Extracellular vesicles helpful for cell repair, preparation method, preparation and application of extracellular vesicles helpful for cell repair

Abstract

The invention relates to an extracellular vesicle for facilitating cell repair, which is formed by inducing stem cell secretion through silicate, contributes to the expression of index factors such as inflammation resistance, tissue regeneration and cell repair, and accelerates the cell growth of wound tissues and the time interval of wound repair.

Description

Technical Field

[0001] The present invention relates to an extracellular vesicle, and in particular to an extracellular vesicle that helps cell repair, a preparation method, a preparation and use thereof. Background Art

[0002] There are many possible factors that may lead to poor wound healing, ranging from failure to properly clean / care for the wound to the patient's physical condition, such as advanced age, malnutrition, diabetes, autoimmune disease, cancer, or bad habits such as smoking and drinking, which are closely related to the speed of wound healing.

[0003] In addition to debridement, the use of antibiotics, wound dressings, and other methods, new treatment methods, including topical application of human growth factors, negative pressure therapy, hyperbaric oxygen therapy, and biotechnology skin transplantation, are expensive and have limitations in the extent to which they can accelerate wound healing. The development of technologies that help accelerate wound healing remains an urgent research goal in related fields. Summary of the invention

[0004] In order to develop a technology that helps accelerate wound healing, the present invention provides an extracellular vesicle that helps cell repair, which is a silicate-induced extracellular vesicle formed by secretion of a stem cell induced by silicate.

[0005] The step of preparing the silicate-induced extracellular vesicles comprises:

[0006] Obtain 1 g of calcium silicate and add it to 10 to 100 ml of a culture solution and soak it for at least 24 hours, collect the clarified liquid and remove the calcium element to prepare a silicate culture solution;

[0007] Cultivating the stem cells in the silicate culture medium; and

[0008] A silicate-induced extracellular vesicle secreted by the stem cells was isolated.

[0009] The culture medium is mesenchymal stem cell medium (MSCM) or Dulbecco's Modified Eagle Medium (DMEM).

[0010] The present invention also provides a preparation for assisting cell repair, comprising a silicate-induced extracellular vesicle (CSEV) formed by secretion of a stem cell induced by silicate.

[0011] The invention comprises a hydrogel for carrying the silicate-induced extracellular vesicles (CSEV).

[0012] The ratio of silicate-induced extracellular vesicles (CSEV) in the hydrogel is between 10 9 -10 14 particles / mL.

[0013] The silicate-induced extracellular vesicle (CSEV) provided by the present invention has a significantly higher surface potential, total protein amount and transmembrane protein content than that of an ordinary extracellular vesicle, which helps to increase the affinity of the silicate-induced extracellular vesicle (CSEV) for receiving cells for endocytosis. Cell experiments and animal experiments have confirmed that the silicate-induced extracellular vesicle (CSEV) easily enters cells through endocytosis, and helps to express index factors such as anti-inflammation, tissue regeneration and cell repair, and accelerates cell growth of wound tissue and the wound repair process. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing changes in the content of calcium ions and silicon ions in the silicate culture solution provided by the present invention;

[0015] Figure 2 This is a diagram of detecting cell activity after stem cell culture provided by the present invention;

[0016] Figure 3A This is a penetration electron microscope image of the extracellular vesicle provided by the present invention;

[0017] Figure 3B This is a detection diagram of the extracellular vesicle nanoparticle tracking analysis provided by the present invention;

[0018] Figure 3C This is a graph of the detection of the surface potential of the extracellular vesicle membrane provided by the present invention;

[0019] Figure 3D This is a graph showing the expression level of extracellular vesicle proteins provided by the present invention;

[0020] Figures 3E to 3G This is a graph showing the expression ratio of the extracellular vesicle transmembrane proteins CD9, CD63 and CD81 provided by the present invention;

[0021] Figure 3H This is an imaging diagram of the extracellular vesicle protein expression provided by the present invention;

[0022] Figure 4A This is a cell activity detection diagram of the diabetic cell model provided by the present invention;

[0023] Figure 4B This is a diagram of detecting active oxygen free radicals in the diabetic cell model provided by the present invention;

[0024] Figure 4CThis is a fluorescence staining image of the diabetic cell model provided by the present invention;

[0025] Figure 4D This is a graph showing the fluorescence expression of the diabetic cell model provided by the present invention;

[0026] Figure 5A This is a diagram of cell activity detection after the diabetic cell model provided by the present invention is subjected to extracellular vesicle reaction;

[0027] Figure 5B This is a diagram showing detection of intracellular reactive oxygen free radicals after the diabetic cell model provided by the present invention has been subjected to an extracellular vesicle reaction;

[0028] Figure 5C This is an imaging diagram of wound healing assay after the diabetic cell model provided by the present invention reacts with extracellular vesicles;

[0029] Figure 5D This is a diagram showing the proportion of cell migration after the diabetic cell model provided by the present invention has been reacted with extracellular vesicles;

[0030] Figure 6 This is a schematic diagram of the hydrogel carrying extracellular vesicles and cell culture provided by the present invention;

[0031] Figure 7 This is a diagram of cell activity detection after the diabetic cell model provided by the present invention is cultured with hydrogel-loaded extracellular vesicles;

[0032] Figure 8 This is a detection diagram of the expression of index factors of the diabetic cell model provided by the present invention after culture;

[0033] 9A to 9C This is a graph showing the expression of miRNA in the diabetic cell model provided by the present invention after culture;

[0034] Fig.10 This is a staining diagram of wound repair sections in the diabetic animal model provided by the present invention. DETAILED DESCRIPTION

[0035] It is understood that the term "extracellular vesicles (EV)" used herein refers to bilayer lipid vesicles secreted by cells into the extracellular space. Extracellular vesicles include exosomes and microvesicles, which are a new form of intercellular communication and a new field of research in physiology, pathology, nanoscience, etc. in recent years. Extracellular vesicles are highly heterogeneous vesicles secreted by cells, and their contents include proteins, mRNA / miRNA, DNA, lipids, etc. They are classified according to their biogenesis, release pathway, size, content, and function, and the purification method is mainly distinguished by particle size.

[0036] It is to be understood that the present invention is described below in conjunction with the description, drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] The present invention provides a method for inducing a stem cell to secrete a silicate-induced extracellular vesicle (CSEV) via silicate. The stem cell is preferably a human stem cell, and the human stem cell may optionally include a human adipose-derived stem cell (ADSCs), a bone marrow-derived stem cell (BMSCs), a dental pulp stem cell (DPSC), or a Wharton's jelly MSC (WJMSC), etc., but is not limited thereto.

[0038] In this embodiment, a human adipose-derived stem cell (ADSCs) is used as a source cell for silicate-induced extracellular vesicle secretion for subsequent description. The human stem cells are cultured in a silicate culture medium with a silicate component added thereto to induce the human adipose-derived stem cells (ADSCs) to secrete the extracellular vesicles. The extracellular vesicles secreted after being induced by the silicate culture medium are further defined herein as the silicate-induced extracellular vesicles (CSEV).

[0039] The method for culturing the human adipose-derived stem cells (ADSCs) is not limited, and any method for culturing the human adipose-derived stem cells (ADSCs) and obtaining the silicate-induced extracellular vesicles (CSEV) is within the scope of the present invention.

[0040] The silicate refers to a silicic acid compound composed of silicon and oxygen (Si x O y ). The silicate compound further comprises one or more metals or hydrogen. In this embodiment, the silicate is calcium silicate.

[0041] The culture medium used for adding the silicate and culturing the human adipose stem cells is not limited, and can be mesenchymal stem cell medium (MSCM, ScienCell Research Laboratories), Dulbecco's Modified Eagle Medium (DMEM) or other common culture mediums used for culturing cells, all of which are within the scope of the present invention.

[0042] Further, the step of culturing the silicate-induced extracellular vesicles (CSEV) comprises:

[0043] Step S1-1, preparing the silicate culture medium: taking 1 g of calcium silicate, adding it into 10-100 ml of mesenchymal stem cell culture medium (MSCM) and soaking it for at least 24 hours, and collecting the clarified liquid to prepare the silicate culture medium.

[0044] Wherein, the collection of the clarified liquid can be achieved by filtering or centrifuging.

[0045] Wherein, during the step of soaking for at least 24 hours, a temperature environment or a shaking table can be provided simultaneously to promote the reaction of calcium silicate in the mesenchymal stem cell culture medium.

[0046] Please refer to Figure 1 , and further analyzed the changes in the content (concertration) of calcium ions and silicon ions in the mesenchymal stem cell culture medium when calcium silicate was added by inductively coupled plasma mass spectrometry (ICP-MS). On day 0, the release of calcium ions can be clearly seen due to the addition of calcium silicate to the mesenchymal stem cell culture medium. After the calcium silicate reacted in the mesenchymal stem cell culture medium for 24 hours and prepared the silicate culture medium, the content of calcium ions and silicon ions can be clearly measured in the prepared silicate culture medium, confirming the effectiveness of the reaction of calcium silicate in the mesenchymal stem cell culture medium.

[0047] Step S1-2, culturing the human adipose-derived stem cells (ADSCs) in the silicate culture medium and culturing in a cell culture incubator at 37° C. containing 5% CO 2 .

[0048] The human adipose-derived stem cells (ADSCs) can be cultured in a T175 culture bottle filled with the mesenchymal stem cell culture medium, and the concentration of the silicate culture medium in the mesenchymal stem cell culture medium is between 5% and 50%.

[0049] Please refer to Figure 2 , further defining the human adipose-derived stem cells (ADSCs) cultured in the silicate culture medium as a cell culture example; culturing the human adipose-derived stem cells (ADSCs) in the mesenchymal stem cell culture medium as a cell culture comparison example. By detecting the cell proliferation effect through cell activity, it can be seen that the number of cells of the human adipose-derived stem cells (ADSCs) cultured in the silicate culture medium is significantly greater than that of the cell culture comparison example from the third day onwards, and it can be inferred that the human adipose-derived stem cells (ADSCs) cultured in the silicate culture medium produce a cell proliferation effect.

[0050] The cell activity detection of cell proliferation effect can be performed by MTT assay, PrestroBlue assay or Alamar Blue assay, etc., by detecting fluorescence or absorbance to analyze cell inventory rate and proliferation effect. In this embodiment, the cell activity detection is achieved by using PrestroBlue assay.

[0051] Step S1-3, separation of the silicate-induced extracellular vesicles (CSEV): The cultured human adipose-derived stem cells (ADSCs) and the silicate culture medium were collected, and a first supernatant was collected after centrifugation at 1500 g for 5 minutes. The first supernatant was then filtered under the condition of a pore size of 0.22 microns (μm) and placed in a concentrated centrifuge tube ( Ultra-15 Centrifugal Filter Unit, Merck), and centrifuge for 15 minutes at 5000 gravitational acceleration (g), the silicate-induced extracellular vesicles (CSEV) will remain in the formed second supernatant. Then, the second supernatant is collected and the principle of polymer precipitation is used to utilize the characteristics of high molecular weight polyethylene glycol that can compete with free water molecules and bind to hydrophobic proteins and lipid molecules, so that the silicate-induced extracellular vesicles (CSEV) with lower solubility are precipitated from the second supernatant. Finally, the supernatant is removed by centrifugation at 1500g for 30 to 40 minutes, and the precipitated high-purity silicate-induced extracellular vesicles (CSEV) can be obtained by re-dissolving with phosphate buffered saline (PBS).

[0052] Experiment 1: Analysis of the properties of silicate-induced extracellular vesicles (CSEV)

[0053] The present invention further compares and analyzes the protein expression, membrane surface electrical properties and particle size of the collected silicate-induced extracellular vesicles (CSEV) with a common extracellular vesicle (EV) obtained after culture in a comparative example, so as to identify the appearance characteristics of the silicate-induced extracellular vesicles (CSEV) and the physiological data of each cell.

[0054] Figure 3A The results of transmission electron microscope (TEM) observations showed that the particle sizes of the ordinary extracellular vesicles (EV) and the silicate-induced extracellular vesicles (CSEV) were not much different, and it can be determined that the characteristics of the two are similar.

[0055] Figure 3B In addition to confirming whether the particle sizes of the common extracellular vesicles (EV) and the silicate-induced extracellular vesicles (CSEV) are similar, nanoparticle tracking analysis (NTA) can also be used to further detect whether the concentrations of the common extracellular vesicles (EV) and the silicate-induced extracellular vesicles (CSEV) are similar, so as to confirm that the common extracellular vesicles (EV) and the silicate-induced extracellular vesicles (CSEV) are both extracellular vesicles.

[0056] Figure 3C The present invention measures the membrane surface potential of the ordinary extracellular vesicle (EV) and the silicate-induced extracellular vesicle (CSEV). From the comparison of the results, it can be seen that the membrane surface potential of the silicate-induced extracellular vesicle (CSEV) is lower than -20mV, preferably between -25mV and -30mV, and its negative value is much greater than that of the ordinary extracellular vesicle (EV).

[0057] Figures 3D to 3H In the present invention, the total protein content of silicate-induced extracellular vesicles (CSEV) and the expression of multiple extracellular vesicle marker proteins (marker) are analyzed by western blot, wherein the marker proteins include transmembrane proteins CD9, CD63 and CD81, and intramembrane proteins Alix, TSG101 and HSP70.

[0058] The results show that the total protein content of the silicate-induced extracellular vesicles (CSEV) is higher than 4 μg / 10 9 , which is significantly higher than that of ordinary extracellular vesicles (EVs).

[0059] It is noteworthy that more than 40% of the silicate-induced extracellular vesicles (CSEVs) expressed transmembrane proteins CD9, CD63, and CD81 (e.g. Figures 3E to 3G ).

[0060] The above test results show that the surface potential, total protein amount and transmembrane protein content of the silicate-induced extracellular vesicles (CSEV) secreted by the human adipose-derived stem cells (ADSCs) after induction by the silicate culture medium are significantly increased, which helps to improve the affinity of the silicate-induced extracellular vesicles (CSEV) to accept cells for endocytosis.

[0061] In order to confirm whether the silicate-induced extracellular vesicles (CSEV) have effects on cell proliferation, recovery and anti-inflammation, the present invention further uses a diabetic cell model commonly used in wound healing, cell proliferation and recovery studies as a test target.

[0062] The diabetic cell model was established by referring to the experimental steps in the literature, and the human dermal fibroblasts (HDF) were cultured in a high glucose culture medium (High Glucose DMEM, HDMEM) to induce the human dermal fibroblasts (HDF) to simulate the cell physiological characteristics of diabetic patients. In the high glucose culture medium (High Glucose DMEM, HDMEM), the concentration of glucose was between 13.5mM and 25mM.

[0063] From Figure 4A and Figure 4B As shown, the proliferation effect of the human skin fibroblasts decreased with the increase of glucose and the number of days in the high-glucose culture medium. Figure 4BThe human skin fibroblast group (HG) cultured in the high glucose medium (High Glucose DMEM, HDMEM) with a glucose concentration of 25mM and the human skin fibroblast group (NG) cultured in a general medium were respectively subjected to intracellular reactive oxygen species (ROS) concentration determination as an indicator of inflammatory response. It can be found that the performance of reactive oxygen species in the human skin fibroblast group (HG) cultured in the high glucose medium (High Glucose DMEM, HDMEM) is significantly higher than that in the human skin fibroblast group (NG) cultured in a general medium. The reduction in the above-mentioned cell proliferation effect and the improvement in the performance of intracellular reactive oxygen species (ROS) can be regarded as the main physiological indicators of the diabetic cell model. The present invention will subsequently perform subsequent experimental descriptions on the diabetic cell model cultured in the high glucose medium (High Glucose DMEM, HDMEM) with a glucose concentration of 25mM.

[0064] Experiment 1, affinity test of diabetic cell model and the silicate-induced extracellular vesicles. The diabetic cell model was treated with the common extracellular vesicles and the silicate-induced extracellular vesicles, and the endocytosis effect of the diabetic cell model on the common extracellular vesicles or the silicate-induced extracellular vesicles was analyzed after 24 hours and 48 hours of reaction. Among them, the group treated with the common extracellular vesicles was a control group (EV), and the group treated with the silicate-induced extracellular vesicles was an experimental group (CSEV).

[0065] Figure 4C The intracellular range of the diabetic cell model can be defined by actin (F-actin) marked by red fluorescence, while the common extracellular vesicles and / or the silicate-induced extracellular vesicles are marked by green fluorescence, and blue fluorescence is used to mark the cell nucleus. From the results, it can be seen that the green fluorescence marking the common extracellular vesicles and / or the silicate-induced extracellular vesicles are all distributed in the intracellular range defined by red fluorescence, which can confirm that the common extracellular vesicles (EV) and / or the silicate-induced extracellular vesicles (CSEV) have indeed entered the diabetic cell model through endocytosis.

[0066] Further observation of the experimental group and the silicate-induced extracellular vesicles showed that the fluorescence of the silicate-induced extracellular vesicles in the diabetic cell model was significantly higher than that of the control group. At 48 hours, the difference in green fluorescence between the control group (EV) and the experimental group (CSEV) was even more obvious. The fluorescence intensity of the green fluorescence was detected and presented in a digital form. Figure 4D In the figure, the difference in the content of silicate-induced extracellular vesicles and the common extracellular vesicles between the experimental group (CSEV) and the control group (EV) at 48 hours can also be clearly compared.

[0067] It was further confirmed that the diabetic cell model had a cell proliferation effect after co-culturing with the silicate-induced extracellular vesicles. Figure 5A Through cell activity detection, it can be seen that the number of cells in the diabetic model cells co-cultured with the silicate-induced extracellular vesicles (experimental group CSEV) has been significantly higher than that of the first control group (Ctl) without any treatment and the second control group (EV) co-cultured with the ordinary extracellular vesicles since the third day. Among them, a significant difference was produced after co-culture to the 7th day. It can be seen that the diabetic cell model produced a cell proliferation effect after co-culture treatment with the silicate-induced extracellular vesicles (CSEV).

[0068] Figure 5B The concentration of reactive oxygen species (ROS) in the cells of the first control group (Ctl), the second control group (EV) and the experimental group (CSEV) was measured as an indicator of inflammatory response. The results show that the experimental group (CSEV) treated with the silicate-induced extracellular vesicles co-cultured in the diabetic cell model has significantly lower intracellular reactive oxygen species than the first control group (Ctl) and the second control group (EV).

[0069] Figure 5C Wound healing assays were performed on the first control group (Ctl), the second control group (EV) and the experimental group (CSEV). Scratches were made on the cell monolayers of each group, and cell images were captured under a microscope at 0 hours and 24 hours to confirm the effect of cell migration as an evaluation indicator of wound healing. The results show that in the cell images of the experimental group (CSEV) 24 hours after the scratch, cells can be seen to be evenly distributed in the scratch area. The data was converted into the cell migration ratio ( Figure 5D ) also found that the effect of cell migration in the diabetic cell model after co-culture with the silicate-induced extracellular vesicles was significantly better than that of the first control group (Ctl) and the second control group (EV).

[0070] The above experimental results can preliminarily confirm that the induction of extracellular vesicles by silicate can promote cell proliferation, cell repair and reduce the production of anti-inflammatory factors.

[0071] Furthermore, the present invention also provides a preparation comprising the silicate-induced extracellular vesicles (CSEV), wherein a hydrogel is used as a carrier to carry the silicate-induced extracellular vesicles (CSEV) therein, so that the preparation can achieve the effect of sustained release of the silicate-induced extracellular vesicles (CSEV).

[0072] The hydrogel may be a three-dimensional network structure formed by cross-linking materials such as collagen, gelatin, hyaluronic acid or chitosan.

[0073] The crosslinking may be physical crosslinking or chemical crosslinking. The physical crosslinking may be achieved by Van der Waals force, ionic attraction, polymer chain entanglement and the like, while the chemical crosslinking may be achieved by covalent bond principle. In this embodiment, the hydrogel is made of collagen as described in the subsequent embodiments.

[0074] The method of carrying the silicate to induce extracellular vesicles in the hydrogel is not limited. In this embodiment, 5x10 9 The silicate-induced extracellular vesicles were mixed with 1 ml of the sterile hydrogel to form a mixed solution, and 50 μL of the mixed solution was extracted into a culture dish and reacted at 37° C. for 30 minutes to perform a gelation reaction.

[0075] The mixed solution may also be subjected to a gelling reaction by adding a photosensitizer or the like through a photocuring technology.

[0076] Experiment 2, compare the release effect of the hydrogel after carrying the common extracellular vesicles and the silicate-induced extracellular vesicles. The common extracellular vesicles and the silicate-induced extracellular vesicles were respectively reacted with the hydrogel and added to PBS for culture, and the liquid in the culture dish was extracted after 0, 1, 2, 3, 5, 7, 10, and 14 hours (h) of culture, and the number of extracellular vesicles in the liquid was measured by NTA to estimate the release amount of the extracellular vesicles. It can be confirmed that the silicate-induced extracellular vesicles secreted by silicate does not affect its release rate in the hydrogel.

[0077] Experiment 3: Compare the effects of the hydrogel loaded with the common extracellular vesicles and the silicate-induced extracellular vesicles on the growth of the diabetic cell model. Figure 6 as well as Figure 7First, the common extracellular vesicles and the silicate-induced extracellular vesicles loaded with the hydrogel were solidified in a culture dish, and then the diabetic cell model was cultured on the hydrogel loaded with the common extracellular vesicles and / or the silicate-induced extracellular vesicles after solidification at a number of 10,000 cells / well, and the serum-free DMEM was added for culture, and the cell proliferation rate was analyzed on the 1st, 3rd, and 7th days respectively. Among them, the group of the hydrogel was used as the first control group (Ctl), the group of the hydrogel loaded with the common extracellular vesicles was used as the second control group (EV), and the group of the hydrogel loaded with the silicate-induced extracellular vesicles was used as the experimental group (CSEV).

[0078] Depend on Figure 7 The results show that the number of cells in the diabetic model cultured with the hydrogel (CSEV) carrying the silicate-induced extracellular vesicles was greater than that of the first control group (Ctl) and the second control group (EV) from the third day. In particular, a significant difference was observed after the seventh day of culture. This result is consistent with the results in Experiment 1. Figure 5A The results echoed each other, confirming that the silicate-induced extracellular vesicles (CSEV) can induce cell proliferation in the diabetic cell model.

[0079] Experiment 4 compared the effects of the hydrogel loaded with the common extracellular vesicles and the silicate-induced extracellular vesicles (CSEV) on the expression of various index factors in the diabetic cell model. Among them, each index factor includes inflammation-related factors TNG-alpha, IL-1 and IL-6; tissue regeneration-related factors Col I, Angiogenin, EGF, FGF-2 and PDGF-BB; anti-inflammatory-related factors IL-8 and IL-10; and collagen factor (ColI) as a wound healing indicator.

[0080] The diabetic model cells were cultured in the hydrogel as the first control group (Ctl), the hydrogel carrying the ordinary extracellular vesicles as the second control group (EV), and the hydrogel carrying the silicate-induced extracellular vesicles as the experimental group (CSEV). The cells were collected and the protein was extracted for quantification. 20 μg of protein was taken out from each group using the cytokine array technology, and the expression amount of each indicator factor in the first control group (Ctl) was used as the baseline value to analyze the second control group (EV) and the experimental group (CSEV).

[0081] Depend on Figure 8The results showed that compared with the second control group (EV), the expression of inflammation-related factors TNG-alpha, IL-1 and IL-6 in the diabetic cell model cultured by the experimental group (CSEV) was significantly reduced; the related factors Col I, Angiogenin, EGF, FGF-2 and PDGF-BB that help tissue regeneration were expressed in large quantities, and the expression of anti-inflammatory related factors IL-8 and IL-10 was also increased, and the expression of collagen factor (Col I) that helps wound healing was also increased. From this result, it can be concluded that the silicate-induced extracellular vesicles (CSEV) can induce the expression of index factors such as anti-inflammatory, tissue regeneration and cell repair in the diabetic model cells.

[0082] RNA sequencing was further used to compare the expression of microRNA (miRNA) in the diabetic model cells cultured in the first control group (Ctl) and the experimental group (CSEV). The small fragment RNA was separated by RNAiso (TaKaRa, Japan) and the subsequent sequence analysis was completed by Shanghai OE Biotech Co., Ltd. The miR-Base v.21 database (http: / / www.mirbase.org / ) was used to compare and identify miRNAs that contribute to angiogenesis, anti-inflammation, and wound healing. The differential expression analysis (DEG) algorithm in R language was used to calculate the miRNAs with significant differences (p<0.05), and three independent repeated experiments were performed for each group. Then, miRanda software was used to predict the targets of miRNAs with significant expression in the animal body with parameters S≥150, ΔG≤-30kcal / mol and 5' primer pairing. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were subsequently used to analyze the gene locations of different miRNA targets.

[0083] By analyzing the log2 fold change in the data, we can get the ratio between the standardized signal values ​​and compare them. Figures 9A to 9CIn the experiment group (CSEV), the diabetic model cells cultured were compared with the expression of each miRNA in the first control group (Ctl). It is worth noting that the diabetic model cells cultured in the experiment group (CSEV) showed significant expression of the index miRNA-31 in angiogenesis, anti-inflammation and wound healing, confirming that the silicate-induced extracellular vesicles (CSEV) can induce the diabetic model cells to produce the expression of index genes (miRNA) such as anti-inflammation, tissue regeneration and wound healing.

[0084] In order to confirm the effects of silicate-induced extracellular vesicles (CSEV) on cell proliferation, recovery and anti-inflammation of real wounds, the present invention further uses a diabetic animal model commonly used in wound healing, cell proliferation and recovery studies as a test target.

[0085] The establishment of the diabetic animal model refers to the experimental steps and methods in the literature, and the experimental animals are induced to produce diabetes by using the chemical agent alloxan monohydrate. The principle of alloxan inducing diabetes is to selectively destroy the beta cells in the pancreas that secrete insulin, resulting in a decrease in insulin content. The alloxan monohydrate used (≥

[0086] 98%, Sigma Aldrich Chemical, Saint Louis, MO, USA) is in powder form. Because it is easy to deteriorate when in contact with air, it is sealed and stored in a refrigerator at a temperature of 2 to 8 degrees Celsius. The drug preparation method is to mix 5.26 grams of the drug with 100 ml of sterile saline to obtain a 5% reagent (pH = 7), filter it using a 0.22μm filter membrane (the above process is performed on ice), use it immediately and take intravenous injection (dose: 100mg / kg). Measure blood sugar once every two days. The goal is to keep its blood sugar above 300mg / dl throughout the experiment. If it is less than one week, give alloxan.

[0087] Experiment 5: Comparison of the effect of silicate-induced extracellular vesicles (CSEV) on wound repair in diabetic animal models. After the diabetic animal model was successfully established, a 1.5 cm 2 The wound was made to a depth reaching the muscle layer as a malignant wound model.

[0088] The group without any treatment was used as the first control group (Ctl), the group using only the hydrogel as a wound dressing was used as the second control group (Col), the wound dressing made of the hydrogel carrying the ordinary extracellular vesicles was used as the third control group (Ev@Col), and the wound dressing made of the hydrogel carrying the silicate-induced extracellular vesicles was used as the experimental group (CSEV@Col). In the second and third weeks after the start of treatment, the wound tissue was removed along the edge of the wound and fixed in 10% formalin. After that, it was paraffin-embedded and made into 6μm tissue sections for hematoxylin-eosin (H&E) staining and picrosirius red staining (PRS). H&E staining can be used to observe and quantitatively analyze the thickness of the wound epithelial tissue, the amount of granulation tissue, and whether the structure of mature skin (such as hair follicles and sebaceous glands) is generated. By using picrosirius red staining, the amount and arrangement of collagen fibers in the wound can be compared, and the repair status of the wound tissue can be evaluated.

[0089] like Fig.10 The results show that the wounds of the experimental group (CSEV@Col) have become completely flat since the second week compared to the other groups. In addition, PRS staining shows that the collagen fibers in the dermis proliferate and are secreted in large quantities by dermal fibroblasts to fill the wound defects and are evenly distributed in red at the wound site (as indicated by arrow A).

[0090] In the third week, the complete muscle texture in the muscle layer was observed in PRS staining (as indicated by arrow B). Through H&E staining, it can be clearly observed that the hair follicles were generated and neatly arranged in the wound tissue after repair (as indicated by arrow C). This proves that the silicate-induced extracellular vesicles (CSEV) contribute to the cell growth and wound repair of the wound tissue in the diabetic animal model.

[0091] The above experimental results can be combined to prove that the silicate-induced extracellular vesicles provided by the present invention have extremely high biosafety and can help wound repair, thereby achieving the effect of promoting wound healing and repair.

Claims

1. An extracellular vesicle that helps cell repair, characterized in that: It is formed by secretion of stem cells induced by silicates.

2. The extracellular vesicles that help cell repair according to claim 1, characterized in that The stem cells are cultured in a silicate culture medium to which a silicate component is added.

3. The extracellular vesicles that help cell repair according to claim 2, characterized in that The stem cells include human adipose stem cells, bone marrow stem cells, dental pulp stem cells or Wharton's jelly mesenchymal stem cells.

4. The extracellular vesicles that aid cell repair according to claim 1, characterized in that The silicate is calcium silicate.

5. The extracellular vesicles that help cell repair according to claim 1, characterized in that The silicate culture medium comprises a culture medium, and the culture medium comprises one of mesenchymal stem cell culture medium (MSCM) and Dulbecco's Modified Eagle Medium (DMEM).

6. The extracellular vesicles that aid cell repair according to claim 1, characterized in that Contains a membrane surface potential less than -15mV.

7. The extracellular vesicles that aid cell repair according to claim 6, characterized in that The membrane surface potential is between -20mV and -30mV.

8. The extracellular vesicles that aid cell repair according to claim 1, characterized in that Contains a total protein content higher than 4μg / 10 9 .

9. The extracellular vesicles that aid cell repair according to claim 1, characterized in that At least 40% of the extracellular vesicles express at least one of a transmembrane protein CD9, a transmembrane protein CD63 or a transmembrane protein CD81.

10. A method for preparing extracellular vesicles that aid in cell repair, characterized in that: The extracellular vesicles are formed by secretion of a stem cell induced by silicate, and the steps include: Obtain 1 g of calcium silicate and add it to 10 to 100 ml of a culture solution for at least 24 hours, and collect the clarified liquid to prepare a silicate culture solution; culturing the stem cells in the silicate culture medium; The extracellular vesicles secreted by the stem cells are isolated.

11. The method according to claim 10, characterized in that: The culture medium includes one of mesenchymal stem cell culture medium (MSCM) and Dulbecco's Modified Eagle Medium (DMEM).

12. A preparation for assisting cell repair, characterized in that: The invention comprises an extracellular vesicle formed by secretion of a stem cell induced by silicate.

13. A preparation for assisting cell repair, characterized in that: The invention comprises a hydrogel carrying an extracellular vesicle formed by secretion of a stem cell induced by silicate.

14. The preparation for assisting cell repair according to claim 13, characterized in that: The ratio of the extracellular vesicles (CSEV) in the hydrogel is between 10 9 -10 14 particles / mL.

15. Use of a preparation for wound repair, characterized in that: The invention comprises an extracellular vesicle formed by secretion of a stem cell induced by silicate.

16. The use as claimed in claim 15, characterized in that The wounds repaired by this preparation are difficult-to-treat wounds caused by diabetes.

Images