Embryo mesenchymal stem cell-derived exosome and preparation method thereof, and application of exosome as protective agent in medicines for improving striae gravidarum of pseudopregnant rats
By injecting with embryonic mesenchymal stem cell-derived exosomes, the problem of difficulty in preventing and treating stretch marks is solved, significantly improving the clinical manifestations of stretch marks and improving the sensitivity and effect of treatment.
Patent Information
- Application Number
- CN202411821738.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-25
AI Technical Summary
Stretch marks are a common skin injury, and the prior art is difficult to effectively prevent and treat, resulting in breakage of skin elastic fibers, reduced hair follicles and atrophy of the skin.
Embryo mesenchymal stem cells were cultured in vitro, their derivation exosomes were isolated and extracted, and the exosomes were injected locally subcutaneously into the abdominal area to prevent and treat the application of stretch marks in pseudo-pregnant mice.
Significantly improve the occurrence and development of stretch marks, reduce the fracture of elastic fibers, promote hair follicles growth, and improve the sensitivity and effect of treatment.
Smart Images
Figure CN119685245A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of skin damage protection, and in particular to an application of embryonic mesenchymal stem cell-derived exosomes in the preparation of drugs for preventing and protecting pseudopregnant mouse stretch marks and stretch marks caused by other reasons, as well as a drug and model construction method. Background Art
[0002] The skin is the outermost organ that protects against damage, pathogens and maintains homeostasis with external factors. Stretch marks (striae gravidarum, SG) are a type of stretch marks (striae diensae, SD) that develop specifically during pregnancy in almost 60-90% of women. They usually appear on the abdomen and generally do not disappear. Over a period of months to years, the skin lesions gradually shrink and are accompanied by hypopigmentation and loss of pigmentation.
[0003] The histopathological manifestations of stretch marks are that the epidermis of early skin lesions may be normal, the dermal blood vessel cavity is dilated, and the surrounding lymphocytes are infiltrated. Under the electron microscope, mast cell degranulation, elastic tissue dissolution, and macrophages phagocytose elastic tissue can be seen. In the late stage, the epidermis becomes thinner, the epidermal ridges become flat, the dermis also becomes thinner, the collagen in the upper dermis decreases, the collagen bundles become thinner and are arranged parallel to the epidermis, and the skin appendages are reduced or even missing. The histological manifestations are similar to scars. In the early stage, the elastic fibers in the superficial dermis are broken and become scarce, the collagen fibers are separated and homogenized, the blood vessel walls are thickened, the lumen is dilated, and there is edema around the blood vessels and lymphocytes infiltration. In the late stage, the epidermis becomes thinner, the spinous cell layer atrophies, the epidermal ridges become flat, the dermis becomes thinner, and the superficial dermis has straight and thin collagen bundles arranged parallel to the skin, the cell nuclei are scarce, and the hair follicles, sweat glands and sebaceous glands also atrophy.
[0004] Stretch marks are also called atrophic striae. Their clinical manifestations are usually multiple, symmetrical, clearly demarcated, and often linear or cord-like lesions along the direction of the skin grain. They are initially red, pink, or violet slightly raised stripes, called red striae (striae rubra), which are usually asymptomatic or accompanied by mild itching. Subsequently, the color turns pale and shiny, the lesions are slightly sunken, the surface is smooth, and there may be fine wrinkles. They are soft to the touch, called white striae (stirae alba), which are asymptomatic and generally do not disappear, but can be alleviated over time. Over a period of months to years, the lesions gradually shrink and are accompanied by hypopigmentation and loss of pigment.
[0005] The pathogenesis of stretch marks varies, mainly including: (1) Increased skin tension: The dermal collagen fiber bundles are severely damaged, especially in the middle to deep dermis, with obvious separation. The dermal collagen fiber bundles are severely damaged, especially in the middle to deep dermis, with obvious separation. (2) Fibroblasts: Compared with normal fibroblasts, the expression of type I and type III procollagen in fibroblasts in stretch mark tissue is significantly reduced, indicating that the disease may have abnormal fibroblast metabolism. (3) Collagenase-II: Fibroblast aberration may increase the secretion of collagenase-2, which causes the occurrence of stretch marks by dissolving type I collagen. (4) Estrogen and estrogen receptors: Estrogen increases skin thickness, elastic fibers in the papillary layer, and skin collagen (mainly I and III), reduces the activity of metalloproteinases in the skin, thereby delaying skin aging, and can also increase skin elasticity by increasing skin water content. Estrogens are essential for collagen synthesis in the dermis of the skin, and they have a clear role in the formation and degradation of the extracellular matrix (type I collagen is the main structural component). (5) Androgens: The disruption of the balance between serum estrogen and androgen levels may have a significant impact on the tissue repair process, while wound healing is accelerated by blocking the action of androgens. The expression of androgens in stretch marks increases, and they have a negative impact on collagen synthesis in stretch marks. The expression of estrogen receptors in stretch marks is about twice that of normal skin, and the expression of androgen and glucocorticoid receptors is also increased, which indicates that the expression of active hormone receptors is higher in skin that is subjected to greater mechanical tension. (6) Glucocorticoids: The surge in glucocorticoids inhibits the activity and proliferation of fibroblasts, reduces the amount of elastic fibers and collagen synthesized by these cells, and thus prevents the complete repair of damaged connective tissue in the dermis. (7) Pigmentation: Capillary dilation and increased melanocytes (affected by androgens). (8) High-risk factors: young age / fast weight gain / large abdominal volume expansion.
[0006] Exosomes are extracellular vesicles with a diameter of about 30-150 nm. They were first discovered in the reticulocytes of mature mammals [1-3]. Extracellular vesicles are vesicles secreted from cells to the outside of cells. According to their diameter and morphological characteristics, they are divided into three categories: exosomes (30-150 nm), microvesicles (100 nm–1 μm), and apoptotic bodies (>2 μm). Exosomes have an endocytic origin. First, the cell membrane indents inward to form early endosomes, which further indent to form multivesicular bodies, also known as late endosomes. Multivesicular bodies contain multiple tiny vesicles. Some multivesicular bodies fuse with lysosomes and are then degraded; the other multivesicular bodies fuse with the cell membrane and release the vesicles contained inside them to the outside of the cell, which are exosomes. Exosomes can be released by different types of living cells, such as macrophages, tumor cells, dendritic cells, mesenchymal stem cells, mast cells, adipocytes, neurons, fibroblasts, etc., and are widely present in various body fluids, such as urine, milk, blood, ascites, saliva, amniotic fluid, synovial fluid, malignant pleural effusion, etc. Studies have shown that unicellular eukaryotic organisms Cryptococcus neoformans and Leishmania can also produce exosomes.
[0007] Small vesicles with biological activity secreted by various tissue cells enter the extracellular environment and are called extracellular vesicles. Under electron microscopy, they are cup-shaped or disc-shaped and have a lipid bilayer structure. Their composition includes nucleic acids, lipids, proteins and other substances. Exosomes contain members of the tetramer cross-linker family (CD9, CD63, CD81), heat shock protein family (HSP70, HSP90), membrane proteins, cytoskeleton proteins (tubulin, actin), transport-essential endosomal sorting complex ALG-2-interacting protein X (ALX), phospholipases, and major histocompatibility complex (MHC) molecules, and some are derived from the selective assembly of protein components in the mother cell. Different proteins are present due to different cell sources and stimulation methods. In addition to proteins, exosomes also contain a large number of nucleotides, such as DNA, mRNA, microRNA, long-chain noncoding RNA (lncRNA) and small interfering RNA (siRNA). After these nucleic acid components are transported to the target cells, they can be expressed in the recipient cells or regulate the gene expression of the recipient cells, causing changes in the biological behavior of the cells and exerting unique biological effects. The lipid components in exosomes mainly include sphingomyelin, cholesterol, phosphatidylcholine, phosphatidylserine and ceramide. The surface of exosomes is rich in phosphatidyl amino acids, which are conducive to the internalization of their receptor cells and play an important role in the biological functions of exosomes. The structure of the lipid bilayer of exosomes has the function of protecting its contents from degradation, maintaining its biological activity and maintaining the morphology of exosomes.
[0008] Studies have shown that exosomes contain a variety of bioactive components, and the lipid bilayer structure of the vesicle membrane can protect them from being degraded during transfer throughout the organism, so they have the function of mediating the transfer of active substances between cells. The active signals carried by exosomes can affect the function of target cells, regulate biological behavior by specifically binding to target cells, and release the active substances contained in them for material transport and signal transmission, participating in information exchange between cells and various pathological and physiological processes.
[0009] The present invention discloses an application of mesenchymal stem cell-derived exosomes in the preparation of drugs for preventing and protecting stretch marks caused by pseudopregnancy and other reasons, and a drug and model construction method, thereby improving the occurrence and development of stretch marks. Summary of the invention
[0010] The purpose of the present invention is to provide an application of embryonic mesenchymal stem cell-derived exosomes in the preparation of drugs for preventing and protecting stretch marks caused by pseudopregnancy mice and stretch marks or atrophy marks caused by other reasons, and a drug and model construction method. The method has high sensitivity in treatment and significant improvement effect.
[0011] The first objective of the present invention is to use embryonic mesenchymal stem cell-derived exosomes in the preparation of drugs for pseudopregnant mouse stretch marks.
[0012] The second purpose of the present invention is that embryonic mesenchymal stem cell-derived exosomes can be used to prepare pseudopregnant mouse stretch marks drugs, thereby improving the occurrence and development of stretch marks.
[0013] The third object of the present invention is to use embryonic mesenchymal stem cell-derived exosomes in the preparation of pseudo-gravida stretch marks drugs, wherein the pseudo-gravida stretch marks include various stretch marks injuries caused by placing absorbent resin balls into the abdominal cavity.
[0014] In order to achieve the above object, the present invention is implemented by the following technical solutions:
[0015] After culturing primary embryonic mesenchymal stem cells in vitro, the supernatant is taken for ultracentrifugation to separate exosomes. The produced exosomes can affect the function of target cells, regulate biological behavior by specifically binding to target cells, and release the active substances contained therein for material transport and signal transmission, participating in information exchange between cells and various pathological and physiological processes, thereby repairing damage.
[0016] A method for preparing exosomes derived from embryonic mesenchymal stem cells comprises the following steps:
[0017] 1) Isolation and culture of mouse embryonic mesenchymal stem cells (eMSCs)
[0018] 1) Usually, mouse embryos at the 14-16 day (E14-E16) stage are selected for the experiment under sterile conditions. The mother mouse is anesthetized, the uterus is removed and the embryo is carefully dissected out. The embryo is washed with PBS to remove attached blood and impurities.
[0019] 2) Cut the mouse embryo (usually the back, limbs and liver tissues) into small pieces (1-2 mm) using small scissors or needles under a microscope. 3 ).
[0020] 3) Add the embryonic fragments to a culture medium containing collagenase II (0.1%-0.2%) and digest in a 37°C water bath for 1-2 hours. Gently shake every 15-20 minutes to promote tissue digestion.
[0021] 4) After digestion, filter the tissue particles using a cell strainer (40-70 μm) to obtain a cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge (500 g, 10 minutes), and remove the supernatant. Wash the cell pellet with sterile PBS to remove enzymes and impurities.
[0022] 5) Further separate mononuclear cells (MNCs) from other cell types by Ficoll density gradient centrifugation (400g, 20 minutes). Collect the MNC layer, remove the Ficoll layer, transfer to a new tube, and wash with culture medium.
[0023] 6) The isolated cells were divided into 1×10 6 The cells / mL density is inoculated into pre-coated culture dishes or culture bottles (you can choose to add 0.1% gelatin or 0.01% fibronectin to the culture medium to promote the attachment of MSCs). The culture medium is DMEM / F-12 (or α-MEM), supplemented with 10%-20% fetal bovine serum (FBS) and antibiotics (penicillin / streptomycin).
[0024] 7) Culture in a 37°C, 5% CO2 incubator. Change the culture medium once a day until the cells reach 70%-80% confluence. Mesenchymal stem cells usually have strong adhesion, and the cells will quickly attach to the bottom of the culture dish to form spindle-shaped or star-shaped cell colonies.
[0025] 8) When the cell confluence reaches 70%-80%, digest the cells with trypsin-EDTA (0.25%). Gently blow the cell suspension to disperse the cells, neutralize the enzyme solution with culture medium, and collect the cells again.
[0026] 9) Subculture the cells into new culture dishes or culture flasks at a ratio of 1:3 and continue culturing. Change the culture medium every 2-3 days.
[0027] 2) Isolation of exosomes using ultracentrifugation-based methods
[0028] 1) Collect the supernatant of embryonic mesenchymal stem cells cultured in vitro, centrifuge at 300*g for 10 min, collect the supernatant into a new centrifuge tube and continue centrifuging at 2000*g for 10 min to remove residual cells.
[0029] 2) Transfer the supernatant from the previous step to a new centrifuge tube and centrifuge at 10,000 × g for 30 min to remove excess cells and cell debris.
[0030] 3) Transfer the collected supernatant to an ultracentrifuge tube, load it into an ultracentrifuge according to the instructions for the corresponding tube and rotor, centrifuge at 120,000*g for 90 minutes, discard the supernatant, and add high-pressure filtered PBS to resuspend and settle.
[0031] 4) The resuspension was centrifuged again at the same centrifugal force and time as in the previous step (120,000*g, 90 min) to wash the precipitate, and the supernatant was discarded.
[0032] 5) Resuspend the pellet with about 100ul PBS and store at -80℃ for later use or use immediately. All the above centrifugation steps were performed at 4℃.
[0033] The invention discloses an application of embryonic mesenchymal stem cell-derived exosomes as protective agents in the preparation of drugs for improving stretch marks in pseudopregnant mice, and a drug and model construction method.
[0034] In the above application, the pseudo-pregnant mouse stretch marks are manifested as rupture of elastic fiber bundles and hyperplasia of hair follicles in the abdominal skin, expansion of the abdominal cavity and promotion of atrophic striae; flattening of the epidermal ridges, thinning of the dermis, reduction of collagen in the upper dermis, thinning of the collagen bundles and their arrangement parallel to the epidermis, and reduction or even loss of skin appendages.
[0035] In the above application, the pseudo-pregnant mouse stretch marks are manifested as rupture of elastic fiber bundles in the abdominal skin and hyperplasia of hair follicles, expansion of the abdominal cavity and promotion of atrophic striae.
[0036] In the above application, the pathogenesis of stretch marks in pseudopregnant mice includes increased skin tension, abnormal fibroblast metabolism, collagenase-II secretion, increased estrogen and its receptors, disorder of estrogen and androgen balance, surge of glucocorticoids, pigmentation and other high-risk factors including rapid weight gain / large expansion of abdominal cavity volume, etc.
[0037] In the above application, the dosage of the embryonic mesenchymal stem cell-derived exosomes is 1 nmol / 20 g body weight, which is injected once every 3 days for a cycle of 21 days. The subjects are Balb / c or C57 mice and other animals with stretch marks. The route of administration is local subcutaneous injection in the abdomen. The treatment method is fast, simple, reasonable and effective.
[0038] In the above application, the pseudo-pregnant mouse stretch marks include various stretch mark injuries caused by placing absorbent resin balls into the abdominal cavity.
[0039] A method for constructing a pseudo-pregnancy mouse stretch mark drug validation model, the method for constructing a pseudo-pregnancy mouse stretch mark drug validation model is characterized in that the model construction method comprises injecting the embryonic mesenchymal stem cell-derived exosome drug into mice after pseudo-pregnancy mouse stretch mark modeling, and the model animal is safe and effective without obvious side effects and complications.
[0040] The method for constructing the above-mentioned injury drug validation model specifically includes the following steps:
[0041] 1) Select 3-4 week old female mice to establish pseudo-pregnant stretch marks model;
[0042] 2) After the ovaries were removed by abdominal surgery, a 1.5 cm longitudinal incision was made in the middle of the abdomen, reaching the peritoneum. The absorbent resin ball was placed into the abdominal cavity, and the peritoneum, abdominal skin, and subcutaneous tissue were sutured, and the wound was cleaned.
[0043] 3) Regular and quantitative intraperitoneal injection of normal saline and estrogen and progesterone, the normal saline injection dose is 1ml / 20g body weight, the estrogen and progesterone injection dose is 1μg / ml E2+10mg / ml P4, each 0.1ml / 20g body weight, the injection time is once a day, and the cycle is 21 days;
[0044] 4) After modeling, the mice were locally injected with the embryonic mesenchymal stem cell-derived exosome drugs subcutaneously in the abdomen, with a dosage of 1 nmol / 20 g body weight. The injection time for the prevention group was once every 3 days, and the injection time for the treatment group was once every 3 days starting from the 12th day after modeling, with a cycle of 21 days;
[0045] 5) The abdominal skin damage and repair of the mice were then detected.
[0046] The method for constructing the above-mentioned pseudo-pregnant mouse stretch mark drug verification model, the detection method is transmission electron microscopy technology, blood biochemical analysis and HE staining.
[0047] The present invention establishes a pseudo-pregnant mouse stretch mark model, and transmission electron microscopy technology, blood biochemical analysis and HE staining quantitative analysis find that compared with the normal group, after simple hormone injection based on modeling, elastic fiber breakage and hair follicle reduction can be observed, while the prevention group has a reduced elastic fiber breakage and an increased hair follicle compared to the hormone group. The treatment group also has a reduced elastic fiber breakage compared to the hormone group, and there is also an increased hair follicle. In summary, the effect of exosomes on abdominal skin may reduce fiber breakage and may stimulate the growth of hair follicles. The above-mentioned technology is used for identification and analysis, and it is determined that the embryonic mesenchymal stem cell-derived exosomes are effective protective agents for the treatment of the injury model; the pseudo-pregnant mouse stretch mark drugs are prepared by embryonic mesenchymal stem cell-derived exosomes to prevent, protect and improve the occurrence and development of stretch marks, thereby playing a protective role against stretch marks or atrophic striae caused by other reasons, and a new treatment method is proposed.
[0048] Therefore, the present invention claims to protect the use of embryonic mesenchymal stem cell-derived exosomes in the preparation of drugs for preventing and protecting stretch marks caused by pseudopregnancy mice and stretch marks or atrophy marks caused by other reasons.
[0049] The use of the exosomes derived from embryonic mesenchymal stem cells to prepare pseudo-pregnant mouse stretch marks drugs in improving stretch marks also falls within the protection scope of the present invention.
[0050] A kind of embryonic mesenchymal stem cell-derived exosomes are used in the preparation of drugs for preventing and protecting stretch marks in pseudo-pregnant mice and stretch marks or atrophic striae caused by other reasons, thereby improving stretch marks or atrophic striae and other injuries. It is safe and effective for model animals, without obvious side effects and complications.
[0051] The method for constructing a pseudo-pregnant mouse stretch mark drug validation model uses the above-mentioned pseudo-pregnant mouse stretch mark drugs, and the model construction method is: locally injecting the embryonic mesenchymal stem cell-derived exosome drugs into the subcutaneous abdomen of the pseudo-pregnant mouse model.
[0052] Most preferably, the exosomes derived from embryonic mesenchymal stem cells are used in the preparation of drugs for pseudo-pregnancy stretch marks, wherein the pseudo-pregnancy stretch marks include but are not limited to various types of stretch mark injuries caused by placing absorbent resin balls into the abdominal cavity.
[0053] Its treatment includes the following steps:
[0054] 1. Establish a pseudo-pregnant mouse stretch mark injury model.
[0055] 2. Drugs prepared by exogenously overexpressing exosomes derived from embryonic mesenchymal stem cells.
[0056] The drug prepared by exosomes derived from embryonic mesenchymal stem cells was locally injected subcutaneously in the abdomen into the pseudo-pregnant mice with stretch marks.
[0057] 3. Analysis of each indicator:
[0058] Transmission electron microscopy was used to detect whether the morphology of exosomes obtained by ultracentrifugation met the standard; blood biochemistry and other tests were used to detect whether the water-absorbent resin balls would have toxic effects on the mouse body after being placed in the abdominal cavity; HE staining was used to detect whether the elastic fiber bundles in the abdominal skin of mice were broken as the abdominal circumference of the mice increased, and whether the preventive injection of embryonic mesenchymal stem cell-derived exosomes and the injection of embryonic mesenchymal stem cell-derived exosomes for treatment later improved the damage to the elastic fibers in the abdominal skin of mice. Overexpression of embryonic mesenchymal stem cell-derived exosomes can partially reverse the above phenomenon and improve stretch mark damage.
[0059] Compared with the prior art, the present invention has the following beneficial effects:
[0060] The present invention discloses the use of exosomes derived from embryonic mesenchymal stem cells as protective agents in the preparation of drugs for improving stretch marks in pseudopregnant mice, as well as methods for constructing drugs and models, and relates to the field of biomedicine. Drugs for improving stretch marks in pseudopregnant mice are prepared by exosomes derived from embryonic mesenchymal stem cells; the method for constructing a protective agent model for verifying stretch marks in pseudopregnant mice includes: subcutaneously injecting exosomes derived from embryonic mesenchymal stem cells into mice after pseudopregnant mice are modeled. The present invention has found that exosomes derived from embryonic mesenchymal stem cells can be used to prepare drugs for improving stretch marks in pseudopregnant mice, thereby improving the damage to abdominal skin fibers caused by breakage, and preventing and protecting abdominal elastic fibers caused by pregnancy and other reasons. The treatment sensitivity of this method is high and the improvement effect is significant. The present invention has good application value and promotion prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0061] Figure 1 An animal model of stretch marks in pseudo-pregnant mice was established.
[0062] Figure 2 To analyze the results of blood biochemistry and observe the toxicological effects of absorbent resin balls placed in the peritoneal cavity on mice.
[0063] Figure 3 Morphological detection of exosomes obtained by ultracentrifugation.
[0064] Figure 4 HE staining of the abdominal skin of mice in the control group, hormone group, prevention group and treatment group was performed to observe the improvement of the damage of elastic fibers in the abdominal skin. Figure 5 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION
[0065] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.
[0066] Example 1
[0067] This embodiment provides a method for preparing exosomes derived from embryonic mesenchymal stem cells:
[0068] 1) Isolation and culture of mouse embryonic mesenchymal stem cells (eMSCs)
[0069] 1) Usually, mouse embryos at the 14-16 day postnatal stage are selected. Under sterile conditions, the mother mouse is anesthetized, the uterus is removed and the embryos are carefully dissected out. The embryos are washed with PBS to remove attached blood and impurities.
[0070] 2) Cut the mouse embryo (usually the back, limbs and liver tissues) into small pieces (1-2 mm) using small scissors or needles under a microscope. 3 ).
[0071] 3) Add the embryonic fragments to a culture medium containing collagenase II (0.1%-0.2%) and digest in a 37°C water bath for 1-2 hours. Gently shake every 15-20 minutes to promote tissue digestion.
[0072] 4) After digestion, filter the tissue particles using a cell strainer (40-70 μm) to obtain a cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge (500 g, 10 minutes), and remove the supernatant. Wash the cell pellet with sterile PBS to remove enzymes and impurities.
[0073] 5) Further separate mononuclear cells (MNCs) from other cell types by Ficoll density gradient centrifugation (400g, 20 minutes). Collect the MNC layer, remove the Ficoll layer, transfer to a new tube, and wash with culture medium.
[0074] 6) The isolated cells were seeded into pre-coated culture dishes or culture flasks at a density of 1×106 cells / mL, and 0.1% gelatin or 0.01% fibronectin was added to the culture medium to promote the attachment of MSCs. The culture medium is DMEM / F-12 (α-MEM can also be selected), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin / streptomycin).
[0075] 7) Culture in a 37°C, 5% CO2 incubator. Change the culture medium once a day until the cells reach 70%-80% confluence. Mesenchymal stem cells usually have strong adhesion, and the cells will quickly attach to the bottom of the culture dish to form spindle-shaped or star-shaped cell colonies.
[0076] 8) When the cell confluence reaches 70%-80%, digest the cells with trypsin-EDTA (0.25%). Gently blow the cell suspension to disperse the cells, neutralize the enzyme solution with culture medium, and collect the cells again.
[0077] 9) Subculture the cells into new culture dishes or culture bottles at a ratio of 1:3 and continue culturing. Change the culture medium every 2 days.
[0078] 2) Isolation of exosomes using ultracentrifugation-based methods
[0079] 1) Collect the supernatant of embryonic mesenchymal stem cells cultured in vitro, centrifuge at 300*g for 10 min, collect the supernatant into a new centrifuge tube and continue centrifuging at 2000*g for 10 min to remove residual cells.
[0080] 2) Transfer the supernatant from the previous step to a new centrifuge tube and centrifuge at 10,000 × g for 30 min to remove excess cells and cell debris.
[0081] 3) Transfer the collected supernatant to an ultracentrifuge tube, load it into an ultracentrifuge according to the instructions for the corresponding tube and rotor, centrifuge at 120,000*g for 90 minutes, discard the supernatant, and add high-pressure filtered PBS to resuspend and settle.
[0082] 4) The resuspension is centrifuged again at the same centrifugal force and time as in the previous step (120,000 x g for 90 min) to wash the precipitate and discard the supernatant.
[0083] 5) Resuspend the pellet with 100ul PBS and store at -80℃ for later use or use immediately. All the above centrifugation steps were performed at 4℃.
[0084] Example 2 Exogenous intraperitoneal placement of absorbent resin balls induces obvious stretch marks in mice, which can be used as an ideal animal model for studying stretch marks in pseudopregnant mice
[0085] 1. Test Method
[0086] Starting from the first day of modeling, the changes in the abdominal circumference and abdominal skin of the mice were observed.
[0087] 2. Test Results
[0088] The amount of water that the absorbent resin ball can absorb in the abdominal cavity is saturated, so in the formal experiment, we adopted a small amount of injection once, injection every other day / interval injection method to ensure the continuous growth of abdominal circumference. In the later stage, we adopted a daily / large amount of injection method to simulate the normal abdominal circumference growth trend in the late pregnancy. It can be concluded that this modeling was successful ( Figure 1 AC). In pseudo-pregnant mice, abdominal fiber bundles were broken and atrophic striae were produced within 21 days of simulated pregnancy. Estrogen (gradually increased, with a significant increase from 1 to 6 days) and progesterone (increased first and then decreased, with the most significant increase on 12 days) were positively correlated with the degree of abdominal expansion during the simulated pregnancy cycle and promoted the production of atrophic striae ( Figure 1 (in DF).
[0089] Example 3: Exogenous intraperitoneal placement of absorbent resin balls has no toxic effect on the mouse body
[0090] 1. Test Method
[0091] The blood biochemical results of mice were analyzed by blood drawing method.
[0092] 2. Test Results
[0093] According to the organ weight, blood routine and biochemical analysis results, it can be seen that the exogenous placement of absorbent resin balls into the abdominal cavity and all surgical operations and injection treatments only caused mild inflammation and had no toxic effects on the mouse body, proving the safety of the experiment ( Figure 2 (in AC).
[0094] Example 4: Ultracentrifugation and extraction of desired exosomes after in vitro culture of primary embryonic mesenchymal stem cells
[0095] 1. Test Method
[0096] Primary embryonic mesenchymal stem cells are cultured in vitro, and the supernatant is taken and ultracentrifuged. The exosomes obtained are needed for the treatment of stretch marks in pseudopregnant mice.
[0097] 2. Test Results
[0098] In order to identify whether the morphology of exosomes obtained by ultracentrifugation meets the standard, we detected their morphological characteristics by negative staining transmission electron microscopy. It has been reported in the literature that the classic exosome morphology under negative staining transmission electron microscopy is a saucer-like membrane structure with a particle size between 30-150nm. As shown in the figure, the exosomes obtained by ultracentrifugation method all have obvious membrane structures under electron microscopy, which are oval saucer-like structures of varying sizes with a diameter within 200nm ( Figure 3 ). The exosomes obtained by the three ultracentrifugation methods have similar structures, but their distribution is uneven, some are in clusters, and some are distributed individually, indicating that the exosomes obtained by the ultracentrifugation method basically conform to the classic exosome morphological characteristics.
[0099] Example 5 Exogenous overexpression of embryonic mesenchymal stem cell-derived exosomes can improve stretch marks in pseudo-pregnant mice
[0100] 1. Test Method
[0101] HE staining of the abdominal skin of mice in the control group, hormone group, prevention group, and treatment group was compared to observe the improvement of the damage to the elastic fibers in the abdominal skin.
[0102] 2. Test Results
[0103] HE staining results showed that compared with the normal group, after simple hormone injection on the basis of modeling, elastic fiber breakage and hair follicle reduction were observed, while the prevention group showed less elastic fiber breakage and more hair follicles compared with the hormone group. The treatment group also showed less elastic fiber breakage and more hair follicles compared with the hormone group ( Figure 4 ). In summary, the effect of exosomes on abdominal skin may reduce fiber breakage and may stimulate the growth of hair follicles.
[0104] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. It is particularly pointed out that for those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.
Claims
1. A method for preparing exosomes derived from embryonic mesenchymal stem cells, characterized in that: The steps include: 1) Isolation and culture of mouse embryonic mesenchymal stem cells (eMSCs) 1) Select mouse embryos at 14-16 days after birth, anesthetize the mother mouse under sterile conditions, remove the uterus and carefully dissect out the embryos; wash the embryos with PBS to remove attached blood and impurities; 2) Cut the mouse embryo into 1-2 mm pieces using small scissors or a needle under a microscope. 3 small pieces; 3) Add the embryo pieces to the culture medium containing collagenase II and digest them in a 37°C water bath for 1-2 hours; Gently shake every 15-20 minutes to promote tissue digestion; 4) After digestion, filter the tissue particles using a 40-70 μm cell strainer to obtain a cell suspension; Transfer the cell suspension to a centrifuge tube, centrifuge, and remove the supernatant; wash the cell pellet with sterile PBS to remove enzymes and impurities; 5) Further separation of mononuclear cells (MNCs) from other cell types by Ficoll density gradient centrifugation; The MNC layer was collected, the Ficoll layer was removed, transferred to a new tube, and washed with culture medium; 6) The isolated cells were divided into 1×10 6 The cells / mL are inoculated into a pre-coated culture dish or culture bottle, and 0.1% gelatin or 0.01% fibronectin is added to the culture medium to promote the attachment of MSCs; the culture medium is DMEM / F-12, supplemented with 10%-20% fetal bovine serum FBS by mass percentage concentration, and 1%-10% antibiotics by mass percentage concentration; the antibiotics are penicillin or streptomycin; 7) Culture in a 37°C, 5% CO2 incubator, and change the culture medium once a day until the cells reach 70%-80% confluence; mesenchymal stem cells usually have strong adhesion, and the cells will quickly attach to the bottom of the culture dish to form spindle-shaped or star-shaped cell colonies; 8) When the cell confluence reaches 70%-80%, digest the cells with trypsin-EDTA at a mass percentage concentration of 0.25%; gently blow the cell suspension to disperse the cells, neutralize the enzyme solution with culture medium, and recollect the cells; 9) Subculture the cells into new culture dishes or culture bottles at a ratio of 1:3 and continue culturing; replace the culture medium every 2-3 days; 2) Isolation of exosomes using ultracentrifugation-based methods 1) Collect the supernatant of mouse embryonic mesenchymal stem cells cultured in vitro, centrifuge, collect the supernatant into a new centrifuge tube and continue centrifugation to remove residual cells; 2) Transfer the supernatant from the previous step to a new centrifuge tube and centrifuge to remove excess cells and cell debris; 3) Transfer the collected supernatant to an ultracentrifuge tube, load it into an ultracentrifuge according to the instructions for the corresponding tube and rotor, centrifuge, discard the supernatant, and add high-pressure filtered PBS to resuspend and settle; 4) The resuspended liquid is centrifuged again at the same centrifugal force and time as in the previous step to wash the precipitate, and the supernatant is discarded; 5) Resuspend the pellet with 100ul PBS and store at -80℃ for later use or use immediately; all the above centrifugation steps are carried out at 4℃.
2. The preparation method according to claim 1, characterized in that: The percentage concentration of the total mass of collagenase II in the culture fluid containing collagenase II to the total mass of the culture medium is 0.1%-0.2%.
3. An embryonic mesenchymal stem cell-derived exosome prepared by the preparation method according to claim 1 or 2.
4. Use of the embryonic mesenchymal stem cell-derived exosomes according to claim 3 as a protective agent in the preparation of drugs for improving stretch marks in pseudopregnant mice.
5. The use according to claim 4, characterized in that: The pseudo-pregnant mouse stretch marks are manifested as rupture of elastic fiber bundles in the abdominal skin and hyperplasia of hair follicles, expansion of the abdominal cavity and promotion of atrophic striae; flattening of the epidermal ridges, thinning of the dermis, reduction of collagen in the upper dermis, thinning of the collagen bundles and their arrangement parallel to the epidermis, and reduction or even loss of skin appendages.
6. The use according to claim 4, characterized in that: The pathogenesis of stretch marks in pseudopregnant mice includes increased skin tension, abnormal fibroblast metabolism, collagenase-II secretion, increased estrogen and its receptors, disorder of estrogen and androgen balance, surge of glucocorticoids, pigmentation and other high-risk factors including rapid weight gain / large expansion of abdominal cavity volume.
7. The use according to claim 4, characterized in that: The dosage of the embryonic mesenchymal stem cell-derived exosomes is 1 nmol / 20 g body weight, which is injected once every 3 days for a cycle of 21 days. The subjects are Balb / c or C57 mice and other animals with stretch marks, and the route of administration is local subcutaneous injection in the abdomen.
8. The use according to claim 4, characterized in that: The pseudo-pregnant mouse stretch marks include various types of stretch mark injuries caused by placing water-absorbent resin balls into the abdominal cavity.
9. A method for constructing a pseudo-pregnant mouse stretch mark drug validation model, characterized in that: The model construction method comprises: injecting the mouse embryonic mesenchymal stem cell-derived exosome drug into mice after pseudo-pregnant mouse stretch marks modeling, and the drug is safe and effective for the model animals without obvious side effects and complications.
10. The method for constructing a disease-related drug validation model according to claim 9, characterized in that: The specific steps include: 1) Select 3-4 week old female mice to establish pseudo-pregnant stretch marks model; 2) After the ovaries were removed by abdominal surgery, a 1.5 cm longitudinal incision was made in the middle of the abdomen, reaching the peritoneum. The absorbent resin ball was placed into the abdominal cavity, the peritoneum, abdominal skin and subcutaneous tissue were sutured, and the wound was cleaned; 3) Regular and quantitative intraperitoneal injection of normal saline and estrogen and progesterone, the normal saline injection dose is 1ml / 20g body weight, the estrogen and progesterone injection dose is 1μg / ml E2+10mg / ml P4, each 0.1ml / 20g body weight, the injection time is once a day, and the cycle is 21 days; 4) After modeling, the mice were locally injected with the embryonic mesenchymal stem cell-derived exosome drugs subcutaneously in the abdomen, with a dosage of 1 nmol / 20 g body weight. The injection time for the prevention group was once every 3 days, and the injection time for the treatment group was once every 3 days starting from the 12th day after modeling, with a cycle of 21 days; 5) Then, the abdominal skin damage and repair of the mice were detected; The detection methods include transmission electron microscopy, blood biochemical analysis and HE staining.