the joint cavity so that the composition contacts the chondrocytes in the joint cavity and enters the chondrocytes.
  • the composition further comprises growth factors derived from blood.
  • Growth factors can be derived from high-concentration platelet-rich plasma (PRP).
  • the growth factor may comprise TGF- ⁇ 1, PDGF-AA, PDGF-AB or PDGF-BB.
  • the high-concentration platelet plasma described here contains 1 ⁇ 10 9 platelets and a variety of growth factors per milliliter.
  • the concentration of TGF- ⁇ 1 can be more than 7767 picograms/ml (pg/ml), and the concentration of PDGF-AA can be It is 9.73 picogram/ml (pg/ml) or more, the concentration of PDGF-AB can be more than 10 picogram/ml (pg/ml), and the concentration of PDGF-BB can be more than 590 picogram/ml (pg/ml) .
  • the ratio of mitochondria to high-concentration platelet plasma may be 1 microgram to 40 micrograms of mitochondria per 10 microliters of high-concentration platelet plasma, that is, 1 microgram: 10 microliters to 40 micrograms: 10 microliters, but Not limited to this.
  • the ratio of mitochondria to high-concentration platelet plasma may be 15 micrograms to 40 micrograms of mitochondria per 10 microliters of high-concentration platelet plasma, that is, 15 micrograms: 10 microliters to 40 micrograms: 10 microliters.
  • the concentration of mitochondria may range from 1000 micrograms to 4000 micrograms per milliliter ( ⁇ g/mL), but is not limited thereto. In other embodiments, the concentration of mitochondria may range from 1500 micrograms to 4000 micrograms per milliliter.
  • the effective dose of mitochondria can be 7.9 mg to 2.11 g per 1 square centimeter of the joint cavity, but it is not limited to this. In other embodiments, the effective dose of mitochondria may be 7.9 mg to 21.1 mg per 1 square centimeter of the joint cavity.
  • the composition may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes a carrier used in any standard medical product or cosmetic product.
  • the carrier may be semisolid or semisolid depending on the form of the composition. liquid.
  • the carrier includes, but is not limited to, hyaluronic acid, gelatin, emulsifier, water, physiological saline, buffered saline or ethanol, and other substances that do not affect the activity of mitochondria.
  • the mitochondria used in the examples of the present invention are taken from human adipose-derived stem cells (ADSC).
  • the stem cell culture medium contains Keratinocyte SFM 1X solution (Gibco), bovine pituitary extract (BPE, Gibco), and fetal bovine serum (HyClone) with a concentration of 10% by weight.
  • the human adipose stem cells were cultured in a petri dish to a cell number of 1.5 ⁇ 10 8 cells, and the cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS).
  • DPBS Dulbecco's Phosphate Buffered Saline
  • the stem cell culture medium was added to stop the reaction. Then, the cells were washed and dispersed, centrifuged at 600g for 10 minutes, and the supernatant was removed. Then, 80 ml of IBC-1 buffer (225mM mannitol, 75mM sucrose, 0.1mM EDTA, 30mM Tris-HCl pH 7.4) was added to the cells, and the cells were ground 15 times on ice in a homogenizer.
  • IBC-1 buffer 225mM mannitol, 75mM sucrose, 0.1mM EDTA, 30mM Tris-HCl pH 7.4
  • the precipitate obtained is mitochondria.
  • IBC-2 buffer 225mM mannitol, 75mM sucrose, 30mM Tris-HCl pH 7.4
  • proteolytic enzyme inhibitor were added to the mitochondrial precipitate, and stored at 4°C.
  • 75 ⁇ M tBHP was used to induce damage to human chondrosarcoma cells (SW-1353).
  • the Alma Blue detection reagent is used to evaluate the repair effect of the composition containing mitochondria on the damage of human chondrosarcoma cells, and it is expressed in terms of cell survival rate.
  • Human chondrosarcoma cells are often used to explore the mechanism of cartilage damage and are a common chondrocyte assessment model.
  • human chondrosarcoma cell culture medium was used to culture human chondrosarcoma cells in a 0% carbon dioxide environment.
  • Human chondrosarcoma cell culture medium contains 90% Leibovitz’s L-15 medium, 2mM L-glutamic acid and 10% fetal bovine serum by weight.
  • Tert-butyl hydroperoxide is an organic peroxide, commonly used as a substance that induces cell oxidative stress damage, aging and cell apoptosis. This experiment uses tBHP as a substance that induces aging or damage to human chondrosarcoma cells.
  • Alamar blue is a detection reagent used to detect cell viability.
  • the resazurin in the detection kit is a redox indicator, which is a non-toxic, dark blue dye that can penetrate cell membranes and has low fluorescence. When resazurin enters healthy cells, it will be reduced to pink and highly fluorescent resorufin due to the reducing environment in living cells.
  • the viability of cells can be evaluated by measuring the light absorption or fluorescence of resorufin. The higher the light absorption or fluorescence value of resorufin, the higher the cell viability. The higher the cell viability, the healthier the cells and the stronger the proliferation ability. The stronger the cell proliferation ability, the more cell mass. Therefore, this experiment uses Alma Blue as an indicator to evaluate cell survival and cell proliferation.
  • the experimental procedure is explained below. First, take human chondrosarcoma cells cultured between 4 to 10 generations for experiments. When the human chondrosarcoma cells are cultured to an eighth full volume of the petri dish, the culture medium in the petri dish is removed and the cells are rinsed with phosphate buffered saline (PBS). Then, 0.25% trypsin was added to the petri dish and the trypsin was allowed to react at 37°C for 5 minutes, and then the human chondrosarcoma cell culture medium was added to stop the trypsin reaction.
  • PBS phosphate buffered saline
  • tBHP was added to the 24-well plate, and the human chondrosarcoma cells were cultured in a culture medium containing tBHP at a concentration of 75 ⁇ M for 4 hours. Then, the human chondrosarcoma cell culture medium containing tBHP is removed, and the culture medium containing the composition of different concentrations of mitochondria is added, and the human chondrosarcoma cells are cultured in the culture medium containing the composition of different mitochondrial concentrations for 20 hours.
  • the concentration of mitochondria in the well disk is 1 ⁇ g/mL, 15 ⁇ g/mL or 40 ⁇ g/mL.
  • Figure 1 is a graph of the cell survival rate of human chondrosarcoma cells treated with 75 ⁇ M tBHP and repaired with the composition of the example.
  • the blank group consisted of human chondrosarcoma cells without tBHP-induced damage.
  • the control group was human chondrosarcoma cells that were injured by tBHP but were not administered with the composition of the example.
  • the test group consisted of human chondrosarcoma cells treated with a composition containing 1 ⁇ g/mL, 15 ⁇ g/mL, or 40 ⁇ g/mL mitochondria treated with tBHP-induced damage.
  • the vertical axis is the number of cells relative to the blank group, expressed in terms of cell survival rate (%).
  • the experimental results showed that the cell survival rate of the test group was greater than that of the control group, and the cell survival rate increased with the increase of mitochondrial concentration, indicating that the composition containing mitochondria of the examples can indeed improve the damage and damage caused by tBHP to chondrocytes. die.
  • tBHP 50 ⁇ M tBHP was used to induce aging of human chondrosarcoma cells.
  • the SA- ⁇ -gal kit is used to evaluate the repair effect of the composition containing mitochondria on the aging caused by tBHP, and it is expressed as the degree of aging.
  • SA- ⁇ -gal Senescence-associated beta-galactosidase
  • the experimental procedure of this experiment is roughly the same as the experimental procedure of experiment one above. The difference is that human chondrosarcoma cells were cultured in a 12-well dish containing human chondrosarcoma cell culture medium at a density of 4 ⁇ 10 4 cells per well for 16 hours. Next, tBHP was added to the 12-well plate, and the human chondrosarcoma cells were cultured in a culture medium containing tBHP at a concentration of 50 ⁇ M for 4 hours.
  • the human chondrosarcoma cell culture medium containing tBHP is removed, and the culture medium containing the composition of different concentrations of mitochondria is added, and the human chondrosarcoma cells are cultured in the culture medium containing the composition of different mitochondrial concentrations for 20 hours.
  • the concentration of mitochondria in the orifice plate is 1 ⁇ g/mL, 15 ⁇ g/mL or 40 ⁇ g/mL.
  • Figure 2 is a graph showing the degree of aging of human chondrosarcoma cells treated with 50 ⁇ M tBHP and repaired with the composition of the example.
  • Figure 3 is a stained photograph of human chondrosarcoma cells treated with 50 ⁇ M tBHP and repaired with the composition of the example.
  • the blank group consisted of human chondrosarcoma cells without tBHP-induced damage.
  • the control group was human chondrosarcoma cells that were injured by tBHP but were not administered with the composition of the example.
  • the test group consisted of human chondrosarcoma cells treated with a composition containing 1 ⁇ g/mL, 15 ⁇ g/mL, or 40 ⁇ g/mL mitochondria treated with tBHP-induced damage.
  • the vertical axis is the degree of aging (%), and the degree of aging is the percentage of the number of aging cells to the total number of cells. ### indicates a significant difference compared to the control group (P ⁇ 0.01).
  • the experimental results show that the aging degree of the test group is less than the aging degree of the control group, and the degree of cell aging decreases as the concentration of mitochondria increases, indicating that the composition containing mitochondria of the examples can indeed improve the aging of cartilage cells caused by tBHP.
  • Mitochondrial function analysis system judges the quality of mitochondrial function by measuring the change of mitochondrial membrane potential.
  • TMRE tetramethylrhodamine ethyl ester
  • FCCP Carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone
  • FCCP Carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone
  • FCCP can be used to eliminate the membrane potential of mitochondria, so it is often used as a control group for mitochondrial inactivation or depolarization.
  • TMRE and FCCP staining the fluorescence intensity is analyzed to know the change of mitochondrial membrane potential, which is used as the basis for judging the function of mitochondria.
  • the supernatant treated with TMRE is detected by flow cytometry, and the proportion of functional mitochondria in all particles in the supernatant can be analyzed according to the TMRE label.
  • the experimental procedure of this experiment is roughly the same as the experimental procedure of experiment one above. The difference is that the human chondrosarcoma cells were cultured in a 6-well dish containing human chondrosarcoma cell culture medium at a density of 1 ⁇ 10 5 cells per well for 16 hours. Next, tBHP was added to the 6-well plate, and the human chondrosarcoma cells were cultured in a culture medium containing tBHP at a concentration of 50 ⁇ M for 4 hours.
  • the human chondrosarcoma cell culture medium containing tBHP is removed, and the culture medium containing the composition of different concentrations of mitochondria is added, and the human chondrosarcoma cells are cultured in the culture medium containing the composition of different mitochondrial concentrations for 20 hours.
  • the concentration of mitochondria in the orifice plate is 1 ⁇ g/mL, 15 ⁇ g/mL or 40 ⁇ g/mL.
  • Fig. 4 is an analysis diagram of mitochondrial membrane potential of human chondrosarcoma cells treated with 50 ⁇ M tBHP and repaired with the composition of the example.
  • the blank group consisted of human chondrosarcoma cells without tBHP-induced damage.
  • the control group was human chondrosarcoma cells that were injured by tBHP but were not administered with the composition of the example.
  • the test group consisted of human chondrosarcoma cells treated with a composition containing 1 ⁇ g/mL, 15 ⁇ g/mL, or 40 ⁇ g/mL mitochondria treated with tBHP-induced damage.
  • the experimental results show that compared with the control group, the test group has more functional mitochondria, and the recovery degree is closer to the blank group as the concentration of mitochondria in the composition increases. Improve the mitochondrial function of chondrocytes. The improvement of mitochondrial function further indicates that the cells have better anti-aging and repair capabilities.
  • MIA monosodium iodoacetate
  • MIA Monosodium iodoacetate
  • Glyceraldehyde 3-phosphate dehydrogenase glyceraldehyde 3-phosphate dehydrogenase
  • OA human osteoarthritis
  • Rotarod analysis is an analysis method to evaluate the balance and coordination ability of mice.
  • One week before the test put the mouse on the rotating wheel for training, so that the mouse can spend more than three minutes on the rotating wheel. After the experimental treatment, the mouse's movement is evaluated by the time on the rotating wheel. ability.
  • High-concentration platelet-rich plasma is a concentrated solution of blood plasma protein that is concentrated with platelet-rich plasma by removing red blood cells after centrifugation. High-concentration platelet plasma is rich in growth factors, so it is considered to be helpful for the repair of cartilage, tendons, ligaments, etc.
  • the high-concentration platelet plasma in this experiment was from Sprague-Dawley (SD) rats. The rats were first anesthetized with 3% pentobarbital sodium at a dose of 30 mg/kg.
  • citrate dextrose acid citrate dextrose
  • anticoagulant syringe of rat whole blood is withdrawn from about 7-8 ml, wherein platelet concentration is about 0.5 ⁇ 10 9 to 1.5x10 9 per ml.
  • the second centrifugation After removing three quarters of the supernatant, the remaining quarter is the high-concentration platelet plasma used in this experiment.
  • the high-concentration platelet plasma described here contains 1 ⁇ 10 9 platelets and a variety of growth factors per milliliter.
  • the types and concentrations of growth factors included are as follows: TGF- ⁇ 1 ( ⁇ 7767pg/ml), PDGF-AA ( ⁇ 9.73pg/ml), PDGF-AB ( ⁇ 10pg/ml) and PDGF-BB ( ⁇ 590pg/ml).
  • mice are divided into cages and given a few days of adaptation period to prevent the mice from being nervous and anxious due to environmental discomfort and affecting the experimental results.
  • MIA the mice were subjected to a rotating wheel test, which was used as the data on day 0 and as a benchmark for subsequent comparison.
  • the mice were given 0.25 ml of 4% cholra hydrate 10 minutes before the operation.
  • isoflurane isoflurane was given to maintain anesthesia to prevent the mice from waking up.
  • 0.1 mg of MIA was dissolved in 10 microliters of normal saline, and a 30G (gauge) needle was used to inject into the joint cavity of the mouse.
  • the composition of the example was injected into the joint cavity of the mouse in the same injection method, and the rotating wheel test was performed on the 7th and 14th day.
  • the maximum length of each test was 1200 seconds. It is 20rpm.
  • the experimental results of the exercise capacity of the mice are shown in Table 4.
  • the control group was mice with degenerative arthritis induced by MIA but not administered with the composition of the example.
  • the PRP group was mice with degenerative arthritis induced by MIA and administered with high-concentration platelet plasma.
  • the test group was mice treated with MIA-induced degenerative arthritis and treated with a composition containing different concentrations of mitochondria and/or 10 microliters of high-concentration platelet plasma.
  • the results of the experiment showed that the exercise capacity of the mice in each group decreased to 30% to 40% on the 7th day, compared with the degenerative arthritis that was not affected on the 0th day.
  • the control group only recovered to 40%, while the exercise capacity of the mice in the PRP group and the test group recovered to more than 50%.
  • mice administered with the composition of Example 6 (15 ⁇ g of mitochondria and 10 ⁇ L of high-concentration platelet plasma)
  • the athletic ability is obviously improved.
  • the recovery of exercise ability in mice means that the pain, inflammation, swelling, or stiffness caused by cartilage damage or degenerative arthritis is relieved and improved.
  • the composition containing mitochondria can improve the exercise ability of animals suffering from degenerative arthritis, and may have a better effect as the concentration of mitochondria increases.
  • a composition containing mitochondria and high-concentration platelet plasma can have a more excellent improvement effect than a composition containing only mitochondria or only high-concentration platelet plasma.
  • the effective dose of mitochondria It can be 7.9 mg to 2.11 g per 1 square centimeter of cartilage tissue, preferably 7.9 mg to 21.1 mg per 1 square centimeter of cartilage tissue.
  • An embodiment of the present invention provides a composition containing mitochondria.
  • mitochondria By providing mitochondria to cartilage, the function of mitochondria in cartilage cells can be improved to improve the repair ability of cartilage cells and repair cartilage damage caused by aging of cartilage cells, thereby improving cartilage damage or degradation. Pain, inflammation, swelling, or stiffness caused by arthritis can improve and treat degenerative arthritis.

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Abstract

本发明一个实施例提供包含线粒体的组合物，藉由对软骨提供线粒体，可通过改善软骨细胞中线粒体的功能以提升软骨细胞的修复能力并修复软骨细胞老化导致的软骨损伤，进而达到改善并治疗退化性关节炎的目的。

Description

含有线粒体的组合物及其修复软骨损伤或改善退化性关节炎的用途 技术领域
本发明是关于线粒体的用途及含有线粒体的组合物，尤其是关于线粒体用于制备修复软骨损伤或改善退化性关节炎的组合物的用途以及含有线粒体及高浓度血小板血浆的组合物。
背景技术
退化性关节炎是人类老化最常见的关节疾病，主要病变的地方在于骨头与骨头之间的关节接触面，也就是所谓的关节软骨。在退化性关节炎病患的X光照片中可观察到软骨受损、关节表面凹凸不平、关节腔变窄以及骨刺产生。退化性关节炎主要的症状包含疼痛、僵硬、肿胀、发炎或变形。传统的退化性关节炎治疗方式包含药物治疗、物理治疗、玻尿酸注射、置换人工关节等。
然而，除了人工关节置换手术外，其他疗法大都只能短暂缓解发炎与疼痛，无法有效改善或延缓病程的进展。因此，如何改善并治疗退化性关节炎为目前的研究目标。
发明内容
根据本发明一个实施例，通过对软骨提供线粒体以修复软骨损伤，进而改善并治疗退化性关节炎。
本发明一个实施例提供一种线粒体用于制备修复软骨损伤的组合物的用途。
本发明一个实施例提供一种线粒体用于制备改善退化性关节炎的组合物的用途。
本发明一个实施例提供一种组合物，包含线粒体及来自血液的生长因子。
本发明一个实施例提供包含线粒体的组合物，藉由对软骨提供线粒体，可通过改善软骨细胞中线粒体的功能以提升软骨细胞的修复能力并修复软骨细胞老化导致的软骨损伤，改善软骨损伤或退化性关节炎所造成的疼痛现象、发炎肿胀反应或僵硬现象，进而达到改善并治疗退化性关节炎的目的。
附图说明
图1为使用75μM的tBHP处理再以实施例的组合物修复的人类软骨肉瘤细胞的细胞存活率的图表。
图2为使用50μM的tBHP处理再以实施例的组合物修复的人类软骨肉瘤细胞的老化程度的图表。
图3为使用50μM的tBHP处理再以实施例的组合物修复的人类软骨肉瘤细胞的染色照片。
图4为使用50μM的tBHP处理再以实施例的组合物修复的人类软骨肉瘤细胞的线粒体膜电位分析图。
具体实施方式
于以下实施方式中详细叙述本发明的详细特征及优点，其内容足以使任何熟习相关技艺者了解本发明的技术内容并据以实施，且根据本说明书所揭露的内容、权利要求书及附图，任何熟习相关技艺者可容易理解本发明相关的目的及优点。以下实施例用于进一步详细说明本发明的观点，但非以任何观点限制本发明的范畴。
线粒体是细胞内进行氧化磷酸化反应及合成三磷酸腺苷(ATP)的场所，除了提供细胞正常代谢所需的能量外，还负责调控细胞内氧化压力处理、讯号传递等功能。与正常的软骨细胞相比，在损伤或老化的软骨细胞中线粒体的功能明显下降，具体表现在ATP产能效率下降、膜电位下降电子传递链的效率变差等。伴随着线粒体的功能下降，各项生物反应会开始衰退，亦即是软骨细胞开始老化，使软骨细胞逐渐无法维持正常的运作，进而出现发炎或关节炎的症状。因此，通过将健康的线粒体提供给软骨细胞，可改善软骨老化现象并提升软骨修复能力，进一步治疗退化性关节炎等老化引起的疾病。
本发明一个实施例提供用于修复软骨损伤或改善退化性关节炎的组合物，包含线粒体。在本实施例中，线粒体的浓度可为每毫升1微克至4000微克(μg/mL)，但不限于此。在另一实施例中，线粒体的浓度可为每毫升1微克至200微克。在又一实施例中，线粒体的浓度可为每毫升1微克至100微克。在再一实施例中，线粒体的浓度可为每毫升1微克至40微克。在其他实施例中，线粒体的浓度可为每毫升15微克至40微克。在本实施例中，线粒体的有效剂量可为每1平方厘米的关节腔7.9毫克至2.11克，但不限于此。在其他实施例中，线粒体的有效剂量可为每1平方厘米的关节腔7.9毫克至21.1毫克。
组合物中包含的线粒体可为外源性线粒体。外源性线粒体为取自不同于施用组合物者的细胞。施用组合物者与提供外源性线粒体者较优选为同属，更优选为同种。在其他实施例中，线粒体可为取自施用组合物者本身的细胞的自体线粒体。提供线粒体的细胞被取出后，可直接从细胞分离出线粒体或将细胞经体外培养增殖后再分离出线粒体。前述提供外源性线粒体或提供自体线粒体的细胞可为脂肪干细胞、单核细胞、胚胎干细胞、间质干细胞、造血干细胞、CD34+干细胞、骨髓干细胞等具有线粒体的细胞。
本实施例的组合物可通过口服、注射等方式给予关节。通过注射的方式将组合物给予关节时，为将组合物注入到关节腔中，使组合物在关节腔中与软骨细胞接触而进入软骨细胞中。
在另一实施例中，组合物进一步包含来自血液的生长因子。生长因子可来自高浓度血小板血浆(platelet-rich plasma，PRP)。生长因子可包含TGF-β1、PDGF-AA、PDGF-AB或PDGF-BB。于此所述的高浓度血小板血浆每毫升含有1×10 9个血小板以及多种生长因子，其中TGF-β1的浓度可为7767皮克/毫升(pg/ml)以上，PDGF-AA的浓度可为9.73皮克/毫升(pg/ml)以上，PDGF-AB的浓度可为10皮克/毫升(pg/ml)以上，PDGF-BB的浓度可为590皮克/毫升(pg/ml)以上。在该组合物中，线粒体与高浓度血小板血浆的比例可为每10微升的高浓度血小板血浆含有1微克至40微克的线粒体，即1微克：10微升至40微克：10微升，但不限于此。在其他实施例中，线粒体与高浓度血小板血浆的比例可为每10微升的高浓度血小板血浆含有15微克至40微克的线粒体，即15微克：10微升至40微克：10微升。在此组合物中，线粒体的浓度可为每毫升1000微克至4000微克(μg/mL)，但不限于此。在其他实施例中，线粒体的浓度可为每毫升1500微克至4000微克。在本实施例中，线粒体的有效剂量可为每1平方厘米的关节腔7.9毫克至2.11克，但不以此为限。在其他实施例中，线粒体的有效剂量可为每1平方厘米的关节腔7.9毫克至21.1毫克。
在其他实施例中，组合物可进一步包含药学上可接受的载体，药学上可接受的载体包含用于任何标准医疗产品或美容产品的载体，依据组合物的形式所述载体可为半固体或液体。举例而言，载体包含但不限于玻尿酸、明胶、乳化剂、水、生理盐水、缓冲盐水或乙醇等不影响线粒体活性的物质。
以下说明如何制备本发明实施例的组合物。
[线粒体萃取]
本发明实施例所使用的线粒体取自人类脂肪干细胞(adipose-derived stem cell，ADSC)。干细胞培养液包含Keratinocyte SFM 1X溶液(Gibco)、牛垂体提取物(bovine pituitary extract，BPE，Gibco)、重量百分浓度10％的胎牛血清(HyClone)。在培养皿中将人类脂肪干细胞培养至细胞数为1.5×10 8个细胞，以杜氏磷酸盐缓冲液(DPBS)冲洗细胞。接着，移除杜氏磷酸盐缓冲液后，加入细胞剥离用的胰蛋白酶(Trypsin)，在37℃下反应3分钟后，再加入干细胞培养液以终止反应。接着，将细胞冲洗下来后打散，以600g离心10分钟，移除上清液。接着，于细胞中加入80毫升的IBC-1缓冲液(225mM甘露醇、75mM蔗糖、0.1mM EDTA、30mM Tris-HCl pH 7.4)，在均质器中于冰上研磨15次。接着，以1000g离心15分钟，将上清液收集至另一离心管，再以9000g离心10分钟，移除上清液。所获得的沉淀物即为线粒体。在线粒体沉淀物中加入1.5毫升的IBC-2缓冲液(225mM甘露醇、75mM蔗糖、30mM Tris-HCl pH 7.4)及蛋白质分解酶抑制剂，并置于4℃下保存。
〔实验一，修复软骨细胞损伤〕
在本实验中，使用75μM的tBHP诱导人类软骨肉瘤细胞(SW-1353)出现损伤。通过阿尔玛蓝检测试剂来评估包含线粒体的组合物对人类软骨肉瘤细胞的损伤的修复效果，并以细胞存活率来表示。
人类软骨肉瘤细胞常被用于探讨软骨损伤的机理，是常见的软骨细胞评估模式。本实验使用人类软骨肉瘤细胞培养液在0％二氧化碳的环境下进行人类软骨肉瘤细胞的培养。人类软骨肉瘤细胞培养液包含90％Leibovitz’s L-15培养基、2mM L-麸酰胺酸及重量百分浓度10％的胎牛血清。
过氧化叔丁醇(tert-butyl hydroperoxide，tBHP)为一种有机过氧化物，常用作为诱导细胞氧化压力损伤、老化与细胞凋亡的物质。本实验使用tBHP作为诱导人类软骨肉瘤细胞老化或损伤的物质。
阿尔玛蓝(Alamar blue)是用于检测细胞活力的检测试剂。检测试剂盒内的刃天青(resazurin)是一种氧化还原指示剂，其为无毒、可穿透细胞膜且低荧光性的深蓝色染料。当刃天青进入健康的细胞中，会因活细胞体内的还原环境而被还原成粉红色且具高荧光性的试卤灵(resorufin)。可藉由测量试卤灵的光吸收值或荧光值来评估细胞的活力。试卤灵的光吸收值或荧光值愈高，表示细胞活力愈高。细胞活力愈高表示细胞愈健康、增生能力愈强。细胞增殖能力愈强，表示细胞量愈多。因此，本实验使用阿尔玛蓝作为评估细胞存活率及细胞增殖率的指标。
以下说明实验流程。首先，取培养代数为4至10代间的人类软骨肉瘤细胞进行实验。将人类软骨肉瘤细胞培养至体积为培养皿的八分满时，移除培养皿中的培养液并使用磷酸盐缓冲液(phosphate buffered saline，PBS)润洗细胞。接着，在培养皿中加入0.25％的胰蛋白酶并使胰蛋白酶在37℃下反应5分钟，再加入人类软骨肉瘤细胞培养液以终止胰蛋白酶的反应。接者，将培养皿中的人类软骨肉瘤细胞及人类软骨肉瘤细胞培养液移至离心管中，以每分钟转速1000(Revolutions Per Minute，rpm)离心5分钟后，移除上清液。接着，再加入新的人类软骨肉瘤细胞培养液至离心管中，进行细胞计数。接着，将人类软骨肉瘤细胞以每孔2×10 4个细胞的密度于盛有人类软骨肉瘤细胞培养液的24孔盘中培养16小时。接着，于24孔盘中加入tBHP，使人类软骨肉瘤细胞在含有浓度为75μM的tBHP的培养液中培养4小时。接着，将含有tBHP的人类软骨肉瘤细胞培养液移除，加入含有不同浓度线粒体的组合物的培养液，使人类软骨肉瘤细胞在含有不同线粒体浓度的组合物的培养液中培养20小时。孔盘中线粒体的浓度为1μg/mL、15μg/mL 或40μg/mL。培养完成后，使用磷酸盐缓冲液清洗人类软骨肉瘤细胞，并将培养液更换为含有阿尔玛蓝的培养液，培养3小时。培养完成后，以OD530/595的波长量测荧光，计算人类软骨肉瘤细胞的细胞存活率。
人类软骨肉瘤细胞的细胞存活率的实验结果如表1及图1所示。图1为使用75μM的tBHP处理再以实施例的组合物修复的人类软骨肉瘤细胞的细胞存活率的图表。空白组为未经tBHP诱导损伤的人类软骨肉瘤细胞。对照组为经tBHP诱导损伤但未施以实施例的组合物的人类软骨肉瘤细胞。试验组为经tBHP诱导损伤且分别施以包含1μg/mL、15μg/mL或40μg/mL的线粒体的组合物处理的人类软骨肉瘤细胞。纵轴为相对于空白组的细胞数，以细胞存活率(％)表示。实验结果显示试验组的细胞存活率皆大于对照组的细胞存活率，且细胞存活率随着线粒体浓度增加而提升，表示实施例的含有线粒体的组合物确实可改善tBHP对软骨细胞造成的损伤及死亡。
〔表1〕
Figure PCTCN2021082094-appb-000001
〔实验二，修复软骨细胞老化〕
在本实验中，使用50μM的tBHP诱导人类软骨肉瘤细胞老化。通过SA-β-gal试剂盒来评估包含线粒体的组合物对tBHP所造成的老化的修复效果，并以老化程度来表示。
在老化的细胞中，老化相关-β-半乳糖苷酶(Senescence-associated beta-galactosidase，SA-β-gal)会被过度表达，因此SA-β-gal可作为细胞衰老的标记之一。本实验使用SA-β-gal试剂盒(Senescenceβ-Galactosidase Staining Kit#9860，Cell Signaling technology)来评估人类软骨肉瘤细胞的老化状态。
本实验的实验流程与上述实验一的实验流程大致相同。差异在于将人类软骨肉瘤细胞以每孔4×10 4个细胞的密度于盛有人类软骨肉瘤细胞培养液的12孔盘中培养16小时。接着，于12孔盘中加入tBHP，使人类软骨肉瘤细胞在含有浓度为50μM的tBHP的培养液中培养4小时。接着，将含有tBHP的人类软骨肉瘤细胞培养液移除，加入含有不同浓度线粒体的组合物的培养液，使人类软骨肉瘤细胞在含有不同线粒体浓度的组合物的培养液中培养20小时。孔盘中线粒体的浓度为1μg/mL、15μg/mL或40μg/mL。培养完成后，使用磷酸盐缓冲液清洗人类软骨肉瘤细胞，并使用SA-β-gal试剂盒进行细胞老化的评估。
人类软骨肉瘤细胞的老化程度的实验结果如表2、图2及图3所示。图2为使用50μM的tBHP处理再以实施例的组合物修复的人类软骨肉瘤细胞的老化程度的图表。图3为使用50μM的tBHP处理再以实施例的组合物修复的人类软骨肉瘤细胞的染色照片。空白组为未经tBHP诱导损伤的人类软骨肉瘤细胞。对照组为经tBHP诱导损伤但未施以实施例的组合物的人类软骨肉瘤细胞。试验组为经tBHP诱导损伤且分别施以包含1μg/mL、15μg/mL或40μg/mL的线粒体的组合物处理的人类软骨肉瘤细胞。纵轴为老化程度(％)，老化程度为老化的细胞数占所有的细胞数的百分比。###表示相较于对照组具有显著差异(P<0.01)。实验结果显示试验组的老化程度皆小于对照组的老化程度，且细胞老化程度随着线粒体浓度增加而降低，表示实施例的含有线粒体的组合物确实可改善tBHP对软骨细胞造成的老化。
〔表2〕
Figure PCTCN2021082094-appb-000002
〔实验三，改善软骨细胞的线粒体功能〕
在本实验中，使用50μM的tBHP诱导人类软骨肉瘤细胞老化，并通过线粒体的膜电位来评估人类软骨肉瘤细胞中线粒体的功能。
线粒体功能分析系通过测量线粒体的膜电位改变来判断线粒体功能的好坏。TMRE(tetramethylrhodamine ethyl ester)是一种带正电的荧光染剂，会聚集在具有活性的线粒体上，故可使用 TMRE来标记健康的线粒体。当线粒体活性较低或呈现去极化现象时线粒体的膜电位会降低，造成TMRE无法保留于线粒体上。FCCP(Carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone)为可跨越线粒体内膜的离子载体，会与质子结合来破坏ATP的合成造成膜电位改变。FCCP可用来消除线粒体的膜电位，因此常被用于线粒体去活性或去极化的对照组。藉由TMRE与FCCP染色处理，分析荧光强度以得知线粒体的膜电位变化，以此作为判断线粒体功能的依据。通过流式细胞仪检测以TMRE处理后的上清液，可根据TMRE标记分析有功能的线粒体占上清液中所有粒子的比例。
本实验的实验流程与上述实验一的实验流程大致相同。差异在于将人类软骨肉瘤细胞以每孔1×10 5个细胞的密度于盛有人类软骨肉瘤细胞培养液的6孔盘中培养16小时。接着，于6孔盘中加入tBHP，使人类软骨肉瘤细胞在含有浓度为50μM的tBHP的培养液中培养4小时。接着，将含有tBHP的人类软骨肉瘤细胞培养液移除，加入含有不同浓度线粒体的组合物的培养液，使人类软骨肉瘤细胞在含有不同线粒体浓度的组合物的培养液中培养20小时。孔盘中线粒体的浓度为1μg/mL、15μg/mL或40μg/mL。培养完成后，使用磷酸盐缓冲液清洗人类软骨肉瘤细胞，并使用TMRE与FCCP进行染色处理，以流式细胞仪进行检测。
人类软骨肉瘤细胞的膜电位分析的实验结果如表3及图4所示。图4为使用50μM的tBHP处理再以实施例的组合物修复的人类软骨肉瘤细胞的线粒体膜电位分析图。空白组为未经tBHP诱导损伤的人类软骨肉瘤细胞。对照组为经tBHP诱导损伤但未施以实施例的组合物的人类软骨肉瘤细胞。试验组为经tBHP诱导损伤且分别施以包含1μg/mL、15μg/mL或40μg/mL的线粒体的组合物处理的人类软骨肉瘤细胞。实验结果显示，相较于对照组，试验组的具有功能的线粒体的数量较多，且恢复程度随着组合物中线粒体浓度增加而更接近空白组，表示实施例的含有线粒体的组合物确实可改善软骨细胞的线粒体功能，线粒体功能提升进一步表示细胞具有较佳的抗老化及修复能力。
〔表3〕
Figure PCTCN2021082094-appb-000003
〔实验四，改善退化性关节炎〕
在本实验中，使用碘乙酸单钠(monosodium iodoacetate，MIA)诱导小鼠产生退化性关节炎，并通过旋转轮测试(rotarod analysis)评估包含线粒体的组合物以及包含线粒体及高浓度血小板血浆的组合物对退化性关节炎小鼠的运动能力的修复效果。本实验使用八周龄、体重约18至22克的C57BL/6雄性小鼠作为实验对象。
碘乙酸单钠(monosodium iodoacetate，MIA)为3-磷酸甘油醛脱氢酶(Glyceraldehyde 3-phosphate dehydrogenase)的活性抑制剂，可诱导出与人类骨关节炎(Osteoarthritis，OA)症状极为相似的软骨损伤，使软骨失去蛋白聚糖基质并诱导软骨细胞死亡。本实验使用MIA作为诱导退化性关节炎的物质。
旋转轮测试(rotarod analysis)为评估小鼠的平衡与协调能力的分析方式。在测试前一周，将小鼠放至旋转轮上训练，使小鼠能在旋转轮上的时间超过三分钟，进行实验处理后，再藉由小鼠在旋转轮上的时间评估小鼠的运动能力。
高浓度血小板血浆(platelet-rich plasma，PRP)为全血经离心后移除红血球成为富含血小板的血浆蛋白浓缩液。高浓度血小板血浆含有丰富的生长因子，因此被认为对软骨、肌腱、韧带等的修复有所帮助。本实验的高浓度血小板血浆来自Sprague-Dawley(SD)大鼠。首先以3％戊巴比妥钠(pentobarbital sodium)麻醉大鼠，剂量为每千克30毫克。接着，以含有柠檬酸右旋糖(acid citrate dextrose)抗凝剂的针筒对大鼠抽取全血约7至8毫升，其中血小板的浓度约为每毫升0.5×10 9至1.5x10 9个。将血液转移至15毫升离心管在室温下以150g进行第一次离心10分钟，将血小板及血浆收集至另一15毫升离心管在室温下以1500g进行第二次离心10分钟，第二次离心后移除四分之三的上清液，剩下的四分之一即为本实验所使用的高浓度血小板血浆。于此所述的高浓度血小板血浆每毫升含有1×10 9个血小板以及多种生长因子，所包含的生长因子的种类及浓度如下：TGF-β1(≥7767pg/ml)、PDGF-AA(≥9.73pg/ml)、PDGF-AB(≥10pg/ml)及PDGF-BB(≥590pg/ml)。
以下说明实验流程。首先，将小鼠分笼并给予数天适应期，避免小鼠因环境不适造成紧张焦虑而影响实验结果。注射MIA前先对小鼠进行旋转轮测试，作为第0天的数据并作为后续比较的基准。进行手术 前10分钟给予小鼠0.25毫升的4％水合氯醛(cholra hydrate)。在手术途中，给予异氟醚(isoflurane)以维持麻醉避免小鼠苏醒。将0.1毫克MIA溶解于10微升的生理盐水中，使用30G(gauge)的针头注入小鼠的关节腔。在注射MIA后的第7天，以同样的注射方式将实施例的组合物注入小鼠的关节腔，并在第7、14天进行旋转轮测试，每次测试最长为1200秒钟，转速为20rpm。
小鼠的运动能力的实验结果如表4所示。对照组为经MIA诱导退化性关节炎但未施以实施例的组合物的小鼠。PRP组为经MIA诱导退化性关节炎且施以高浓度血小板血浆的小鼠。试验组为经MIA诱导退化性关节炎且分别施以包含不同浓度的线粒体及/或10微升的高浓度血小板血浆的组合物处理的小鼠。实验结果显示，相较于第0天尚未受退化性关节炎影响，在第7天时各组别的小鼠的运动能力下降至30％至40％。在第14天时，对照组仅恢复至40％，而PRP组及试验组的小鼠的运动能力皆恢复至50％以上。此外，相较于PRP组(10微升的高浓度血小板血浆)及实施例四(15μg的线粒体)，施以实施例六的组合物(15μg的线粒体及10μL的高浓度血小板血浆)的小鼠的运动能力明显提升。小鼠运动能力的恢复表示软骨损伤或退化性关节炎所造成的疼痛现象、发炎肿胀反应或僵硬现象受到舒缓及改善。由上述结果可知含有线粒体的组合物可改善患有退化性关节炎的动物的运动能力，且随着线粒体浓度提高可具有较佳的效果。此外，含有线粒体及高浓度血小板血浆的组合物相较于仅含有线粒体或仅含有高浓度血小板血浆的组合物可具有更优异的改善效果。
文献记载小鼠关节腔中软骨组织的面积约为1896.91平方微米。本实验证实对小鼠的关节腔给予15微克至40微克的线粒体，可有效改善软骨损伤或退化性关节炎所造成的疼痛现象、发炎肿胀反应或僵硬现象，因此，经换算，线粒体的有效剂量可为每1平方厘米的软骨组织7.9毫克至2.11克，较佳为每1平方厘米的软骨组织7.9毫克至21.1毫克。
〔表4〕
Figure PCTCN2021082094-appb-000004
本发明一个实施例提供包含线粒体的组合物，藉由对软骨提供线粒体，可通过改善软骨细胞中线粒体的功能以提升软骨细胞的修复能力并修复软骨细胞老化导致的软骨损伤，改善软骨损伤或退化性关节炎所造成的疼痛现象、发炎肿胀反应或僵硬现象，进而达到改善并治疗退化性关节炎的目的。

Claims (13)

  1. 线粒体用于制备修复软骨损伤的组合物的用途。
  2. 线粒体用于制备改善退化性关节炎的组合物的用途。
  3. 如权利要求1所述的用途，其中，修复软骨损伤为修复软骨细胞老化导致的软骨损伤。
  4. 如权利要求1所述的用途，其中，修复软骨损伤为通过改善软骨细胞中线粒体的功能以提升软骨细胞的修复能力。
  5. 如权利要求1所述的用途，其中，所述组合物改善软骨损伤所造成的疼痛现象或发炎肿胀反应。
  6. 如权利要求2所述的用途，其中，改善退化性关节炎为修复因关节处的软骨细胞老化导致的软骨损伤。
  7. 如权利要求2所述的用途，其中，改善退化性关节炎为通过改善关节处的软骨细胞中线粒体的功能以提升关节处的软骨细胞的修复能力。
  8. 如权利要求2所述的用途，其中，所述组合物改善退化性关节炎所造成的疼痛现象或发炎肿胀反应。
  9. 如权利要求1或2所述的用途，其中，所述组合物中的线粒体的浓度为每毫升1微克至4000微克(μg/mL)。
  10. 如权利要求1或2所述的用途，其中，所述组合物进一步包含高浓度血小板血浆。
  11. 一种组合物，包含线粒体及来自血液的生长因子。
  12. 如权利要求11所述的组合物，其中，所述生长因子来自高浓度血小板血浆。
  13. 如权利要求11所述的组合物，其中，每10微升的高浓度血小板血浆含有1微克至40微克的线粒体。
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