WO2023123215A1

WO2023123215A1 - Uses of extracellular vesicles

Info

Publication number: WO2023123215A1
Application number: PCT/CN2021/143111
Authority: WO
Inventors: 施松涛; 寇晓星; 唐健霞; 傅钰
Original assignee: 医微细胞生物技术(广州)有限公司
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-07-06

Abstract

Provided are the uses of extracellular vesicles. Further specifically provided are the uses of extracellular vesicles in the aspects of adjusting the ovarian function, prolonging the life of mammals or preventing aging, treating or preventing metabolic inflammatory syndromes, etc. In another aspect, also provided is the use of an apoptotic vesicle detection reagent in the preparation of an obesity detection reagent or kit.

Description

Applications of Extracellular Vesicles

technical field

The disclosure belongs to the field of biomedicine, and specifically relates to the application of extracellular vesicles.

Background technique

Female precocious puberty is caused by the premature activation of ovarian follicular development, and the developmental pattern of puberty occurs despite the female's chronological age (before the age of 8 years). Premature ovarian maturation will lead to premature follicle development and excessive discharge of follicles. From the perspective of hormone level, premature ovarian maturation will lead to elevated estrogen. Excessive estrogen will lead to precocious puberty, obesity and excessive ovulation. Complications include uterine fibroids and endometriosis.

Premature ovarian failure (Prematrue ovarian failure, POF) refers to the premature decline of ovarian function in women before the age of 40. The specific manifestations include some symptoms of ovarian function decline, such as menstrual cycle disorder, abnormal menstrual flow, reproductive tract inflammation, and accompanied by a A series of menopausal symptoms, such as loose skin, easy insomnia, emotional irritability, etc., seriously affect the normal life of women.

Chemotherapy has been widely used in the treatment of various malignant tumors and autoimmune diseases, but patients who receive chemotherapy must face some serious side effects. For female patients, the irreversible damage caused by chemotherapy to ovarian tissue is still very worthwhile. Concerns. The main symptoms of premature ovarian failure are: the volume of bilateral ovaries is significantly smaller than normal, the apoptosis of granulosa cells, follicular atresia, and the decrease of estrogen level. Hot flashes, osteoporosis, sexual dysfunction and infertility are all the consequences of chemotherapy-induced premature ovarian failure. At present, hormone replacement therapy is more or less used for chemotherapy-induced POF, but this method cannot actually restore ovarian morphology and function.

In terms of precocious ovarian puberty, although GnRH-a treatment is widely used clinically as the gold standard, it may have side effects such as headache, rash, and gastrointestinal discomfort, and there is still a lack of a drug or preparation that is more suitable for maintaining the homeostasis of the human body. In terms of premature ovarian failure, estrogen supplementation therapy is the most commonly used method. In principle, the reduced estrogen is produced by supplementing ovarian failure, but this method can only improve symptoms, and cannot fundamentally restore ovarian function. Dependence that leads to side effects that worsen symptoms after drug discontinuation. At present, there is no drug or biological agent that can simultaneously treat premature ovarian puberty and premature ovarian failure in clinical treatment.

With the advent of the global aging society, the health of the elderly and the prevention and treatment of senile diseases have become important public health issues facing the international community. my country is the most populous country in the world, and it is also the country with the largest elderly population. "Aging society" is a serious problem facing our country. In order to solve the troubles brought about by the aging society, anti-aging medicine began to rise. Explore mechanisms of aging. Finding new targets and new treatments for anti-aging has become a major issue in life sciences.

Aging is the lifelong deterioration of physiological integrity caused by external stimuli and internal processes. Body aging is the final result of cell aging, and aging is also a manifestation of insufficient stem cells and body cells. To delay aging and maintain homeostasis in the body, continuous tissue renewal and regeneration are required.

Extracellular vesicles (EVs) are nanoscale carriers secreted by cells that contain proteins, nucleic acids and various cytokines. Extracellular vesicles can act on target cells in an endocrine or paracrine manner, and play an important role in the process of intercellular material transfer and information exchange. Studies have found that the information exchange mediated by extracellular vesicles plays an important regulatory role in the physiological or pathological processes of the body, involving immune regulation, tumor growth, angiogenesis, and damage repair. Current research in this field is mainly focused on exosomes. Exosomes are extracellular vesicles with a diameter of about 30-150 nm, which contain components such as RNA, lipids, and proteins. Exosomes are widely involved in various physiological/pathological regulation of the body, and can be used for diagnosis, treatment and prognosis assessment of various diseases. So far, mesenchymal stem cells (MSCs) are considered to be the cells with the strongest ability to produce exosomes. Numerous studies have found that MSCs-derived exosomes can mimic the biological functions of MSCs, and play an important regulatory role in promoting cell growth and differentiation, and repairing tissue defects. Therefore, MSCs-derived exosome-based cell vesicle therapy has achieved remarkable development in recent years. However, there are still many problems in the current exosome-based cell vesicle therapy, mainly in the complex and time-consuming process of exosome extraction and purification, high requirements for equipment and reagents, and low yield of physiological exosomes And so on, these defects limit the clinical transformation and application of exosome therapy.

Contents of the invention

In one aspect, the present disclosure provides the use of inducible extracellular vesicles in the preparation of a preparation for regulating ovarian function.

In some embodiments, said modulating ovarian function comprises:

improve premature ovarian development; or

Improve premature ovarian failure.

In some embodiments, the premature ovarian development is selected from immunologically induced premature ovarian development.

In some embodiments, the premature ovarian failure is selected from immune, drug-induced or age-induced premature ovarian failure.

In some embodiments, the formulation is a formulation for the treatment or prevention of precocious puberty.

In some embodiments, the formulation is a formulation for treating or preventing menstrual irregularities.

In some embodiments, the formulation is a formulation to treat or prevent hyperovulation.

In some embodiments, the formulation is a formulation for treating or preventing premature ovarian failure.

The IEVs in the embodiments of the present disclosure is the abbreviation of induced extracellular vesicles, which may be called induced vesicles or induced extracellular vesicles (Induced extracellular vesicles, IEVs). Inducible extracellular vesicles refer to a type of subcellular product that is intervened or induced to induce apoptosis when precursor cells (such as stem cells) survive normally. Usually this type of subcellular product has a membrane structure, expresses apoptotic markers, and partially contains genetic material DNA. The inventors found that inducible extracellular vesicles are a class of substances that are distinguished from cells and conventional extracellular vesicles (such as exosomes, etc.). In some embodiments, the normal surviving cells are, for example, non-apoptotic cells, non-senescent cells, non-senescent cells with stagnant proliferation, non-resuscitated cells after cryopreservation, non-malignant cells with abnormal proliferation cells or non-damaged cells, etc. In some embodiments, the normally viable cells are obtained from the cells when the cells are 80-100% confluent during cell culture. In some embodiments, the normally viable cells are obtained from logarithmic phase cells. In some embodiments, the normal living cells are obtained from primary culture and subculture cells derived from human or mouse tissue. In some embodiments, the normally viable cells are obtained from established cell lines or strains. In some embodiments, the precursor cells are obtained from earlier cells.

In some embodiments, the agent is selected from agents that reduce ovary size, reduce ovary weight, or reduce ovary to body weight ratio.

In some embodiments, the formulation is a formulation that modulates the physiological cycle of the ovary. In some specific embodiments, when mice are used as the experimental subject, the physiological cycle is the estrous cycle; when humans are used as the experimental subject, the physiological cycle refers to the menstrual cycle.

In some embodiments, the formulation is a formulation that modulates serum estradiol levels. In one aspect, the present disclosure provides a method for regulating estradiol content in the body, comprising the following steps: 1) detecting the serum estradiol content of the subject; 2) comparing the serum estradiol content of the subject with the normal control Comparison of the serum estradiol content of the sample; 3) Based on the deviation of the serum estradiol content of the subject and the normal control sample, the diagnosis of abnormal serum estradiol content; 4) The subject diagnosed with abnormal serum estradiol content , give inducible extracellular vesicle therapy.

In some embodiments, the formulation treats or prevents premature ovarian failure by modulating the Wnt signaling pathway.

In some embodiments, the preparation treats or prevents premature ovarian failure by regulating the expression of β-Catenin-active.

In some embodiments, the preparation is selected from pharmaceutical preparations or health product preparations.

In some embodiments of the present disclosure, it was found that MRL/lpr mice (MRL/lpr mice are a type of Fas receptor mutant mice) exhibit enlarged ovaries, secondary follicular And mature follicles increase, long-term in estrus and other symptoms of premature ovarian maturity. After injection of IEVs, these characteristics of MRL/lpr mice can be restored to normal levels, indicating that IEVs can improve the symptoms of precocious ovary.

In some specific embodiments of the present disclosure, in a premature ovarian failure model established with cyclophosphamide (CP), injection of IEVs can reduce follicular atresia in the POF model and restore the ovarian morphology of mice. At the same time, through the research on the expression of β-Catenin-active in the mice of POF group, it was found that the expression of β-Catenin-active in the Wnt signaling pathway increased in the mice of CP group, and the injection of IEVs could down-regulate the expression of β-Catenin-active. The expression level reached the normal level, indicating that IEVs can improve premature ovarian failure by regulating the expression of β-Catenin-active.

In one aspect, the present disclosure provides a method of modulating ovarian function, administering inducible extracellular vesicle therapy. Inducible extracellular vesicles can be administered to a subject diagnosed with abnormal ovarian function, or a subject suspected of requiring regulation/enhancement of ovarian function. Administration can be systemic or topical.

In one aspect, the present disclosure can administer inducible extracellular vesicle therapy to a subject diagnosed with abnormal ovarian function after the following diagnosis: the diagnosis includes: 1) detecting the subject’s ovarian function; 3) Based on the deviation between the ovarian function of the subject and the normal control sample, abnormal ovarian function is diagnosed; 4) The subject diagnosed with abnormal ovarian function is given an inductive Extracellular vesicle therapy.

In one aspect, the present disclosure provides the use of inducible extracellular vesicles in the preparation of a preparation for extending the lifespan of a mammal or treating or preventing aging.

In some embodiments, the inducible extracellular vesicles prolong the lifespan of mammals or treat or prevent aging by restoring the proliferation and/or differentiation of damaged cells.

In some embodiments, the preparation is a preparation for treating or preventing obesity in the elderly.

In some embodiments, the preparation is a preparation for treating or preventing aging hair loss.

In some embodiments, the formulation is a formulation for treating or preventing splenomegaly.

In some embodiments, the formulation is a formulation for treating or preventing osteoporosis, bone loss or bone aging.

In some embodiments, the inducible extracellular vesicles are used to reduce body weight in an elderly individual.

In some embodiments, the inducible extracellular vesicles are used to reduce hair loss. In one embodiment of the present disclosure, the induced extracellular vesicles derived from bone marrow mesenchymal stem cells can significantly improve hair loss in 24-month-old mice, and have a more significant effect than bone marrow mesenchymal stem cells themselves.

In some embodiments, the inducible extracellular vesicles are used to reduce spleen weight or volume.

In some embodiments, the inducible extracellular vesicles are used to increase bone density.

In some embodiments, the inducible extracellular vesicles are used to increase bone volume fraction.

In one aspect, the present disclosure provides the use of inducible extracellular vesicles in the preparation of a preparation for treating or preventing senile hair loss.

In one aspect, the present disclosure provides a method for treating/controlling senile hair loss by administering inducible extracellular vesicles. Inducible extracellular vesicles can be administered to a subject diagnosed with senile alopecia. Administration can be systemic or topical.

In one aspect, the present disclosure provides the use of inducible extracellular vesicles in the preparation of antiaging, and/or repairing, and/or regenerative preparations for skin and/or skin appendages.

In some embodiments, the skin is epidermis, dermis, or subcutaneous tissue.

In some embodiments, the skin appendage is hair, hair, sweat glands, sebaceous glands, fingernails, or toenails.

In some embodiments, the formulation is a formulation for treating or preventing hair loss, or a formulation for promoting hair regeneration.

In some embodiments, the formulation is a formulation that promotes the repair and/or regeneration of skin wounds or scars.

In one aspect, the present disclosure provides the application of the detection reagent of apoptotic vesicle in the preparation of obesity detection reagent or kit.

Apoptotic extracellular vesicles (apoptotic extracellular vesicles, apopEVs): A large number of apoptotic vesicles are produced during the natural apoptosis of cells in the body. Apoptotic vesicles contain a wide variety of signaling molecules such as proteins, lipids, and nucleic acids, and can also Mediate material transfer and signal exchange between cells.

Normal extracellular vesicles (EVs) refer to nanometer-sized small bodies with membrane structure that are spontaneously secreted by cells during normal culture or under physiological conditions in vivo. Vesicles (Microvesicles, MV) and exosomes (Exosomes) contain a variety of RNA, protein and other signaling molecules.

In some embodiments, the present disclosure makes a breakthrough finding that apoptotic vesicles are reduced in white adipose tissue of obese mice, whether in the induced obesity group, the senile obesity group, the leptin-deficient group or the Fas-deficient obesity Both groups showed a relative reduction in apoptotic vesicles in white adipose tissue. The inventor speculates that the occurrence of obesity may be related to the reduction of apoptotic vesicles in the body. Fortunately, after the inventor injected the mice with the inducible extracellular vesicles of the present disclosure, the number of apoptotic vesicles was restored, and it was found that the inducible vesicles had a significant effect on the induced obesity group, the elderly obesity group, and the leptin-deficient group. Both the Fas-deficient obesity group and the Fas-deficient obesity group had a good therapeutic effect. Therefore, the inducible extracellular vesicles of the present disclosure are particularly suitable for the treatment of obesity.

In some embodiments, the apoptotic vesicle detection reagent is selected from one or more of the reagents for detecting the number of apoptotic vesicles, surface markers of apoptotic vesicles, and the total amount of apoptotic vesicle proteins .

In some embodiments, the detection reagent is selected from flow cytometry reagents and/or kits, Western blot reagents and/or kits, BCA quantitative reagents and/or kits, nanoparticle tracking analysis reagents and/or kits one or more of. In some embodiments, Western blot can detect surface markers of apoptotic vesicles, nanoparticle tracking analysis can detect the concentration of apoptotic vesicles, and BCA method can detect the total protein content of apoptotic vesicles.

In one aspect, the present disclosure provides a method for detecting obesity, the method comprising,

S1. Detecting the level of apoptotic vesicles in the adipose tissue of the subject;

S2. Comparing the level of apoptotic vesicles in the subject with the level of apoptotic vesicles in a normal control sample;

S3. According to the comparison result of step S2, the level of apoptotic vesicles in the subject is lower than that of the normal control sample, indicating that the subject suffers from or is at risk of developing obesity.

Detecting the level of apoptotic vesicles in the adipose tissue of the subject can be performed by means commonly used in the prior art, or by the above-mentioned detection reagents.

In one aspect, the present disclosure provides an obesity detection system, the system comprising:

1) A detection component for apoptotic vesicles;

2) Data processing components;

3) Result output component;

In some embodiments, the detection components of the apoptotic vesicle include flow cytometer, western blot electrophoresis tank and imaging system, high-speed centrifuge, microplate reader, BCA quantitative kit, and nanoparticle tracking analysis kit. one or more.

In some embodiments, the data processing component is configured to a. receive the test data of the test sample and the normal control sample; b. store the test data of the test sample and the normal control sample; c. compare the same type The test data of the sample to be tested and the normal control sample; d. According to the comparison result, responding to the probability or possibility of the subject suffering from obesity.

In some embodiments, the result output component is used to output the probability or likelihood that the subject suffers from obesity.

In some embodiments, the judging criterion of the data processing component is: judging the obese specimen and the normal specimen according to the boundary value.

In some embodiments, the cut-off value of the apoptotic vesicle level in the fat sample is 12-15*10 ⁶ apoptotic vesicles per 0.05 ug of adipose tissue, and the apoptotic vesicle level of the fat sample is less than The threshold value of the apoptotic vesicle level is judged as an obese specimen, and the apoptotic vesicle level of the fat sample is greater than or equal to the threshold value of the apoptotic vesicle level is judged as a normal specimen.

In some embodiments, the sample tested is selected from adipose tissue; in some embodiments, the sample tested is selected from white adipose tissue.

In one aspect, the present disclosure provides a method for treating/managing obesity by administering inducible extracellular vesicles for treating/managing. Inducible extracellular vesicles can be administered to subjects diagnosed with obesity, or suspected of requiring modulation of metabolic function. Administration can be systemic or topical.

In some embodiments, the administration of inducible extracellular vesicles can be determined by the detection of apoptotic vesicles. When the level of apoptotic vesicles in the adipose sample of the subject is less than the threshold value of the level of apoptotic vesicles, it is judged as an obese sample, and the inducible extracellular vesicles are administered.

In some embodiments, the administration of inducible extracellular vesicles can be determined by detecting the number of apoptotic vesicles. When the number of apoptotic vesicles in the adipose specimen of the subject is ≤12-15*10 ⁶ /0.05ug adipose tissue, the inducible extracellular vesicles are administered.

In one aspect, the present disclosure provides a method for treating/controlling obesity, which comprises the following steps: 1) detecting the number of apoptotic vesicles in the adipose tissue of the subject; The number is compared with the number of apoptotic vesicles in the adipose tissue of the normal control sample; 3) based on the deviation of the number of apoptotic vesicles in the adipose tissue of the test object and the normal control sample, obesity is diagnosed; 4) the obesity is diagnosed Subjects were treated with inducible extracellular vesicles. Administration can be systemic or topical.

In one aspect, the present disclosure provides the use of inducible extracellular vesicles in the preparation of a preparation for treating or preventing metabolic inflammatory syndrome. In one aspect, the present application provides a method for treating metabolic inflammatory syndrome, comprising administering inducible extracellular vesicles. Inducible extracellular vesicles can then be administered to a subject diagnosed with metabolic inflammatory syndrome. Administration can be systemic or topical.

In some embodiments, the metabolic inflammatory syndrome includes one or more of obesity, atherosclerosis, and diabetes.

In some embodiments, the obesity includes at least one of induced obesity, senile obesity, leptin-deficient obesity, and Fas-deficient obesity.

In some embodiments, the leptin-deficient obesity is selected from childhood obesity.

In some embodiments, the diabetes is selected from type 2 diabetes.

In some embodiments, the formulation is a weight loss formulation.

In some embodiments, the agent is an agent that inhibits one or more of the p-Akt, p-Erk, p-P50 pathways.

In some embodiments, the preparation is a preparation that inhibits adipogenic differentiation of adipose-derived mesenchymal stem cells.

In some embodiments, the preparation is a preparation for treating obesity or atherosclerosis by inhibiting triglyceride synthesis.

In some embodiments, the preparation is a preparation for promoting the secretion of high cholesterol to treat atherosclerosis.

In one aspect, the present disclosure provides an obesity treatment system comprising:

1) A detection component for apoptotic vesicles;

2) Data processing components;

3) Result output component;

4) Administering the inducible extracellular vesicles to a subject diagnosed as obese.

In some embodiments, the inducible extracellular vesicles are vesicles produced by induction of apoptosis by external factors when stem cells are in normal survival.

In some embodiments, the inducible extracellular vesicles are produced by inducing apoptosis of stem cells, and the induction method includes adding Staurosporum, ultraviolet irradiation, starvation method, or heat stress method.

In some embodiments, the stem cells are mesenchymal stem cells.

In some embodiments, the mesenchymal stem cells are selected from blood mesenchymal stem cells, bone marrow mesenchymal stem cells, urine mesenchymal stem cells, oral cavity mesenchymal stem cells, fat mesenchymal stem cells, placental mesenchymal stem cells One or more of umbilical cord mesenchymal stem cells, periosteal mesenchymal stem cells, and skin mesenchymal stem cells.

In some embodiments, the mesenchymal stem cells are selected from blood mesenchymal stem cells, bone marrow mesenchymal stem cells, fat mesenchymal stem cells, umbilical cord mesenchymal stem cells, oral cavity mesenchymal stem cells, skin mesenchymal stem cells one or more of.

In some embodiments, the stem cells are selected from one or both of blood stem cells and bone marrow mesenchymal stem cells.

In some embodiments, the inducible extracellular vesicles are produced by adding staurosporine to induce apoptosis of mesenchymal stem cells.

In some embodiments, the concentration of the staurosporine is greater than or equal to 1 nM; preferably, 1-15000 nM; preferably, 200-10000 nM; preferably, 250-1000 nM; preferably, 500 nM -1000nM. In addition, the concentration of staurosporine can be 280-9000nM; 230-8500nM; 500-1000nM; 500-900nM; 500-800nM.

In some embodiments, the inducible extracellular vesicles have a diameter of 0.45 μm or less. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.45 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.1-0.45 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.1-0.35 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.15-0.35 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.15-0.3 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.15-0.2 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.4 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.38 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.35 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.32 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.3 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.25 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.22 μm. In some embodiments, the diameter of the inducible extracellular vesicle is 0.15-0.22 μm. In some embodiments, the diameter of the inducible extracellular vesicles may also be 0.15-0.45 μm, or 0.2-0.3 μm.

In some embodiments, the inducible extracellular vesicle has the marker Syntaxin 4. In some embodiments, the inducible extracellular vesicles highly express the marker Syntaxin 4. In some embodiments, the expression of the marker Syntaxin 4 of the inducible extracellular vesicles is higher than that of MSCs or exosomes. In some embodiments, the expression level of the marker Syntaxin 4 is 3-6 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the expression level of the marker Syntaxin 4 is 3.5-5 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the expression level of the marker Syntaxin 4 is 4.45 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the markers also include one or more of Annexin V, Flotillin-1, Cadherin 11 and Integrin alpha 5. In some embodiments, the marker is a combination of Syntaxin 4, Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5. In some embodiments, the inducible extracellular vesicles highly express markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5. In some embodiments, the expression levels of the inducible extracellular vesicle markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 are higher than those of MSC or exosomes. In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 in the inducible extracellular vesicles are relative to the markers in the exosomes derived from mesenchymal stem cells The expression levels were 1-2 times, 2-3 times, 1-3 times and 3-4 times, respectively. In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 in the inducible extracellular vesicles relative to the markers in exosomes derived from mesenchymal stem cells are respectively 1.5-2 times, 2.5-3 times, 1.5-2.5 times and 3.5-4 times. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 in the inducible extracellular vesicles are expressed relative to markers in exosomes derived from mesenchymal stem cells They are 1.76 times, 2.81 times, 2.41 times and 3.68 times respectively. The inducible extracellular vesicles described in this disclosure are essentially different from exosomes. For example, compared with exosomes, the inducible extracellular vesicles IEVs described in this disclosure highly express Syntaxin 4, and its Annexin V, The expression levels of Flotillin-1, Cadherin 11, and Integrin alpha 5 were significantly higher than those in exosomes (see Example 3). In addition to differences in the expression of markers, the inducible extracellular vesicle IEVs also exhibit characteristics different from those of stem cells and other extracellular vesicles such as exosomes in terms of function or therapeutic effect. In some embodiments, the inducible extracellular vesicles also express CD29, CD44, CD73, CD166; and do not express CD34, CD45. In some embodiments, the inducible extracellular vesicle also expresses one or more of CD9, CD63, CD81, and C1q.

In some embodiments, the drug is an injection, an oral preparation or an external preparation.

In some embodiments, the drug is an injection.

In some embodiments, the drug is an intravenous, intramuscular, subcutaneous, or intrathecal injection.

In some embodiments, the medicament further includes a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical carrier includes one or more of diluents, excipients, fillers, binders, disintegrants, surfactants and lubricants.

In some embodiments, a method for preparing the inducible extracellular vesicles is also provided, comprising the following steps:

1) Cultivate mesenchymal stem cells in vitro, when the cell confluence is 80%-90%, wash with PBS 2-5 times;

2) adding the mesenchymal stem cells prepared in step 1) to a serum-free medium containing 500-1000 nM staurosporine, incubating at 37° C. for 16-24 hours, and collecting the cell supernatant;

3) Centrifuge the cell supernatant collected in step 2) at 500-15000g for 5-30 minutes at 4°C, and collect the supernatant;

4) Centrifuge the cell supernatant collected in step 3) at 1500-2500g for 5-30 minutes at 4°C, and collect the supernatant;

5) Centrifuge the cell supernatant collected in step 4) at 10,000-30,000g for 15-60 minutes at 4°C, and the resulting precipitate is extracellular vesicles;

Preferably, a washing step of the induced extracellular vesicles is also included;

Preferably, the cleaning step is as follows: 6) resuspend the induced extracellular vesicles prepared in step 5) with PBS, and centrifuge at 10000-30000g for 15-60 minutes at 4°C, and the obtained precipitate is induced cells outer vesicle.

In the present disclosure, the "mesenchymal stem cell" refers to a kind of pluripotent stem cell, which has all the common characteristics of stem cells, namely self-renewal and multilineage differentiation ability. The mesenchymal stem cells can be derived from bone marrow, fat, blood (for example: foreign blood), synovium, bone, muscle, lung, liver, pancreas, oral cavity, craniomaxillofacial (for example: deciduous teeth, dental pulp, dental Peripheral membrane, gingiva, apical tooth papilla, etc.) and amniotic fluid, umbilical cord, that is, the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells, fat mesenchymal stem cells, synovial mesenchymal stem cells, bone mesenchymal stem cells Stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, pancreas mesenchymal stem cells, amniotic fluid mesenchymal stem cells, umbilical cord mesenchymal stem cells, etc.

In the present disclosure, the "mammal" is selected from mice, rats, dogs, cats, rabbits, monkeys or humans.

In this disclosure, "prevention" means that when used for a disease or condition, the drug reduces the frequency or delays the onset of symptoms of the medical condition in a subject compared to a subject not administered the drug .

In the present disclosure, the "treatment" means to relieve, alleviate or improve the symptoms of diseases or disorders, to improve the underlying symptoms caused by metabolism, to inhibit diseases or symptoms, such as to prevent the development of diseases or disorders, to relieve diseases or disorders, to cause Regression of a disease or disorder, alleviation of a condition caused by a disease or disorder, or arrest of symptoms of a disease or disorder.

"Detection" in the present disclosure is the same as "diagnosis", and besides the early diagnosis of the disease, it also includes the diagnosis of the middle and late stages of the disease, and also includes disease screening, risk assessment, prognosis, disease identification, diagnosis of disease stage and therapeutic effect. Target selection.

Herein, early diagnosis refers to the possibility of finding the disease before metastasis, preferably before morphological changes of tissues or cells can be observed.

In this disclosure, "sample" is the same as "specimen".

Description of drawings

Fig. 1 is the diagram of flow cytometry in embodiment 1.

Figure 2 is a technical roadmap for preparing IEVs in Example 2.

Figure 3 is the statistical result of the number of IEVs produced by MSCs (106 MSCs) analyzed by flow cytometry.

Figure 4A-Figure 4D is the diameter detection of IEVs particles: Figure 4A is the scattered light intensity of IEVs analyzed by the standardized small particle microspheres produced by Bangs Laboratories, showing the particle diameter distribution of IEVs; Figure 4B is the observation by transmission electron microscope (TEM) IEVs, showing the particle diameter distribution of IEVs; Figure 4C is nanoparticle tracking analysis (NTA), showing the particle diameter distribution of IEVs; Figure 4D is the particle size detection of IEVs at the single vesicle level by nano flow detection technology, showing the particles of IEVs diameter distribution.

Figure 5A-Figure 5K are the analysis results of surface membrane proteins of IEVs by flow cytometry.

Figure 6A-Figure 6D is the content analysis of IEVs: Figure 6A is the quantitative analysis results of MSCs, MSCs-Exosomes, MSCs-IEVs proteomics by DIA quantitative technology; Figure 6B is the heat map drawn for screening the specifically highly expressed proteins of IEVs ; Figure 6C is the result of GO enrichment analysis of differential proteins for IEVs expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 molecules; Figure 6D is western blot verification of MSCs, MSCs-Exosomes, MSCs-IEVs expressing Annexin V, results of Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4.

Figure 7 shows that the ovary weight of MRL/lpr mice from 12 weeks to 20 weeks was significantly higher than that of C57BL/6 wild-type mice, and the ratio of ovary to body weight was significantly higher than that of C57BL/6 wild-type mice, all after injection of IEVs Significantly lowered.

Figure 8 shows that the ovary morphology of LPR mice at 20 weeks was significantly larger than that of C57BL/6 wild-type mice. After injection of IEVs, the ovary morphology became smaller, but still larger than that of C57BL/6 wild-type mice.

Figure 9 shows the changes of ovarian follicles at all levels after injection of IEVs in C57BL/6 wild-type mice, MRL/lpr mice, and MRL/lpr mice from 12 weeks to 20 weeks. The red arrow points out the case of follicular atresia.

Figure 10 shows the statistical data of the number of follicles at all levels in the ovaries of C57BL/6 wild-type mice, MRL/lpr mice, and MRL/lpr mice injected with IEVs from 12 weeks to 20 weeks.

Fig. 11 shows the change of the estrous cycle of C57BL/6 wild-type mice, MRL/lpr mice, and MRL/lpr mice injected with IEVs in 20 weeks; in the figure: preestrus (Proestrus, P), estrus (Estrus, E) , late estrus (Metestrus, M), interestrus (Diestrus, D).

Figure 12 shows the change of estradiol in serum after C57BL/6 wild-type mice, MRL/lpr mice, and MRL/lpr mice were injected with IEVs from 12 weeks to 20 weeks.

Figure 13 shows that PKH26 and AIE were used to mark IEVs, and PKH26-IEVs and AIE-IEVs could reach ovarian stromal cells and follicular theca cells of MRL/lpr mice 1 day after injection.

Figure 14 shows the results of Western blot analysis of β-Catenin/β-Catenin-active by extracting ovarian proteins from 20W MRL/lpr mice and C57BL/6 mice, comparing the treatment group with or without IEVs injection.

In Fig. 15, A: Ovary section of wild-type mouse, all levels of follicles and corpus luteum can be seen; B: After 100 mg/kg CP injection for 7 days, the granulosa cell layer in the primordial follicle and primary follicle of the mouse ovary disappeared, and the follicle Atresia (indicated by the red arrow), the number of mature follicles decreased; C: 7 days after the injection of IEVs into the mice in the CP group, the number of ovarian atresia follicles decreased (indicated by the red arrow), but no mature follicles were seen. D: Enlarged part of panel A, showing mature follicles of normal ovary; E: Enlarged part of panel B, showing a large number of atretic follicles formed by primitive follicles and primary follicles (indicated by red arrows). F: Partial enlargement of Figure C, it can be seen that the number of atresia follicles in the CP+IEVs group was significantly lower than that in the CP group (indicated by the red arrow).

Figure 16 shows the changes of estradiol in serum of different groups.

Figure 17 shows that using PKH26 to label IEVs, PKH26-IEVs can reach ovarian stromal cells and follicular theca cells of POF mice 1 day after injection.

In Fig. 18, A/B/C: in wild-type mouse ovary, mesenchymal cells (indicated by red arrows) and corpus luteum granulosa cells (indicated by white arrows) have low expression of β-Catenin-active; D/E/F: In the ovaries of mice in the CP group, the expression of β-Catenin-active in the mesenchymal cells (indicated by the red arrow) and corpus luteum granulosa cells (indicated by the white arrow) increased; In the CP group, the expression of β-Catenin-active in the interstitial cells (indicated by the red arrow) and corpus luteum granulosa cells (indicated by the white arrow) decreased, which was consistent with the wild type.

Figure 19 is the Kaplan-Meier survival curve showing that injection of IEVs can significantly prolong the lifespan of mice.

Figure 20 shows that multiple injections of IEVs derived from BMMSCs significantly reduced the body weight and hair loss of aged mice.

Figure 21 shows that multiple injections of IEVs derived from BMMSCs significantly reduced the spleen volume and weight of aged mice.

Fig. 22A is a microCT image of different treatment groups with multiple injections of IEVs derived from BMMSCs.

Figure 22B is a statistical analysis showing that multiple injections of MSCs-derived IEVs significantly increased bone mineral density and bone volume fraction in aged mice.

Figure 23A is the statistical analysis results of CFU-F and BrdU stained with toluidine blue and BrdU stained with different treatments.

Figure 23B shows the ability of BMMSCs under different treatments to form mineralized nodules by Alizarin Red staining, and oil red O staining shows the number of adipocytes in MSCs.

Figure 24A is a schematic diagram of the dynamic metabolism of IEV on the skin surface in Example 20.

Figure 24B shows that IEVs migrate gradually from the subcutaneous tissue to the dermis and epidermis over time.

FIG. 24C shows that PKH26-IEV was found in the hair follicles of the plucked hairs from the mice on day 7.

Fig. 25A shows the distribution of IEV in the whole body of mice on

day

1, 3 and 7 detected by in vivo imaging technology.

Figure 25B shows the distribution of IEV in various organs of mice detected by in vivo imaging technology.

Figure 25C is the comparison of the distribution of IEV in various organs of the mice on the 7th day with that of the control group.

Figure 25D is a schematic diagram of the dynamic metabolism of IEV in the colon on

days

1, 3, and 7; wherein M represents the muscularis mucosa, and V represents the villi layer.

Figure 25E shows a schematic diagram of IEV metabolism in mouse nails and incisors at day 3.

Fig. 26A is a schematic diagram of back hair regeneration of mice treated differently in Example 21 on day 0, day 10, and day 14.

Fig. 26B is a schematic diagram of the statistical analysis of the area of hair regeneration on the back of the mice treated with different treatments in Example 21 on day 0, day 10, and day 14.

Figure 27 shows the promotion effect of IEV and MSC treatment on wound healing in Example 22.

28A-28F are the effects of IEVs injection on obesity in the experiment of using high-fat diet to induce obesity in mice.

Figures 29A-29D show the effect of IEVs on obesity in aging mice.

Figures 30A-30D, 31A-31C show the effect of IEVs on obesity in OB mice.

Figures 32A-32D show the effect of IEVs on obesity in Lpr mice.

Figure 33A-Figure 33F are in vitro experiments to detect the effect of IEVs on the synthesis of triglyceride (triglyceride, TG) in adipocytes: Figure 33A and 33B show that after IEVs treatment, TG in adipocytes and in the supernatant were significantly reduced; Figure 33C , 33D show that lipid droplets in adipocytes after IEVs treatment are significantly reduced; Figure 33E is Western blot detection, IEVs treatment enhances the p-Akt and p-Erk pathways that inhibit lipogenesis, and inhibits the p-Akt and p-Erk pathways that promote lipogenesis -P50 pathway; Figure 33F shows that after IEVs treatment, the expressions of adipogenic-related proteins LPL and PPARγ were significantly reduced.

Figure 34 shows the addition of PKH-26-labeled IEVs (red fluorescence) into adipocytes. As time goes by, IEVs enter adipocytes and increase in size while lipid droplets become smaller.

Figure 35A shows the expression level of the brown fat induction marker protein UCP1 detected by in vitro experiments, and the Western blot results showed that compared with the untreated group, the protein content of UCP1 was significantly increased after IEVs treatment.

Figure 35B shows the expression level of UCP1 detected by in vivo experiments. Compared with the untreated group, the protein expression level of UCP1 in brown adipose tissue was significantly up-regulated after intravenous injection of IEVs in obese mice fed with high-fat diet.

Figure 36 shows that after treatment with IEVs and exosomes in Comparative Example 2, there were significantly fewer lipid droplets and smaller adipocytes (Figure 36A); triglycerides in adipocytes (Figure 36B) and in the supernatant were significantly reduced (Figure 36C).

Figure 37 shows that compared with exsosomes from the same source, the whitening tendency of brown adipose tissue in the IEVs treatment group is reduced, and the iWAT cells are smaller. The Western blot results of brown adipose tissue also show that the exosome group does not have the effect of inducing brown adipose tissue.

Figure 38 shows that the area and severity of aortic arch atherosclerotic plaques were significantly relieved after IEVs treatment.

Figure 39 shows the effects of IEVs on various biochemical indicators in mice with atherosclerosis.

Fig. 40 is a flow chart of experimental processing of IEVs for treating diabetes.

Figures 41A-41C are the treatment of diabetes with IEVs: high-dose IEVs has the best effect on controlling fasting blood sugar (Figure 41A), the best effect on improving glucose tolerance (Figure 41B), and high-dose IEVs can significantly reduce fasting blood sugar in type 2 diabetic mice (41C ).

Figure 42 is IEVs treatment of Sjögren's syndrome: A.IEVs treatment of Sjögren's syndrome (Sjögren's syndrome) salivary flow rate; B.IEVs treatment of Sjögren's syndrome submandibular gland HE staining results; C. Treatment of Sjögren's syndrome on B Effects on cells.

Figure 43 is the in vivo procoagulant effect of IEVs in hemophilia A mice.

Figure 44A-Figure 44D is the change of various coagulation factor levels after injecting IEVs to the hemophilia A mice: Figure 44A is the change of blood coagulation factor VIII; Figure 44B is the change of vWF factor; Figure 44C is the tissue factor ( TF) changes; Figure 44D shows the changes in prothrombin.

Figure 45A-Figure 45B shows the effects of blocking IEVs with PS and TF on the therapeutic effect of IEVs in vivo in the hemophilia A mouse model.

Figure 46 is a comparison of the therapeutic effects of IEVs and Exosomes derived from the same MSCs on hemophilia A mice.

Note: In the accompanying drawings, WT is wild-type mice; HA group is hemophilia A mouse model; HA+IEVs is hemophilia A mouse model treated with IEVs; HA+PS-IEVs is hemophilia A mouse model was given PS-negative IEVs; HA+TF-IEVs was a hemophilia A mouse model given TF-negative IEVs; HA+Exosomes was a hemophilia A mouse model given Exosomes.

Detailed ways

The technical solution of the present disclosure is further described through specific examples below, and the specific examples do not represent limitations on the protection scope of the present disclosure. Some non-essential modifications and adjustments made by others based on the concepts of the present disclosure still belong to the protection scope of the present disclosure.

STS in the present disclosure is staurosporine.

"Comprising" or "comprising" is intended to mean that compositions (eg, media) and methods include the listed elements, but do not exclude other elements. "Consisting essentially of" when used to define compositions and methods means excluding other elements of any significance to the combination for the stated purpose. Accordingly, a composition consisting essentially of the elements defined herein does not exclude other materials or steps which do not materially affect the basic and novel characteristics of the claimed disclosure. "Consisting of" means trace elements and substantial method steps excluding other constituents. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

As used herein, the term "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and/or" means that any one of the listed items can be used alone, or any combination of two or more listed items can be used . For example, if a composition, combination, configuration, etc. is described as comprising (or comprising) components A, B, C and/or D, the composition may comprise A alone; B alone; C alone; D alone ; Combination of A and B; Combination of A and C; Combination of A and D; Combination of B and C; Combination of B and D; Combination of C and D; Combination of A, B and C A combination; a combination comprising A, B, and D; a combination comprising A, C, and D; a combination comprising B, C, and D; or a combination of A, B, C, and D.

The isolation and culture of embodiment 1 BMMSCs

According to the guidance of the Animal Ethics Committee, the mice were killed with excessive CO ₂ , and under aseptic conditions, the tibia and femur were removed, and the muscles and connective tissues attached to them were peeled off, and the metaphysis was further separated to expose the bone marrow cavity. Extract the PBS with a volume fraction of 10% fetal bovine serum into a sterile syringe to repeatedly rinse the bone marrow cavity, filter through a cell strainer with a pore size of 70 μm, centrifuge at 500g for 5min, remove the supernatant and collect the cell pellet at the bottom, resuspend in PBS, and centrifuge again at 500g After 5 min, the final cell pellet was collected. Then, the cells were sorted by flow cytometry, and bone marrow mesenchymal stem cells (BMMSCs) were sorted out by using CD34- and CD90+ as sorting criteria. Finally, the cells were resuspended in Dex(-) medium and inoculated on a 10cm diameter cell culture dish, and cultured at 37°C and 5% CO ₂ . After 24 hours, the non-adherent cells in the supernatant were sucked off, washed with PBS, and then added with Dex(-) medium to continue culturing. One week later, an equal amount of Dex(+) culture medium was added, and dense colonies of primary BMMSCs could be seen after another week. Trypsin was used to incubate and digest BMMSCs at 37°C, and then passage and expand. After that, the Dex (+) culture medium was changed every 3 days, and passage was performed after overgrowing. Subsequent experiments were performed using P2 generation BMMSCs.

Wherein, the composition of Dex (-) culture solution is as shown in Table 1, and the composition of Dex (+) culture solution is as shown in Table 2:

Table 1 Components of Dex(-) culture medium

Table 2 Dex (+) culture solution formula table

The purity of isolated BMMSCs was assessed by flow cytometry analysis of surface markers. For the identification of surface markers, after trypsinization to collect P2 BMMSCs, wash them once with PBS, resuspend the cells at a density of 5×10 ⁵ /mL in PBS containing 3% FBS, add 1 μL of PE fluorescence-conjugated CD29, CD44, CD90, CD45 and CD34 antibodies were not added to the blank group. Incubate at 4°C in the dark for 30 minutes, wash with PBS twice, and test on the machine. The test results are shown in Figure 1.

The preparation of the IEV of embodiment 2 BMMSC source

The MSCs (MSCs derived from bone marrow) cultured to the second generation in Example 1 were continued to be cultured with the medium (Dex(+) culture fluid) in Example 1 until the cells were confluent 80%-90%, washed with PBS for 2 Add the serum-free medium containing 500nM STS (the medium is the addition of 500nM STS to the medium in Example 1), incubate at 37°C for 16-24h, collect the cell supernatant, and centrifuge at 800g at 4°C for 10 Minutes, the supernatant was collected and centrifuged at 2000g at 4°C for 10 minutes, and the supernatant was collected again and centrifuged at 16000g at 4°C for 30 minutes, and the obtained precipitate was IEV. Resuspend the pellet in 500 μl PBS, and centrifuge again at 16,000 g for 30 minutes at 4°C to obtain the washed IEV.

The preparation route is shown in Figure 2.

Comparative Example 1 Isolation and extraction of exosomes derived from the same MSC

The MSCs (bone marrow-derived MSCs, BMMSCs) cultured to the second generation in Example 1 were continued to be cultured with the medium in Example 1 until the cells were confluent 80%-90%, rinsed twice with PBS, and added serum-free Medium, incubated at 37°C for 48h, and the cell supernatant was collected for the isolation and extraction of exosomes.

The extraction steps include: centrifuge at 800g for 10 minutes—collect the supernatant—centrifuge at 2000g for 10 minutes—collect the supernatant—centrifuge at 16,000g for 30 minutes—collect the supernatant—centrifuge at 120,000g for 90 minutes—remove the supernatant and resuspend in sterile PBS Precipitation—centrifuge again at 120,000g for 90 minutes, remove the supernatant, collect exosomes at the bottom, and resuspend in sterile PBS.

The analysis of embodiment 3 IEVs

(1) Quantification of IEVs and analysis of membrane proteins

The IEVs obtained in Example 2 were quantitatively analyzed by flow cytometry, and the measurement time points were 1h, 4h, 8h, 16h and 24h. The results showed that ¹⁰⁶ MSCs were induced to 1h, 4h, 0.76×10 ⁸ , 1.29×10 ⁸ , 1.95×10 ⁸ , 2.48×10 ⁸ , and 3.14×10 ⁸ IEVs can be produced after 8h, 16h and 24h respectively. After 24h, a single MSC can produce 300 IEVs (Figure 3).

In addition, flow cytometry found that the particle diameter distribution of IEVs was concentrated below 1 μm, accounting for 94.97% (Fig. 4A).

The results of side scattered light (SSC) analysis also showed that the scattered light intensity of IEVs was concentrated in the range below 1 μm (Fig. 4B).

Further, the scattered light intensity of IEVs was analyzed by standardized small particle microspheres (0.2 μm, 0.5 μm, 1 μm) produced by Bangs Laboratories, and the results showed that the particle diameters of IEVs were all below 0.2 μm (Figure 4C).

The results observed by transmission electron microscope (TEM) were similar to those detected by flow cytometry, most of the vesicles had a diameter of 200 nm or less (Fig. 4D).

The results of nanoparticle tracking analysis (NTA) were consistent with those observed by transmission electron microscopy, and the average particle diameter of IEVs was 169 nm (Fig. 4E).

The particle size detection at the single vesicle level was performed using the most advanced nano flow detection technology, and the results also showed that the average particle diameter of IEVs was 100.63nm (Fig. 4F).

The surface membrane proteins of the IEVs extracted in Example 3 were analyzed by flow cytometry, and the results showed that the IEVs derived from MSCs could express surface proteins similar to MSCs, that is, CD29, CD44, CD73, and CD166 were positive, and CD34 and CD45 were negative. At the same time, IEVs can express the ubiquitous surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles (as shown in Figures 5A-5K).

(2) Content analysis of IEVs

The proteomic quantitative analysis of MSCs, MSCs-Exosomes (extracted in Comparative Example 1), and MSCs-IEVs (obtained in Example 2) was completed by protein DIA quantitative technology. The results showed that the protein content expression of MSCs-Exosomes and MSCs-IEVs had a high overlap with that of parent cells, and 170 proteins were specifically highly expressed in IEVs (Fig. 6A). Through bioinformatics analysis, IEVs specifically and highly expressed proteins were screened, and a heat map was drawn (Figure 6B). Further combined with the GO enrichment analysis results of differential proteins, it was confirmed that IEVs could specifically and highly express Annexin V, Flotillin-1, and Cadherin 11 , Integrin alpha 5 and Syntaxin 4 molecules (Fig. 6C). Compared with Exosomes derived from the same MSCs, the expression levels of five characteristic molecules of IEVs were significantly up-regulated, specifically: the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in IEVs were compared with The expression levels of corresponding markers in exosomes were 1.76 times, 2.81 times, 2.41 times, 3.68 times and 4.45 times, respectively. Finally, the western blot technique was used to verify again, and the results were consistent with the quantitative analysis results of DIA (Fig. 6D).

MSCs-Exosomes: refers to exosomes derived from MSCs.

MSCs-IEVs: refers to IEVs derived from MSCs.

The MSCs in the content analysis described therein are the same cell line as the MSCs from which exosomes and IEVs were extracted.

General experimental method of embodiment 4-7

1. Experimental materials

6 normal wild-type C57BL/6 and 12 MRL/lpr mice were divided into three groups, i.e. C57BL/6 group (wild-type control group), MRL/lpr group, MRL/lpr+IEVs group, 6 in each group Only.

IEVs were prepared in Example 2, and MSCs were BMMSCs prepared in Example 1.

2. Experimental method

2.1 Handling of mice

Six 8-week-old MRL/lpr mice in the MRL/lpr+IEVs group were injected with 4×10 ⁸ IEVs into the tail vein, and after 4 weeks, two samples were collected from each of the three groups; the remaining 4 mice in the MRL/lpr+IEVs group continued to be After intravenous injection of 4×10 ⁸ IEVs per animal, two samples were collected from each of the three groups after 4 weeks; the remaining 2 mice in the MRL/lpr+IEVs group continued to be injected with 4×10 ⁸ IEVs through the tail vein for 4 weeks (period Vaginal smear, detection of estrous cycle) three groups of two each;

That is, the experiment set up three time gradients: 12 weeks, 16 weeks, and 20 weeks. Each time gradient included the C57BL/6 group (wild-type control group), the MRL/lpr group, and the MRL/lpr+IEVs group. Each group had 2 Only. The 2 mice in the MRL/lpr+IEVs group in the 12-week gradient were injected with IEVs once, the 2 mice in the MRL/lpr+IEVs group in the 16-week gradient were injected with IEVs twice, and the 2 mice in the 20-week gradient Two mice in MRL/lpr+IEVs group were injected with IEVs three times.

Materials: The mice were weighed and recorded before the mice were killed; the blood was collected from the eyeballs after the mice were anesthetized with pentobarbital, and the supernatant was centrifuged after sedimentation to measure the sex hormones; after the ovaries were weighed, they were fixed in paraformaldehyde, embedded in paraffin, Paraffin sections were performed and stained with HE.

2.2 Staining, sectioning, and how to count the number of follicles at all levels

After the ovaries were fixed and embedded, they were serially sectioned in paraffin. After HE staining, the follicles of all levels of the ovary were counted every 5 slices, and the follicles that wrapped the oocytes with a single layer of flat granulosa cells were regarded as primordial follicles. The follicles that envelop the oocyte with multiple layers of cuboidal granulosa cells but do not form a follicular lumen are called primary follicles; follicle); the number of granulosa cells increases and the cavity becomes larger, which is called antral follicle; the follicle forms a cumulus, and the follicle that is about to be discharged is called preovulatory follicle. The luminal follicles and preovulatory follicles are both mature follicles.

2.3 PKH26 and AIE marker IEVs

PKH26 labeling: The IEVs suspended in the EP tube were centrifuged to obtain a pellet, and the IEVs were resuspended using the diluent in the PKH26 staining kit (sigma). The working concentration of PKH26 dye was prepared according to the concentration of IEVs/μl PKH26 derived from 2×10 ⁸ cells, and the working concentration of PKH26 dye was obtained by dissolving the PKH26 dye master solution in 250 μl diluent. Mix 250 μl of PKH26 dye and IEVs resuspension and blow evenly, react at room temperature for 5 minutes, neutralize with fetal bovine serum, centrifuge at 16,000g for 30 minutes to obtain IEVs pellet, resuspend and wash with PBS, centrifuge at 16,000g for 30 minutes to obtain IEVs pellet, wash with PBS Resuspend ready for injection.

AIE labeling: use AIE kit, dilute AIE Stock solution with serum-containing medium, and prepare working staining solution (5uM) 5ml/dish. Stain BMMSCs at 37°C in the dark for 1 hour, remove the supernatant, wash off excess dye with PBS, add 6ml of serum-free medium, and irradiate for 4.5-5 hours under an LED incandescent lamp (0.71mw/cm ² ) (observe cell morphology changes under a microscope) , collected by pipetting, and gradient centrifugation to obtain stained IEVs.

Example 4 BMMSCs-derived IEVs reduce the ovarian size, weight, and ratio of ovarian weight to body weight in MRL/Lpr mice

As shown in Figure 7, the ovary weight of MRL/lpr mice at 12-20 weeks was significantly higher than that of C57BL/6 wild-type mice, and the ratio of ovary to body weight was significantly higher than that of C57BL/6 wild-type mice. , the ratio of ovaries to body weight were significantly down-regulated after injection of IEVs. As the age of the mice increases (12 weeks to 20 weeks), the ratio of the ovaries to the body weight of the mice in the MRL/lpr+IEVs group gradually returns to the level of the wild-type control mice, and the situation of premature ovarian maturity is reversed, indicating that with There was a trend towards better IEVs treatment as the mice got older.

As shown in Figure 8, the ovarian morphology of MRL/lpr mice at 20 weeks was significantly larger than that of C57BL/6 wild-type mice. /6 wild-type mice have larger ovaries.

Example 5 Effect of IEVs derived from BMMSCs on ovarian follicles of MRL/Lpr mice

As shown in Figure 9 and Figure 10, compared with C57BL/6 wild-type mice, the number of follicles in MRL/lpr mice at 12 weeks increased at all levels, showing a precocious trend; after IEVs injection (ie MRL/lpr+IEVs group mice) , the number of follicles at all levels was restored (at the same level as the C57BL/6 group), indicating that IEVs can alleviate the tendency of premature ovarian maturation.

Compared with C57BL/6 wild-type mice, the number of primitive and primary follicles in MRL/lpr mice at 16-20 weeks decreased, and a large number of follicular atresia occurred, but the number of secondary follicles and mature follicles was still higher, showing a tendency of premature aging; After IEVs injection (that is, MRL/lpr+IEVs group mice), the number of primary and primary follicles increased compared with the MRL/lpr mouse group, and the number of secondary and mature follicles decreased, showing that premature ovarian failure was relieved the trend of.

The results of this example show that IEVs have a therapeutic effect on both precocious ovarian puberty and premature ovarian failure, and for individuals showing either precocious ovarian puberty or premature ovarian failure, IEVs injection can restore the state of the ovary to a normal level.

Example 6 Effect of IEVs derived from BMMSCs on the estrous cycle of MRL/Lpr mice

MRL/lpr mice were tested for estrous cycle by vaginal smear, and MRL/lpr was in estrus most of the time in two estrous cycles (Figure 11). After IEVs injection, MRL/lpr returned to normal estrous cycle.

Example 7 Effect of IEVs derived from BMMSCs on serum estradiol in MRL/Lpr mice

12-20 week MRL/lpr mouse serum estradiol concentration is detected, as shown in Figure 12, MRL/lpr mouse estradiol content is significantly higher than C57BL/6 wild-type mice; IEVs injection treatment group (ie MRL/lpr+IEVs group mice), with the change of time, the serum estradiol content of mice in the MRL/lpr+IEVs group decreased from 16 weeks to 20 weeks, indicating that after multiple injections of IEVs, the secretion of ovarian estrogen was excessive The symptoms of premature ovarian failure have been relieved, and IEVs may restore the symptoms of premature ovarian failure by regulating ovarian estrogen secretion.

The results of Examples 4-7 show that MRL/lpr mice have precocious ovaries. As a model of precocious ovaries in the present disclosure, after injecting IEVs, they can reduce the size, weight, and weight of ovaries in MRL/Lpr mice. Ratio of ovarian weight to body weight, reducing secondary follicles and mature follicles, restoring abnormal estrous cycle and serum estradiol levels, improving premature ovarian maturation.

Example 8 The targeting effect of IEVs derived from BMMSCs on MRL/lpr mice

As shown in Figure 13, the results of immunofluorescence showed that using PKH26 and AIE to label IEVs, PKH26-IEVs and AIE-IEVs could reach the ovarian stromal cells and follicular theca cells of MRL/lpr mice 1 day after tail vein injection, indicating that IEVs regulate ovarian dysfunction by targeting the ovary.

Example 9 IEVs derived from BMMSCs reduce the abnormal increase of Wnt signaling pathway in the ovary of MRL/lpr mice by regulating β-Catenin-active

The 20W MRL/lpr mouse ovarian protein was extracted and analyzed by Western blot. The results are shown in Figure 14.

Compared with C57BL/6 mice, the content of β-Catenin-active was significantly increased. After injection of IEVs, the content of β-Catenin-active in MRL/lpr mice decreased significantly, indicating that IEVs injection can effectively reduce the abnormal increase of Wnt signaling pathway in the ovaries of MRL/lpr mice. However, there was no significant difference in the content of β-Catenin in the ovaries of MRL/lpr mice before and after IEVs injection.

General experimental method of embodiment 10-12

1. Experimental materials

Eighteen C57BL/6 mice were divided into three groups, WT group (wild-type control group), CP group (cyclophosphamide-treated group), CP+IEVs group, 6 mice in each group.

Cyclophosphamide (Cylcophosphamide, CP)

IEVs were prepared in Example 2.

2. Experimental method

2.1 Handling of mice

C57BL/6 mice were treated differently: wild type (WT) without treatment; CP group: intraperitoneally inject C57BL/6 mice once with 100 mg/kg CP dose; CP+IEVs group: intraperitoneally inject 100 mg/kg CP dose C57BL/6 mice were injected once, and seven days later, the IEV dose of 4×10 ⁸ mice was injected into the tail vein once, and the samples were collected seven days later.

Materials: Blood was collected from the eyeballs of mice anesthetized with pentobarbital, centrifuged after precipitation, and the supernatant was taken for estradiol determination; ovaries were fixed in paraformaldehyde, embedded in paraffin, paraffin-sectioned, and stained with HE; ovaries were collected in paraformaldehyde After being fixed and dehydrated with sucrose, they were frozen and embedded for cryosection and immunofluorescence.

After the ovaries were fixed and embedded, they were serially sectioned in paraffin. After HE staining, the follicles of all levels of the ovary were counted every 5 slices, and the follicles that wrapped the oocytes with a single layer of flat granulosa cells were regarded as primordial follicles. The follicles that envelop the oocyte with multiple layers of cuboidal granulosa cells but do not form a follicular lumen are called primary follicles; follicle); the number of granulosa cells increases and the cavity becomes larger, which is called antral follicle; the follicle forms a cumulus, and the follicle that is about to be discharged is called preovulatory follicle. Luminal follicles and preovulatory follicles are both mature follicles.

2.3 PKH26 and AIE marker IEVs

Establishment of POF model

As shown in Figure 15, the granulosa cell layer in the primordial follicle and primary follicle of the ovary of mice in the CP group disappeared, the follicle atresia (indicated by the red arrow), and the number of mature follicles decreased, indicating that the POF model was successfully established.

Example 10 Effect of IEVs derived from BMMSCs on POF mouse ovarian follicles

Seven days after injecting IEVs into mice in the CP group, ovarian atresia follicles decreased (indicated by the red arrow), but no mature follicles were seen. (Figure 13)

The experimental results indicated that IEVs could restore the ovarian morphology of mice in the CP group.

Example 11 Effect of IEVs derived from BMMSCs on serum estradiol in POF mice

As shown in Figure 16, the concentration of serum estradiol in mice established by CP injection decreased. After injecting IEVs to the mice in the CP group, it can be seen that the serum estradiol of the mice recovered somewhat compared with that in the CP group.

Example 12 The targeting effect of IEVs derived from BMMSCs on POF mice

As shown in Figure 17, the results of immunofluorescence showed that PKH26-IEVs were labeled with PKH26, and PKH26-IEVs could reach the ovarian stromal cells and follicular theca cells of POF mice 1 day after tail vein injection, indicating that IEVs regulate ovarian function by targeting the ovary abnormal.

Example 13 IEVs derived from BMMSCs improve premature ovarian failure by regulating β-Catenin-active

As shown in Figure 18, A/B/C: in wild-type mouse ovary, mesenchymal cells (red arrow indication) and corpus luteum granulosa cells (white arrow indication) β-Catenin-active expression levels are low; D/E/ F: In the ovary of mice in the CP group, the expression of β-Catenin-active in the mesenchymal cells (indicated by the red arrow) and the granulosa cells of the corpus luteum (indicated by the white arrow) increased; G/H/I: in the ovary of the mice in the CP+IEVs group , compared with the CP group, the expression of β-Catenin-active in the mesenchymal cells (indicated by the red arrow) and luteal granulosa cells (indicated by the white arrow) decreased, which was consistent with the wild type.

The results showed that the expression of β-Catenin-active in the Wnt signaling pathway increased in the CP group mice, and the injection of IEVs could down-regulate the expression of β-Catenin-active to the normal level, indicating that IEVs can regulate the expression of β-Catenin-active. Expression improves premature ovarian failure.

General Experimental Procedures for Examples 14-19

1. Experimental materials

60 normal wild-type C57 mice were divided into 3 groups, namely control group (Control), IEV group and MSC group, with 20 mice in each group.

The IEV was prepared in Example 2, and the MSC was the BMMSC prepared in Example 1.

2. Experimental method

From the age of 8 months, the three groups of mice were injected with PBS, IEV (prepared in Example 2), and MSC (prepared in Example 1) through the tail vein, once a month. Among them, each mouse in the IEV group was resuspended with 1×10 ⁶ IEVs in 200 μL PBS, mixed well, placed on ice, and injected through the tail vein within 30 minutes; each mouse in the MSC group was injected with the same amount of cells (1×10 ⁶ MSCs); mice in the control group were injected with an equal volume of solvent (PBS, 200ul/mouse).

Example 14 Multiple injections of IEVs derived from BMMSCs significantly prolong the lifespan of mice

Groups of mice were prepared using the general experimental method described above.

The status and death of the mice were continuously monitored. And Kaplan-Meier survival analysis was performed. Among them, the log-rank test was used to analyze the statistical difference between the control group and each treatment group, and a P value less than 0.05 was considered to be statistically different.

The results of this embodiment show that injecting both IEVs and MSCs can improve the survival rate of aged mice. After Kaplan-Meier survival analysis, the IEV group and the MSC group have significantly prolonged the lifespan of the mice compared with the control group ( Figure 19).

Example 15 Multiple injections of IEVs derived from BMMSCs significantly reduced the body weight of aged mice

At the age of 24 months, the body weight of mice in each group was weighed for statistical analysis. The results of this example showed that both IEV injection and MSC injection significantly reduced the body weight of aged mice ( FIG. 20A ).

Example 16 Multiple injections of IEVs derived from BMMSCs significantly reduced hair loss in aged mice

At the age of 24 months, mice were photographed to record the state of hair, and the photos showed that IEV injection significantly reduced the phenomenon of mouse hair loss ( FIG. 20B ). Hair loss was almost completely restored in the IEV-injected treatment group compared to the MSC-injected treatment group. There was no significant improvement in hair loss in the MSC-injected group.

Example 17 Multiple injections of BMMSCs-derived IEVs significantly reduced spleen volume and weight in aged mice

At the age of 24 months, 5 mice were randomly selected from each group, and the spleens of the mice were taken after sacrifice, and the mice were photographed and weighed. The results showed that IEV injection significantly reduced spleen volume and weight in mice (Fig. 21). However, mice injected with MSCs lost less spleen volume and weight than those treated with IEV.

Example 18 Multiple injections of IEVs derived from BMMSCs significantly enhanced bone mineral density in aged mice

At the age of 24 months, 5 mice were randomly selected from each group, and the femurs of the mice were taken after sacrifice, and microCT was used to analyze BMD, BV/TV and other related indicators of bone mineral density and bone volume.

MicroCT analysis: After fixing mouse femurs in 4% PFA, femurs were analyzed using a high-resolution Scanco MicroCT50 scanner (Scanco Medical AG). Samples were scanned at 20 kVp and 200 μA using a voxel size of 20 μm. Data sets were analyzed using Amira 5.3.1 software (Visage Imaging) to reconstruct images and measure bone mineral density.

The results showed that both IEV and MSC injections could enhance bone mineral density BMD and bone volume fraction BV/TV in aged mice (Fig. 22A, 22B).

Example 19 Multiple injections of BMMSCs-derived IEVs significantly enhanced the function of BMMSCs in aged mice

At the age of 24 months, 5 mice were randomly selected from each group, and their BMMSCs were isolated and cultured.

Isolation of BMMSCs: A single suspension of bone marrow-derived all nucleated cells (ANCs, 15×10 ⁶ ) from femurs was seeded in 100 mm dishes (Corning) and incubated at 37° C. with 5% CO ₂ . After 24 hours, non-adherent cells were removed and adherent cells were cultured for an additional 14 days in α-minimal essential medium (α-MEM, Invitrogen) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine (Invitrogen), 55 μM 2-mercaptoethanol (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). These adherent single colonies were passaged at passage 1 with frequent medium changes to eliminate potential hematopoietic cell contamination. Determination of colony forming units-fibroblasts (CFU-F): 1×10 ⁶ monosuspension BMMSCs were seeded in 60 mm culture dishes (Corning). After 16 days, cultures were washed with PBS and stained with 1% toluidine blue solution containing 2% paraformaldehyde (PFA, Sigma-Aldrich). Cell clusters were counted under a microscope and those with more than 50 cells were considered colonies.

Cell Proliferation Assay: Proliferation of BMMSCs was assessed using a bromodeoxyuridine (BrdU) incorporation assay. Briefly, BMMSCs were seeded on 12-well plates at 1 × ¹⁰⁴ cells per well and cultured for 24 hours, then the cultures were incubated with BrdU solution (1:100) (Invitrogen) for 24 hours and incubated according to the manufacturer. Instructions for staining with BrdU staining kit (Invitrogen). BrdU-positive and total cells were counted in 10 images per sample. The number of BrdU-positive cells is expressed as a percentage of the total number of cells. For each experimental group, the BrdU assay was repeated with five independent samples.

In vitro osteogenic differentiation: BMMSCs were cultured under osteoinductive conditions including 2 mM β-glycerophosphate (Sigma-Aldrich), 100 μM L-ascorbic acid, 2-phosphate (Wako) and 10 nM dexamethasone (Sigma-Aldrich) in growth medium. -Aldrich). After 3 weeks of induction, matrix mineralization was detected by staining with 1% Alizarin Red S (Sigma-Aldrich), and the stained positive area was quantified using NIH ImageJ software and displayed as a percentage of the total area.

In vitro adipogenic differentiation: For adipogenic induction, 500 nM isobutylmethylxanthine (Sigma-Aldrich), 60 μM indomethacin (Sigma-Aldrich), 500 nM hydrocortisone (Sigma-Aldrich), 10 μg/ml with insulin ( Sigma-Aldrich) and 100nM L-ascorbyl phosphate. This was added to regular mouse BMMSC growth medium. After 7 days of induction, cultured cells were stained with Oil Red O (Sigma-Aldrich), and positive cells were quantified under a microscope and displayed as the percentage of the number of positive cells to the total cells.

Compared to 6-month-old mice, 24-month-old mice had reduced MSC self-renewal capabilities, including clonogenicity (CFU-F colony-forming unit fibroblasts), and proliferation (BrdU bromodeoxyuridine) Decreased; decreased osteogenic capacity, increased adipogenesis. These all reflect the impairment of MSC ability, and the MSC function of mice injected with IEV was significantly improved compared with the non-injected group (Fig. 23A, 23B).

Example 20 IEVs can be excreted through skin and hair

Take 4×10 ⁶ IEVs prepared in Example 2 and mark them with DIR, resuspend them in 200 microliters of PBS, and inject them into nude mice BALB/c-nu/nu systemically through the tail vein, observe them for 1, 3, and 7 days and use live imaging The instrument detects the distribution of IEVs on the skin surface, and the results are shown in Figures 24A-24C.

Figure 24A shows that IEVs can reach the skin surface, and the number is the largest on the 3rd day, and basically disappears on the 7th day, showing the dynamic metabolic process of IEVs on the skin surface (Figure 24A). Immunofluorescence results showed that after systemic injection of C57 mice, PKH26-IEV gradually moved from the subcutaneous tissue to the dermis and epidermis over time. On the 7th day, a large number of IEVs were observed in the stratum corneum of the skin surface, suggesting that the systemically injected IEVs could be excreted with the exfoliation of the stratum corneum of the skin ( FIG. 24B ). At the same time, PKH26-IEVs were found in the hair follicles of the plucked hairs from the mice on day 7, indicating that the systemically injected IEVs could also be metabolized as the hairs fell off (Fig. 24C).

In addition, in vivo imaging data and immunofluorescence results show that IEVs are distributed in large numbers in the liver, spleen, bone marrow, lymph and other organs in vivo, but not in the heart, kidney, and brain, but are distributed in the colon, teeth, and nails, further proving that IEVs can Excreted with metabolism (Figure 25A-Figure 25E).

This example shows that IEVs can be excreted through skin and hair, indicating that injection or increasing the content of IEVs in the body is safe.

Example 21 IEV can promote hair regeneration

Seven-week-old female C57 mice (telogen phase) were depilated, and PBS, IEVs, MSCs and 2% Minoxidil were injected subcutaneously, respectively. Comparing the hair regeneration area on the back of the mice on the 10th day and the 14th day, the experimental results showed that IEVs and MSCs had a significant effect on promoting hair regeneration compared with the control group. , both IEVs and MSCs had a more obvious effect of promoting hair regeneration (Fig. 26A-26B).

Example 22 IEVs can promote wound healing

A 1cm×1cm full-thickness wound was made on mice, and the promotion effect of systemic injection of MSCs and IEVs on wound healing was detected. The results showed that both IEVs and MSCs could promote wound healing ( FIG. 27 ), with no significant difference.

Combining Examples 20, 21, and 22, metabolic studies of IEV show that it is excreted through the skin and skin appendages and has positive effects on these tissues.

Example 23 Effect of IEVs on Induced Mouse Obesity

High fat diet (high fat diet, HFD) was used to induce obesity in mice, and IEVs derived from BMMSCs were injected through the tail vein, once a week, for a total of 4 injections, each dose was 4× ¹⁰⁶ IEVs, and the IEVs were observed effect on obesity.

The results showed that the disclosed study found that obese mice had relatively reduced apoptotic vesicles in white adipose tissue, and the reduction of apoptotic vesicles in white adipose tissue was significantly alleviated after mice were treated with IEVs ( FIG. 28E ).

In addition, the weight of obese mice was significantly reduced after IEVs injection (Figure 28A), and the fat distribution of mice was reconstructed using microCT (pink in Figure 28B is the constructed whole body fat distribution), and the body fat rate was found to decrease significantly after IEVs treatment (Figure 28B), The content of triglycerides in the blood circulation was significantly reduced (Figure 28C), and the accumulation of white fat was significantly reduced (Figure 28D). Histological examination showed that IEVs treatment could significantly alleviate the increase of adipocyte volume in the abdominal white adipose tissue of obese mice (Figure 28F ).

Example 24 Effect of IEV on Obesity in Aging Mice

With age, the body's basal metabolic rate decreases, the balance of lipogenesis and lipolysis is broken, and white fat accumulation or even obesity often occurs in the abdominal cavity.

Experimental process: C57BL6 mice were injected with IEVs derived from BMMSCs once a month from the 7th month, with a dose of 1×10 ⁶ IEVs each time, for a total of 18 injections. In the control group, only the same amount of PBS was injected into the tail vein every month. Eight-week-old C57BL6 mice were used as normal mice for control.

Aging mice had weight gain and accumulation of white fat in the abdominal cavity. The experimental results showed that there was a relative reduction of apoptotic vesicles in white adipose tissue in aging mice, and the reduction of apoptotic vesicles in white adipose tissue was significantly alleviated after aging mice were treated with IEVs (Fig. 29D).

In addition, BMMSCs-derived IEVs treatment can significantly inhibit weight gain (Figure 29A) and abdominal white fat accumulation (Figure 29B), and IEVs treatment can significantly inhibit the content of triglycerides in blood circulation (Figure 29C).

Example 25 The effect of IEV on OB mice

OB mice are leptin-deficient mice, which are commonly used to study obesity.

Experimental process: OB mice were injected with IEVs derived from BMMSCs through the tail vein after their body weight reached 45 g, once a week, for a total of 4 injections, each with a dose of 4×10 ⁶ IEVs.

The experimental results showed that OB mice had relatively reduced apoptotic vesicles in white adipose tissue, and the reduction of apoptotic vesicles in white adipose tissue was significantly alleviated after OB mice were treated with IEVs ( FIG. 30D ).

In addition, OB mice were treated with IEVs, and it was found that IEVs treatment could significantly inhibit the weight gain (Figure 30A) and the accumulation of abdominal white fat (Figure 30C), and the IEVs treatment could significantly inhibit the content of triglycerides in the blood circulation (Figure 30B) .

Next, 6-week-old OB mice were locally injected with BMMSCs-derived IEVs in the perigonadal fat of the peritoneal cavity, and one dose was two 10cm dish cultured BMMSCs 100% confluent induced IEVs. After 2 weeks, it was found that the body weight growth rate of OB mice after local injection of IEVs was significantly lower than that of the control group (Figure 31A). MicroCT analysis of body fat rate found that the body fat rate of OB mice with local injection of IEVs decreased (Figure 31B). The triester content was significantly reduced (FIG. 31C).

Example 26 Effect of IEVs on Lpr mice

Apoptotically impaired Lpr mice, ie mice deficient in Fas, were also studied.

Experimental process: Lpr mice were injected with IEVs derived from BMMSCs through the tail vein, once a week, for a total of 4 injections, and each dose was 4×10 ⁶ IEVs.

The results showed that Lpr mice had relatively reduced apoptotic vesicles in white adipose tissue, and the reduction of apoptotic vesicles in white adipose tissue was significantly alleviated after Lpr mice were treated with IEVs ( FIG. 32D ).

In addition, the inventors found that Lpr mice also had the accumulation of white fat in the abdominal cavity and the increase of triglyceride in the blood. Treat Lpr mice with IEVs, and found that IEVs treatment can significantly inhibit the accumulation of abdominal white fat (Figure 32A, 32B), and IEVs treatment can significantly inhibit the content of triglycerides in blood circulation (Figure 32C).

Combining the results of Examples 23-26, this disclosure makes a breakthrough discovery for the first time that whether it is for induced obesity, senile obesity, leptin-deficient obesity, or Fas-deficient obesity, apoptotic vesicles are present in individual white adipose tissue. Compared with normal individuals, there is a decrease in the number of apoptotic vesicles, indicating that apoptotic vesicles can be used as an indication of obesity; after IEVs treatment was given to these obese individuals, the number of apoptotic vesicles recovered, indicating that IEVs have a therapeutic effect on obesity.

Example 27 The treatment and mechanism of IEVs on obesity in mice

1.IEVs can significantly inhibit lipogenesis in adipocytes

Experimental process: The skin fibroblasts of C57BL6 mice born within 1-3 days were obtained by tissue culture, and after passage to the P3 generation, they were plated on a six-well plate, and after 100% confluence, they were induced with adipogenic induction medium to become adipocytes for 7 days, and then added as a source of BMMSCs The IEVs.

(1) After IEVs were added into the adipogenic induction solution, the adipocytes were continued to be cultured. After 3 days, the cell culture medium and adipocytes were extracted respectively, and the cell culture supernatant and the intracellular supernatant were obtained respectively, and the cell culture supernatant and the cell culture supernatant were detected. Triglyceride content in the supernatant. The results showed that after the adipocytes were treated with IEVs derived from BMMSCs, triglyceride (triglyceride, TAG) in the adipocytes (adipocytes) and in the adipocyte culture supernatant were significantly reduced, indicating that IEVs can significantly inhibit the lipogenesis of adipocytes (FIGS. 33A, 33B). Lipid droplets in adipocytes treated with IEVs were significantly reduced (Fig. 33C, 33D).

Note: 1:1 IEVs equal ratio refers to using IEVs collected from a six-well plate with 100% fusion of BMMSCs to treat adipocytes in a six-well plate, and 1:2 IEVs equal ratio refers to using two six-well plates with 100% fusion IEVs collected from BMMSCs were treated with adipocytes in a six-well plate.

(2) Western blot detection found that IEVs treatment of the adipocytes enhanced the p-Akt and p-Erk pathways that inhibit lipogenesis, and inhibited the p-P50 pathway that promotes lipogenesis ( FIG. 33E ).

(3) IEVs (F-IEVs) derived from adipose-derived mesenchymal stem cells (F-IEVs) and IEVs (B-IEVs) derived from bone marrow mesenchymal stem cells were used to pretreat adipose-derived mesenchymal stem cells to induce adipogenic differentiation, and it was found that F-IEVs and The B-IEVs treatment group could inhibit the adipogenic differentiation of adipose-derived mesenchymal stem cells, showing that the expressions of adipogenic-related proteins LPL and PPARγ were significantly reduced ( FIG. 33F ).

Note:

Preparation method of IEVs derived from adipose-derived mesenchymal stem cells: similar to that of BMMSC-derived IEVs. When the P3 generation adipose-derived mesenchymal stem cells were 100% confluent, wash them twice with PBS, add serum-free medium containing 500nM STS (the medium is the addition of 500nM STS to the medium in Example 1), and incubate at 37°C for 16 -24h, collect the cell supernatant, centrifuge at 800g at 4°C for 10 minutes, collect the supernatant and centrifuge at 2000g at 4°C for 10 minutes, collect the supernatant again and centrifuge at 16000g at 4°C for 30 minutes, and the obtained precipitate is IEVs. Resuspend the pellet in 500 μl PBS, and centrifuge again at 16,000 g for 30 minutes at 4°C to obtain the washed IEVs.

F-IEVs 1:1 refers to treating adipocytes in a six-well plate with IEVs collected from 100% confluent adipose-derived mesenchymal stem cells in a six-well plate;

F-IEVs 1:5 refers to the treatment of adipocytes in a six-well plate with IEVs collected from 100% confluent adipose-derived mesenchymal stem cells in five six-well plates;

B-IEVs 1:1 refers to the treatment of adipocytes in a six-well plate with IEVs collected from 100% confluent BMMSCs in a six-well plate;

B-IEVs 1:5 refers to the treatment of adipocytes in a six-well plate with IEVs collected from 100% confluent BMMSCs in five six-well plates.

(4) Add PKH-26-labeled IEVs (red fluorescence) to adipocytes at a ratio of 1:5, that is, after the IEVs collected from 100% confluent BMMSCs in five six-well plates were stained with PKH-26, one six-well plate was treated. Adipocytes in a well plate. Observe the dynamic changes of lipid droplets in adipocytes in the same field of view. And as time goes by, IEVs enter adipocytes and increase, while lipid droplets become smaller (Figure 34, the same color in Figure 34 represents the dynamic changes of the same adipocyte, the contents of the upper and lower figures are consistent, and the red fluorescent markers are not marked in the lower figure IEVs are placed in the picture so that the size changes of the lipid droplets can be seen clearly).

2.IEVs can significantly promote the browning of adipocytes

Experimental process: The skin fibroblasts of C57BL6 mice born within 1-3 days were obtained by tissue culture, and after passage to the P3 generation, they were plated on a six-well plate, and after 100% confluence, they were induced with adipogenic induction medium to become adipocytes for 7 days, and then added as a source of BMMSCs Add 1um rosiglitazone to the IEVs at the same time to induce browning of adipocytes.

In vitro experiments were performed to detect the expression level of the brown fat-induced marker protein UCP1 to explore the effect of IEVs on the browning of adipocytes. The results of Western blot showed that compared with the untreated group, the protein content of UCP1 was significantly increased after IEVs treatment, indicating that IEVs can significantly promote the browning of adipocytes (Figure 35A, the IEV treatment group in the figure is all IEVs derived from BMMSC).

At the same time, in vivo experiments found that compared with mice fed normal food, the expression of UCP1 in the brown adipose tissue (BAT) of obese mice fed with high-fat diet was significantly decreased, and the IEVs (10 ⁹ /g) given tail vein injection In obese mice, the protein expression of UCP1 in brown adipose tissue was significantly up-regulated, indicating that IEVs can significantly promote the browning of adipose tissue in vivo (Figure 35B).

Comparative example 2 IEVs (derived from BMMSC) and exosomes with the same number of cells compared the lipogenesis and lipolysis of adipocytes

IEVs (obtained in Example 2) and exosomes (obtained in Comparative Example 1) with the same cell number were extracted, and their effects on lipogenesis and lipolysis of adipocytes were compared in vitro. The results showed that after IEVs and exosomes treatment, there were significantly fewer lipid droplets and smaller adipocytes (Fig. 36A). Triglycerides in adipocytes and supernatants were significantly reduced. Compared with exosomes treatment group, the reduction of lipid droplets was more obvious in the IEVs treatment group, and the reduction of triglycerides in adipocytes and supernatants was more obvious, with statistical differences (FIG. 36B, 36C), indicating that IEVs are better than exosomes in inhibiting lipogenesis in adipocytes.

Comparative example 3 Comparison of the browning effect of IEVs (derived from BMMSC) and exosomes with the same number of cells on adipocytes

Compared with exosomes derived from the same MSCs (obtained in Comparative Example 1), IEVs derived from BMMSCs have a unique effect of promoting the browning of adipose tissue: in vivo experiments found that compared with IEVs injected with the same amount of exosomes (Exo), the Exo group The brown adipose tissue in the IEVs group still had a whitening tendency, and the subcutaneous adipocytes (iWAT) became larger, while the whitening tendency of the brown adipose tissue in the IEVs group decreased, and the iWAT cells became smaller (Fig. 37A). The results of Western blot of brown adipose tissue also showed that the Exo group did not have the effect of inducing the browning of adipose tissue, showing that the content of UCP1 did not increase significantly compared with the high-fat-induced obesity group (Figure 37B).

Example 28 Effect of IEVs on atherosclerotic mice

High cholesterol diet was used to induce atherosclerosis in apoE ^-/- mice, and IEVs were injected through the tail vein to observe the effect of IEVs on atherosclerosis. The results showed that the area and severity of aortic arch atherosclerotic plaques were significantly relieved after IEVs treatment (Fig. 38). HE staining of the aortic arch showed that a large number of aortic foam-like cells in atherosclerotic mice gathered, protruding to the intima surface, surrounding fibrous tissue increased, and atheromatous plaque protruded into the lumen to cause stenosis. Cells were reduced (FIG. 38A). Lipid droplets stained with Oil Red O showed a wide range of lipid accumulation in the aorta of atherosclerotic mice, and the aortic lipid accumulation in the IEVs treatment group was significantly reduced, and the area of atherosclerotic fibrous plaque was significantly reduced (Figure 38B).

Example 29 Effects of IEVs on Biochemical Indexes in Atherosclerotic Mice

Analyzing the biochemical indicators of atherosclerotic mice and IEVs after treatment, it was found that triglyceride (TAG) in blood of atherosclerotic mice after IEVs treatment was significantly reduced (Fig. 39A), although total cholesterol (TC) in blood after IEVs treatment ) content did not change significantly (Figure 39B), but the content of high cholesterol (HDL) in the blood increased significantly after IEVs treatment (Figure 39C), suggesting that IEVs may treat atherosclerosis by reducing lipid synthesis and promoting the secretion of high cholesterol.

Example 30 The impact of IEVs on type 2 diabetes

Studies have found that bone marrow mesenchymal stem cells can improve blood sugar and glucose tolerance in type 2 diabetes to a certain extent.

The inventors compared the control effects of bone marrow mesenchymal stem cells and their derived IEVs on type 2 diabetes. As shown in the flow chart of Figure 40, wild-type C57BL6 mice were fed high-fat diet for 6 weeks, and the mice were given a single injection of ¹⁰⁶ MSCs and a single high-dose IEVs (8× ¹⁰⁶ , 2 dishes) at 10W. Or 4 low-dose IEVs (4×10 ⁶ , 1dish) tail vein injection. We found that compared with the MSCs group, high-dose IEVs had the best effect on controlling fasting blood glucose (Figure 41A) and improving glucose tolerance (Figure 41B). We further used type 2 diabetes model db mice to test the treatment of IEVs on type 2 diabetes Effect, compared with the non-intervention group, high-dose IEVs can significantly reduce the fasting blood glucose of type 2 diabetic mice, with a statistical difference (Fig. 41C).

Test example 1

(1) Detection steps or methods: take 8-week-old Sjögren syndrome (SS) model mice, inject MSCs and IEVs through the tail vein system, collect samples 4 weeks after injection, detect saliva flow rate, collect salivary gland samples and perform paraffin section HE Staining and B cell marker B220 staining.

(2) Results: As shown in Figure 42A-Figure 42C, the results show that comparing the effect of mouse bone marrow mesenchymal stem cells and IEVs derived from them on the salivary flow rate of Sjögren's syndrome (Sjögren's syndrome), mesenchymal stem cell therapy After the salivary flow rate recovered slightly, the salivary flow rate did not improve after IEVs treatment (*p<0.05 compared with WT group, ###p<0.001 compared with MSCs group). IEVs injection did not change the inflammatory infiltration and B cell accumulation in salivary glands.

Test example 2

The in vitro coagulation-promoting effect of the IEVs obtained in Example 2 and the Exosomes extracted from Comparative Example 1 was detected by an in vitro coagulation test. The results are shown in Table 3. IEVs can significantly shorten the coagulation time of most plasma in vitro, and the coagulation-promoting effect is better than that of Exosomes.

However, for plasma lacking factors II, V, and X, IEVs cannot exert in vitro procoagulant effect, indicating that the in vitro procoagulant effect of IEVs is more concentrated in the upstream of the common coagulation pathway.

table 3

Using hemophilia A mice (deficiency of coagulation factor VIII) as a model, 9×10 ⁸ IEVs were injected through the tail vein to observe the procoagulant effect of IEVs in vivo. The results shown in Figure 43, IEVs treatment can significantly improve the bleeding tendency of hemophilia mice, and the therapeutic effect can be maintained stably for 14 days.

The experimental results show that IEVs can play a significant role in promoting coagulation in vitro. Moreover, the injection in vivo can significantly improve the bleeding tendency, and can be used to improve the bleeding tendency caused by hemophilia A. At the same time, the levels of various coagulation factors in mouse plasma were detected, and it was found that coagulation factor VIII, vWF factor, tissue factor (tissue factor, TF) and prothrombin (prothrombin) did not change significantly (Figure 44A, Figure 44B, Figure 44C , Figure 44D).

In the hemophilia A mouse model, normal IEVs, PS-negative IEVs and TF-negative IEVs were injected respectively, and the tail-cutting experiment was performed 7 days later. The results shown in Figure 45A and Figure 45B showed that the blocking of PS and TF did not affect the in vivo The treatment effect preliminarily shows that the mechanism of IEVs treating hemophilia mice has nothing to do with PS and TF. In previous literature reports, the coagulation-promoting effect of extracellular vesicles is highly dependent on the PS and TF on their surface, while the in vivo experimental results of IEVs are inconsistent with previous studies, which suggests that in the in vivo environment, IEVs may have a new mechanism of action. Coagulant effect.

For the hemophilia A mouse model, the same MSCs-derived IEVs (obtained in Example 2) and Exosomes (extracted in Comparative Example 1) were injected (9×10 ⁸ ), and the results showed that IEVs can significantly The bleeding tendency of mice was corrected, while Exosomes had no obvious therapeutic effect (Fig. 46).

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the present disclosure is defined by the appended claims. We therefore claim protection for all inventions that come within the scope and spirit of these claims.

Claims

Application of inducible extracellular vesicles in the preparation of preparations for regulating ovarian function.
The application according to claim 1, wherein said regulation of ovarian function comprises:

improve premature ovarian development; or

Improve premature ovarian failure;

Preferably, said premature ovarian development is selected from premature ovarian development caused by immunity;

Preferably, the premature ovarian failure is selected from immune, drug-induced or age-induced premature ovarian failure.
The use according to any one of the preceding claims, wherein the preparation is a preparation for treating or preventing precocious puberty;

Preferably, the preparation is a preparation for treating or preventing irregular menstruation;

Preferably, the preparation is a preparation for treating or preventing hyperovulation;

Preferably, the preparation is a preparation for treating or preventing premature ovarian failure;

Preferably, the premature ovarian failure is selected from immune, drug-induced or age-induced premature ovarian failure.
Use according to any one of the preceding claims, characterized in that the preparation is selected from preparations which reduce the size of the ovary, reduce the weight of the ovary or reduce the ratio of ovary to body weight;

Preferably, the preparation is a preparation for regulating ovarian physiological cycle;

Preferably, the preparation is a preparation for regulating serum estradiol content;

Preferably, the preparation treats or prevents premature ovarian failure by regulating the Wnt signaling pathway;

Preferably, the preparation treats or prevents premature ovarian failure by regulating the expression of β-Catenin-active;

Preferably, the preparation is selected from pharmaceutical preparations or health product preparations.
Application of inducible extracellular vesicles in the preparation of preparations for prolonging the lifespan of mammals or treating or preventing aging.
The use according to any one of the preceding claims, characterized in that the inducible extracellular vesicles prolong the lifespan of mammals or treat or prevent aging by restoring the proliferation and/or differentiation of damaged cells.
The use according to any one of the preceding claims, wherein the preparation is a preparation for treating or preventing obesity in the elderly;

Preferably, the preparation is a preparation for treating or preventing aging hair loss;

Preferably, the preparation is a preparation for treating or preventing splenomegaly;

Preferably, the preparation is a preparation for treating or preventing osteoporosis, bone loss or bone aging;

Preferably, the preparation is selected from pharmaceutical preparations or health product preparations.
Use according to any one of the preceding claims, wherein the inducible extracellular vesicles are used for reducing body weight in elderly individuals;

Preferably, the inducible extracellular vesicles are used to reduce senile hair loss;

Preferably, said inducible extracellular vesicles are used to reduce spleen weight or volume;

Preferably, said inducible extracellular vesicles are used to increase bone density;

Preferably, said inducible extracellular vesicles are used to increase bone volume fraction.
Application of inducible extracellular vesicles in preparing preparations for treating or preventing senile hair loss;

Preferably, the preparation is selected from pharmaceutical preparations or health product preparations.
Use of inducible extracellular vesicles in preparing anti-aging, and/or repairing, and/or regenerative preparations for skin and/or skin appendages;

Preferably, the skin is epidermis, dermis, or subcutaneous tissue;

Preferably, the skin appendages are hair, hair, sweat glands, sebaceous glands, fingernails, or toenails;

Preferably, the preparation is a preparation for treating or preventing hair loss, or a preparation for promoting hair regeneration;

Preferably, the preparation is a preparation that promotes the repair and/or regeneration of skin wounds or scars;

Preferably, the preparation is selected from pharmaceutical preparations or health product preparations.
Application of the detection reagent of apoptosis vesicle in the preparation of obesity detection reagent or kit.
The application according to claim 11, wherein the detection reagent of the apoptotic vesicle is selected from reagents for detecting the number of apoptotic vesicles, surface markers of apoptotic vesicles, and the total amount of apoptotic vesicle proteins one or more of

Preferably, the detection reagent is selected from one of flow cytometry reagents and/or kits, Western blot reagents and/or kits, BCA quantitative reagents and/or kits, nanoparticle tracking analysis reagents and/or kits one or more species.
A detection method for obesity, characterized in that said method comprises,

S1. Detecting the level of apoptotic vesicles in the subject's sample;

S2. Comparing the level of apoptotic vesicles in the subject with the level of apoptotic vesicles in a normal control sample;

S3. According to the comparison result of step S2, the level of apoptotic vesicles in the subject is lower than that of the normal control sample, indicating that the subject suffers from or is at risk of developing obesity.
A detection system for obesity, characterized in that said system comprises:

1) A detection component for apoptotic vesicles;

2) Data processing components;

3) Result output component;

Preferably, the detection components of the apoptotic vesicles include one or more of a flow cytometer, western blot electrophoresis tank and imaging system, a high-speed centrifuge, a microplate reader, a BCA quantitative kit, and a nanoparticle tracking analysis kit. various;

Preferably, the data processing component is configured to a. receive the test data of the sample to be tested and the normal control sample; b. store the test data of the sample to be tested and the normal control sample; c. compare the same type of samples to be tested And the test data of the normal control sample; d. According to the comparison result, responding to the probability or possibility of the subject suffering from obesity;

Preferably, the result output component is used to output the probability or possibility of the subject suffering from obesity;

Preferably, the judging standard of the data processing component is: judging the obesity specimen and the normal specimen according to the boundary value;

Preferably, the threshold value of the apoptotic vesicle level in the fat sample is 12-15*10 6 apoptotic vesicles per 0.05 ug of adipose tissue, and the apoptotic vesicle level of the fat sample is less than the apoptotic vesicle If the cutoff value of the apoptotic vesicle level is judged as an obese specimen, if the apoptotic vesicle level of the fat sample is greater than or equal to the cutoff value of the apoptotic vesicle level, it is judged as a normal specimen.
The application according to claim 11 or 12, or the detection method according to claim 13, or the detection system according to claim 14, wherein the sample to be detected is selected from adipose tissue; more preferably white adipose tissue.
Application of inducible extracellular vesicles in the preparation of preparations for treating or preventing metabolic inflammatory syndrome;

Preferably, the metabolic inflammatory syndrome includes: one or more of obesity, atherosclerosis, and diabetes;

Preferably, the obesity includes at least one of induced obesity, senile obesity, leptin-deficient obesity, and Fas-deficient obesity;

Preferably, the leptin-deficient obesity is selected from childhood obesity;

Preferably, the diabetes is selected from type 2 diabetes;

Preferably, the formulation is a formulation for reducing body weight;

Preferably, the preparation is a preparation that inhibits one or more of p-Akt, p-Erk, and p-P50 pathways;

Preferably, the preparation is a preparation that inhibits adipose-derived differentiation of adipose-derived mesenchymal stem cells;

Preferably, the preparation is a preparation for treating obesity or atherosclerosis by inhibiting the synthesis of triglycerides;

Preferably, the preparation is a preparation for promoting the secretion of high cholesterol to treat atherosclerosis;

Preferably, the preparation is selected from pharmaceutical preparations or health product preparations.
A treatment system for obesity, characterized in that the system comprises:

1) A detection component for apoptotic vesicles;

2) Data processing components;

3) Result output component;

4) administering the inducible extracellular vesicles to a subject diagnosed with obesity;

Preferably, the detection components of the apoptotic vesicles include one or more of a flow cytometer, western blot electrophoresis tank and imaging system, a high-speed centrifuge, a microplate reader, a BCA quantitative kit, and a nanoparticle tracking analysis kit. various;

Preferably, the data processing component is configured to a. receive the test data of the sample to be tested and the normal control sample; b. store the test data of the sample to be tested and the normal control sample; c. compare the same type of samples to be tested And the test data of the normal control sample; d. According to the comparison result, responding to the probability or possibility of the subject suffering from obesity;

Preferably, the result output component is used to output the probability or possibility of the subject suffering from obesity;

Preferably, the judging standard of the data processing component is: judging the obesity specimen and the normal specimen according to the boundary value;

Preferably, the threshold value of the apoptotic vesicle level in the fat sample is 12-15*10 6 apoptotic vesicles per 0.05 ug of adipose tissue, and the apoptotic vesicle level of the fat sample is less than the apoptotic vesicle If the cutoff value of the apoptotic vesicle level is judged as an obese specimen, if the apoptotic vesicle level of the fat sample is greater than or equal to the cutoff value of the apoptotic vesicle level, it is judged as a normal specimen.
The application according to any one of the preceding claims, wherein the inducible extracellular vesicles are vesicles produced by inducing apoptosis through external factors when stem cells are in normal survival;

Preferably, the inducible extracellular vesicles are produced by inducing apoptosis of stem cells, and the induction method includes adding staurosporum, ultraviolet irradiation, starvation method, or heat stress method;

Preferably, the stem cells are selected from mesenchymal stem cells;

Preferably, the mesenchymal stem cells are selected from blood mesenchymal stem cells, bone marrow mesenchymal stem cells, urine mesenchymal stem cells, oral cavity mesenchymal stem cells, fat mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord mesenchymal stem cells One or more of mesenchymal stem cells, periosteal mesenchymal stem cells, and skin mesenchymal stem cells;

Preferably, the mesenchymal stem cells are selected from blood mesenchymal stem cells, bone marrow mesenchymal stem cells, fat mesenchymal stem cells, umbilical cord mesenchymal stem cells, oral cavity mesenchymal stem cells, skin mesenchymal stem cells one or more kinds;

Preferably, the stem cells are selected from one or both of blood stem cells and bone marrow mesenchymal stem cells.
The application according to any one of the preceding claims, wherein, preferably, the extracellular vesicles of stem cells are produced by adding staurosporine to induce apoptosis of mesenchymal stem cells.
The use according to any one of the preceding claims, wherein the concentration of the staurosporine is greater than or equal to 1 nM;

Preferably, it is 1-15000nM;

Preferably, it is 200-10000 nM; preferably, it is 250-1000 nM; preferably, it is 500-1000 nM.
The application according to any one of claims 1-4, 5-7, wherein the diameter of the inducible extracellular vesicles is 0.45 μm or less;

Preferably, the diameter of the inducible extracellular vesicle is 0.05-0.45 μm;

Preferably, the diameter of the inducible extracellular vesicle is 0.1-0.45 μm;

Preferably, the diameter of the inducible extracellular vesicle is 0.1-0.35 μm;

Preferably, the diameter of the inducible extracellular vesicle is 0.15-0.35 μm;

Preferably, the diameter of the inducible extracellular vesicle is 0.15-0.3 μm;

Preferably, the diameter of the inducible extracellular vesicle is 0.15-0.2 μm.
The application according to any one of the preceding claims, wherein the preparation is an injection, an oral preparation or an external preparation;

Preferably, the preparation is an injection;

Preferably, the formulation is intravenous injection, intramuscular injection, subcutaneous injection or intrathecal injection;

Preferably, the preparation also includes a pharmaceutically acceptable carrier;

Preferably, the pharmaceutical carrier includes one or more of diluents, excipients, fillers, binders, disintegrants, surfactants and lubricants.