WO2021185377A1 - 促进毛发再生的组合物、富线粒体血浆、其制造方法及其用途 - Google Patents

促进毛发再生的组合物、富线粒体血浆、其制造方法及其用途 Download PDF

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WO2021185377A1
WO2021185377A1 PCT/CN2021/082117 CN2021082117W WO2021185377A1 WO 2021185377 A1 WO2021185377 A1 WO 2021185377A1 CN 2021082117 W CN2021082117 W CN 2021082117W WO 2021185377 A1 WO2021185377 A1 WO 2021185377A1
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mitochondria
plasma
cells
composition
hair
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French (fr)
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郑汉中
许智凯
曾惠卿
郑安玲
翁琬婷
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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 CN202180022370.3A priority Critical patent/CN115315248A/zh
Publication of WO2021185377A1 publication Critical patent/WO2021185377A1/zh
Priority to US17/948,764 priority patent/US20230016499A1/en

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Definitions

  • the present invention relates to a composition containing mitochondria and its use, and in particular to the use of a composition containing mitochondria to promote hair regeneration.
  • Hair follicles are important tissues for growing hair.
  • the activation cycle of hair follicles will go from the telogen phase through the growth phase (anagen) to the decay phase (catagen), and finally return to the telogen phase, during which the hair grows and falls off.
  • Hair follicles are most active during the growth period.
  • Hair follicle dermal papilla cells Hair Follicle Dermal Papilla Cell, HFDPC located at the root of hair follicles proliferate in large numbers during the growth phase and transmit signals to surrounding stromal cells to promote the proliferation and differentiation of stromal cells into hair.
  • hair follicles When hair follicles are damaged or aging, it will affect hair growth. Specific manifestations include fragile hair, slow growth, sparse hair volume, and even no longer hair growth. Hair not only has a beautiful effect, but also has heat insulation, warmth and protection effects depending on the location. If an animal lacks hair, it will not only affect its appearance (for example, baldness), but also face many inconveniences and dangers, such as eyelashes to protect the eyes and hair to adjust the head temperature.
  • hair regeneration by administering mitochondria to hair follicles, hair regeneration can be promoted.
  • An embodiment of the present invention provides a composition for promoting hair regeneration, including mitochondria.
  • An embodiment of the present invention provides a mitochondrial-rich plasma composition comprising mitochondria and plasma.
  • An embodiment of the present invention provides a use of a composition containing mitochondria for promoting hair regeneration.
  • An embodiment of the present invention provides a use of a mitochondrial-rich plasma composition for promoting hair regeneration.
  • An embodiment of the present invention provides a method for producing mitochondrial-rich plasma, comprising: separating plasma and a plurality of cells from blood; removing a plurality of mitochondria from the plurality of cells; and mixing the plasma with the plurality of mitochondria , The formation of this mitochondrial-rich plasma.
  • the growth of cells in the hair follicles can be promoted to promote hair regeneration.
  • damaged cells it can improve the survival rate of damaged cells; for aging cells, it can improve the aging degree of cells; for cells damaged by free radicals, it can improve cell oxidative damage.
  • applying or injecting the composition containing mitochondria on the skin around the hair follicle can improve the inflammation of the hair follicle.
  • Fig. 1 is a flowchart of a method for manufacturing mitochondrial-rich plasma in an embodiment.
  • Figure 2 shows the effect of different concentrations of tBHP on the cell viability of HFDPC.
  • Figure 3 shows the effect of different concentrations of H 2 O 2 on the cell viability of HFDPC.
  • Figure 4 shows the use of the composition of the Example to improve the cell survival rate of HFDPC damaged by tBHP.
  • Figure 5 shows the use of the composition of the example to enhance the cell survival rate of HFDPC damaged by H 2 O 2.
  • Figure 6 shows the effect of different concentrations of H 2 O 2 on the aging degree of HFDPC.
  • Fig. 7 shows the use of the composition of the example to improve the aging degree of HFDPC.
  • Figure 8 shows the use of the composition of the example to improve the oxidative damage of HFDPC.
  • Figure 9 shows the hair growth on the back of mice treated with the composition of the example.
  • Fig. 10 shows a frozen section image of the mouse of Fig. 9.
  • Fig. 11 shows the amount of macrophages present in the section of Fig. 10.
  • Figure 12 shows the hair growth on the back of mice treated with the composition of the example.
  • Fig. 13 shows a frozen section image of the mouse of Fig. 12.
  • Fig. 14 shows the amount of macrophages present in the section of Fig. 13.
  • Fig. 15 shows the hair growth before and after applying the composition of the example to the scalp.
  • Figure 16 shows the pixel analysis of the scalp in the photo of Figure 15.
  • 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.
  • abnormalities of mitochondria in hair follicle cells can cause problems with hair follicle activation and hair growth. Therefore, the inventors repaired mitochondria in hair follicle cells by mitochondrial replacement, which can improve hair follicle damage and promote hair regeneration.
  • the composition for promoting hair regrowth includes mitochondria.
  • the composition can contact the hair follicle or the skin around the hair follicle through application, injection or direct dripping to promote hair regeneration.
  • the concentration of mitochondria can range from 5 ⁇ g/ml to 200 ⁇ g/ml, but is not limited to this. In other embodiments, the concentration of mitochondria may be 40 ⁇ g/ml to 200 ⁇ g/ml.
  • the solvent contained in the composition may be a solvent that allows mitochondria to maintain function and form, such as water, physiological saline, phosphate solution, various salt buffers, mannitol solution, sucrose solution, plasma, serum, platelet rich Plasma etc.
  • Mitochondria can be the mitochondria of animal cells, but not limited to this.
  • the cells that provide mitochondria may include adipose stem cells, monocytes, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, CD34+ stem cells, bone marrow stem cells, muscle cells, liver cells, kidney cells, skin cells, nerve cells, blood cells, etc. cell.
  • the mitochondria used in the following experiments are described here.
  • the mitochondria used in the examples were 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 10% fetal bovine serum (HyClone).
  • Keratinocyte SFM (1X) solution Gibco
  • BPE bovine pituitary extract
  • HyClone 10% fetal bovine serum
  • the human adipose stem cells were cultured in a petri dish to a cell number of 1.5 ⁇ 10 8 cells, and then the human adipose stem cells in the petri dish were washed with Dulbecco's Phosphate Buffered Saline (DPBS).
  • DPBS Dulbecco's Phosphate Buffered Saline
  • the human adipose stem cells were washed from the petri dish and then dispersed, centrifuged at 600g for 10 minutes, and then the supernatant was removed. Then, the human adipose stem cells left after centrifugation and 80 ml of IBC-1 buffer (225mM mannitol, 75mM sucrose, 0.1mM EDTA, 30mM Tris-HCl pH 7.4) were added to the homogenizer and placed on ice.
  • IBC-1 buffer 225mM mannitol, 75mM sucrose, 0.1mM EDTA, 30mM Tris-HCl pH 7.4
  • the human adipose stem cells are ground 15 times in the homogenizer. Next, centrifuge the ground human adipose stem cells at 1000g for 15 minutes, collect the supernatant in another centrifuge tube, and centrifuge the collected supernatant again at 9000g for 10 minutes to remove the supernatant obtained after the centrifugation again ,
  • the precipitate obtained is mitochondria.
  • 1.5 ml of IBC-2 buffer (225mM mannitol, 75mM sucrose, 30mM Tris-HCl pH 7.4) and proteolytic enzyme inhibitor were added to the mitochondrial precipitate, and stored at 4°C.
  • the composition for promoting hair regrowth may include mitochondria and plasma.
  • the concentration of mitochondria can range from 5 ⁇ g/ml to 200 ⁇ g/ml, but is not limited to this. In other embodiments, the concentration of mitochondria may be 40 ⁇ g/ml to 200 ⁇ g/ml.
  • the number of platelets in the plasma may be less than 1 ⁇ 10 4 /microliter, and the volume percentage concentration of plasma may be 5%.
  • the plasma used in the following experiment is explained.
  • draw fresh blood to a blood collection tube or centrifuge tube containing anticoagulant centrifuge the blood in the tube at 1500-2000g for 10 minutes to separate the blood to form a layer of red blood cells, a layer of colloids, a layer of white blood cells (buffy coat) and plasma Floor.
  • the separated plasma layer was collected to obtain the plasma used in the examples.
  • the number of platelets is less than 1 ⁇ 10 4 / ⁇ l.
  • the mitochondrial-rich plasma composition includes mitochondria and plasma.
  • the plasma may contain growth factors.
  • the growth factor may comprise TGF- ⁇ 1, PDGF-AA, PDGF-AB or PDGF-BB.
  • the concentration of TGF- ⁇ 1 can be 3.1 milligrams per milliliter (mg/mL) or more
  • the concentration of PDGF-AA can be 9.5 nanograms/ml (ng/mL) or more
  • the concentration of PDGF-AB can be Above 91.7 ng/mL (ng/mL)
  • the concentration of PDGF-BB can be above 79.3 ng/mL (ng/mL).
  • the composition can contact the hair follicle or the skin around the hair follicle through application, injection or direct dripping to promote hair regeneration.
  • the weight of mitochondria may be greater than 5 micrograms.
  • FIG. 1 is a flowchart of a method for manufacturing Mitochondria Rich Plasma (MRP) of the embodiment.
  • the method for manufacturing mitochondrial rich plasma includes: removing plasma and cells from blood Separation (S10); removing mitochondria from the cells (S20); and mixing plasma and mitochondria to form the mitochondrial-rich plasma (S30).
  • the step (S10) of separating plasma and cells from the blood may include: layering the blood to form a red blood cell layer, a buffy coat and a plasma layer; and removing the white blood cell layer and the plasma layer.
  • the step of removing mitochondria from the cell (S20) may include removing mitochondria from the cells in the white blood cell layer.
  • the step (S30) of mixing plasma and mitochondria may include mixing the plasma layer and mitochondria.
  • the step (S20) of removing mitochondria from the cells may include mixing the cells with the extraction buffer to form a mixture; using a syringe and needle to aspirate the mixture to rupture the cells into cell fragments and release mitochondria; and Separate cell debris and mitochondria, and collect mitochondria.
  • the method for producing mitochondrial-rich plasma used in the following experiments is described here.
  • draw 8 to 10 ml of blood to the CPT tube (BD CPT TM Cell Preparation Tube, REF362761), but not limited to this, any blood collection tube or centrifuge tube containing anticoagulant can be used.
  • the blood was centrifuged at 1500 g for 10 minutes to separate the blood to form a red blood cell layer, a colloid layer, a white blood cell layer (buffy coat), and a plasma layer.
  • the white blood cell layer contained monocytes and platelets.
  • the plasma layer and white blood cell layer were taken out into a new centrifuge tube, the plasma layer and white blood cell layer were centrifuged at 400 g for 5 minutes to separate the cells and plasma, and the separated plasma was collected in another centrifuge tube. Then, 1.5 ml of extraction buffer was added to the cells, and the cells were aspirated with a 23G needle and syringe to rupture the monocytes and platelets and release mitochondria.
  • the extraction buffer can be a sodium chloride solution with an osmotic pressure of 42.8 mOsm/L.
  • the aspirated cells were centrifuged at 400 g for 5 minutes to separate the cell debris and mitochondria, and the supernatant containing the mitochondria was collected.
  • the way of releasing mitochondria is not limited to this, and surfactants (detergent) can also be used to help cells release mitochondria, such as CHAPS detergent, Triton X-100, NP40, etc.
  • surfactants detergent
  • the mitochondrial-containing supernatant is mixed with the plasma collected above to obtain about 5 to 8 ml of mitochondrial-rich plasma.
  • the weight of mitochondria can be greater than 5 micrograms.
  • HFDPC Human Hair Follicle Dermal Papilla Cell
  • the culture conditions are as follows. HFDPC is cultured in Follicle Dermal Papilla Growth Medium (PromoCell, C-26501) and placed in an incubator at 37°C and 5% carbon dioxide. When the HFDPC is cultured until its volume is nine minutes full of the petri dish, the culture medium in the petri dish is removed and the cells are rinsed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the phosphate buffer was removed, 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 culture medium was added to stop the trypsin reaction. Then, the supernatant was removed by centrifugation at 1000 rpm for 5 minutes, and new culture medium was added to count the cells, and subculture the cells according to the experimental requirements.
  • Hydrogen peroxide (H 2 O 2 ) and tert-butyl hydroperoxide (tBHP) are both peroxides, which are commonly used as substances that induce cell damage, oxidation, aging, and apoptosis.
  • H 2 O 2 and tBHP are used to cause damage to HFDPC, and the damage of HFDPC is explored through the Alma Blue detection reagent, which is expressed as survival rate (also known as cell survival rate).
  • 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 cell proliferation rate or cell proliferation can be evaluated by measuring the light absorption or fluorescence value of resorufin. The higher the light absorption value or fluorescence value of resorufin, the higher the cell mass, the higher the cell proliferation or survival rate. The higher the proliferation rate or survival rate of the cell, the healthier the cell and the stronger the proliferation ability. Therefore, this experiment uses Alma Blue as an indicator to evaluate the cell proliferation rate or cell survival rate.
  • the experiment procedure is explained below.
  • the HFDPC was subcultured and cultured in a 24-well plate at a density of 5 ⁇ 10 4 cells/500 microliters ( ⁇ L) per well for 8 hours. Next, remove the supernatant in the well plate and rinse the cells with phosphate buffered saline (PBS), then remove the supernatant and add 500 ⁇ L of cell culture medium to incubate for 8 hours.
  • PBS phosphate buffered saline
  • Figure 2 shows the effect of different concentrations of tBHP on the cell viability of HFDPC.
  • Figure 3 shows the effect of different concentrations of H 2 O 2 on the cell viability of HFDPC.
  • the blank group is the group without peroxide added.
  • the control group is the group to which peroxide has been added.
  • the survival rate is the number of cells after culture relative to the number of cells before culture. * Indicates a significant difference relative to the blank group (P ⁇ 0.05).
  • the experiment procedure is explained below. Please refer to the experimental procedure of Experiment 1, add H 2 O 2 or tBHP to the well plate, and incubate the cells in the presence of 300 ⁇ M H 2 O 2 or 300 ⁇ M tBHP for 4 hours, then remove the supernatant from the well plate
  • the composition of the example was added and cultured for 8 hours.
  • the well disk contained 40 ⁇ g of mitochondria and 5% plasma.
  • Figure 4 shows the use of the composition of the Example to improve the cell survival rate of HFDPC damaged by tBHP.
  • Figure 5 shows the use of the composition of the Example to improve the cell survival rate of HFDPC damaged by H 2 O 2.
  • the blank group is the group without peroxide, plasma and mitochondria.
  • the control group was the group without mitochondria added.
  • the survival rate is the number of cells after culture relative to the number of cells before culture. * Indicates a significant difference relative to the control group (P ⁇ 0.05), ** indicates a highly significant difference relative to the control group (P ⁇ 0.01).
  • composition containing mitochondria helps to improve the survival rate of cells, indicating that it can improve the damage caused by H 2 O 2 and tBHP to help cell repair.
  • the composition containing mitochondria and plasma can further improve the cell survival rate, which means that the effect of improving the damage caused by H 2 O 2 and tBHP and helping cell repair is more obvious.
  • SA- ⁇ -gal Senescence-associated beta-galactosidase
  • SA- ⁇ -gal kit Senescence ⁇ -Galactosidase Staining Kit#9860, Cell Signaling technology
  • the experiment procedure is explained below.
  • the HFDPC was subcultured in a 12-well plate and cultured at a density of 4 ⁇ 10 4 cells/1 ml per well for 8 hours. Then, H 2 O 2 of different concentrations was added to the well plate, and the cells were cultured in the presence of H 2 O 2 for 4 hours, where the concentration of H 2 O 2 in the well plate was 100 ⁇ M, 300 ⁇ M, and 500 ⁇ M, respectively. After the culture is completed, the cells are washed with phosphate buffer and the SA- ⁇ -gal kit is used to evaluate cell aging.
  • Figure 6 shows the effect of different concentrations of H 2 O 2 on the aging degree of HFDPC.
  • the blank group is the group without adding H 2 O 2 .
  • the control group is a group supplemented with H 2 O 2 .
  • the degree of aging is the percentage of the number of aging cells per unit area to the number of all cells. * Indicates a significant difference relative to the blank group (P ⁇ 0.05), ** indicates a highly significant difference relative to the blank group (P ⁇ 0.01), and *** indicates a very significant difference relative to the blank group (P ⁇ 0.001).
  • the experiment procedure is explained below. Please refer to the experimental procedure of Experiment 3, add H 2 O 2 to the well plate, let the cells incubate in the presence of 300 ⁇ M H 2 O 2 for 4 hours, remove the supernatant from the well plate and use phosphate buffer The cells were washed, and the composition of the example was added and cultured for 8 hours. The well plate contained 40 ⁇ g of mitochondria and 5% plasma. After the culture is completed, the cells are washed with phosphate buffer and the SA- ⁇ -gal kit is used to evaluate cell aging.
  • Fig. 7 shows the use of the composition of the example to improve the aging degree of HFDPC.
  • the blank group is the group without H 2 O 2 , plasma and mitochondria.
  • the control group was the group without mitochondria added.
  • the degree of aging is the percentage of the number of aging cells per unit area to the number of all cells. * Indicates a significant difference relative to the control group (P ⁇ 0.05), ** indicates a highly significant difference relative to the control group (P ⁇ 0.01), *** indicates a very significant difference relative to the control group (P ⁇ 0.001).
  • composition containing mitochondria helps to reduce the degree of cell aging, which means that it can effectively improve the aging of cells to help cell repair.
  • composition containing mitochondria and plasma can further reduce the degree of cell aging, which means that the effect of improving cell aging and helping cell repair is more obvious.
  • DCFDA is a fluorescent substance with four benzene rings.
  • DCFDA can pass through the cell membrane and be removed by the intracellular esterase (esterase) to remove the ethyl carboxyl group and stay in the cell.
  • DCFDA can react with free radicals in cells to emit fluorescence. The more free radicals there are, the greater the oxidative pressure and the stronger the fluorescence intensity produced. Therefore, DCFDA is often used as a fluorescent substance to detect free radicals. This experiment uses DCFDA to detect fluorescence to evaluate the oxidative pressure of cells.
  • HFDPC was cultured in 96-well plates at a density of 2.5 ⁇ 10 4 cells/200 microliters per well for 8 hours.
  • tBHP was added to the well plate, and the cells were cultured in the presence of tBHP for 4 hours, where the concentration of tBHP in the well plate was 300 ⁇ M.
  • the supernatant in the well plate was removed and the cells were washed with phosphate buffer solution, and the composition of the example was added to culture for 8 hours, wherein the well plate contained 1, 15, 40 ⁇ g of mitochondria.
  • wash the cells with phosphate buffer solution and use a culture medium containing 25 ⁇ M DCFDA to react at 37° C. for 30 minutes.
  • the cells were washed with phosphate buffer and new cell culture medium was added, and the fluorescence was measured at the wavelength of OD485/535.
  • Figure 8 shows the use of the composition of the example to improve the oxidative damage of HFDPC.
  • the blank group is the group without addition of peroxide and mitochondria.
  • the control group was the group without mitochondria added.
  • the vertical axis is the relative fluorescence intensity, which represents the fluorescence intensity relative to the blank group, which is set as 1 for the blank group. ** indicates a highly significant difference relative to the control group (P ⁇ 0.01).
  • composition containing mitochondria helps to reduce the amount of free radicals in the cells, which means that it can effectively reduce the oxidative stress and improve the oxidative damage to the cells to help cell repair.
  • concentration of mitochondria increases, the amount of free radicals decreases more significantly, indicating that the effect of improving oxidative damage also increases.
  • mice were used for animal experiments to observe whether the mice had skin and hair growth conditions treated with the composition of the examples after the mice were injured by tBHP.
  • the skin of the back of the mice was taken for freezing tissue embedding treatment.
  • the skin tissue is taken into a 4% formaldehyde fixative solution, and shaken in a spirit level at 4° C. for 4 to 6 hours.
  • the skin tissue was transferred to a 15% sucrose (Sucrose) solution and shaken on a level instrument at 4°C for 6 to 8 hours.
  • sucrose sucrose
  • the skin tissue was moved into the 30% sucrose solution, and the skin tissue was shaken on a leveler at 4° C. overnight.
  • OCT frozen embedding solution model: 23-730-571, Thermo Fisher Scientific
  • the embedded tissue is stored at -80°C.
  • the skin tissue is frozen sectioned, the section thickness is about 15-20 microns, and the sectioned tissue is stored at -80°C.
  • the completed sectioned tissue is fluorescently stained.
  • the sliced tissue was taken out from -80°C and then placed at room temperature for warming, and then rinsed twice with PBS for 5 minutes each.
  • the tissue was rinsed twice with 1% bovine serum albumin (BSA) prepared in PBS-T (PBS containing 0.4% triton X-100), each for 10 minutes.
  • BSA bovine serum albumin
  • PBS-T PBS containing 0.4% triton X-100
  • 5% BSA prepared in PBS-T was used for blocking, and it was allowed to interact with the tissue at room temperature for 30 minutes to block non-specific substances and improve antibody specificity and sensitivity.
  • the tissue was rinsed twice with 1% BSA prepared in PBS-T for 10 minutes each.
  • F4/80 antibody (#MA5-16624, Thermo Fisher Scientific) was used to prepare it in 1% BSA PBS-T at a ratio of 1:50, and it was allowed to act on the tissue for 1.5 hours at room temperature.
  • the tissue was rinsed twice with 1% BSA prepared in PBS-T for 10 minutes each.
  • use the goat anti-mouse secondary antibody complex Alexa (goat anti rat secondary conjugation Alexa ab150160, abcam) was prepared in 1% BSA in PBS-T at a ratio of 1:200, and reacted with the tissue for 1 hour at room temperature.
  • the tissue was rinsed twice with 1% BSA prepared in PBS-T for 10 minutes each.
  • DAPI D1306, Thermo Fisher Scientific
  • use a fluorescence microscope to take pictures.
  • Figure 9 shows the hair growth on the back of mice treated with the composition of the example.
  • Fig. 10 shows a frozen section image of the mouse of Fig. 9.
  • Fig. 11 shows the amount of macrophages present in the section of Fig. 10.
  • the blank group is a group that has not been treated with tBHP and the composition of the examples.
  • the control group was a group that was not treated with the composition of the example.
  • Example 8-1 is a group treated with a composition containing 40 micrograms of mitochondria.
  • Example 8-2 is a group treated with a composition containing 40 micrograms of mitochondria and a plasma concentration of 5% by volume. *** indicates a very significant difference compared to the control group (P ⁇ 0.001).
  • mice in the control group are redder, indicating that the skin and hair follicles of the mice are inflamed.
  • mice treated with a composition containing mitochondria or a composition containing mitochondria and plasma showed no redness on the back and a darker color, indicating that the inflammation has been improved and the hair follicles have been damaged due to reduced damage. Start to grow hair.
  • blue indicates stained cell nuclei, and the blue area presents a hair follicle pattern; red indicates stained macrophages, and the red area indicates the area where macrophages aggregate, and also indicates the location of inflammation.
  • the density of red in the control group is significantly greater than that in the example, indicating that the mice in the control group are damaged and inflamed due to tBHP-induced hair follicle damage and macrophages are concentrated in the hair follicles.
  • the red density at the hair follicles was significantly reduced, indicating that the macrophages at the hair follicles were significantly reduced, and inflammation was also significantly improved.
  • Figure 11 and Table 8 present the analysis of the slice image in Figure 10 with Image J quantitative software.
  • the amount of macrophages is the amount of fluorescence in a unit area analyzed by the Image J quantitative software, expressed as the product of the area and the fluorescence intensity. The greater the amount of macrophages, the more severe the inflammation. It was further confirmed by fluorescence analysis that the amount of macrophages in mice treated with a composition containing mitochondria or a composition containing mitochondria and plasma decreased significantly, indicating a significant improvement in inflammation. In addition, the composition containing mitochondria and plasma has a more excellent effect of improving inflammation.
  • mice were used to conduct animal experiments to observe whether the mice had skin and hair growth conditions treated with the composition of the examples after the mice were injured by tBHP.
  • the composition used in this experiment is mitochondrial-rich plasma.
  • Figure 12 shows the hair growth on the back of mice treated with the composition of the example.
  • Fig. 13 shows a frozen section image of the mouse of Fig. 12.
  • Fig. 14 shows the amount of macrophages present in the section of Fig. 13.
  • the blank group is a group that has not been treated with tBHP and the composition of the examples.
  • the control group was a group that was not treated with the composition of the example.
  • Example 9 is a group treated with a composition containing mitochondrial-rich plasma. ** indicates a highly significant difference relative to the control group (P ⁇ 0.01).
  • mice in the control group are redder, indicating that the skin and hair follicles of the mice are inflamed.
  • mice treated with the composition containing mitochondrial plasma have no redness on their backs and a darker color, indicating that the inflammation has been improved and the hair follicles have started to grow hair due to the slowing down of damage.
  • red indicates stained cell nuclei
  • the blue area presents the shape of hair follicles
  • red indicates stained macrophages
  • the red area indicates the area where macrophages aggregate, and also indicates the location of inflammation.
  • the red density of the control group is significantly greater than that of the example and the blank group, which means that the mice in the control group are damaged and inflamed due to tBHP-induced hair follicle damage and macrophages are concentrated in the hair follicles.
  • Mice treated with a composition containing mitochondrial-rich plasma showed a significant reduction in the red density at the hair follicles, indicating that the macrophages at the hair follicles were significantly reduced, and the inflammation phenomenon was also significantly improved.
  • Figure 14 and Table 9 present the analysis of the slice image in Figure 13 with Image J quantitative software. It was further confirmed by fluorescence analysis that the amount of macrophages in mice treated with the composition containing mitochondrial-rich plasma decreased significantly, indicating a significant improvement in inflammation.
  • micro-needles were used to create micro-wounds on the scalp, and a composition containing mitochondrial-rich plasma was applied to the scalp. Take an appropriate amount of the composition containing mitochondrial-rich plasma to evenly cover the affected area, and the smearing range is about 25 to 49 square centimeters. Before application and on the 60th day after application, observe the hair growth condition and perform pixel analysis.
  • Figure 15 shows the hair growth before and after applying the composition of the example to the scalp.
  • the left image is before application, and the right image is 60 days after application.
  • Figure 16 shows the pixel analysis of the scalp in the photo of Figure 15.
  • the pixels on the vertical axis represent the number of hairs per unit area, and the density of hairs is represented by pixels.
  • the growth of cells in the hair follicles can be promoted to promote hair regeneration.
  • damaged cells it can improve the survival rate of damaged cells; for aging cells, it can improve the aging degree of cells; for cells damaged by free radicals, it can improve cell oxidative damage.
  • applying or injecting the composition containing mitochondria on the skin around the hair follicle can improve the inflammation of the hair follicle.

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Abstract

本发明实施例提供一种促进毛发再生的组合物、富线粒体血浆、其制造方法及其用途。包含线粒体的组合物可提高受损毛囊细胞的存活率、改善毛囊细胞的老化、改善毛囊细胞的氧化损伤及改善毛囊发炎现象，以达到促进毛发再生的目的。

Description

促进毛发再生的组合物、富线粒体血浆、其制造方法及其用途 技术领域
本发明系关于包含线粒体的组合物及其用途，尤其是关于包含线粒体的组合物于促进毛发再生的用途。
背景技术
毛囊是生长毛发的重要组织。毛囊的活化循环会从休止期(telogen)经由生长期(anagen)再进入衰退期(catagen)，最后回到休止期，毛发则在此循环中生长并脱落。毛囊在生长期最为活跃。位于毛囊根部的毛囊真皮乳头细胞(Hair Follicle Dermal Papilla Cell，HFDPC)在生长期大量增生，并传递讯号给周围的基质细胞，以促进基质细胞增殖并分化成毛发。
当毛囊受损或老化时，会影响毛发生长。具体表现包含毛发脆弱、生长缓慢、毛发量稀少，甚至不再生长毛发。毛发不仅有美观作用，依部位的不同，毛发还具有隔热、保暖及保护作用。动物若缺乏毛发，不仅会对外观造成影响(例如秃头)，还会面临许多不便与危险，例如睫毛可以保护眼睛、头发可以调节头部温度等。
发明内容
根据本发明一个实施例，透过对毛囊给予线粒体，可促进毛发再生。
本发明一个实施例提供一种促进毛发再生的组合物，包含线粒体。
本发明一个实施例提供一种富线粒体血浆的组合物，包含线粒体及血浆。
本发明一个实施例提供一种含线粒体的组合物用于促进毛发再生的用途。
本发明一个实施例提供一种富线粒体血浆的组合物用于促进毛发再生的用途。
本发明一个实施例提供一种制造富线粒体血浆的方法，包含：将血浆及多个细胞自血液中分离；从所述多个细胞中取出多个线粒体；以及混合该血浆与所述多个线粒体，形成该富含线粒体血浆。
根据本发明一个实施例，藉由对毛囊给予线粒体或富线粒体血浆，可帮助毛囊中的细胞生长，以促进毛发再生。对于受损的细胞而言，可提高受损细胞的存活率；对于老化的细胞而言，可改善细胞的老化程度；对于受自由基损伤的细胞而言，可改善细胞的氧化损伤。除此之外，将含线粒体的组合物涂敷或注射于毛囊周围的皮肤，可改善毛囊发炎现象。
附图说明
图1为实施例的富线粒体血浆的制造方法的流程图。
图2显示不同浓度的tBHP对HFDPC的细胞存活率的影响。
图3显示不同浓度的H 2O 2对HFDPC的细胞存活率的影响。
图4显示使用实施例的组合物提升受到tBHP损害的HFDPC的细胞存活率。
图5显示使用实施例的组合物提升受到H 2O 2损害的HFDPC的细胞存活率。
图6显示不同浓度的H 2O 2对HFDPC的老化程度的影响。
图7显示使用实施例的组合物改善HFDPC的老化程度。
图8显示使用实施例的组合物改善HFDPC的氧化损伤。
图9显示使用实施例的组合物处理的小鼠背部的毛发生长状况。
图10显示图9的小鼠的组织冷冻切片图。
图11显示图10的切片图中巨噬细胞的存在量。
图12显示使用实施例的组合物处理的小鼠背部的毛发生长状况。
图13显示图12的小鼠的组织冷冻切片图。
图14显示图13的切片图中巨噬细胞的存在量。
图15显示使用实施例的组合物涂敷于头皮前后毛发生长的情况。
图16显示图15的照片中头皮的像素分析。
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。在毛发生长的过程中，毛囊细胞内线粒体异常会导致毛囊活化与毛发生长发生问题。因此，发明人通过线粒体置换的方式修补毛囊细胞内的线粒体，能改善毛囊的受损并促进毛发再生。
根据本发明一个实施例，促进毛发再生的组合物包含线粒体。组合物可通过涂敷、注射或直接滴入的方式接触毛囊或毛囊周围的皮肤，以促进毛发再生。在此组合物中，线粒体的浓度可为5微克/毫升至200微克/毫升，但不以此为限。在其他实施例中，线粒体的浓度可为40微克/毫升至200微克/毫升。组合物中所包含的溶剂可为使线粒体可维持功能与形态的溶剂，例如为水、生理盐水、磷酸盐溶液、各种盐类缓冲液、甘露醇溶液、蔗糖溶液、血浆、血清、富血小板血浆等。
线粒体可取自动物细胞的线粒体，但不以此为限。提供线粒体的细胞可包含脂肪干细胞、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、肌肉细胞、肝脏细胞、肾脏细胞、皮肤细胞、神经细胞、血球细胞等具有线粒体的细胞。
于此说明下述实验中所使用的线粒体。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)。首先，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，再以杜氏磷酸盐缓冲液(DPBS)冲洗培养皿中的人类脂肪干细胞。接着，移除培养皿中的杜氏磷酸盐缓冲液后，在培养皿中加入细胞剥离用的胰蛋白酶(Trypsin)，并在37℃下反应3分钟后，再加入干细胞培养液以终止反应。接着，将人类脂肪干细胞自培养皿冲洗下来后打散，以600g离心10分钟后，移除上清液。接着，将离心后留下的人类脂肪干细胞及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)加入均质器中，并在冰上的均质器中对人类脂肪干细胞进行研磨15次。接着，以1000g将研磨后的人类脂肪干细胞离心15分钟，将上清液收集至另一离心管，再以9000g将收集的上清液再次离心10分钟，移除再次离心后得到的上清液，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。
根据本发明另一实施例，促进毛发再生的组合物可包含线粒体及血浆。在此组合物中，线粒体的浓度可为5微克/毫升至200微克/毫升，但不以此为限。在其他实施例中，线粒体的浓度可为40微克/毫升至200微克/毫升。在此组合物中，血浆中的血小板数目可小于1×10 4个/微升，且血浆的体积百分浓度可为5％。
于此说明下述实验中所使用的血浆。首先，抽取新鲜血液至含有抗凝血剂的采血管或离心管，将管中的血液以1500至2000g离心10分钟使血液分层，形成红血球层、胶体层、白细胞层(buffy coat)及血浆层。接着，收集所分离出的血浆层以获得实施例所使用的血浆。由上述方式获得的血浆中，血小板数目小于1×10 4个/微升。
根据本发明另一实施例，富线粒体血浆的组合物包含线粒体及血浆。血浆可包含生长因子。生长因子可包含TGF-β1、PDGF-AA、PDGF-AB或PDGF-BB。在该组合物中，TGF-β1的浓度可为3.1毫克/毫升(mg/mL)以上，PDGF-AA的浓度可为9.5纳克/毫升(ng/mL)以上，PDGF-AB的浓度可为91.7纳克/毫升(ng/mL)以上，PDGF-BB的浓度可为79.3纳克/毫升(ng/mL)以上。组合物可通过涂敷、注射或直接滴入的方式接触毛囊或毛囊周围的皮肤，以促进毛发再生。在该组合物中，线粒体的重量可大于5微克。
根据本发明另一实施例，请参考图1，图1为实施例的富线粒体血浆(Mitochondria Rich Plasma，MRP)的制造方法的流程图，富线粒体血浆的制造方法包含：将血浆及细胞自血液中分离(S10)；从细胞中取出线粒体(S20)；以及混合血浆与线粒体，形成该富含线粒体血浆(S30)。将血浆及细胞自血液中分离的步骤(S10)中，可包含：使血液分层，形成红血球层、白细胞层(buffy coat)及血浆层；以及取出白细胞层及血浆层。从细胞中取出线粒体的步骤(S20)中，可包含从白细胞层中的细胞中取出线粒体。混合血浆与线粒体的步骤(S30)中，可包含混合血浆层与线粒体。此外，从细胞中取出线粒体的步骤(S20)中，可包含将细胞与萃取缓冲液混合，以成为混合物；使用针筒及针头对混合物进行抽吸，使细胞破裂成细胞碎片并释放线粒体；以及分离细胞碎片及线粒体，并收集线粒体。
于此说明下述实验中所使用的富线粒体血浆的制造方法。首先，抽取8至10毫升的血液至CPT管(BD 
Figure PCTCN2021082117-appb-000001
CPT TM Cell Preparation Tube，REF362761)，但不限于此，可使用任何含有抗凝血剂的采血管或离心管。接着，将血液以1500g离心10分钟使血液分层，形成红血球层、胶体层、白细胞层(buffy coat)及血浆层，其中白细胞层含有单核细胞及血小板。接着，将血浆层及白细胞层取出至新的离心管中，将血浆层及白细胞层以400g离心5分钟使细胞及血浆分离，并将分离的血浆收集至另一离心管。接着，加入 1.5毫升的萃取缓冲液至细胞，以23G针头及针筒抽吸细胞，使单核细胞及血小板破裂进而释放线粒体。萃取缓冲液可为渗透压为42.8mOsm/L的氯化钠溶液。接着，将经抽吸处理的细胞以400g离心5分钟使细胞碎片与线粒体分层，并收集含有线粒体的上清液。释放线粒体的方式不限于此，亦可使用表面活性剂(detergent)来帮助细胞释放线粒体，例如CHAPS detergent、Triton X-100、NP40等。接着，将含有线粒体的上清液与前述收集的血浆混合，获得富线粒体血浆约5至8毫升。由上述方式获得的富线粒体血浆中，线粒体的重量可大于5微克。
于此说明下述实验所使用的细胞，实验一至实验五使用人类毛囊真皮乳头细胞(Human Hair Follicle Dermal Papilla Cell，HFDPC)作为探讨毛囊活化的细胞模式。培养条件如下，将HFDPC培养于毛囊真皮乳头细胞培养基(Follicle Dermal Papilla Growth Medium，PromoCell，C-26501)，并置于37℃、5％二氧化碳环境的培养箱中。将HFDPC培养至其体积为培养皿的九分满时，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，移除磷酸盐缓冲液，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，再加入培养液以终止胰蛋白酶的反应。接着，以1000rpm离心5分钟移除上清液，并加入新的培养液，进行细胞计数，依实验需求进行细胞继代培养。
〔实验一，细胞损伤〕
过氧化氢(Hydrogen peroxide，H 2O 2)及过氧化叔丁醇(tert-butyl hydroperoxide，tBHP)皆为过氧化物，常用作为诱导细胞损伤、氧化、老化与凋亡的物质。在本实验中，使用H 2O 2及tBHP对HFDPC造成损伤，并透过阿尔玛蓝检测试剂探讨HFDPC的损伤情况，以存活率(亦称为细胞存活率)来表示。
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增殖率或存活率(cell proliferation)。试卤灵的光吸收值或荧光值愈高，表示细胞量越多，细胞的增殖率或存活率愈高。细胞的增殖率或存活率愈高也表示细胞愈健康、增殖能力愈强。因此，本实验使用阿尔玛蓝作为评估细胞增殖率或细胞存活率的指标。
以下说明实验流程。将HFDPC继代后培养于24孔盘中，以每孔5×10 4个细胞/500微升(μL)的密度培养8小时。接着，移除孔盘中的上清液并使用磷酸盐缓冲液(phosphate buffered saline，PBS)润洗细胞，再移除上清液并加入500μL细胞培养液培养8小时。接着，在孔盘中加入不同浓度的H 2O 2或tBHP，使细胞在H 2O 2或tBHP的存在下培养4小时，其中H 2O 2及tBHP于孔盘中的浓度分别为100μM、300μM、500μM及100μM、200μM、300μM。接着，移除孔盘中的上清液并加入含有阿尔玛蓝的培养液，在37℃下培养3至4小时。培养完成后，以OD560/590的波长测量荧光，计算细胞的存活率。
实验结果请参考表1、表2、图2及图3。图2显示不同浓度的tBHP对HFDPC的细胞存活率的影响。图3显示不同浓度的H 2O 2对HFDPC的细胞存活率的影响。空白组为未添加过氧化物的组别。对照组为添加有过氧化物的组别。存活率为培养后的细胞数相对于培养前的细胞数。*表示相对空白组具有显著差异(P<0.05)。
〔表1〕
  tBHP(μM) 存活率(％)
空白组1 - 98.17±8.1
对照组1-1 100 88.59±22.1
对照组1-2 200 82.69±21.7
对照组1-3 300 59.46±28.7
〔表2〕
  H 2O 2(μM) 存活率(％)
空白组2 - 101.83±8.4
对照组2-1 100 88.9±28.4
对照组2-2 300 63.05±26.9
对照组2-3 500 61.14±14.0
由实验结果可知，tBHP及H 2O 2皆会对HFDPC造成损伤。并且，损伤的程度随着浓度增加而更加明显。以下实验使用tBHP及H 2O 2诱导HFDPC损伤及老化。
〔实验二，提高受损细胞的存活率〕
在本实验中，使用300μM的H 2O 2及300μM的tBHP对HFDPC造成损伤，再使用实施例的组合物进行修复。并且，透过阿尔玛蓝检测试剂探讨实施例的组合物对HFDPC的修复情形，以存活率(亦称为细 胞存活率)来表示。
以下说明实验流程。请参考实验一的实验流程，在孔盘中添加H 2O 2或tBHP，使细胞在300μM的H 2O 2或300μM的tBHP的存在下培养4小时后，移除孔盘中的上清液并加入实施例的组合物培养8小时，其中孔盘中含有40μg的线粒体及体积百分浓度为5％的血浆。接着，移除孔盘中的上清液并加入含有阿尔玛蓝的培养液，在37℃下培养3至4小时。培养完成后，以OD560/590的波长量测荧光，计算各细胞的存活率。
实验结果请参考表3、表4、图4及图5。图4显示使用实施例的组合物提升受到tBHP损害的HFDPC的细胞存活率。图5显示使用实施例的组合物提升受到H 2O 2损伤的HFDPC的细胞存活率。空白组为未添加过氧化物、血浆及线粒体的组别。对照组为未添加线粒体的组别。存活率为培养后的细胞数相对于培养前的细胞数。*表示相对对照组具有显著差异(P<0.05)，**表示相对对照组具有高显著差异(P<0.01)。
〔表3〕
Figure PCTCN2021082117-appb-000002
〔表4〕
Figure PCTCN2021082117-appb-000003
由实验结果可知，含有线粒体的组合物有助于提升细胞存活率，表示可改善H 2O 2及tBHP所造成的损伤情况以帮助细胞修复。并且，含有线粒体及血浆的组合物可进一步提升细胞存活率，表示在改善H 2O 2及tBHP所造成的损伤以及帮助细胞修复的效果更加明显。
〔实验三，细胞老化〕
在本实验中，使用不同浓度的H 2O 2对HFDPC诱导老化损伤，并透过SA-β-gal试剂盒来探讨HFDPC的老化程度，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)来评估细胞的老化程度。
以下说明实验流程。将HFDPC继代后培养于12孔盘中，以每孔4×10 4个细胞/1毫升的密度培养8小时。接着，在孔盘中加入不同浓度的H 2O 2，使细胞在H 2O 2的存在下培养4小时，其中H 2O 2于孔盘中的浓度分别为100μM、300μM、500μM。培养完成后，使用磷酸盐缓冲液清洗细胞并使用SA-β-gal试剂盒进行细胞老化的评估。
实验结果请参考表5及图6。图6显示不同浓度的H 2O 2对HFDPC的老化程度的影响。空白组为未添加H 2O 2的组别。对照组为添加有H 2O 2的组别。老化程度为在单位面积中老化细胞的数量占所有细胞的数量的百分比。*表示相对空白组具有显著差异(P<0.05)，**表示相对空白组具有高显著差异(P<0.01)，***表示相对空白组具有非常显著差异(P<0.001)。
〔表5〕
  H 2O 2(μM) 老化程度(％)
空白组5 - 29±6.1
对照组5-1 100 42±2.5
对照组5-2 300 47±6.9
对照组5-3 500 58±6.4
由实验结果可知，H 2O 2会对HFDPC造成老化损伤。并且，老化程度随着浓度增加而更加明显。以下 实验使用300μM的H 2O 2诱导HFDPC老化。
〔实验四，改善细胞老化〕
在本实验中，选用300μM的H 2O 2对HFDPC造成老化损伤，再使用实施例的组合物进行修复。并且，透过SA-β-gal试剂盒探讨实施例的组合物对老化HFDPC的影响，并以老化程度(％)来表示。
以下说明实验流程。请参考实验三的实验流程，在孔盘中添加H 2O 2，使细胞在300μM的H 2O 2的存在下培养4小时后，移除孔盘中的上清液并使用磷酸盐缓冲液清洗细胞，再加入实施例的组合物培养8小时，其中孔盘中含有40μg的线粒体及体积百分浓度为5％的血浆。培养完成后，使用磷酸盐缓冲液清洗细胞并使用SA-β-gal试剂盒进行细胞老化的评估。
实验结果请参考表6及图7。图7显示使用实施例的组合物改善HFDPC的老化程度。空白组为未添加H 2O 2、血浆及线粒体的组别。对照组为未添加线粒体的组别。老化程度为在单位面积中老化细胞的数量占所有细胞的数量的百分比。*表示相对对照组具有显著差异(P<0.05)，**表示相对对照组具有高显著差异(P<0.01)，***表示相对对照组具有非常显著差异(P<0.001)。
〔表6〕
Figure PCTCN2021082117-appb-000004
由实验结果可知，含有线粒体的组合物有助于降低细胞老化程度，表示可有效改善细胞的老化情况以帮助细胞修复。并且，含有线粒体及血浆的组合物可进一步降低细胞老化程度，表示在改善细胞老化以及帮助细胞修复的效果更加明显。
〔实验五，改善细胞氧化损伤〕
在本实验中，选用300μM的tBHP对HFDPC诱导氧化损伤，再使用实施例的组合物进行修复。并且，透过双氯荧光黄乙酸乙酯(2’,7’-Dichlorodihydrofluorescein diacetate，DCFDA)探讨实施例的组合物对HFDPC的氧化压力的影响，并以荧光强度表示。
DCFDA为一种荧光物质，具有四个苯环。DCFDA可通过细胞膜并受细胞内的酯解酶(esterase)作用移除乙羧基而停留在细胞中。DCFDA可与细胞内的自由基物质反应而发出荧光，自由基存在量愈多，表示氧化压力愈大，所产生的荧光强度愈强。因此，DCFDA常被用作为检测自由基的荧光物质。本实验使用DCFDA检测荧光来评估细胞的氧化压力。
以下说明实验流程。将HFDPC继代后培养于96孔盘中，以每孔2.5×10 4个细胞/200微升的密度培养8小时。接着，在孔盘中加入tBHP，使细胞在tBHP的存在下培养4小时，其中tBHP于孔盘中的浓度为300μM。接着，移除孔盘中的上清液并使用磷酸盐缓冲液清洗细胞，再加入实施例的组合物培养8小时，其中孔盘中含有1、15、40μg的线粒体。培养完成后，使用磷酸盐缓冲液清洗细胞，并使用含有25μM的DCFDA的培养液，在37℃下反应30分钟。反应结束后，使用磷酸盐缓冲液清洗细胞并加入新的细胞培养液，以OD485/535的波长量测荧光。
实验结果请参考表7及图8。图8显示使用实施例的组合物改善HFDPC的氧化损伤。空白组为未添加过氧化物及线粒体的组别。对照组为未添加线粒体的组别。纵轴为相对荧光强度，表示相对于空白组的荧光强度，以空白组订为1。**表示相对对照组具有高显著差异(P<0.01)。
〔表7〕
Figure PCTCN2021082117-appb-000005
由实验结果可知，含有线粒体的组合物有助于降低细胞中自由基存在量，表示可有效降低氧化压力并改善细胞受到的氧化损伤以帮助细胞修复。并且，随着线粒体的浓度增加，自由基存在量下降更加明显，表示改善氧化损伤的效果也随之增加。
〔实验六，小鼠实验〕
在本实验中，使用tBHP与小鼠进行动物实验，观察小鼠在受到tBHP诱导损伤后，有无使用实施例的组合物处理的皮肤及毛发生长状况。
以下说明实验流程。选用35天(Day 35)大的C57BL/6公鼠，使用气麻机以2％异氟醚(isoflurane)进行麻醉。使用电动剃刀将小鼠背部的毛发剃除，并使用75％酒精进行消毒。使用胰岛素注射针(BD VeoTMinslin syringes)取100μL的300μM tBHP于小鼠背部进行皮下注射。每天注射一次，连续注射5天。接着，以皮下注射的方式分别给予100μL的实施例的组合物。每天注射一次，连续注射3天。注射结束6天后(Day 49)观察皮肤及毛发生长状况。
接着，将小鼠牺牲后，取小鼠背部的皮肤进行冷冻组织包埋处理。详细来说，将皮肤组织取至4％甲醛(Formaldehyde)固定液中，在4℃下于水平仪摇荡4至6小时。移除4％甲醛固定液后，将皮肤组织移入15％蔗糖(Sucrose)溶液中，在4℃下于水平仪摇荡6至8小时。移除15％蔗糖溶液后，将皮肤组织移入30％蔗糖溶液中，在4℃下于水平仪摇荡一晚。将皮肤组织自溶液中取出后移至包埋盒，加入OCT冷冻包埋液(型号：23-730-571，Thermo Fisher Scientific)进行包埋处理。包埋完成的组织保存于-80℃中。将皮肤组织进行冷冻切片，切片厚度约为15至20微米，切片的组织保存于-80℃中。
接着，将完成切片的组织进行荧光染色。详细来说，将切片的组织自-80℃取出后置于室温进行回温，再以PBS润洗两次，每次5分钟。接着，使用配制于PBS-T(含有0.4％triton X-100的PBS)中的1％牛血清白蛋白(Bovine serum albumin，BSA)进行组织润洗两次，每次10分钟。接着，使用配制于PBS-T中的5％BSA进行封闭(blocking)，在室温下与组织作用30分钟，以封闭非特异性物质并提高抗体特异性及灵敏度。接着，使用配制于PBS-T中的1％BSA进行组织润洗两次，每次10分钟。接着，使用F4/80抗体(#MA5-16624，Thermo Fisher Scientific)以1：50的比例配制于1％BSA的PBS-T中，在室温下与组织作用1.5小时。接着，使用配制于PBS-T中的1％BSA进行组织润洗两次，每次10分钟。接着，使用羊抗鼠二抗复合物Alexa
Figure PCTCN2021082117-appb-000006
(goat anti rat secondary conjugation Alexa
Figure PCTCN2021082117-appb-000007
ab150160，abcam)以1：200的比例配制于1％BSA的PBS-T中，在室温下与组织作用1小时。接着，使用配制于PBS-T中的1％BSA进行组织润洗两次，每次10分钟。接着，使用DAPI(D1306，Thermo Fisher Scientific)在室温下与组织反应5分钟，进行细胞核染色。最后，使用荧光显微镜拍照。
实验结果请参考图9至图11及表8。图9显示使用实施例的组合物处理的小鼠背部的毛发生长状况。图10显示图9的小鼠的组织冷冻切片图。图11显示图10的切片图中巨噬细胞的存在量。空白组为未施以tBHP及实施例的组合物处理的组别。对照组为未施以实施例的组合物处理的组别。实施例8-1为施以含40微克的线粒体的组合物处理的组别。实施例8-2为施以含40微克的线粒体及体积百分浓度5％血浆的组合物处理的组别。***表示相对对照组具有非常显著差异(P<0.001)。
〔表8〕
Figure PCTCN2021082117-appb-000008
从图9的照片可看出，相较于空白组与实施例，对照组的小鼠的背部较红，表示小鼠的皮肤及毛囊产生发炎现象。相较于对照组，给予含有线粒体的组合物或含有线粒体及血浆的组合物处理的小鼠，其背部没有发红现象且颜色呈现较黑，表示发炎现象得到改善，且毛囊有因损伤减缓而开始生长毛发。
在图10中，蓝色表示被染色的细胞核，蓝色区域呈现出毛囊型态；红色表示被染色的巨噬细胞，红色区域表示巨噬细胞聚集的区域，亦表示发炎的位置。从图10的切片图可看出，对照组的红色的密度明显大于实施例，表示对照组的小鼠因tBHP诱导毛囊受损发炎，巨噬细胞集中在毛囊处。给予含有线粒体的组合物或含有线粒体及血浆的组合物处理的小鼠，在毛囊处的红色密度明显减少，表示在毛囊处的巨噬细胞明显减少，亦表示发炎现象得到明显改善。
图11及表8呈现以Image J定量软件对图10的切片图进行分析。巨噬细胞存在量为以Image J定量软件分析单位区域内的荧光表现量，以面积与荧光强度的乘积表示。巨噬细胞存在量愈多，表示发炎现象愈严重。由荧光分析进一步证实，给予含有线粒体的组合物或含有线粒体及血浆的组合物处理的小鼠，其巨噬细胞存在量明显下降，表示发炎现象明显改善。并且，含有线粒体及血浆的组合物具有更优异的改善发炎现象的效果。
〔实验七，富线粒体血浆的小鼠实验〕
在本实验中，使用tBHP与小鼠进行动物实验，观察小鼠在受到tBHP诱导损伤后，有无使用实施例 的组合物处理的皮肤及毛发生长状况。
实验流程请参考实验六。本实验所使用的组合物为富线粒体血浆。
实验结果请参考图12至图14及表9。图12显示使用实施例的组合物处理的小鼠背部的毛发生长状况。图13显示图12的小鼠的组织冷冻切片图。图14显示图13的切片图中巨噬细胞的存在量。空白组为未施以tBHP及实施例的组合物处理的组别。对照组为未施以实施例的组合物处理的组别。实施例9为施以含富线粒体血浆的组合物处理的组别。**表示相对对照组具有高显著差异(P<0.01)。
〔表9〕
Figure PCTCN2021082117-appb-000009
从图12的照片可看出，相较于空白组与实施例，对照组的小鼠的背部较红，表示小鼠的皮肤及毛囊产生发炎现象。相较于对照组，给予含有富线粒体血浆的组合物处理的小鼠，其背部没有发红现象且颜色呈现较黑，表示发炎现象得到改善，且毛囊有因损伤减缓而开始生长毛发。
在图13中，蓝色表示被染色的细胞核，蓝色区域呈现出毛囊型态；红色表示被染色的巨噬细胞，红色区域表示巨噬细胞聚集的区域，亦表示发炎的位置。从图13的切片图可看出，对照组的红色的密度明显大于实施例与空白组，表示对照组的小鼠因tBHP诱导毛囊受损发炎，巨噬细胞集中在毛囊处。给予含有富线粒体血浆的组合物处理的小鼠，在毛囊处的红色密度明显减少，表示在毛囊处的巨噬细胞明显减少，亦表示发炎现象得到明显改善。
图14及表9呈现以Image J定量软件对图13的切片图进行分析。由荧光分析进一步证实，给予含有富线粒体血浆的组合物处理的小鼠，其巨噬细胞存在量明显下降，表示发炎现象明显改善。
〔实验八，富线粒体血浆的头皮实验〕
在本实验中，以微针在头皮上创造出微型伤口，使用含有富线粒体血浆的组合物涂抹于头皮上。取用适当量的含有富线粒体血浆的组合物，使其均匀覆盖患部，涂抹范围约25至49平方厘米。在涂抹前及涂抹后第60天，观察毛发生长状况并进行像素分析。
实验结果请参考表10、图15及图16。图15显示使用实施例的组合物涂敷于头皮前后毛发生长的情况，左图为涂抹前，右图为涂抹后第60天。图16显示图15的照片中头皮的像素分析。纵轴的像素代表单位面积中毛发的数量，以像素表示毛发的密度。
〔表10〕
时间 像素
涂抹前 167.2±13.7
涂抹前后第60天 188.4±8.9
由图15及图16可看出，在将实施例的组合物涂敷于头皮经过60天后，毛发的密度有所提升，表示含有富线粒体血浆的组合物确实有助于毛发的生长。
根据本发明一个实施例，藉由对毛囊给予线粒体或富线粒体血浆，可帮助毛囊中的细胞生长，以促进毛发再生。对于受损的细胞而言，可提高受损细胞的存活率；对于老化的细胞而言，可改善细胞的老化程度；对于受自由基损伤的细胞而言，可改善细胞的氧化损伤。除此之外，将含线粒体的组合物涂敷或注射于毛囊周围的皮肤，可改善毛囊发炎现象。
【附图标记说明】
S10、S20、S30…步骤

Claims (13)

  1. 一种促进毛发再生的组合物，包含线粒体。
  2. 如权利要求1所述的组合物，其中，所述线粒体的浓度为5微克/毫升至200微克/毫升。
  3. 如权利要求1所述的组合物，进一步包含血浆，所述血浆中的血小板数目小于1×10 4个/微升。
  4. 如权利要求3所述的组合物，其中，所述血浆的体积百分浓度为5％。
  5. 一种富线粒体血浆的组合物，包含线粒体及血浆。
  6. 如权利要求5所述的组合物，其中，所述富线粒体血浆中的所述线粒体的重量大于5微克。
  7. 如权利要求1-4中任一项所述的包含线粒体的组合物用于促进毛发再生的用途。
  8. 如权利要求5或6所述的富线粒体血浆的组合物用于促进毛发再生的用途。
  9. 如权利要求7或8所述的用途，其中，将所述组合物以涂敷、注射或直接滴入的方式接触毛囊或毛囊周围的皮肤。
  10. 如权利要求7或8所述的用途，其中，所述组合物提高受损人类毛发细胞的存活率、改善人类毛发细胞的老化或改善人类毛发细胞的氧化损伤。
  11. 一种制造如权利要求5或6所述的富线粒体血浆的方法，包含：
    将血浆及多个细胞自血液中分离；
    从所述多个细胞中取出多个线粒体；以及
    混合所述血浆与所述多个线粒体，形成所述富含线粒体血浆。
  12. 如权利要求11所述的方法，其中，
    将血浆及多个细胞自血液中分离的步骤包含：
    使所述血液分层，形成红血球层、白细胞层(buffy coat)及血浆层；以及
    取出所述白细胞层及所述血浆层；
    从所述多个细胞中取出多个线粒体的步骤包含从所述白细胞层中的所述多个细胞中取出所述多个线粒体；
    混合所述血浆与所述多个线粒体的步骤包含混合所述血浆层与所述多个线粒体。
  13. 如权利要求11所述的方法，其中，所述富线粒体血浆中的所述多个线粒体的重量大于5微克。
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