TW201609115A - Composition using exogenous mitochondria as effective ingredient, application thereof, and method for repairing cells - Google Patents

Abstract

This invention discloses a composition using exogenous mitochondria as effective ingredient, application thereof, and a method for repairing cells, wherein the composition provides a high degree of safety; and, by administering an effective amount of the composition to an individual, the exogenous mitochondria can be delivered intact to the interior of cells so as to achieve the effects of repairing damaged cells and improving or preventing cell aging.

Description

Exogenous mitochondria as a composition of active ingredients, use thereof and method of repairing cells

The present invention relates to a method for resisting aging and repairing damaged cells of mitochondria, and particularly to a composition comprising an exogenous mitochondria as an active ingredient, a use thereof, and a method for repairing cells.

The mitochondria are responsible for providing cellular energy in the cell and producing adenosine triphosphate (ATP). The mitochondria will change dynamically due to differences in intracellular energy requirements or cellular pressure, and are not always in a single mitochondrial state. Specifically, when the cellular energy requirement increases, the mitochondria will continue to fission to rapidly produce adenosine triphosphate; conversely, when the cell is starved, the mitochondria will fuse to reduce energy. Production and utilization to maintain normal physiological functions of cells. In addition, when the mitochondria suffers from a decrease in membrane potential, mitochondrial DNA mutation (mtDNA), etc., a fusion reaction is also performed, and the damaged granules are replaced by homologous recombination. Body DNA, and when the mutated DNA in the mitochondria accumulates too much to be repaired, the mitochondria are cleared by autophagosome, leaving only normal mitochondria (Lamb CA et al ., 2013). Therefore, when the cells have too many damaged mitochondria and cannot be removed, the cells will move toward apoptosis (Mukhopadhyay S et al ., 2014).

Mitochondrial defects and dysfunction are associated with many diseases, such as Leber's hereditary optic neuropathy (Mitochondrial Enephalomyopathy, Lactic Acidosis, and Strokelike Episodes, MELAS), myocardial epilepsy Myoclonic Epilepsy Associated With Ragged-Red Fibers (MERRF) and the like. In addition, neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, and Parkinson's disease are also associated with dysregulation of mitochondrial division (Ghavami S et al ., 2014). In addition, aging or ageing causes the mutated DNA in the mitochondria to grow and accumulate, leading to the development of related diseases, such as age-related macular degeneration (AMD) (Brennan LA et al ., 2014; Jarrett SG et al ., 2010) and skin aging, etc. (Blatt T et al ., 2005; Makrantonaki E et al ., 2007).

In order to delay the aging of the skin, people will use many beauty care products, such as hyaluronic acid, vitamin A and vitamin C, antioxidants, sunscreens, etc., or seek medical beauty clinics to improve aging, such as laser, botulinum, electroporation Leather and so on. However, the existing cosmetic methods cannot fundamentally improve or delay the occurrence of skin aging. Other studies have confirmed that injection of mesenchymal stem cells can effectively improve aging phenomena such as skin wrinkles, and stem cell therapy is considered to be an opportunity to delay skin aging. However, in fact, stem cells are not easy to obtain, and a large amount of culture takes too much time and money, and stem cell transplantation has the risk of mutation or tumor rejection. Therefore, there is currently no method that is highly safe and can effectively and effectively improve skin aging.

In 1989, it was pointed out that when exogenous mitochondria were co-cultured with cells, mitochondria could enter the cells via direct injection or membrane fusion, and the cells with gene-deficient mitochondria returned to normal. Function (King MP et al ., 1988; King MP et al ., 1989). Many studies have also confirmed that simply co-culturing cells with mitochondria does not allow mitochondria to enter cells (Chang JC et al ., 2013; Spees JL et al. , 1989), and thus it is not known to directly give exogenous sources. Whether the mitochondria can enter the cell. As can be seen from the prior art, different cells may have different abilities to ingest foreign mitochondria. Therefore, before the mitochondria enters the cell, the researchers are unable to adjust the experimental conditions to correlate with the mitochondria. The experiment was repeated.

Furthermore, although the cells are exposed to foreign substances such as bacteria via phagocytosis, the foreign substances ingested by the phagocytic pathway form a phagolysosome with the liposome. Degradation of foreign matter. Therefore, it is generally believed that exogenous mitochondria cannot remain in the cell via phagocytosis and repair endogenous mitochondria. The recent research utilizes a cell-penetrating peptide as disclosed in U.S. Patent No. 8,486,034, or a liposome-coated mitochondria as disclosed in U.S. Patent No. 20130022666, to facilitate the fusion of the mitochondria with the cell membrane for easy access. In the cell, increase the oxidative respiration of cells. However, although the above method can promote the feeding of mitochondria into cells, the vectors used, such as transmembrane peptides and liposomes, may cause rupture of mitochondria and cell membrane, resulting in damage and target of mitochondria. Cell toxicity.

The main object of the present invention is to provide a composition comprising an effective amount of exogenous mitochondria.

A second object of the invention is to provide the use of the composition for repairing damaged mitochondria or improving cell aging.

Another object of the present invention is to provide a method for repairing cells by administering an effective amount of exogenous mitochondria into a body, and allowing the exogenous mitochondria to be completely delivered into the cells, To achieve the effect of repairing damaged cells, improving or preventing cell aging.

In order to achieve the above object, an embodiment of the present invention discloses a composition, An exogenous mitochondria is included, as well as at least one pharmaceutically or cosmetically acceptable carrier.

Preferably, the composition further comprises an adjuvant, and the adjuvant is serum, plasma, complement or a combination of at least the above two components.

Preferably, the exogenous granulation system is obtained by extraction from cells.

Preferably, the exogenous granulocyte system is obtained from the cells by centrifugal purification.

In another embodiment of the invention, the use of the above pharmaceutical composition is for improving the aging of skin cells.

In yet another embodiment of the invention, the use of the above composition is for repairing damaged cells.

A method for repairing cells according to an embodiment of the present invention, which comprises administering an effective amount of exogenous mitochondria to a body, allowing the exogenous mitochondria to enter the cell, replacing the damaged or Aging mitochondria.

Preferably, the exogenous mitochondria are pretreated with at least one component selected from the group consisting of serum, plasma and complement prior to administration to the individual.

The first picture is the result of the relative position of the ingested mitochondria and intracellular lysosome after one hour of co-culture with BHK cells stained with red fluorescein and green fluorescent lysotracker. .

The second graph is the result of the relative position of the ingested mitochondria and intracellular lysosomes after the mitochondria with the red fluorescein and the BHK cells were co-cultured for four hours.

In the third panel, A and B, the exogenous mitochondria are engulfed by the cells by scanning electron microscopy. The box in the A graph indicates the mitochondria being phagocytized at low magnification; The arrow points to the mitochondria that are being swallowed by the pseudopods of the cells at high magnification.

The fourth figure is the result of observing the mitochondria into BHK cells after culturing the mitochondria with red fluorescein and BHK cells stained with phalloidin-FITC for four hours. Mark 10 μm .

The fifth panel shows the results of red mitochondria entering BHK cells after treatment of BHK cells with antibiotic actinomycin D (ActD). The white bars indicate 10 μm .

The sixth graph is the fourth and fifth graphs, with or without the treatment of AcD BHK cells, with statistical results for the proportion of cells with exogenous mitochondria.

Figure 7A shows the relative position of the ingested mitochondria and intracellular mitochondria after four hours of co-culture with exogenous red mitochondria and BHK cells with green fluorescent mitochondria. As a result, the arrow indicates yellow fluorescence, indicating that the exogenous and endogenous mitochondria overlap in the intracellular location.

FIG seventh line B is a C3 to complement after four hours cocultured than 10 μ g / mL and treated mitochondria derived BHK cells with green fluorescence of mitochondria, the mitochondrial uptake and somatic cell The result of the relative position of the mitochondria, wherein the arrow indicates yellow fluorescence, indicating that the exogenous and endogenous mitochondria overlap in the intracellular location.

Figure 7C shows the red linear region in the seventh panel B observed by a conjugate focal microscope, indicating the red fluorescence of the exogenous mitochondria and the overlap of the green fluorescent signals representing the endogenous mitochondria.

Figure 8 is a view of the appearance of an untreated mitochondria by an electron microscope.

Figure 8B shows the appearance of serum-treated mitochondria by electron microscopy.

Figure 8C shows the appearance of the mitochondria treated with C3 complement by electron microscopy.

Figure 8D shows the appearance of the mitochondria treated with Pep-1 transmembrane peptide by electron microscopy.

Figure IX is the result of the mitochondria entering HUVEC cells after co-culture of exogenous mitochondria with HUVEC cells.

The ninth panels B to D are the results of SA β -gal staining of each group of HUVEC cells treated with different treatments.

The tenth figure shows the case where the exogenous mitochondria with red fluorescent protein enter the mouse dermal fibroblasts by a conjugate focal microscope.

The eleventh panels A to D are images of the skin surface of the first to fourth groups of nude mice after microscopic observation and treatment with exogenous mitochondria.

The twelfth figure is the result of roughness analysis of the epidermis wrinkles of each group of nude mice after treatment with exogenous mitochondria.

Fig. 13A to D are the results of skin tissue sections of nude mice of each group stained with Masson's trichome.

The meaning of the technical and scientific terms used in the description and claims of the present invention are the same as those of ordinary skill in the art to which the present invention pertains. In case of conflict, the content of the present invention shall prevail.

By "effective amount" is meant the amount of the compound or active ingredient required to produce the desired effect, expressed as a percentage by weight of the composition. As the invention belongs to It is known to those of ordinary skill in the art that this effective amount will vary depending on the route of administration desired to effect a particular effect. Generally, the active ingredient or compound may be present in the compositions in an amount of from about 1% to about 100% by weight of the composition, preferably from about 30% to about 100%.

The term "carrier acceptable in pharmacy or cosmetic products" is used to encompass any standard used in pharmaceutical or cosmetic products, and the carrier may be solid, semi-solid or liquid depending on the type of composition. For example, carriers include, but are not limited to, gelatin, emulsifiers, hydrocarbon mixtures, water, glycerin, physiological saline, buffered saline, lanolin, paraffin, beeswax, dimethicone, ethanol.

The term "composition" is used to include an effective amount of the desired compound or active ingredient to produce a particular effect, and at least one carrier. As is well known to those of ordinary skill in the art to which the present invention pertains, the form of the composition may vary depending on the route of administration desired to cause a particular effect, such as a lozenge, a powder, an injection, etc., and the carrier is also It is solid, semi-solid or liquid as the composition is shaped.

The term "administration" refers to the way in which a substance is delivered to a specific part of a body, to a specific cell, to a specific target, or to its contact with an individual. In general, the route of administration includes, but does not Limited to oral, smear, spray, inhalation, injection, etc.

In the following, in order to explain the advantages of the present invention, the present invention will be described in detail by way of example only. The scope and significance.

Example 1: Firefly cursor grading line body

Transfection of red fluorescent protein DsRed or green fluorescent protein with mitochondria signal peptide In the baby hamster kidney fibroblast cells (BHK-21 cells, hereinafter referred to as BHK cells), through the screening of G418 antibiotics and flow cytometry, RedM- which can continuously express red fluorescein was obtained. BHK cells or GFP-BHK cells.

Example 2: Separation of mitochondria from BHK cells

When the number of BHK cells was raised to 2× ^{10 8} , the cell culture dish was washed with SEH buffer (0.25 M sucrose, 0.5 mM EGTA and 3 mM HEPES-NaOH, pH 7.2), and centrifuged at 1000 x g for 3 minutes. After removing the supernatant, 2 ml of SEH buffer was added, ground in a Dounce homogenizer for about 15 times, and operated on ice to reduce damage to cells and mitochondria. After milling is complete, the homogenate was centrifuged at 1000x g centrifuged for 15 minutes, the precipitate was removed, and then centrifuged at 9000xg 10 minutes and finally the pellet in 50 μ L of buffer solution SEH, inhibitor of proteolytic enzymes was added, Store at 4 ° C.

Example 3: Determining the way in which mitochondria enter cells

In this example, in order to track the movement path of the mitochondria into the cell, the relationship between the position of the mitochondrial movement and the lysosome is observed at different times by adding the exogenous mitochondria.

First, mitochondria were calibrated with red fluorescent protein DsRed, and BHK cells were transfected with a green fluorescent lysosomal dye (LysoTracker) to determine the location of intracellular lysosomes. 5 μ g of the addition to give a red fluorescent protein source calibration mitochondria, lysosomes and stain the treated BHK cells co-incubated at room temperature, and incubated for one hour at the time of four hours, the conjugated confocal microscopy observation The case where the exogenous mitochondria enters the BHK cell and its relative position to the lysosome, the results are shown in the first and second figures.

As can be seen from the first figure, the exogenous mitochondria with red fluorescence were distributed on the periphery of BHK cells after one hour of culture. As can be seen from the second figure, after four hours of cultivation, part The lysosomal dye signal with red fluorescence and exogenous mitochondria overlaps with green fluorescence. From the above results, it can be seen that the exogenous mitochondria enters the cell and the lysosome is located at the same position in the cell, and it is inferred that the exogenous granulocyte system enters the cell via phagocytosis.

Further, the situation in which the exogenous mitochondria enters the BHK cell is observed by a scanning electron microscope, as shown in the third panel A and B, wherein the third graph A is the observation result at a low magnification, and is within The box shows the mitochondria that are being phagocytosed by the cells; and the third panel B shows the results observed at high magnification, with the arrows indicating the mitochondria being swallowed by the pseudopods of the cells. Therefore, the results of the third panel show that the BHK cell line coats the exogenous mitochondria by extending the pseudopodia, confirming that the pathway of exogenous mitochondria into the cell is phagocytosis.

Furthermore, BHK cells were stained with phalloidin-FITC to calibrate actin in cells and to display cell types. After culturing the above-mentioned stained BHK cells with the exogenous mitochondria at 37 ° C for 4 hours, a large number of mitochondrial bodies were found in the cells, as shown in the fourth figure. However, antibiotics actinomycin D 20 μ M of (abbreviation of ActD) treated BHK cells, BHK cells make the phagocytosis is suppressed, it can be found exogenous mitochondria completely unable to enter the cell, as shown in FIG fifth. The number of exogenous mitochondria into the above-mentioned cells according to different treatments was counted, and the results are shown in the sixth figure.

From the above results, it is understood that when the exogenous mitochondria are simply administered to the cells, the cells take up the mitochondria by phagocytosis, allowing the mitochondria to enter the cells.

Example 4: Serum lines help mitochondria enter cells

The diluted commercially available fetal bovine serum (GIBCO) is mixed with the serum-derived mitochondria of the red protein for one hour, the serum in the supernatant is removed by centrifugation, and the precipitated mitochondria are taken as SEH. The buffer is dissolved back to its original volume. BHK cells with phalloidin (phalloidin)-FITC staining, by which the dye specifically binds to F-actin, defines the boundary of the cell membrane.

The BHK cells were divided into four groups, wherein the first group was a blank group; the second group was mixed and diluted 1000-fold serum; the third group was mixed and diluted 500-fold serum; and the fourth group was mixed and diluted 100-fold serum. The mitochondria were extracted from the above SEH solution, and co-cultured with each group of BHK cells at 37 ° C for 4 hours, and the fluorite with red fluorescence was observed by a laser conjugated focal microscope. In the case, and the number of red mitochondria contained in a single cell was analyzed, the results are shown in Table 1 below, wherein Table 1 was analyzed by the statistical method of one-way ANOVA test. The asterisk indicates that the p- value is less than 0.05, representing a statistically significant difference from the first set of control groups.

From the results of the above Table 1, it is known that in the presence of serum, the number of cells having exogenous mitochondria and their entry into a single cell are significantly increased compared to those without serum. It can be seen that the treatment of exogenous mitochondria or cell lines by serum helps to enhance the outside. The efficiency of the source mitochondria into the cell.

Example 5: The supplement system helps the mitochondria enter the cell

The exogenous mitochondria labeled with red fluorescent protein are mixed with a predetermined concentration of C3 complement for one hour, and the C3 complement in the supernatant is removed by centrifugation, and the SEH buffer of the exogenous mitochondria after precipitation is buffered. The solution is dissolved back to its original volume.

The first group is an untreated group. The second to fifth groups were set at a concentration of 0.1 μ g / mL, than 1 μ g / mL, 10 μ g / mL and 20 μ g / mL of C3 complement (Sigma-Aldrich) treatment mitochondria 5 μ g, after co-cultivation for 4 hours at 37 ° C, the red fluorescence in each group of cells was observed by a laser conjugated focus microscope, and the exogenous nature of each group of cells was calculated by flow cytometry. The ratio of mitochondria and further quantitative statistics. The situation in which the exogenous mitochondria enters the cell and fuses with the endogenous mitochondria is as shown in the seventh panel A to C.

Referring to Figures 7A and B, each of the arrows in the figure is yellow fluorescent, indicating that the exogenous mitochondria and the endogenous mitochondria overlap within the cell. Therefore, as can be seen from the results of the seventh graph, the exogenous granulocyte system ingested by the cells and the original mitochondria in the cell are located at the same position in the cell, with or without the treatment of complement, showing exogenous mitochondria and The mitochondria are fused to each other and can escape the phagolysosome and enter the cytoplasm.

Furthermore, by the results of the flow cytometry analysis, on the first group without C3 complement treatment, on average, about 26.16±4.75% of the cells were detected to have red fluorescence; the second group was averaged. Approximately 43.43±3.5% of the cells were detected to have red fluorescence; in the cells of the third group, an average of approximately 65.13±7.5% of the cells were detected to have red fluorescence; in the cells of the fourth group On average, approximately 78.97±13.35% of the cells were detected to have red fluorescence; in the cells of the fifth group, approximately 80% of the cells were detected to have red fluorescence; and, the second to fifth groups There was a significant difference between the lines and the first group ( p < 0.05).

It is confirmed by the above results that by administering complement to the exogenous mitochondria, the proportion of the exogenous mitochondria into the cell can be significantly increased, and the exogenous mitochondria can be extracted from the endogenous granules. Body fusion, in addition, as the concentration of the complement is increased, the number of exogenous mitochondria entering the cell is also increased.

Example 6: Serum or complement does not destroy the isolated mitochondria

The separated outside the endogenous mitochondrial divided into four groups, each group of 5 μ g. Wherein the first set of blank lines for the group, the second group of lines 100-fold dilution of fetal calf serum in the process of exogenous mitochondria, the third group based complement C3 concentration 10 μ g / mL of the exogenous treatment The mitochondria, the fourth group treated the exogenous mitochondria with a 100 nM cell penetrating peptide Pep-1. Each group was cultured at 37 ° C for 4 hours, and the appearance of each group of exogenous mitochondria was observed by a transmission electron microscope, and the results are shown in Figs. 8 to 8D.

As can be seen from the results of the eighth graph, the appearance of the mitochondria of the second and third groups is similar to the appearance of the first mitochondria without any treatment, respectively. Compared with the first group, the exogenous mitochondria treated by the transmembrane peptide in the fourth group were significantly enlarged and had a rupture. Therefore, serum or complement is less toxic than the transmembrane peptide, and does not destroy the appearance of the mitochondria, but maintains the integrity of the mitochondria after entering the cell.

Example 7: Culture human umbilical cord endothelial cells

Human umbilical vascular endothelial cells (hereinafter referred to as HUVEC cells) were purchased from Hsinchu Food Industry Development Research Institute. HUVEC cells were cultured in M199 medium supplemented with 10% fetal bovine serum, 0.1% heparin, and 0.03% endothelial cell growth supplement. HUVEC cells can It was grown on a 0.1 weight percent gelatin-coated petri dish.

Example 8: Granules can delay cell aging

The mitochondria are first extracted from the human fibroblast HS68 as a source of exogenous mitochondria. Each group of 5 μ g. After the mitochondria were treated with complement, the mitochondria were stained with a red fluorescent mitochondrial tracking dye (Mitotracker).

In addition, primary cultured HUVEC cells were treated with hydrogen peroxide to age. The HUVEC cells (8×10 ⁵ cell/well) cultured to the 8th passage were treated with 100 μM hydrogen peroxide at 37° C. for 2 hours, washed with phosphate buffer to remove hydrogen peroxide, and used as normal cell culture solution. After one day of cultivation, it is divided into three groups, wherein the first group is a blank group without mitochondria, the second group is added with the mitochondria without complement treatment, and the third group is added with the complement treatment. Granules. After 4 hours of culture in each of the cell lines, SA β -gal (Senescence-associated β- galatosidase) staining and Ki67 and BrdU staining were performed.

The dyeing process of Ki67 and BrdU is generally known in the art and is generally known to those skilled in the art, and thus will not be described herein.

The SA β- gal staining process is as follows: the cells are first washed with phosphate buffer, fixed with 2% paraformaldehyde, 0.2% glutaraldehyde for 5 minutes, and then stained at 37 ° C. For 12 hours, wherein the staining solution contains 1 mg/mL of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (5-bromo-4-chloro-3-indolyl -β-D-galactoside, BCIG or X-gal), 40 mM citric acid/phosphate buffer (pH 6.0), 5 mM potassium ferricyanide, 5 mM ferrocyanide Sodium ferricyanide, 150 mM sodium chloride and 2 mM magnesium dichloride. Then, the cells were stained with 0.5% eosin and observed under a microscope.

The results after staining with SA β -gal are as shown in the ninth panels A to D. It can be seen from Figure 9A that the exogenous granulocyte system can enter HUVEC cells. As can be seen from the ninth panels B to D, the HUVEC cell line of the first group was significantly stained with SA β -gal. The HUVEC cells of the second group were stained with SA β- gal, but the number of stained HUVEC cells in the second group was significantly lower than that of the first group. Compared to the first or second group, the HUVEC cells in the third group were almost uninfected with SA β- gal. Further, the staining results were statistically analyzed, and it was found that about 85±12.3% of the cells in the first group were stained, and about 60.1±6.8% of the cells in the second group were stained, while in the third group of cells. 25±6.2% of the cells were stained.

Furthermore, by counting the staining results of Ki67 and BrdU, it was found that the ratio of Ki67 and BrdU in the first group of HUVEC cells was 13.3% and 13%. Compared with the first group, the proportion of Ki67 and BrdU in the HUVEC cells of the second group increased by 35% and 33%, respectively. The proportion of Ki67 and BrdU in HUVEC cells in the third group was the highest, 71% and 59.6%, respectively. Moreover, when the administered mitochondria are treated with fetal bovine serum, the same efficacy as in the case of complement treatment can be achieved.

From the above results, it can be seen that the entry of exogenous mitochondria into the cell line can effectively reduce the degree of cell aging, increase cell growth, and increase the efficiency of cell replication, and can be increased as the number of exogenous mitochondria entering the cell increases. Significantly reduce the degree of cell aging, increase the cells in the split state to increase cell replication and growth. Accordingly, by administering the pharmaceutical composition containing the exogenous mitochondria disclosed in the present invention to a body, the degree of cell aging can be effectively improved or retarded, and the granules are promoted in the pharmaceutical composition. When entering the components of a cell, such as Serum, plasma or complement can significantly improve its efficacy.

Example 9: Animal Experiment (1)

Cell extracts from RedM-BHK with red fluorescent mitochondrial of, and be dealt with in 100 dilution of fetal calf serum or 10 μ g / mL C3 complement. The natural aging nude mice of 48 weeks old were injected into the subcutaneous tissue of the nude mice. After one hour, the whole skin of the nude mice was taken, fixed with 4% paraformaldehyde for 5 minutes, and then allowed to stand. The 0.1 M phosphate buffer was allowed to sink to the sample, and then the sample was infiltrated and embedded with an ice embedding agent (OCT), and frozen sections were sliced to a thickness of about 12 μm . After the film was mounted, it was observed with a conjugated focus microscope, and the results are shown in the tenth figure.

The tenth picture shows the dermal layer area of nude mice after mitochondrial transplantation. The blue fluorescence is the DAPI-stained fibroblast nuclei, and the red fluorescent system is the mitochondria isolated from the RedM-BHK cells. The results of the tenth graph show that the mitochondria can enter the dermis after transplantation. In the fibroblasts.

Example 10: Separation of mitochondria from liver cells

First, the mice were sacrificed after deep anesthesia, and the mice were perfused with physiological saline until the blood in the liver was removed. About 1 cubic centimeter of liver tissue was taken out, about 6 ml of SEH buffer was added, and after grinding through a tissue grinder, 1000 x g was centrifuged, centrifuged for 15 minutes, and the supernatant was taken. Further, a sucrose solution having a concentration of 55%, 40%, and 30% was sequentially added to the centrifuge tube to obtain a 30 to 55% sucrose gradient centrifuge tube. The supernatant obtained by the centrifugation step was applied to the upper layer of the gradient centrifuge tube, and centrifuged at 35,000 rpm for 30 minutes to form a white-permeable layer between the layers of 40% and 55%. Aspirate about 1 ml of the permeable layer and collect it in a 15 ml centrifuge tube, add 5 ml of SEH buffer, centrifuge 13000 x g, centrifuge for 3 minutes, remove the supernatant, and repeat the above centrifugation step three times. Finally, back to 200 μL of SEH buffer The mitochondrial precipitate was added to the inhibitor of proteolytic enzyme and stored at 4 °C.

Example 11: Animal Experiment (2)

Thirty-two 48-week-old natural-aged nude mice were divided into 4 groups, 8 in each group, and treated under different conditions for 12 weeks. The first group was treated with no treatment, and the second group was injected with 1000 per week. μ g of liver mitochondria to each of the mice, the third group of weekly injections of 1000 μ g by the liver mitochondria complement to each of the mice of treatment, the fourth group is based weekly injections of 1000 μ g sera The liver mitochondria were treated to each of the mice. The subcutaneous injection method of the second group to the fourth group of mice is to inject about 5,000 μg/mL of liver mitochondria into 20 points on the back of each nude mouse, and inject 0.01 ml at each point, the total injection amount. It is 0.2 ml. In order to remove the effects of simple complement and serum on wrinkles, the complement and serum remaining in the supernatant were removed by high-speed centrifugation twice before the application of complement and serum-treated mitochondria.

After the test was completed, the whole skin of each of the nude mice of each group was photographed, tissue frozen sectioned and stained as described in Example 9. The skin photographing results are shown in Fig. 11 to Fig. A to D, and the skin wrinkle roughness of each of the nude mice is further analyzed. The results are shown in Fig. 12, wherein * indicates significant difference from the first group. difference. The skin tissues of each of the nude mice of each group were sectioned and stained by Masson's trichrome to reveal the content of the dermal collagen layer, and the results are shown in Fig. 13 to Figs.

From the results of the twelfth figure, the wrinkles observed in the second to fourth groups in which the exogenous mitochondria were injected were significantly lower than those in the first group in which no treatment was performed, among which the third group and the fourth group were significantly lower. The wrinkles on the skin of nude mice were slightly milder than those in the second group. Furthermore, as can be seen from the results of the thirteenth graph, the thickness of the first epidermis layer is the thickest, and the thickness of the epidermis layer of the second group to the fourth group is lower than that of the first group, and the staining of the collagen layer is respectively compared. The first group becomes deeper.

Based on the results of the eleventh to thirteenth images, it can be seen that the exogenous granule system disclosed in the present invention can enter living cells, can effectively reduce the generation of wrinkles, and enhance the ability to produce collagen in epidermal fibroblasts. Moreover, since serum or complement can promote the entry of foreign mitochondria into the cell, the source granule system has a more anti-aging ability than the serum or complement treatment. It can be seen that the pharmaceutical composition containing the exogenous mitochondria disclosed in the present invention can indeed achieve the effect of delaying or improving skin aging.

It can be seen from the above examples that the exogenous mitochondria and the pharmaceutical composition having the active ingredient thereof have the following advantages: First, the use of the exogenous granule system can overcome the conventional technique and Exclusion caused by cell transplantation; Second, exogenous mitochondria are obtained from general cell lines or living organisms, have a wide range of sources, and are not harmful to human health. For example, conventional cell transplantation techniques may cause cancer. Or the occurrence of tumors; Third, the exogenous mitochondria can directly enter the cell, and fuse with the endogenous mitochondria to replace the damaged mitochondria in the aged cells or damaged cells, thereby reducing the oxidative stress of the cells and restoring the cells. The function of normal function, and can provide long-term direct protection of cells; Fourth, exogenous mitochondria can completely enter cells after treatment with serum or complement, and avoid peptides or liposomes such as liposome The cytotoxicity caused by the treatment; fifth, the exogenous granulation system can fundamentally improve the phenomenon of wrinkles and skin aging, and effectively promote the increase of collagen synthesis .

Accordingly, the pharmaceutical composition disclosed in the present invention is highly safe, and by casting By administering an effective amount of the pharmaceutical composition to a body and entering the cells through the exogenous mitochondria, the function of repairing the damaged cells of the mitochondria and improving the aging phenomenon can be achieved.

The above is only the detailed description of the present invention by the examples, and any simple modifications or changes made to the embodiments of the specification should be made without departing from the spirit of the invention. The resulting hunter.

references

Lamb CA, Yoshimori T, Tooze SA (2013) The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol 14: 759-774.

Mukhopadhyay S, Panda PK, Sinha N, Das DN, Bhutia SK (2014) Autophagy and apoptosis: where do they meet? Apoptosis 19: 555-566.

Ghavami S, Shojaei S, Yeganeh B, Ande SR, Jangamreddy JR, et al. (2014) Autophagy and apoptosis dysfunction in neurodegenerative disorders. Prog Neurobiol 112: 24-49.

Brennan LA, Kantorow M (2009) Mitochondrial function and redox control in the aging eye: role of MsrA and other repair systems in cataract and macular degenerations. Exp Eye Res 88: 195-203.

Jarrett SG, Lewin AS, Boulton ME (2010) The importance of mitochondria in age-related and inherited eye disorders. Ophthalmic Res 44: 179-190.

Blatt T, Lenz H, Koop U, Jaspers S, Weber T, et al. (2005) Stimulation of skin's energy metabolism provides multiple benefits for mature human skin. Biofactors 25: 179-185.

Makrantonaki E, Zouboulis CC (2007) Molecular mechanisms of Skin aging: state of the art. Ann N Y Acad Sci 1119: 40-50.

King MP, Attardi G (1988) Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA. Cell 52: 811-819.

King MP, Attardi G (1989) Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246: 500-503.

Chang JC, Liu KH, Li YC, Kou SJ, Wei YH, et al. (2013) Functional recovery of human cells harbouring the mitochondrial DNA mutation MERRF A8344G via peptide-mediated mitochondrial delivery. Neurosignals 21: 160-173.

Spees JL, Olson SD, Whitney MJ, Prockop DJ (2006) Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci U S A 103: 1283-1288.

Claims (8)

A composition comprising exogenous mitochondria and at least one pharmaceutically or cosmetically acceptable carrier. The composition according to claim 1, further comprising an adjuvant selected from the group consisting of serum, plasma, complement and at least a combination of the above two components. The composition of claim 1, wherein the exogenous granulation system is extracted from cells. The composition according to claim 1, wherein the exogenous granulation system is obtained from a cell by a method of centrifugal purification. A use according to the composition of claim 1 for improving or preventing aging of skin cells. A use according to the composition of claim 1 for repairing damaged cells of mitochondria. A method of repairing cells by administering an effective amount of exogenous mitochondria to a body, allowing the exogenous mitochondria to enter the cell, replacing the damaged or aged mitochondria. The method of anti-cell aging according to claim 7, wherein the exogenous mitochondria are pre-exposed to at least one of a group consisting of serum, plasma and complement before administration to the individual. deal with.