CN116440162A

CN116440162A - Biological product with mammal heart system anti-aging effect and preparation method and application thereof

Info

Publication number: CN116440162A
Application number: CN202210009606.6A
Authority: CN
Inventors: 陈熹; 朱卿; 骆阳; 潘金梦; 陈骁锐; 吴舟怡; 张辰宇
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2022-01-06
Filing date: 2022-01-06
Publication date: 2023-07-18

Abstract

The invention provides a biological product with an anti-aging effect on a mammalian heart system, wherein the effective component of the biological product comprises exosomes for extracting blood of an animal body, and a preparation method and application thereof. The invention has the characteristics and advantages that: the biological product with the anti-aging effect of the mammalian heart system, the preparation method and the application thereof in preparing the product with the anti-aging effect of the mammalian heart system, which are provided by the invention, contain exosomes and miRNA extracted from blood, so that the content of the exosomes and the quantity of the exosomes are ensured, and meanwhile, the exosomes in the blood are the combined bodies, so that the biological product can more effectively realize various effects, has simple and wide sources, and is favorable for long-term repeated and massive use.

Description

Biological product with mammal heart system anti-aging effect and preparation method and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a biological product with an anti-aging effect of a mammalian heart system, a preparation method thereof and application thereof in preparing a product with the anti-aging effect of the mammalian heart system.

Background

Aging refers to the phenomenon that the physiological and psychological adaptive capacity of the body to the environment is progressively reduced and gradually tends to die. Aging can be divided into two categories: physiological aging and pathological aging. The former refers to physiological degenerative processes occurring after maturity, and the latter is an senile change due to various external factors including various diseases. Physiological changes in the aging process of the human body are mainly reflected in the loss of tissue cells and constituent substances of the body, the slowing of the metabolic rate of the body and the hypofunction of the body and organs. Aging is unavoidable but it is possible to delay aging.

Aging of the organism is mainly affected by genetic, environmental, cellular genes and other factors. Genetic factors determine the highest longevity of various animals. At the same time, the external environment (such as air quality, water quality, etc.) and the internal environment of the body can have a significant impact on aging. In addition, if most of important cells of the body are healthy, the life of the body is correspondingly prolonged.

The existing medical and biological researches find that the aging of the organism is the comprehensive expression of the physiological function decline and disorder of the organism in the degeneration period, is an irreversible life process and is influenced by various internal and external factors. The delayed and progressive processes of mitochondrial disease suggest that mitochondrial function deteriorates with age. Under normal physiological state, the body's own defense system can timely remove oxygen free radicals generated in the energy metabolism process. In the aging process of the organism, the oxidation resistance defense system is weakened, and the free radicals in mitochondria cannot be effectively cleared and accumulated, so that oxidative damage of mitochondria, including biomembrane damage, mtDNA damage and the like, is caused. Numerous studies have demonstrated that aging is closely related to the reduction of mitochondrial oxidative phosphorylase activity and the accumulation of mutated mtDNA in tissues at the end of division. The types of mutations associated with age-related events are mainly deletions and associated with oxidative damage. mtDNA mutations in somatic cells increase with age and are positively correlated with the extent of aging. Accumulation of mtDNA mutations can induce a variety of senile diseases.

The heart is the central organ of a vertebrate, and the main function of the heart is to promote blood flow, provide sufficient blood flow to organs and tissues to supply oxygen and various nutrients (such as water, inorganic salts, glucose, proteins, various water-soluble vitamins, etc.), and carry away metabolic end products (such as carbon dioxide, urea, uric acid, etc.), so that cells maintain normal metabolism and function.

Aging of the body induces changes in the structure and function of the atria, ventricles, valves, cardiac muscles, pericardium, cardiac conduction systems and vasculature. With some important alterations in the pathways involved in cell growth and signal transduction, such alterations will be in accelerating or reducing the age-dependent changes in the heart. Vascular aging is composed of endothelial cell dysfunction, vascular remodeling, plaque formation. With age, cellular oxidative stress increases, telomeres shorten, and mitochondrial dysfunction occurs, which are the pathophysiological processes that occur in heart aging.

Exosomes (exosomes) are small vesicles of about 30-150nm diameter secreted by living cells, with a typical lipid bilayer structure; in cell culture supernatants, serum, plasma, saliva, urine, amniotic fluid, and other biological fluids; the exosomes carry important information such as various proteins, lipids, RNA and the like, not only play an important role in substance and information transfer among cells, but also are expected to become early diagnosis markers of various diseases.

Exosomes secreted by cells play an indispensable role in cellular vesicle transport by carrying biomolecules (proteins, miRNAs, DNA and other non-coding RNAs). The function of exosomes depends on the cell type from which it is derived, and can be involved in a number of processes in the immune response of the body, antigen presentation, cell migration, cell differentiation, tumor invasion, etc.

In the existing technical research for anti-aging, there are some related technical documents using exosomes, which mainly focus on three aspects:

1. after a certain type of cells are cultured in vitro, exosomes are separated to generate an anti-aging effect, and the cell sources are mainly embryonic stem cells, umbilical cord/placenta mesenchymal stem cells, bone marrow mesenchymal stem cells, bovine placental cells, nerve stem cells, dental pulp stem cells, adipose-derived stem cells and the like, for example, the invention patent of publication No. CN113041208A (application of embryonic stem cell exosomes in preparation of whitening and anti-aging drugs or cosmetics);

2. the exosomes are used as a transfer carrier to load certain anti-aging substances or modify certain small molecular substances, including active peptides, transdermal peptides, small molecular peptides, NAMPT genes and the like, as in the invention patent of publication No. CN112980801A, a preparation method of NAMPT gene modified mesenchymal stem cell exosomes;

3. The exosomes are combined with a substance to form a composition for use in anti-aging, as in the invention patent of publication number CN111225659a, anti-aging composition comprising extracellular vesicles of lactic acid bacteria origin.

However, the above-mentioned techniques have the disadvantages of complex operation, single application range, poor continuous effect of anti-aging effect, etc.

Disclosure of Invention

The invention aims to provide a biological product with the anti-aging effect of a mammalian heart system, a preparation method thereof and application thereof in preparing a product with the anti-aging effect of the mammalian heart system.

The above object of the present invention can be achieved by the following technical solutions:

a biological product with the anti-aging effect of the heart system of a mammal comprises exosomes extracted from the blood of the mammal.

Blood is the only tissue in contact with all organs, carries a large number of cells of various types related to the organism, and the exosomes which can be produced are also a complex combination, are extracted from blood, and have the most wide application; meanwhile, the blood is used as a liquid substance containing exosomes, the extraction method is relatively simple, the total amount of exosomes which can be extracted by the blood is obviously higher than that of other organs/tissues as the maximum-scale liquid substance in the body, and the blood is more suitable for large-scale, long-term and repeated application.

Further, the above biological product having an anti-aging effect on the heart system of a mammal, wherein the exosomes are exosomes extracted from blood or plasma of a young mammal.

Exosomes extracted directly from blood of young animals are actually a collection of exosomes comprising exosomes secreted by various cell types, closely linked to each other, and co-act. Since young animals are in a high-speed growth and development stage, the number and activity of exosomes in blood are far higher than those of the old animals, and the content (such as miRNA and the like) contained in exosomes is also significantly higher than those of the old animals. Exosomes in the blood of older animals, many of which are progressively inactivated or reduced in association with anti-aging, antioxidant, vitality-stimulating factors, with age, thereby triggering the onset of aging and aging-related diseases. The exosomes contained in the blood of young animals can comprehensively exert antagonism against aging and diseases related to aging through the contained contents, thereby realizing the corresponding effects of resisting aging and prolonging life.

Further, the biological product with the anti-aging effect of the heart system of the mammal, wherein the young mammal animal body refers to an animal body with the growth age of less than or equal to 17% of the average life span of the species; preferably an animal body of growth age equal to 12% -16.5% of the average life span of the species.

Since the total period of the animal ages is different, and the ages are one of the most intuitive standards in the research on the aging mechanism of the animal, the invention also defines the concept of 'young' and 'old' by adopting the age proportion. Since the final object of the present invention is to inhibit aging of human body with biological product having anti-aging effect of mammal heart system, and to produce life-prolonging effect while antagonizing related diseases due to aging, the present invention has been made at the beginning of experiment, i.e. to determine the exosome use effect of human body by animal experiment, and this has also been determined to have correlation between ages of various experimental animals and human body.

Through early query, in order to more accurately express the concepts of "young animals", "old animals" described in the present invention, the applicant has queried the following 2 relevant documents, and determined the age definitions of experimental mice by the document description: that is, "young" means mice within 4 months of the month of age, and "old" means mice with 18 months or more of the month of age.

1. Related document 1:

experimental animal and human age-related research progress, journal of comparative medicine in china, 2019, 29 (11): 116-122, chen, sudan, noble Wen Juan, sun Xiaorong.

In this connection, correlation between the years of a laboratory animal such as a mouse and a human is described, and the years of the laboratory animal are shown in table 1 in fig. 1 as calculated from the human ages.

2. Related document 2:

《THE MOUSE IN BIOMEDICAL RESEARCH,2ND EDITION》。

here, the correspondence between different growth stages of mice and humans is described, and statistics are performed using C57BL/6J as an example, as shown in FIG. 2.

In connection with the above document, the applicant has finally optimized and confirmed, after a number of experiments, that the term "young animal" as used in the present invention refers to an animal having a growth age of 17% or less of the average life of the species, preferably an animal having a growth age of 12% -16.5% of the average life of the species; "aged animal" means an animal having a growth age of 62% or more of the average life of the species, preferably an animal having a growth age of 62% to 95% of the average life of the species.

The above biological product with mammal heart system anti-aging effect, wherein the exosome is extracted from the same mammal as the biological product (or from different mammal, i.e. cross species).

The term "donor animal" as used herein refers to a young animal of unlimited species from which exosomes/mirnas can be isolated/extracted from their in vivo blood/plasma; "recipient animal" refers to an older animal body of an unlimited species that can receive exosomes/mirnas isolated/extracted from blood/plasma in a "donor animal.

In 2007, valadi et al found that exosomes secreted by murine mast cells could be captured by human mast cells and that the mRNA components carried by them could be translated into proteins into the cytosol, not just mRNA, but micrornas transferred by exosomes were also biologically active and could target modulation of mRNA levels in cells after entering target cells. Subsequent studies have found that exosomes are "trans-species" in nature, i.e., exosomes produced in animals such as mice are available to the same or similar cells of humans, thereby effecting trans-species transfer of the content of the exosomes. The characteristic of the exosomes greatly increases the diagnosis and treatment effects of the exosomes on the gene level of various diseases of human bodies, and meanwhile, as the exosomes are of single micro vesicle structures, the exosomes among different species cannot generate corresponding immune response, namely, the trans-species rejection reaction is abandoned, and the source of the gene preparation is greatly expanded.

Based on the above-mentioned research results, in the specific implementation process of the present invention, young animals of various animals can be used as donor animals, old animals of various animals can be used as recipient animals, and especially, the old human population can be used as recipient animals, so as to realize the "rejuvenation" effect consistent with the same species.

Further, the above biological product with the anti-aging effect on the heart system of the mammal, the extraction method of the exosomes comprises the following steps: extraction is carried out by combining ultracentrifugation with an exosome kit.

Further, the above biological product with the anti-aging effect on the heart system of the mammal, the extraction method of the exosomes specifically comprises the following steps:

s1, obtaining animal blood, and separating to obtain plasma;

s2, if the blood plasma obtained in the S1 is not used immediately, storing at-80 ℃;

s3, separating and purifying the plasma in the step S1 or the step S2 by adopting an overspeed differential centrifugation method, and centrifuging to obtain supernatant and precipitate containing crude extract exosomes;

s4, extracting exosomes by adopting an exosome separation kit to obtain an exosome end product;

if the exosome end product obtained in step S4 is not used immediately, it is stored at 4℃and used within 14 days, or at-80 ℃.

In recent years, various commercial exosome extraction kits have appeared on the market, including filtration of impurity components through specially designed filters, separation and purification by Size Exclusion Chromatography (SEC), precipitation of exosomes by compound precipitation, and the like. The kits do not need special equipment, and the extraction efficiency and the purification effect are gradually improved along with the continuous updating of products.

The ultra-separation method (ultra-centrifugation method or differential centrifugation) is the most commonly used exosome purification means, and adopts low-speed centrifugation and high-speed centrifugation to alternatively perform, so that exosome vesicle particles with similar sizes can be separated. The super-separation method is popular because of simple operation, the obtained exosomes are more in quantity, but the process is time-consuming, the recovery rate is unstable, and the purity is questioned; furthermore, repeated centrifugation may also cause damage to the exosomes, thereby reducing their quality.

The method provided in this patent combines exosome kit with ultracentrifugation mode to extract exosome. The method of extraction is combined, the damage of ultracentrifugation to exosomes is overcome, and the recovery stability and the recovery purity are further increased by extracting with the aid of the kit, so that the effect is better than that of extracting by using ultracentrifugation or the kit alone.

Further, the above biological product with the anti-aging effect on the heart system of the mammal has the exosome particle size range of 20-150 nm, and the preferable exosome particle size range of 50-100 nm.

In a biological product with anti-aging effect on mammalian heart system, the exosomes comprised in the range of 20-150 nm are required, including but not limited to 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 nm, preferably in the range of 50-100 nm. Through research on correlation between exosome particle size and transfer efficiency, it is found that exosome particle size lower than 20 nm particle size or higher than 150 nm particle size can seriously affect the transfer efficiency of exosome. Research in the prior art shows that in theory, the smaller the particle size of an exosome is, the faster the propagation speed is, and the growth speed of cells is influenced by the acceleration of the propagation speed, so that the growth speed is accelerated; however, in the technology, not only the transfer function of exosomes is needed, but also the quantity of exosomes and the content of exosomes are required to reach a certain standard, so that the exosomes with the too low particle size cannot reach the standard through detection of the content of exosomes, that is, the exosomes with the too low particle size or the too high particle size cannot achieve the effect required by the technology. The applicant finds through experiments that the exosomes with the particle size ranging from 20 to 150 nanometers can realize corresponding content transfer and reach a certain concentration, so that the 'rejuvenation' effect required by the invention is realized, and particularly the exosomes with the particle size ranging from 50 to 100 nanometers have the highest 'rejuvenation' efficiency and the best 'rejuvenation' effect.

The second invention provides a preparation method of a biological product with the anti-aging effect of the heart system of the mammal, which comprises the following steps:

1) Obtaining animal blood and separating to obtain plasma;

2) If the plasma obtained in 1) is not used immediately, storing at-80 ℃;

3) Separating and purifying the plasma in the step 1) or the step 2) by adopting an overspeed differential centrifugation method, and obtaining supernatant and precipitate containing crude extract exosomes after centrifugation;

4) Extracting exosomes by adopting an exosome separation kit to obtain an exosome end product;

if the exosome end product obtained in step 4) is not used immediately, it is stored at 4℃and used within 14 days or at-80 ℃.

The third invention aims at providing another biological product with the anti-aging effect of the heart system of a mammal, wherein miRNA is miR-144, miR-149 and/or miR-455 (namely, three types of combinations of miR-144+miR-149+miR-455, miR-144 is used singly, miR-149 is used singly, miR-144+miR-149 is used in combination, miR-149+miR-455 is used in combination, miR-144+miR-455 is used in combination), and miR-144+miR-455 is used in combination) is provided; the miRNA is contained in the exosomes described above.

Preferably, the group of mirnas may be mirnas contained in the exosomes and co-administered with the exosomes to act together, or may be directly administered after being extracted alone.

The exosomes contain a variety of different molecules, such as proteins, lipids, DNA, mRNA and miRNA. MicroRNA (miRNA) is a class of endogenous, small RNAs of about 20-24 nucleotides in length, several miRNAs can also regulate the same gene. The expression of a gene can be finely regulated by a combination of several miRNAs. It is speculated that mirnas regulate one third of the genes in humans.

The applicant finds that the three miRNAs can generate certain antagonism on the aging of animals singly or through the way of the content of the exosome.

The fourth invention is to provide the application of the biological product with the anti-aging effect of the mammalian heart system in preparing products with the anti-aging effect of the mammalian heart system.

The biological product with the anti-aging effect of the heart system of the mammal has the action mechanism that the exosomes in the aged animals can be replaced, replaced or activated by a large amount of exosomes extracted from blood of the young animals, so that the basic function is improved from the cellular level, the aging effect is slowed down, and the life-prolonging effect is realized.

Further, for the above-mentioned applications, the administration of the product having an anti-aging effect on the heart system of a mammal includes, but is not limited to: oral, topical, intramuscular, intravenous and/or intravenous instillation.

The administration mode can adopt a plurality of conventional administration modes, not limited to the listed modes, but because the exosome source is blood, the exosome replacement and activation effects are better, mainly intravenous injection and intravenous drip methods, and after a certain amount of exosome of the blood of young animals is intravenous injected, the exosome replacement process in the young animals can be realized at the fastest speed (through the whole body flow of the blood); experiments show that the optimal choice among intravenous injection and intravenous drip modes is the intravenous drip mode, and the intravenous injection has the defect that the short-time massive injection of extracted exosomes can cause the excessive concentration of local exosomes in the body, the unpredictable effect is probably caused, and the total dosage of the intravenous injection is necessarily lower than that of the intravenous drip. The intravenous drip has the advantages that the flow of blood in the vein is real-time, meanwhile, the drip liquid is diluted in the intravenous drip process under the normal condition, so that the problem of overhigh local concentration of an exosome in the body can be effectively prevented, and more exosomes can be injected into the body in a certain period due to the adjustable intravenous drip speed, so that the anti-aging effect of the exosomes is exerted for a longer time. Meanwhile, the local 'rejuvenation' effect on different parts of the organism can be realized by oral administration, blood reentry, direct application on skin and hair through external application of skin and application mode of intramuscular injection on musculoskeletal.

Further, in the above application, when the active ingredient of the product having an anti-aging effect on the heart system of the mammal is exosomes, the administration period is divided into short-term administration and long-term administration; the short-term administration is to administer the product with the anti-aging effect of the heart system of the mammal once every 1-3 days by intravenous injection, 170-190 mug of each dose is continuously administered for 6-8 times; the long-term administration is that the product with the anti-aging effect of the heart system of the mammal is administered by intravenous injection once every 6-8 days, and the dosage is 170-190 mug;

preferably, the short-term administration is to administer the product having an anti-aging effect on the heart system of a mammal once every 1 day by intravenous injection, 180 μg per dose, 7 consecutive administrations; long term administration is to administer the product with anti-aging effect on the mammalian cardiac system intravenously every 7 days at a dose of 180 μg.

The two application period modes designed by the applicant are short-term application and long-term application, the effects of the two application period modes are not very different, and the two application period modes can be correspondingly adjusted according to the reaction of a test body.

Furthermore, in the application, when the effective component of the product with the anti-aging effect of the heart system of the mammal is miRNA, the plasmid with the over-expressed miRNA is firstly constructed, then the anti-aging and life-prolonging product containing the plasmid is applied, the application period is 1-3 times of intravenous injection every 6-8 days, and the application dosage is 4-6mg/kg every time.

Further, the above-mentioned application, said product with anti-aging action of mammal heart system also includes other preparation or method which can be combined with biological product, including but not limited toLimited to the enhancement of NAD in vivo ⁺ Methods of coenzyme levels, addition of nicotinamide riboside NR/nicotinamide mononucleotide NMN formulations, addition of rapamycin formulations.

The biological product (exosomes extracted from blood of young animals or exosome content miRNAs) with the anti-aging effect of the heart system of the mammal provided by the application can be used singly, can be used in combination with other methods and preparations with the functions of aging inhibition or oxidation inhibition, can also be used in combination with other methods for inhibiting the aging or oxidation, realizes complementation and interaction on the preparations or methods, and can realize better effects.

The invention has the characteristics and advantages that:

the biological product with the anti-aging effect of the mammalian heart system, the preparation method and the application thereof in preparing the product with the anti-aging effect of the mammalian heart system, which are provided by the invention, contain exosomes and miRNA extracted from blood, so that the content of the exosomes and the quantity of the exosomes are ensured, and meanwhile, the exosomes in the blood are the combined bodies, so that the biological product can more effectively realize various effects, has simple and wide sources, and is favorable for long-term repeated and massive use.

The exosome sources can be selected from the blood of young animals, exosomes extracted from the blood of young animals are used for replacing, displacing or activating exosomes in the old animals, so that the content of the exosomes and exosomes in the old animals is improved, the effect of stimulating the living activities of the old animals can be realized, the in-vivo mechanism of the organism against diseases caused by aging can be effectively stimulated, and the' returning to the old is realized to a certain extent.

The extraction method of the animal blood exosome in the technology combines the advantages of the kit method and the ultracentrifugation method, overcomes the inherent defects in the two methods by combining the two methods, improves the control and accuracy of the extraction process of the animal blood exosome, and reduces the extraction difficulty.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a comparative table of the ages of experimental animals calculated from the ages of humans described in the prior art.

FIG. 2 is a diagram showing the correspondence between different growth stages of a mouse and human, as shown by way of example in C57 BL/6J.

FIG. 3 is a graph showing the results of preparation and isolation of exosomes.

FIG. 3A shows the results of detection of the mouse exosome marker protein in young/old mice; of these, CD9, CD63, ALIX and TSG101 are exosome marker proteins, ALBUMIN is a plasma marker protein, and the abscissa shows the plasma group (young/old), exosome group (young/old) and supernatant group (young/old), respectively.

FIG. 3B is a nanoSight test of exosome size in young mice, with the abscissa indicating exosome size (nm) and the ordinate indicating the concentration of mExo-Y (particles/ml).

FIG. 3C is a NanoSight test result of exosome particle size of aged mice, with the abscissa indicating exosome particle size (nm) and the ordinate indicating the concentration of mExo-A (particles/ml).

FIG. 3D shows the results of detection of the marker protein of the umbilical cord blood exosomes; of these, CD9, CD63, ALIX and TSG101 are exosome marker proteins, ALBUMIN is a plasma marker protein, and three columns show the plasma group, umbilical cord blood exosome group (UCB-EXO) and supernatant group (EXO-Free), respectively.

FIG. 3E shows the results of transmission electron microscopy of young/aged mouse exosomes, which were mExo-O and mExo-Y.

Figure 4 shows the first part of the data of the change of the whole body organ tissue of the mice after "replacement" of exosomes.

Fig. 4A shows the change in appearance of the back (left panel) and the abdomen (right panel) of the mice after injection of the young mouse exosomes, wherein the young mice, the aged mice injected with PBS blank control, and the aged mice injected with the young mouse exosomes were respectively from left to right.

Fig. 4B is a photograph of a beta-galactosidase (beta-gal) staining of a heart tissue senescence marker of aged mice after injection of the exosomes of the young mice, from left to right, of the young mice, of the aged mice injected with PBS blank, of the aged mice injected with the exosomes of the young mice, respectively.

Fig. 4C is a photograph of BrdU staining of a cardiac tissue proliferation marker of an aged mouse after injection of the aged mouse exosomes, from left to right, of the aged mouse, the aged mouse injected with PBS blank, and the aged mouse injected with the aged mouse exosomes, respectively.

FIG. 4D shows the detection and quantification of β -galactosidase (β -gal), a marker of aging in heart tissue of aged mice after injection of their exosomes, wherein the ordinate is recorded as a comparison of β -galactosidase of aged mice, and the detection values of aged mice, aged mice injected with PBS blank, and aged mice injected with their exosomes are recorded from left to right.

FIG. 4E shows the quantitative results of the staining of the aged mouse heart tissue proliferation marker BrdU after injection of the aged mouse exosomes, wherein the ordinate is recorded as a comparison of the aged mouse BrdU, and the detection values of the aged mouse, the aged mouse injected with PBS blank, and the aged mouse injected with the aged mouse exosomes are recorded from left to right.

Figure 5 shows the second part of the data of the change in tissue of organs throughout the body of the mice after "replacement" of exosomes.

Fig. 5A shows the results of quantitative detection of Reactive Oxygen Species (ROS) in heart tissue of aged and aged mice after injection of their exosomes, wherein the ordinate is recorded as a comparison of reactive oxygen species in aged and aged mice, and the detection values of aged and aged mice injected with PBS blank, and aged mice injected with their exosomes are recorded from left to right, respectively.

FIG. 5B shows the results of detection and quantification of advanced glycation end products (AGEs) of heart tissue of aged mice after injection of their exosomes, wherein the ordinate is recorded as a comparison of AGEs of aged mice, and the detection values of aged mice, PBS blank-injected aged mice, and aged mice injected with their exosomes are recorded from left to right, respectively.

Fig. 5C is a photograph of lipofuscin (lipofuscin) staining of heart tissue of an aged mouse after injection of an exosome of a young mouse, wherein the aged mouse is a young mouse, an aged mouse injected with PBS blank, and an aged mouse injected with an exosome of a young mouse, respectively, from left to right.

FIG. 5D is a western blot image of the heart tissue senescence markers P21 of young and old mice, with the upper row shown as the P21 detection result and the lower row shown as the beta-actin internal control detection result; the left column shows the detection results of young mice, and the right column shows the detection results of old mice.

FIG. 5E is a western blot image of the aged mouse heart tissue senescence marker P21 after injection of the young mouse exosomes, wherein the upper row shows the detection result of P21 and the lower row shows the internal reference result of beta-actin; the left column shows the results of the detection of aged mice injected with PBS (blank control), and the right column shows the results of the detection of aged mice injected with exosomes of young mice.

FIG. 5F shows the result of western blot quantification of heart tissue senescence marker P21 (mRNA level) in young and old mice, with left data being the result of young mice and right data being the result of old mice.

FIG. 5G shows the result of western blot quantification of cardiac tissue senescence marker P21 (mRNA level) after injection of young mouse exosomes, with the left data being the result of detection of aged mice injected with PBS (blank control) and the right data being the result of detection of aged mice after injection of young mouse exosomes.

FIG. 6 shows a third part of a graph of change data of tissues of organs of the whole body of a mouse after "replacement" of exosomes.

Fig. 6A shows changes in heat production of mice after injection of the young mouse exosomes (left: statistical graph, right: line graph), in which data of young mice, PBS (blank control), and aged mice injected with the young mouse exosomes are shown from left to right, respectively.

FIG. 6B shows changes in oxygen consumption of mice after injection of the young mouse exosomes (left: statistical plot, right: line plot), wherein the statistical plot shows data of young mice, PBS (blank control), and aged mice injected with the young mouse exosomes, respectively, from left to right.

Fig. 6C shows changes in respiratory exchange rate of mice after injection of young mouse exosomes (left: statistical plot, right: line plot), wherein the statistical plot shows data of young mice, PBS (blank control), and aged mice injected with young mouse exosomes, respectively, from left to right.

Fig. 6D shows the activity change of the mice after injection of the young mouse exosomes (left: statistical graph, right: line graph), wherein the statistical graph shows the data of the young mice, PBS (blank control), and the aged mice injected with the young mouse exosomes, respectively, from left to right.

FIG. 6E shows changes in carbon dioxide production of mice after injection of the young mouse exosomes (left: statistical graph, right: line graph), in which data of young mice, PBS (blank control), and aged mice injected with the young mouse exosomes are shown from left to right, respectively.

Fig. 7 shows the relevant effect data for mirnas.

FIG. 7A is a Venn diagram showing the overlap of circulating miRNAs, which increase or decrease continuously with aging in human and mouse plasma;

the human body exosomes with the age increase comprise miR-130, miR-23a, miR-221, miR-29a, miR-29c and miR-34a, the mouse body exosomes comprise miR-23b, miR-27b, miR-183a, miR-29c and miR-34a, and the overlapping parts of the two are miR-29a, miR-29c and miR-34a;

the human body exosomes with age reduction are miR-144, miR-149 and miR-455, the mouse body exosomes are miR-7, let-7a, let-7f, miR-144, miR-149 and miR-455, and the overlapping parts of the two are miR-144, miR-149 and miR-455.

FIG. 7B is a photograph of a Western Blot representation: PGC 1-alpha protein levels in cells after transfection of scrRNA, miR-29a/29C/34a mimc (mimics) or miR-144/149/455 mimc are shown, where NE-4C is a neural stem cell-like cell and C2C12 is a mouse myoblast cell.

FIGS. 7C-7H are ATP production rate, mitochondrial complex V activity, relative mtDNA content after transfection (left to right) of scrRNA, miR-29a/29C/34a or miR-144/149/455 mic into cells; wherein, (C and F) show ATP production rates after transfection of scrRNA, miR-29a/29C/34a or miR-144/149/455mimic into the cell; (D and G) show mitochondrial complex V activity following transfection of scrRNA, miR-29a/29c/34a or miR-144/149/455mimic into cells; (E and H) show the relative mtDNA content after transfection of scrRNA, miR-29a/29c/34a or miR-144/149/455mimic into the cell.

Fig. 7I is a western bolt representation: PGC 1-alpha protein levels in hippocampus and muscle of aged mice injected with PBS and young exosomes are shown.

Fig. 7J is a western bolt representation: PGC 1-alpha protein levels in cells (NE-4C is a neural stem cell-like cell, C2C12 is a mouse myoblast) after 24 hours of co-culture with PBS or young exosomes are shown.

Fig. 7K is a western bolt representative picture: shows PGC 1-alpha protein levels in cells (NE-4C is a neural stem cell-like cell, C2C12 is a mouse myoblast) after transfection of scrRNA, young exosomes plus scrRNA, or young exosomes plus antisense oligonucleotides of miR-144, miR-149 and miR-455.

FIGS. 7L-7M are OCR (Oligomycin Oligomycin, FCCP mitochondrial oxidative phosphorylation uncoupler, rotenone-antimycin A) in cells after 24 hours of treatment with scrRNA, young exosomes plus scrRNA or young exosomes plus miR-144/149/455 antisense oligonucleotides.

FIGS. 7N-7S are ATP production rate, mitochondrial complex V activity, relative mtDNA content of cells after 24 hours of treatment with (left to right) scrRNA, young exosomes plus scrRNA or young exosomes plus miR-144/149/455 antisense oligonucleotides; wherein (N and Q) ATP production rate of the cells after 24 hours of treatment with scrRNA, young exosomes plus scrRNA or young exosomes plus miR-144/149/455 antisense oligonucleotides; (O and R) mitochondrial complex V activity of cells after 24 hours of treatment with scrRNA, young exosomes plus scrRNA or young exosomes plus miR-144/149/455 antisense oligonucleotides; (P and S) relative mtDNA content of cells after 24 hours of treatment with scrRNA, young exosomes plus scrRNA or young exosomes plus miR-144/149/455 antisense oligonucleotides.

FIGS. 7T and U are representative pictures of EdU (5-ethynyl-2' -deoxyuridine) (T is NE-4C neural stem cell-like cells and U is C2C12 mouse myoblasts): representative microscopic views of EdU positive cells in cells after 24 hours treatment with scrRNA, young exosomes plus scrRNA, or young exosomes plus miR-144/149/455 antisense oligonucleotides, where EDU is a 5-ethynyl-2 'deoxyuridine staining pattern, DAPI is a 4', 6-diamidino-2-phenylindole staining pattern, and Merge is a co-staining pattern.

Fig. 7V is a western bolt representation: protein levels of senescence marker P21 in cells are shown, wherein the upper row shows the detection result of P21, and the lower row shows the internal detection result of β -actin, from left to right, P21 protein levels in cells (NE-4C is neural stem cell-like cells, C2C12 is mouse myoblasts) after transfection of scrRNA, young exosomes plus scrRNA, or young exosomes plus antisense oligonucleotides of miR-144, miR-149 and miR-455, respectively.

FIG. 8 shows the results of short-term administration of P21western blot in which the detected organ tissue was heart tissue, PBS was injected into aged mice on the left side, and young mouse exosomes were injected into aged mice on the right side.

Fig. 9 shows the result of long-term administration of P21western blot, in which the detection organ tissue was cardiac tissue in sequence, PBS was injected to aged mice on the left side, and young mouse exosomes were injected to aged mice on the right side.

Fig. 10 shows graphs of data of changes in heart tissue of mice after "replacement" of exosomes, from left to right, respectively, data of young mice, data of PBS injection to aged mice (blank), exosomes of young mice injected to aged mice, and the results suggest significant recovery of heart function in aged mice after exosomes of young mice were injected.

FIG. 10A is a representative image of cardiac color Doppler ultrasound after injection of exosomes in young mice;

FIG. 10B shows the change in cardiac Ejection Fraction (EF) after injection into the exosomes of young mice;

FIG. 10C shows the change in Fractional Shortening (FS) of the heart after injection of exosomes in young mice;

FIG. 10D shows the change in left ventricular mass after injection of exosomes in young mice;

FIG. 10E shows the change in left ventricular volume during systole after injection of exosomes in young mice;

fig. 10F shows the change in volume of the left ventricle in diastole following injection of exosomes in young mice.

Fig. 11 shows rejuvenation data (in vivo experiments) after injection of young human exosomes into aged mice.

FIG. 11A is a schematic diagram of an experimental procedure for extracting exosomes sEV from blood in young human bodies of 19-24 years old, injecting the exosomes sEV into old mice of 21 months old, and injecting the exosomes into the old mice 7 times every 2 weeks for performing behavioral tests and metabolic analyses respectively;

FIGS. 11B-D shows the results of a water maze test with PBS on the left side and exosomes on the right side;

Fig. 11B. Latency, wherein the abscissa is days and the ordinate is latency(s);

FIG. 11C shows the number of passes, wherein the ordinate is the number of passes;

FIG. 11D station quadrant dwell time(s);

FIG. 11E shows the results of the associative condition fear test, with the ordinate being the percentage (%) of rest time;

FIG. 11F shows the results of the fatigue treadmill test, with the ordinate being the exhaustion time (min);

FIG. 11G hippocampus and muscle ATP production rates, left hand hippocampus, right hand muscle;

FIG. 11H hippocampal and muscle mitochondrial complex V activity, left hippocampus, right muscle;

FIG. 11I, hippocampus and muscle mitochondrial DNA (mtDNA) content, left side is hippocampus, right side is muscle;

FIG. 11J-M shows the results of tissue electron microscopy with PBS injection in the upper row and exosomes injection in the lower row;

FIG. 11J, hippocampal tissue electron microscopy pictures;

FIG. 11K is a picture of a muscle tissue electron microscope;

FIG. 11L hippocampal mitochondrial statistics;

FIG. 11M, muscle tissue mitochondrial statistics;

fig. 11N-O: muscle tissue Succinate Dehydrogenase (SDH) staining results, n.picture (upper row PBS injected to aged mice, lower row exosomes injected to aged mice), o.statistics results.

Figure 12 shows rejuvenation data after co-culturing young human exosomes with cells in vitro (in vitro experiments).

FIG. 12A is a schematic diagram of an experimental procedure in which exosomes sEV are extracted from blood in young humans aged 19-24 years and injected into neural stem cell-like cells NE-4C and mouse myoblasts C2C12, and after co-culturing for 24 hours, metabolic analysis and senescent phenotype analysis are performed respectively; the method comprises the steps of carrying out a first treatment on the surface of the

FIGS. 12B and 12E. ATP production rates (B. Neural stem cell-like cells NE-4C, E. Mouse myoblasts C2C 12);

FIGS. 12C and 12F mitochondrial Complex V Activity nmol/min/10 ⁴ cell (C. Neural stem cell-like cell NE-4C, F. Mouse myoblast C2C 12);

FIGS. 12D and 12G. Mitochondrial DNA content (D. Neural stem cell-like cells NE-4C, G. Mouse myoblasts C2C 12);

FIG. 12H-K SeaHorse Instrument analysis of neural Stem cell-like cell NE-4C experimental results pmol/min/10 ⁵ cell (h. line graph, i. basal respiration rate, j. Atp-related respiration rate, k. maximum respiration rate);

FIG. 12L-O. analysis of the experimental results pmol/min/10 by a seahorse instrument of mouse myoblasts C2C12 ⁵ cell (l. line graph, m. basal respiration rate, n.atp-related respiration rate, o. maximum respiration rate);

FIG. 12P. Neurostem cell-like cell NE-4CP21 mRNA content;

FIG. 12Q. Detection of proliferation potency (EdU staining, DAPI staining and Merge co-staining) of neural stem cell-like cells NE-4C;

FIG. 12R. Mouse myoblast C2C12P21 mRNA content;

FIG. 12S. Mouse myoblasts C2C12 proliferation potency (EdU staining, DAPI staining and Merge co-staining) was tested.

Fig. 13-17 show graphs comparing the effects of three mirnas alone and any two of them used in combination.

Fig. 13 shows a western bolt representation of miRNA: protein levels of senescence marker P21 in cells are shown, wherein the upper row shows the detection result of P21, the lower row shows the internal detection result of beta-actin, and P21 protein levels in cells transfected with scrRNA, miR-144mimic, scrRNA, miR-149mimic, scrRNA, miR-455mimic (NE-4C is a neural stem cell-like cell, and C2C12 is a mouse myoblast) from left to right, respectively.

FIG. 14 shows the ATP production rate, mitochondrial complex V activity, relative mtDNA content following transfection of NE-4C cells (left to right) with scrRNA (control), combined or alone with miR-144/149/455mimic into the cells.

FIG. 15 shows ATP production rate, mitochondrial complex V activity, relative mtDNA content following transfection of C2C12 cells (left to right) scrRNA (control), combined or independent transfection of miR-144/149/455mimic into cells.

FIG. 16 shows the ATP production rate, mitochondrial complex V activity, relative mtDNA content of NE-4C cells after 24 hours of treatment with (left to right) scrRNA (control 1), young exosomes plus scrRNA (control 2), young exosomes plus miR-144/149/455 antisense oligonucleotides alone or in combination.

FIG. 17 shows the ATP production rate, mitochondrial complex V activity, relative mtDNA content of cells after 24 hours of C2C12 cell treatment with (left to right) scrRNA, young exosomes plus scrRNA, young exosomes, or miR-144/149/455 antisense oligonucleotides alone or in combination.

As is apparent from fig. 14 to 17, the effect of the co-administration of the three exosomes is significantly better than that of either alone or two.

FIGS. 18 to 20 show the results of the immunoreaction detection.

FIG. 18 shows results of biochemical analysis of liver and kidney functions of aged mice after injection of PBS, young mouse exosomes and young human exosomes (data from left to right are respectively PBS-injected aged mice, blood exosomes of aged mice injected with young mice, blood exosomes of young human injected with aged mice). Among them, FIG. 18A, serum glutamic pyruvic transaminase (ALT), FIG. 18B, glutamic oxaloacetic transaminase (AST), FIG. 18C, alkaline phosphatase (ALP), FIG. 18D, bilirubin (TBIL), FIG. 18E, serum Creatinine (CREA) and FIG. 18F, blood Urea Nitrogen (BUN) levels.

From the figure, it can be seen that after the blood exosomes of the young are injected into the aged mice, the biochemical analysis results of the liver and kidney functions of the aged mice are not changed significantly, that is, the immune response which causes liver and kidney injury is not substantially generated by the cross-species injection of the exosomes.

Fig. 19 shows the results of detection of inflammatory cytokine levels in aging mice after PBS injection, young mouse exosomes, and young human exosomes (data from left to right for aged mice injected with PBS, aged mice injected with young mouse blood exosomes, and aged mice injected with young human blood exosomes, respectively).

Wherein, FIG. 19A. IL-6, FIG. 19B. TNF- α levels.

As can be seen from the figure, the detection result of inflammatory cytokine levels in aged mice is not significantly changed after the young human blood exosomes are injected into the aged mice, that is, the immune response causing inflammation onset is not substantially generated by the "cross-species" injection of exosomes.

FIG. 20 shows the results of complement level detection in aged mice after injection of PBS, young mouse exosomes, and young human exosomes (data from left to right for aged mice injected with PBS, aged mice injected with young mouse blood exosomes, and aged mice injected with young human blood exosomes, respectively). Which is a kind of

Serum complement 3 and serum complement 4 levels are included.

As can be seen from the figure, the complement level detection results of aged mice were not significantly changed after the young human blood exosomes were injected into the aged mice, that is, the "cross-species" injection of exosomes did not substantially develop an immune response that would cause complement C3/C4 abnormality.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1:

Exosomes are exosomes extracted from blood or plasma of young mammals.

Young mammalian animal body means an animal body having a growth age of 17% or less of the average life span of the species; preferably an animal body of growth age equal to 12% -16.5% of the average life span of the species.

"young animal" means an animal having a growth age of 17% or less of the average life of the species, preferably an animal having a growth age of 12% to 16.5% of the average life of the species; "aged animal" means an animal having a growth age of 62% or more of the average life of the species, preferably an animal having a growth age of 62% to 95% of the average life of the species.

A biological product with mammal heart system antiaging effect is prepared by extracting exosomes from the same mammal as the biological product (or different mammal, i.e. cross-species administration).

As can be seen from the in vivo and in vitro experiments of fig. 11-12, the donor animal (young human) and the recipient animal (aged mouse) can be of different species, and the donor animal exosomes across the species can also produce corresponding anti-aging effects on the recipient animal.

The exosome extraction method comprises the following steps: extraction is carried out by combining ultracentrifugation with an exosome kit.

The extraction method of exosomes specifically comprises the following steps:

s1, obtaining animal blood, and separating to obtain plasma;

The exosomes in the biological product with the anti-aging effect of the mammalian heart system have a particle size in the range of 20-150 nm, preferably 50-100 nm.

In a biological product with anti-aging effect on mammalian heart system, the exosomes comprised in the range of 20-150 nm are required, including but not limited to 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 nm, preferably in the range of 50-100 nm.

A method for preparing a biological product with the anti-aging effect of a mammalian heart system, wherein the biological product is the biological product with the anti-aging effect of the mammalian heart system, and the preparation method comprises the following steps:

1) Obtaining animal blood and separating to obtain plasma;

2) If the plasma obtained in 1) is not used immediately, storing at-80 ℃;

The other biological product with the anti-aging effect of the heart system of the mammal comprises a group of miRNAs, wherein the miRNAs are miR-144, miR-149 and/or miR-455 (namely, miR-144+miR-149+miR-455 combination, miR-144 single use, miR-149 single use, miR-455 single use, miR-144+miR-149 combination, miR-149+miR-455 combination and miR-144+miR-455 combination); the miRNA is contained in the exosomes described above.

Use of a biologic having an anti-aging effect on the mammalian heart system for the preparation of a product having an anti-aging effect on the mammalian heart system.

The administration of products having an anti-aging effect on the mammalian cardiac system include, but are not limited to: oral, topical, intramuscular, intravenous and/or intravenous instillation.

When the effective component of the product with the anti-aging effect of the heart system of the mammal is exosome, the application period of the product is divided into short-term application and long-term application; the short-term administration is to administer the product with the anti-aging effect of the heart system of the mammal once every 1-3 days by intravenous injection, 170-190 mug of each dose is continuously administered for 6-8 times; the long-term administration is that the product with the anti-aging effect of the heart system of the mammal is administered by intravenous injection once every 6-8 days, and the dosage is 170-190 mug;

When the effective component of the product with the anti-aging effect of the heart system of the mammal is miRNA, the miRNA over-expressed plasmid is firstly constructed, then the anti-aging and life-prolonging product containing the plasmid is applied, the application period is 1-3 times of intravenous injection every 6-8 days, and the application dosage is 4-6mg/kg each time.

Products having anti-aging effects on mammalian cardiac systems include other agents or methods that can be used in combination with biological products, including but not limited to, increasing NAD in vivo ⁺ Methods of coenzyme levels, addition of nicotinamide riboside NR/nicotinamide mononucleotide NMN formulations, addition of rapamycin formulations.

From fig. 1 to fig. 12 provided by the invention, it can be seen that injection of exosomes of young mice and mirnas contained therein can effectively restore the appearance of old mice, delay/antagonize aging of main organs of the whole body, restore mitochondrial function, restore cellular respiratory capacity, and simultaneously, trans-species injection of exosomes can also produce corresponding effects.

Example 2:

1. animal body selection:

1. age selection:

young animals: selecting an animal body having a growth age of 17% or less of the average life span of the species; preferably an animal body having a growth age equal to 12% to 16.5% of the average life span of the species;

the young laboratory mice used in this example were mice within 4 months of the age of a month (approximately equivalent to human age within 13.15 years);

aged animals: selecting an animal body with a growth age of 62% or more of the average life span of the species, preferably an animal body with a growth age of 62% -95% of the average life span of the species;

the aged mice used in the experiment in this example were mice 18 months or more old (approximately 59 years or more old as human age).

The comparison table of the ages of the experimental animals calculated by the ages of the human beings is shown in fig. 1, and the corresponding relations between the different growth stages of the mice and the human beings are shown in fig. 2.

In the prior studies, other relevant records exist, such as the correlation of changes in DNA methylation with aging (Stubbs, T.M., bonder, M.J., stark, AK.et al. Multi-tissue DNA methylation age predictor in mouse. Genome Biol 18,68 (2017), https:// doi.org/10.1186/s 13059-017-1203-5). The study generated the most comprehensive set of matched single base resolution methylation sets so far in mice of multiple tissues and ages. The results allow the study of the correlation between DNA methylation changes and aging. The study first established a mouse epigenetic clock that estimated age based on methylation status of 329 discrete CG sites throughout the mouse genome. This new epigenetic clock behaves like a human epigenetic clock and can be used to evaluate (epigenetic) age in unrelated methylation datasets. Specifically, the mouse clock had an age correlation of 0.839 at all tissues and ages tested, and a Mean Absolute Error (MAE) in the test data of 3.33 weeks. Thus: 329 loci of the mouse epigenetic clock performed better than loci corresponding to the human Horvath clock locus in the mouse genome in predicting age of the mouse sample; the mouse epigenetic clock has prediction errors and requires experiments and data sets to evaluate its accuracy in aged mice.

The above-mentioned DNA methylation study is more accurate in time description of animal body aging, and is fully applicable to the specific limitation of young animals and old animals in the technology of the present invention, and is believed to necessarily give more accurate corresponding stages of body functions, but since each tissue in the animal body is changed at any time, the technology does not need to strictly limit the growth stage, but only uses age or methylation degree as a way of judging whether the exosomes are "young" or "elderly" animal bodies, so the limitation and description of the present invention still adopts the mode of limiting the "young" or "elderly" animal bodies by the ages of the animal bodies.

2. Animal selection:

mice used in this example were purchased from Vetolihua laboratory animal technologies Inc.

2. Blood source, plasma extraction and preservation:

1. blood source:

the blood sources in this embodiment may be selected from human, mouse, umbilical cord blood, and the specific blood taking process is respectively:

1) Blood was taken from a person:

(1) Preparing a test tube: taking test tubes with proper quantity and specification for standby.

(2) Checking the syringe: the disposable injector package is opened, the needle is held by the left hand, the needle is held by the right hand, the needle is tightly connected with the needle cylinder, the inclined surface of the needle is aligned with the graduation of the needle cylinder, and the plug is pulled to check whether blocking and air leakage exist. Finally, the air in the injector is exhausted for standby.

(3) Vein selection: the person to be sampled (18-20 years old) takes a sitting position, and the forearm is horizontally straightened and placed on the table pillow. The puncture site is exposed, and the elbow vein which is easy to fix and obvious to see is selected.

(4) And (3) disinfection: the skin is disinfected from the selected venipuncture position from inside to outside and clockwise by using a 30g/L iodine tincture cotton swab, and after the iodine tincture volatilizes, the iodine trace is wiped off by using a 75% ethanol cotton swab in the same method, and the skin is dried.

(5) Pulse band pressing: the upper end of the blood sampling part is pricked with a tourniquet or tourniquet, and the sampled person holds the fist tightly after repeatedly making the fist several times, so that the vein is full and exposed, and the puncture is convenient.

(6) Puncturing: the needle sterile cap is taken down, the lower end of the venipuncture part is fixed by the thumb of the left hand, the syringe needle cylinder is held by the thumb and the middle finger of the right hand, the needle lower seat is fixed by the index finger, the needle inclined surface and the needle cylinder scale are upwards, the needle and the skin form an angle of 30 degrees along the vein trend to make the needle and the skin form an inclined line to quickly puncture the skin, and then the vein wall is pierced forward at an angle of 5 degrees to enter the vein cavity. After blood return, the needle head is inserted a little in order to avoid the needle head from sliding out when blood is collected; however, deep prick is not necessary to avoid hematoma and the tourniquet is removed immediately.

(7) Drawing blood: the syringe is fixed by the left hand, the inner core of the syringe is slowly drawn to the required blood volume, and then the needle hole is pressed by the sterilized dry cotton ball, so that the syringe is quickly pulled out. The sampled person continues to press the pinhole for several minutes to prevent bleeding.

(8) Bleeding and mixing evenly: the syringe needle is taken down, and blood is slowly injected into the anticoagulation tube along the tube wall, so that hemolysis and foam generation are prevented. The anticoagulants are mixed gently, the shaking test tube is forbidden, and the test tube plug is closed for standby.

2) Mice were bled:

(1) Preparing a proper amount of 1.5Ml centrifuge tube containing 3.5% sodium citrate according to the requirement;

(2) The left thumb and the index finger grasp the skin behind the ears and the neck of the mouse, and the tail is fixed by the small finger;

(3) The middle finger lightly presses the left forelimb of a young mouse (2 months old) on the heart part of the sternum, the ring finger presses on the abdomen, the thumb is twisted, and the blood taking side eye skin is lightly pressed, so that the eyeball congestion is prominent;

(4) Clamping eyeballs by using elbow forceps;

(5) Twisting the thumb and the index finger according to the requirements, so that blood vertically flows into the centrifuge tube from the eyebox at different speeds;

(6) Simultaneously, the heart part of the mouse is lightly pressed by the middle finger of the left hand so as to accelerate the blood pumping speed of the heart;

(7) When the blood is completely discharged, the mice are killed by dislocation, the tube caps of the centrifuge tube are closed, and the tube caps are turned upside down for 8 times at 180 degrees, so that the blood and the anticoagulant are fully mixed.

3) Cord blood taking:

(1) Checking whether the blood collection tube and the injector are damaged or not in the validity period. Before delivery, opening the injector according to a sterile principle for standby;

(2) After the fetus is delivered, the navel is broken conventionally;

(3) Disinfecting the umbilical cord: rapidly wiping from the broken end of the umbilical cord to the broken end of the placenta by using iodophor gauze, sterilizing for 2 times, wherein the sterilization range is 10-15cm from the broken end of the umbilical cord, and removing the blood sheep water embryo and manure; after disinfection, the gauze is used for supporting the broken end of the umbilical cord, and the filling part of the umbilical vein is exposed for puncture;

(4) Umbilical vein puncture blood collection: taking a 30ml sterile injector, removing the needle cap, and taking blood by puncturing umbilical vein at a small angle with the inclined surface of the needle head facing downwards or the side surface of the needle head at a position 3-5cm away from the broken end of the umbilical cord; if the umbilical cord is relatively straight, the needle can move forward by 1-2cm along the blood vessel wall after blood is seen, and then the puncture needle is fixed by hemostatic forceps or hands; after 30ml of cord blood is extracted, the needle head is removed and placed in the sharp instrument box, 30ml of cord blood in the injector is immediately injected into 3 10ml EDTA blood collection tubes of which the tube caps are removed, 3 tube caps are closed, and the 180-degree upper and lower layers are reversed for 8 times, so that the cord blood and the anticoagulant are fully mixed.

2. Plasma extraction:

centrifuging within 6 hours after blood collection, placing the blood collection tube into a centrifuge, centrifuging for 5-10min at 1500g (or 3000 rpm), wherein the blood collection tube can see the blood plasma at the uppermost layer, the blood cells at the bottom layer, and the centrifugation temperature is room temperature or 4 ℃.

3. Plasma preservation:

the upper plasma layer was carefully transferred to a clean Eppendoff tube and stored at-80 ℃ for long term use.

3. Extracting and preserving exosomes:

1. exosome extraction:

(1) Melting the plasma in a water bath at 37 ℃ and diluting with PBS of equal volume;

(2) Centrifuging at 500 Xg at 4deg.C for 5 min, collecting supernatant, centrifuging at 3,000Xg at 4deg.C for 25 min, collecting supernatant, centrifuging at 12,000Xg at 4deg.C for 60 min, collecting supernatant;

(3) Centrifuging the supernatant at 4deg.C for 70 min at 120,000Xg, carefully removing the supernatant for use, and dissolving the precipitate with PBS;

(4) Half of the supernatant was extracted according to the instructions of the total exosome isolation kit (from plasma) (Invitrogen, 4484450, ma, usa) and 0.2 fold supernatant volume of exosome precipitation reagent was added to the supernatant;

(5) Rapidly vortex, shake and mix evenly, incubate for 10 minutes at room temperature;

(6) If the plasma is derived from mice, it is centrifuged at 10,000Xg for 30 minutes at 4 ℃; if the plasma is of human origin, it is centrifuged at 10,000Xg for 10 minutes at room temperature; the supernatant was carefully removed, centrifuged at 10,000Xg for 30 seconds at 4℃and the supernatant carefully removed and the pellet dissolved in PBS.

2. Exosome preservation:

the exosome can be prepared into biological product with mammal heart system antiaging effect, and can be stored at 4deg.C for 2 weeks and-80deg.C for long term.

Figure 3 shows the various relevant data of the extracted exosomes.

4. The biological product application mode is as follows:

the exosomes extracted directly or after storage of the exosomes of young animals can be administered to the "aged animals" by a variety of administration methods, including oral administration, external application to the skin, intramuscular injection, intravenous instillation, and the like. The oral administration mode has relatively poor effect, but the administration mode is relatively convenient; the external application mode of the skin has better effect on skin and hair, but relatively poorer absorption effect; the intramuscular injection administration mode has better activation effect on musculoskeletal but has poorer effect on other parts of the body; intravenous injection and intravenous drip are the optimal choices in all administration modes, and the exosomes or activated substances (miRNA and the like) are activated by blood and transported to various parts of the whole body, so that the comprehensive 'rejuvenation' of the organism can be effectively realized.

Fig. 4-6 show that the external urinary body of the young animal provided by the invention has an anti-aging effect by comparing data before and after injecting the external urinary body of the young animal through detection of organ tissues of the animal body and detection of various functions of the animal body.

Fig. 10 shows comparative data of the verification of the preparation of exosomes with anti-aging effect on mammalian heart system, which is evident on the heart system.

5. Biologic application cycle:

1) Short-term application:

the biological product prepared from the exosome was injected into other mice by intravenous injection 7 times at a dose of 180 μg every other day. The results of the biochemical detection of the tissue are shown in FIG. 8.

2) Long-term application:

the biological product prepared from the exosome was injected into other mice intravenously at a dose of 180 μg once a week, and the results of the biochemical detection of the tissue are shown in fig. 9.

From the data graph results, it can be seen that the corresponding anti-aging effect can be generated both in short-term application and in long-term application, and the application effects of the two are approximate.

Example 3:

another related biologic:

another biological product contains miRNA contained in the exosomes as an active ingredient; the miRNA is miR-144, miR-149 and/or miR-455.

Fig. 7 shows the selection process of miRNA and the effect of the miRNA on resisting aging and prolonging life in exosomes, and as can be seen from the comparison of the data in fig. 7, the miR-144, miR-149 and/or miR-455 contained in exosomes have a strong aging resisting effect, and the main effective components possibly having the aging resisting effect for young animal exosomes are deduced.

Example 4:

the exosome biologicals were prepared in the same manner as in example 2 and used in combination with other methods or formulations:

1. the method comprises the following steps:

NAD ⁺ coenzyme can catalyze various cellular metabolic functions through cellular redox reaction, and is converted into NADH through cellular redox reaction, so that NAD is increased ⁺ The horizontal method can prolong the health life of mice, and the method can be combined with the biological product of the invention.

2. In combination with the formulation NR/NMN:

1) Operation in conjunction with NR/NMN:

based on the NAD ⁺ Coenzyme method, simultaneously based on NAD ⁺ Is not taken up by cells and therefore cannot be directly replenished, usually by adding its precursor and using NAD ⁺ Ways of synthesizing pathways to increase NAD in vivo ⁺ Level of: two of the most common tests are Nicotinamide Riboside (NR) and Nicotinamide Mononucleotide (NMN), and therefore, design experiments were used in conjunction with exosomes, specifically as follows:

A. blood is taken from veins of young mice, exosomes in the blood are extracted by using a currently commercially available exosome extraction kit, and then ultra-high speed centrifugation is performed to obtain exosome biological products;

B. mixing the powdery mouse grains with NR/NMN dissolved in water to prepare pellets, and drying the pellets for 48 hours under a laminar flow hood to obtain an NR/NMN biological product;

C. The biological product prepared from the exosome is injected into other mice by intravenous injection according to a dosage of 180 mug once per week, and NR/NMN biological product is fed to the mice according to a dosage of 400 mg/kg/day; D. through long-time experiments, the biological product can delay the aging process (beta-galactosidase content and P21 protein content) of various main tissues (heart, liver, brain, spleen, lung, kidney, muscle and testis) of an injection subject. This degree of delay was clearly observable compared to the control group with PBS solution as control.

2) NR/NMN combination results:

the applicant compares the results of the data of NR/NMN alone, the data of exosomes alone and the data of the combination of the two, wherein the test data of the exosomes alone is superior to the test data of the NR/NMN alone, and the test data of the combination of the two is obviously superior to the test data of the combination of the two, so that the co-administration of the NR/NMN and the exosomes produces the interrelated promotion effect.

3. In combination with the formulation rapamycin:

1) Operation in combination with rapamycin:

rapamycin is a macrolide compound, first discovered in 1960 as an antifungal drug, and later discovered to regulate cell growth and various cellular processes including autophagy, ribosomal biogenesis, protein synthesis and turnover, and lipid, nucleotide and glucose metabolism in mammalian cells. Administration of rapamycin starting at an age of 9 months or 20 months can extend the median and maximum life span of multiple mouse strains. Therefore, the design experiment is combined with exosomes, and is specifically as follows:

B. dissolving rapamycin in DMSO to 100mg/ml, then further diluting in 5% PEG-400/5% Tween-80 to a final concentration of 1.2mg/ml, and sterile filtering to obtain rapamycin biological product;

C. the biological product prepared from the exosome is injected into other mice by intravenous injection according to a dosage of 180 mug once per week, and the rapamycin biological product is injected into the mice by intraperitoneal injection according to a dosage of 8mg/kg once per day;

D. through long-time experiments, the biological product can delay the aging process (beta-galactosidase content and P21 protein content) of various main tissues (heart, liver, brain, spleen, lung, kidney, muscle and testis) of an injection subject. This degree of delay was clearly observable compared to the control group with PBS solution as control.

2) Rapamycin combination results:

the applicant compares the results of rapamycin alone, exosome alone and exosome alone with the results of rapamycin alone, and the results of rapamycin alone and exosome alone are significantly better than the results of rapamycin alone, so that it can be demonstrated that the co-administration of rapamycin and exosome produces a correlation of promotion.

Example 5:

the preparation method of the exosome biological product is the same as that of the embodiment 2, and the exosome biological product is used for immune reaction, and specifically comprises the following three aspects:

1. effects on complement activation:

complement (C) is a group of enzymatically active, thermolabile globulins found in human and vertebrate serum and tissue fluids, mostly synthesized by liver parenchymal cells, monocytes, macrophages. Normally, complement components of the circulation are present as inactive enzyme precursors, and immune complexes or other factors activate complement. Among them, complement 3 (C3) is the complement component with the highest content in serum, and is mainly synthesized by macrophages and liver, and is cleaved into two fragments of C3a and C3b under the action of C3 convertase, which play an important role in both the classical complement activation pathway and the alternative complement activation pathway. In addition, complement 4 (C4) is an early component of complement classical pathway activation, and C4 is synthesized mainly in hepatocytes and macrophages, playing a role in preventing immune complex precipitation, activating complement, neutralizing virus, and promoting phagocytosis. Therefore, experiments were designed to detect complement expression levels, specifically as follows:

A. Taking blood from veins of young mice and venous blood of young people, extracting exosomes in the blood by using a currently commercially available exosome extraction kit, and then performing ultra-high speed centrifugation to obtain exosome biological products;

B. the biological product prepared from the exosome is injected into other mice by intravenous injection according to the dosage of 180 mug;

C. after tail vein exosomes were injected, mouse serum was extracted, and each group was tested for levels of serum complement 3 (MU 30594, biosvamp), complement 4 (MU 30595, biosvamp) using ELISA kit to verify that complement activation was absent.

The test results are shown in fig. 20.

2. Inflammatory cytokine level detection:

inflammatory cytokines refer to various cytokines involved in inflammatory reactions, and function to induce T-cell activation proliferation, differentiation, and the like. Among the many inflammatory cytokines, TNF- α, IL-6, IL-1β, monocyte chemotactic proteins (IL-8, MCP-1), etc. play a major role. Wherein, TNF-alpha is the earliest and most important inflammatory medium in the inflammatory reaction process, and can activate neutrophils and lymphocytes, increase vascular endothelial cell permeability, regulate other tissue metabolic activities and promote the synthesis and release of other cytokines; IL-6 can induce B cell differentiation and antibody production, and induce T cell activation proliferation and differentiation, and participate in immune response of organism, and is a trigger of inflammatory response. MCP-1 is representative of the β subfamily and chemotactic monocytes. Thus, experiments were designed to detect inflammatory cytokine levels, specifically as follows:

C. after tail vein exosomes were injected, mouse serum was extracted, individual groups of TNF- α (ab 208348, abcam) and IL-6 (ab 100712, abcam) levels were detected using ELISA kit, and monocyte chemotactic protein-1 (MCP-1) and keratinocyte-derived chemokine (KC) levels were detected using cell count bead array mouse Flex kit (BD Biosciences) to verify if inflammatory effects were caused.

The test results are shown in fig. 19.

3. Biochemical analysis of liver and kidney function:

liver function blood biochemical assays are used for the preliminary assessment of liver injury, including the determination of serum glutamate pyruvate transaminase and glutamate oxaloacetate transaminase (ALT and AST), alkaline phosphatase (ALP) and bilirubin (TBIL and DBIL) levels. Serum Creatinine (CREA), blood Urea Nitrogen (BUN) and Uric Acid (UA) are used as indicators of kidney damage and have been used as gold standard biomarkers for assessing acute kidney damage. Biochemical analysis of liver and kidney function is a basic test for diagnosing blood diseases or the presence or absence of inflammatory anemia or coagulation problems in the body. Thus, experiments were designed to detect inflammatory cytokine levels, specifically as follows:

C. after the tail vein is injected with exosomes, the serum of the mice is extracted, and the biochemical indexes of the liver and kidney functions of each group are detected to verify whether the liver and kidney are damaged.

The test results are shown in fig. 18.

As can be seen from the above-described test and test results, the extraction source of exosomes and the administration target of the biological product may be the same mammal or different mammals, and the mutual administration of exosomes between different mammals does not cause an immune response, thereby being safe and reliable.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A biological product having an anti-aging effect on the heart system of a mammal, wherein the effective component of the biological product having an anti-aging effect on the heart system of a mammal comprises exosomes extracted from blood of a mammal.

2. The biological product having an anti-aging effect on the heart system of a mammal according to claim 1, wherein the exosomes are exosomes extracted from blood or plasma of a young mammal.

3. The biologic having an anti-aging effect on the cardiac system of a mammal according to any one of claims 1 to 2, wherein the source of extraction of the exosomes is the same mammal as the subject to which the biologic is administered.

4. A biologic according to any one of claims 1-3, having an anti-ageing effect on the mammalian heart system, wherein the exosomes are extracted by the following method: extraction is carried out by combining ultracentrifugation with an exosome kit.

5. The biological product with anti-aging effect of mammalian heart system according to claim 4, wherein the exosome extraction method specifically comprises:

s1, obtaining animal blood, and separating to obtain plasma;

s5, if the exosome end product obtained in the step S4 is not used immediately, the exosome end product is stored at 4 ℃ and used within 14 days, or is stored at-80 ℃.

6. A biologic having an anti-aging effect on the cardiac system of a mammal according to claims 1-3, wherein the grain size of the exosomes in the biologic is in the range of 20-150 nm, preferably in the range of 50-100 nm.

7. A method for preparing a biologic having an anti-aging effect on the heart system of a mammal, wherein the biologic is a biologic having an anti-aging effect on the heart system of a mammal according to any one of claims 1 to 6, comprising the steps of:

1) Obtaining animal blood and separating to obtain plasma;

2) If the blood plasma obtained in S1 is not used immediately, storing at-80deg.C;

3) Separating and purifying the plasma in the step S1 or the step S2 by adopting an overspeed differential centrifugation method, and obtaining supernatant and precipitate containing crude extract exosomes after centrifugation;

5) If the exosome end product obtained in step S4 is not used immediately, it is stored at 4℃and used within 14 days, or at-80 ℃.

8. A biological product with an anti-aging effect of a mammalian heart system, which is characterized in that the effective components of the biological product with the anti-aging effect of the mammalian heart system comprise a group of miRNAs, wherein the miRNAs are miR-144, miR-149 and/or miR-455; the miRNA is comprised in the exosome of any one of claims 1-7.

9. Use of a biologic according to any one of claims 1-8 having an anti-ageing effect on the mammalian heart system for the preparation of a product having an anti-ageing effect on the mammalian heart system.

10. The use according to claim 9, wherein the means of administration of the product having an anti-ageing effect on the mammalian heart system includes, but is not limited to: oral, topical, intramuscular, intravenous and/or intravenous instillation.

11. The use according to claim 9, wherein when the active ingredient of the product having an anti-aging effect on the heart system of a mammal is exosomes, the administration cycle thereof is divided into short-term administration and long-term administration; the short-term administration is to administer the product with the anti-aging effect of the heart system of the mammal once every 1-3 days by intravenous injection, 170-190 mug of each dose is continuously administered for 6-8 times; the long-term administration is to administer the product with the anti-aging effect of the heart system of the mammal by intravenous injection every 6-8 days, and each dosage is 170-190 mug.

12. The use according to claim 9, wherein when the active ingredient of the product with the anti-aging effect of the heart system of the mammal is miRNA, the plasmid with the miRNA over-expression is constructed, and then the anti-aging and life-prolonging product containing the plasmid is applied, the application period is 1-3 times of intravenous injection every 6-8 days, and the application dosage is 4-6mg/kg every time.

13. The use according to any one of claims 9 to 12, wherein the product having an anti-ageing effect on the mammalian heart system further comprises other agents or methods which may be used in combination with the biologic including, but not limited to, methods of increasing nad+ coenzyme levels in vivo, addition of nicotinamide riboside NR/nicotinamide mononucleotide NMN formulations, addition of rapamycin formulations.